Antibody and Antigen in Heparin-Induced Thrombocytopenia

Peter M. Newman

Submitted as part of the requirement for the degree of

Doctor of Philosophy

School of Pathology

University of New South Wales

1999 Table of Contents

Table of Figures...... 7

Acknowledgments ...... 10

Publications arising from this thesis...... 11

Abbreviations ...... 12

Summary...... 15

Chapter 1 - Literature Review ...... 17

Heparin-induced thrombocytopenia ...... 17 Historical aspects ...... 17 Type I HIT...... 19 Type II HIT...... 20 Clinical observations...... 21 Thrombocytopenia ...... 21 Incidence of HIT ...... 21 Thrombosis...... 23 Clinical diagnosis...... 25 Normal platelet physiology ...... 27 Pathophysiology of HIT ...... 29 An immune basis for HIT ...... 29 A HIT antibody ...... 30 Role of the platelet FcγRII in HIT...... 30 Fc-receptor polymorphism ...... 31 Heparin is not the HIT antigen...... 32 Platelet HIT antigens...... 33 HIT antibodies against PF4-heparin...... 34 Are anti-PF4-heparin antibodies pathogenic?...... 37 HIT antibodies against IL-8, NAP-2 ...... 38 Epitopes on PF4 ...... 38 Role of T cells in HIT ...... 40 Mechanism of thrombosis...... 40 2 A paradigm for HIT...... 42 Dissenting views ...... 45 Laboratory diagnosis of HIT ...... 46 Platelet aggregation...... 46 14C-Serotonin Release Assay ...... 48 Variations of functional assays eg. HIPAA, Platelet Microparticles ...... 48 ELISA...... 49 Comparisons of HIT assays...... 50 Prevention of HIT...... 51 Prevention through early diagnosis ...... 51 Prevention by using alternative anticoagulants...... 51 Treatment of HIT ...... 52 Treatment with LMWH...... 53 Treatment with Oral Anticoagulants...... 53 Treatment with Orgaran ...... 54 Treatment with Ancrod ...... 55

Heparin ...... 57 Clinical use of heparin ...... 57 Other side effects...... 58 Structure and synthesis of heparin...... 58 Mode of anticoagulant action...... 61 Pharmacokinetics of heparin ...... 62 Effect of heparin on platelets ...... 63

PF4...... 64 PF4 in the Plasma...... 65 Plasma Kinetics of PF4 ...... 65 Physiological role of PF4 ...... 66 PF4 in Inflammation and Immune regulation ...... 66 PF4 and coagulation...... 67 Regulation of Platelet production by PF4 ...... 68 Platelet associated PF4...... 68 Endothelial associated PF4 ...... 69

3 PF4 and Angiogenesis...... 70 Structure of PF4 ...... 70

Interaction between PF4 and heparin...... 74 In vitro molecular interactions ...... 74 In vivo release of PF4 by heparin...... 76

Other drug-induced and immune thrombocytopenias ...... 77 Quinine/quinidine induced thrombocytopenia (QIT)...... 77 Idiopathic/Immune thrombocytopenic purpura (ITP) ...... 78 Insights into the HIT antigen offered by the antiphospholipid syndrome...... 78

Key Questions...... 80 Why does HIT only occur in a few percent of patients? ...... 80 How does the HIT antibody bind and activate platelets?...... 81 What is the best way to diagnose HIT?...... 81 What is the best way to treat HIT?...... 81 Are the anti-PF4-heparin antibodies pathogenic? ...... 82 What is the HIT epitope(s) ? ...... 82

Chapter 2 - IgG binding to PF4-heparin complexes in the fluid phase and cross-reactivity with low molecular weight heparin and heparinoid...... 83 Introduction ...... 83 Methods...... 84 Patients...... 84 Purification of PF4 ...... 85 Biotinylation of PF4...... 85 SDS-PAGE...... 86 Coomassie blue stain...... 86 Colloidal coomassie blue stain...... 87 Electrotransfer to membranes from SDS-PAGE gels ...... 87 Fluid-Phase Enzyme Immunoassay (EIA) ...... 87 Heparin-PF4 Antibody ELISA...... 89 Functional Assays ...... 90

4 Results...... 91 PF4 Purification ...... 91 Biotinylation of PF4...... 92 Fluid phase EIA...... 93 Heparin-PF4 Antibody ELISA...... 95 Effect of heparins on the coating of PF4 to microtitre wells ...... 96 Cross-reactivity ...... 97 Clinical outcomes of HIT patients treated with danaparoid...... 100 Discussion ...... 101

Chapter 3 - Further characterisation of antibody and antigen in HIT 107 Introduction ...... 107 Methods...... 108 Patients...... 108 ELISA...... 108 Fluid-phase Enzyme Immunoassay...... 109 Depletion of HIT antibodies...... 109 Total IgG and anti-adenovirus IgG assays ...... 110 Affinity purification of HIT anti-PF4-heparin IgG ...... 110 Purification of Normal IgG ...... 111 Micro-dialysis ...... 111

Preparation of F(ab’)2...... 112 Preparation of Fab...... 112 Iodination of Proteins...... 114 Binding studies of anti-PF4-heparin IgG ...... 114 Results...... 117 HIT antibodies recognise PF4-heparin and PF4 alone in ELISA...... 117 Effect of sulfoMBS activation of Covalink ...... 118 HIT IgG does not bind to native PF4 in the fluid-phase ...... 122 Depletion of HIT plasma by heparin-PF4-agarose and PF4-agarose...... 124

Purity of affinity-purified HIT IgG, Fab and F(ab’)2 ...... 129 Importance of antibody bivalency in binding of HIT IgG to PF4-heparin...... 131 Binding studies of affinity purified anti-PF4-heparin IgG...... 133

5 Discussion ...... 135

Chapter 4 - Binding of purified anti-PF4-heparin antibodies to platelets and the resulting platelet activation...... 145 Introduction ...... 145 Methods...... 148 Purification of HIT IgG...... 148 IgG binding during platelet aggregation...... 149 Recording platelet aggregation...... 150 PF4 release and expression during platelet activation ...... 153 PF4 Assay...... 153 Inhibition of HIT IgG binding to activated platelets by sheep anti-PF4 ...... 154 HIT IgG binding to thrombin activated platelets ...... 155 Results...... 155 HIT IgG binding during platelet aggregation...... 155 Role of platelet activation ...... 158 Role of platelet FcγRII ...... 159 Specificity of affinity purified HIT IgG ...... 164 Release of PF4 from platelets ...... 165 Increased expression of PF4 on the platelet surface following activation ...... 166 Role of heparin...... 167 Discussion ...... 169

Chapter 5 - Overview...... 175

Appendix 1 - Technical details of amplifier and ADC...... 180 Linearity of amplifier ...... 180 Circuit diagram...... 181

References...... 184

6 Table of Figures

Figure 1 - Models of platelet activation by HIT IgG ...... 44 Figure 2 - Typical platelet aggregation profile caused by HIT plasma and heparin..... 47 Figure 3 - Fundamental sugar moieties of heparin ...... 59 Figure 4 - Structure of heparin ...... 60 Figure 5 - Tertiary and quaternary structure of PF4 ...... 73 Figure 6 - Space-filling model of human PF4 showing putative heparin-binding and HIT antibody-binding sites...... 76 Figure 7 - Schematic representation of the fluid-phase assay...... 88 Figure 8 - SDS-PAGE demonstrating purification of PF4 ...... 91 Figure 9 - Antibody confirmation of PF4 ...... 92 Figure 10 - Successful biotinylation of PF4 ...... 93 Figure 11 - Optimisation of heparin/LMWH/heparinoid concentration in fluid-phase EIA ...... 94 Figure 12 - Fluid-phase EIA for HIT antibodies ...... 95 Figure 13 - ELISA for HIT antibodies ...... 96 Figure 14 - Effect of different heparin-like anticoagulants on the binding of PF4 to microtitre wells...... 97 Figure 15 - Cross-reactivity of HIT antibodies with PF4 complexed to heparin-like anticoagulants...... 98 Figure 16 - Correlation of HIT antibody cross-reactivity ...... 99 Figure 17 - Serial platelet counts of representative HIT patients treated with danaparoid (Orgaran)...... 101 Figure 18 - Micro-dialysis unit...... 112 Figure 19 - Miniature protein A column...... 113 Figure 20 - Time-course for the binding of affinity-purified HIT IgG to PF4±heparin115 Figure 21 - ELISA ...... 118 Figure 22 - Reactions involved in covalently crosslinking a protein to Covalink microtitre wells using sulfoMBS...... 120 Figure 23 - Effect of sulfoMBS activation of ELISA wells on binding of HIT antibody to PF4 and PF4-heparin ...... 121 Figure 24 - Fluid-phase EIA...... 123

7 Figure 25 - Effect of varying PF4 concentration on the fluid-phase EIA...... 124 Figure 26 - Depletion of anti-PF4-heparin antibodies...... 125 Figure 27 - Elution of HIT anti-PF4-heparin antibody from PF4-agarose ...... 126 Figure 28 - Depletion of antibodies recognising solid-phase PF4 without heparin .... 127 Figure 29 - Non-specific depletion of total IgG and anti-adenovirus IgG by agarose beads...... 128 Figure 30 - Depletion of protein G purified HIT IgG by agarose-PF4±heparin ...... 129

Figure 31 - SDS-PAGE of HIT anti-PF4-heparin IgG, Fab and F(ab’)2...... 130 Figure 32 - IgG Fragmentation ...... 131

Figure 33 - Binding of HIT IgG, Fab and F(ab’)2 to PF4-heparin ...... 132 Figure 34 - Equilibrium binding isotherm of affinity purified HIT IgG to PF4±heparin ...... 133 Figure 35 - Competitive inhibition of affinity purified HIT 125I-IgG binding by unlabelled IgG...... 134 Figure 36 - Model for the effect of IgG bivalency and antigen immobilisation on HIT antibody binding to PF4...... 139 Figure 37 - Representation of HIT IgG binding to PF4-heparin...... 141 Figure 38 - Models for the binding of HIT IgG to platelets...... 146 Figure 39 - Recording of platelet aggregation ...... 150 Figure 40 - Photograph of Amplifier and analog to digital converter ...... 152 Figure 41 - Binding of affinity purified anti-PF4-heparin HIT IgG to platelets during heparin-induced platelet aggregation...... 157 Figure 42 - Binding of HIT 125I-IgG to platelets without unlabelled HIT IgG ...... 159 Figure 43 - Effect of blocking platelet Fc receptor on the binding of HIT IgG during platelet aggregation...... 161

Figure 44 - Binding of F(ab’)2 to platelets aggregated with collagen plus heparin .... 162 Figure 45 - Inhibition of HIT-induced platelet aggregation by IV.3 ...... 163 Figure 46 - Effect of Fc receptor blockade on the binding of HIT IgG to thrombin activated platelets...... 164 Figure 47 - Inhibition of HIT IgG binding to platelets by anti-PF4...... 165 Figure 48 - Release of PF4 following platelet activation...... 166 Figure 49 - Expression of PF4 on the platelet surface following activation...... 167

8 Figure 50 - Heparin dependence in the binding of HIT IgG to collagen aggregated platelets...... 168 Figure 51 - Dynamic model of platelet activation by HIT IgG...... 174 Figure 52 Calibration curve for amplifier and analog to digital converter...... 181 Figure 53 - Schematic Diagram of Amplifier Circuit ...... 183

9 Acknowledgments

I would like to thank my supervisor Professor Beng Chong who provided guidance and encouragement but also let me take my own path. I am grateful to all the staff and students at the Centre for Thrombosis and Vascular Research for their friendship and making my time there so enjoyable. Drs Philip Hogg and Kathy Quinn provided valuable practical advice on experiments and techniques.

There are many people and organisations without whom this project could not have been undertaken. Specifically, I would like to thank the HIT patients who provided plasma and their referring doctors; the NSW Red Cross Blood Transfusion Service who donated expired platelet packs for the purification of PF4; the haematology laboratory at Prince of Wales Hospital for running platelet counts and Ms Sue Evans who performed routine HIT assays.

There are others who made my life a lot easier. Ms Rebecca Swanson generously performed some of the fluid-phase assays; Mrs Bernadette O’Reilly, as Prof. Chong’s secretary, was always most helpful.

This research was supported by a grant from the National Health and Medical Research Council (Australia). I received an Australian Postgraduate Award (Industry) scholarship from the Australian government in conjunction with Gradipore Ltd.

Last, but not least, I would like to thank Gina McCredie for all her support and encouragement.

10 Publications arising from this thesis

Newman, P.M., R.L. Swanson, and B.H. Chong. (1998). Heparin-induced thrombocytopenia: IgG binding to PF4-heparin complexes in the fluid phase and cross-reactivity with low molecular weight heparin and heparinoid. Thromb. Haemost. 80: 292-297.

Newman, P.M. and B.H. Chong. (1999). Further characterisation of antibody and antigen in heparin-induced thrombocytopenia. Br. J. Haematol. 107: 303-309.

Newman, P.M. and B.H. Chong. (2000). Heparin-induced thrombocytopenia: New evidence for the dynamic binding of purified anti-PF4-heparin antibodies to platelets and the resulting platelet activation. Blood. 96: 182-187.

11 Abbreviations

ACD acid citrate dextrose anticoagulant ADC analogue to digital converter ADP adenosine diphosphate APC activated protein C aPL antiphospholipid (+ protein) antibody APS antiphospholipid (+ protein) antibody syndrome APTT activated partial thromboplastin time ATIII antithrombin III

β2GPI β2-glycoprotein I βTG β thromboglobulin BSA bovine serum albumin DAG 1,2-diacylglycerol DVT deep vein thrombosis EDTA ethylenediaminetetraacetic acid EIA enzyme immunoassay ELISA enzyme-linked immuno-sorbent assay

ETP EDTA + theophylline + PGE1 anticoagulant

F(ab’)2 divalent antigen binding fragment of IgG Fab monomeric antigen binding fragment of IgG Fc complement binding fragment of IgG FcγRII platelet Fc receptor for IgG. Also known as CD32. FITC fluorescein isothiocyanate GAG glycosaminoglycan GM-CSF granulocyte macrophage colony stimulating factor GPIa/IIa glycoprotein Ia/IIa complex GPIb/IX/V glycoprotein Ib/IX/V GPIIb/IIIa glycoprotein IIb/IIIa HBS HEPES buffered saline HCII heparin cofactor II HEPES N-[2-Hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid]

12 HIPA or HIPAA heparin-induced platelet activation assay HIT immune (type II) heparin-induced thrombocytopenia HLA human leucocyte antigen HRP horseradish peroxidase enzyme IL-8 interleukin 8

IP3 inositol 1,4,5-triphosphate ITP idiopathic/immune thrombocytopenic purpura IV.3 a monoclonal antibody specific for FcγRII Kd dissociation constant kD kilodalton LMWH low molecular weight heparin mAb monoclonal antibody NAP-2 neutrophil activating peptide 2 NHS N-hydroxysuccinimide NMR nuclear magnetic resonance OD optical density PBMC peripheral blood mononuclear cells PBS phosphate buffered saline PBS-tween phosphate buffered saline with 0.05% tween-20 detergent PF4 platelet factor 4 PF4-heparin complex of platelet factor 4 and heparin PF4±heparin PF4 either with or without heparin

PGD2 prostaglandin D2

PGE1 prostaglandin E1

PGI2 prostaglandin I2

PIP2 phospatidylinositol bisphosphate PIPES 1,4-Piperazinediethanesulfonic acid PPP platelet poor plasma PRP platelet rich plasma PVDF polyvinylidene difluoride QIT quinine/quinidine induced thrombocytopenia RIA radioimmunoassay SD standard deviation 13 SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SE standard error SRA 14C-serotonin release assay for HIT TCR T-cell receptor TMB 3,3’,5,5’-tetramethylbenzidine Tris Tris(hydroxymethyl)aminomethane UH unfractionated heparin vWF von Willebrand factor

14 Summary

Immune heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy and is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. Early diagnosis of HIT is important to reduce morbidity and mortality. I developed an enzyme immunoassay that detects the binding of HIT IgG to PF4-heparin in the fluid phase. This required techniques to purify and biotinylate PF4. The fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. I was able to detect anti-PF4-heparin IgG in 93% of HIT patients. I also investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 88% of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration.

While HIT patients possess antibodies to PF4-heparin, I observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4- heparin HIT IgG, I provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd=7-30nM) and polystyrene adsorbed PF4 alone (Kd=20-70nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. I conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and thus most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (sub-optimal) promotes recognition of this epitope.

15 Under conditions that are more physiological and sensitive than previous studies, I observed that affinity-purified HIT IgG will cause platelet aggregation upon the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. I quantitated the binding of affinity-purified HIT 125I-IgG to platelets as they activate in a plasma milieu. Binding of the HIT IgG was dependent upon heparin and some degree of platelet activation. Blocking the platelet Fcγ receptor-II with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. I conclude that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to induce platelet aggregation. The Fab region of HIT IgG binds to PF4-heparin that is on the surface of activated platelets. I propose that only then does the Fc portion of the bound IgG activate other platelets via the Fc receptor. My data support a dynamic model of platelet activation where released PF4 enhances further antibody binding and more release.

16 Chapter 1 - Literature Review

Heparin-induced thrombocytopenia

Heparin induced thrombocytopenia (HIT) is a potentially serious side-effect of heparin therapy. It is associated with, and probably caused by, antibodies directed against complexes of a platelet protein and heparin. Paradoxically, despite having low platelet counts, patients with HIT are at increased risk of life-threatening thrombosis. Laboratory diagnosis is based on the ability of HIT plasma to activate normal platelets in the presence of heparin or by detection of the pathogenic antibodies with ELISA. Treatment of HIT primarily involves cessation of heparin therapy and administration of an alternative anticoagulant.

Historical aspects

Early experiments concerning the effect of heparinisation on platelets produced conflicting reports. In 1930 Howell and Macdonald reported that six successive days of heparinisation in a dog had no effect on platelet counts 1. In the 1940’s a number of reports indicated that a single dose of heparin can cause an immediate but transient thrombocytopenia in dogs 2-4. Whether this effect also occurred in humans was disputed 3,4 Gollub and Ulin 5 provided convincing evidence that it did but Davey and Lander 6 suggested that it was an in vitro artefact because the reduction in platelet counts was not reflected in the levels of circulating radiolabelled platelets. These early studies did not view the thrombocytopenia caused by heparin as being a particularly significant problem. It was of interest because it may help to understand the anticoagulant mechanism of heparin or explain why excessive bleeding was a common side effect.

In a clinical setting, thrombocytopenia associated with heparinisation was first reported in the early sixties, in patients undergoing heart surgery requiring extra-corporeal circulation 7-9. However, the thrombocytopenia may have been a result of the blood being traumatised by the perfusion equipment and platelets adhering to foreign surfaces during the perfusion procedure 8. Thrombocytopenia also correlated with hypothermia 7

17 so in these reports it is difficult to directly attribute it to heparin. In 1969 Natelson et al. 10 described a patient who developed thrombocytopenia 1-2 weeks into heparinisation for pulmonary thromboemboli. Concurrently, the patient was diagnosed with consumption coagulopathy (disseminated intravascular coagulation) which was attributed to his prostate adenocarcinoma. The platelet count increased when heparin was withdrawn and fell when re-administered. A crude in vitro assay using the patients platelet rich plasma, indicated that heparin reduced the platelet count. However, the mechanism for the heparin induced thrombocytopenia remained elusive.

The first report of thrombotic complications of heparin therapy was made by Weismann and Tobin in 1958 11. They reported 10 cases where patients developed arterial thromboembolism while receiving heparin. The embolisms appeared 7-15 days into heparin therapy. When removed surgically, the emboli were pale/grey in colour and composed of fibrin, platelets and leucocytes but almost devoid of erythrocytes. The size and shape of the emboli suggested that the origin was in the aorta. Weismann and Tobin presumed that heparinisation dislodged a pre-existing aortal thrombi. They did not consider the possibility that heparin may have caused the thrombi. While they recognised the desirability of stopping heparin therapy, they believed that heparinisation needed to continue to prevent further thrombosis. They proposed lowering the dose of heparin to prevent the dislodgment of any thrombi. Weismann and Tobin made no mention of thrombocytopenia in the patients they describe.

Rhodes et al. 12 were the first to report a link between thrombosis and delayed-onset thrombocytopenia which developed in two men receiving heparin. Both patients initially responded to heparin with increased clotting times. However, heparin resistance developed despite increasing dosages and culminated in myocardial infarction and thrombocytopenia after 8-10 days of heparinisation. The patients remained hypercoagulable despite continuation of heparin therapy. Only when heparin was discontinued did the thrombocytopenia resolve. When the patients were re-challenged with heparin, two weeks later, thrombocytopenia rapidly returned.

Rhodes et al. believed that the mechanism for the delayed-onset heparin-induced thrombocytopenia was immunological because fractionation of serum from one patient identified a complement-fixing IgG antibody as the causative agent 12. They also

18 recognised that this was unlikely to be the same mechanism that produced the acute transient thrombocytopenia observed by others.

During the latter half of the 1970’s there were many reports describing thrombocytopenia with thrombosis associated with heparin therapy 13-20. Frequently, withdrawal of heparin coincided with a return to normal platelet counts while continued heparinisation was often fatal. However, there were also instances where thrombocytopenia developed during heparin therapy but spontaneously resolved despite maintenance of the same heparin dose 21-26. This confusion began to clarify when Nelson et al. 21 recognised two types of heparin induced thrombocytopenia. A “common mild thrombocytopenia” occurred 2-4 days into heparin therapy and resolved spontaneously despite continued heparinisation. In contrast, a less common “sporadic severe thrombocytopenia” occurred after at least a week of heparin and was characterised by recurrent thrombosis. Chong 27 proposed that the mild transient thrombocytopenia be called Type I heparin-induced thrombocytopenia, while the severe disease be called Type II heparin-induced thrombocytopenia.

This thesis will focus on the more serious, Type II HIT. Clearly, however, distinguishing between the mild and severe forms requires an understanding of both.

Type I HIT

Type I HIT, also known as non-immune HIT, is a common and mild thrombocytopenia caused by heparin that resolves despite continued heparinisation. The thrombocytopenia develops after 2-5 days of heparin therapy 21,25,26. Platelet counts are typically between 150x109/l (the bottom of the normal range) and 100x109/l 21,25,28. Platelet levels return to normal in 1-5 days, even with continued heparin administration 21,29,30. The incidence of Type I HIT is difficult to estimate from the literature. Reasons for this include differing definitions of thrombocytopenia, inconsistencies in the frequency of platelet counts and the failure of many early reports to distinguish between Type I and Type II HIT. When Type I HIT (mild and transient thrombocytopenia) can be identified, its incidence still varies from 2% to 31% 15,21,25,31-33. 15% would appear to be a reasonable estimate.

The mechanism of Type I HIT is thought to be due to the direct action of heparin on platelets 28,34,35 and the early reports of thrombocytopenia following a single 19 experimental dose of heparin 2-5 are often cited as evidence. However, such experiments produced immediate thrombocytopenia which lasted only a few hours4,5. This immediate thrombocytopenia has not been widely reported in a clinical setting, probably because platelet counts are rarely performed straight after heparin injection in humans 35. Nevertheless, a weak pro-aggregating effect of heparin on platelets has been well documented and heparin enhances the pro-aggregating effects of some platelet activators 34,36-38. Perhaps a few days of heparinisation is required to sufficiently deplete platelets for an observable Type I thrombocytopenia or an underlying condition (eg. infection, trauma) may causes the release of platelet aggregating factors which act in concert with heparin. Platelets from patients requiring hospitalisation in an intensive care unit are likely to show greater aggregation due to heparin than platelets from healthy individuals 36. It has also been suggested that Type I HIT may be an artefact, due to heparin inducing platelet aggregates which are counted as a single cell by an automated platelet counter 29,39,40. However, this has been excluded as a cause in some studies by also counting direct blood smears 15.

Patients with Type I HIT are asymptomatic and so require no specific treatment 28. However, careful monitoring is advisable to confirm resolution of thrombocytopenia and ensure that it is not, the more serious, Type II HIT.

Type II HIT

Type II HIT is also known as immune HIT41, heparin associated thrombocytopenia (HAT) 42,43, heparin induced thrombocytopenia (HITP) 44,45, heparin-induced thrombocytopenia/ thrombosis (HITT) 46-48, heparin-induced thrombocytopenia syndrome (HITS) 49-51, and white clot syndrome 52,53. I shall follow the suggestion of Warkentin et al. 54 and use the simple abbreviation “HIT” to refer to this severe form of the disease and only explicitly refer to it as “Type II” or “immune” HIT when required to distinguish it from Type I HIT.

20 Clinical observations

Thrombocytopenia HIT has occurred in patients given heparin for both the treatment and prophylaxis of thrombosis 46,52,55,56. The thrombocytopenia in HIT typically develops 6-14 days into heparin therapy 16,20,30,33,57-60 but may occur more quickly, particularly if there has been previous exposure to the drug 20,58,59. Platelet counts are often below 60x109/l 61, although it is also possible for platelet counts to be within the normal range but to have dropped substantially from pre-heparin levels. In fact, there is a variety of definitions as to what constitutes thrombocytopenia. Some authors define thrombocytopenia as platelet counts below 150x109/l 26,33,56, others use a more stringent value of 100x109/l 15,20,22,59,62 or may also include situations where counts drop by 50% 54. There are even reports of vascular surgery patients who apparently developed HIT without a substantial fall in platelets counts and whose platelets remained above 150x109/l 63. These patients developed unexplained thrombotic complications while receiving heparin and possessed HIT antibodies when assessed by HIPA and 14C-serotonin release assays. The degree of thrombocytopenia is typically more severe in case reports than prospective studies 64, which presumably reflects a tendency to publish more extreme cases. Interestingly, the degree of thrombocytopenia in HIT does not appear to be related to the dose of heparin 15,16,27. Although, higher doses of heparin may be associated with increased risk of thrombosis complicating HIT 58. Platelet production is presumed to be normal or elevated based on the observation that megakaryocytes are normal or elevated 18,35. The thrombocytopenia is thought to be caused by shortened platelet survival 18 that occurs when activated platelets form aggregates and are removed by the spleen or reticuloendothelial system. Although, significant thrombocytopenia can still occur in patients who have had their spleen removed 65.

Incidence of HIT There is considerable variation in the reported incidence of HIT 61. Reasons for this are unclear but may, in part, be caused by differing definitions of thrombocytopenia 66, failure to distinguish Type I HIT from Type II 30,60,67, different batches/sources of heparin 61 and more recently the use of ELISA to detected HIT antibodies without clinical symptoms 46,56. Currently, the best estimate of the frequency of HIT caused by 21 unfractionated heparin is about 3%. This is based on a review of the literature by Warkentin and Kelton 68, and a large prospective study by Warkentin et al. 33 where HIT was defined as two consecutive platelet counts below 150x109/l occurring after 5 days of heparin and was confirmed with a 14C-serotonin release assay. The risk of HIT does not appear to be related to age, sex or race 62.

HIT is not only observed with high dose heparin (20,000-40,000 U/day) but also with low dose heparin (5,000 U heparin every 8-12 hrs) 44,58,69-71 and when doses as low as 250-500 U/day are used to flush catheters 58,72-74. The lower doses of heparin appear to be associated with a lower risk of HIT and milder thrombocytopenia 75 although this is contradicted by others 59.

It has been suggested that bovine lung heparin is more likely to cause HIT than heparin from porcine mucosa 22,26,30,55,75,76. However, the small number of HIT cases observed in prospective clinical trials means that the differences are rarely statistically significant 39,61,77,78, particularly when thrombocytopenia is defined as a platelet count reproducibly below 100x109/l 66. Some studies which reported a statistically significant difference between bovine and porcine heparin may have had inflated numbers of HIT cases because they did not distinguish between Type I and Type II HIT 55,76. Interest in resolving this issue appears to have waned in favour of assessing the relative risk of HIT in patients treated with unfractionated heparin (UH) versus LMWH.

The risk of HIT with LMWH appears to be substantially lower than that for unfractionated heparin. A large clinical trial, involving 333 patients, reported by Warkentin et al. failed to detect any instances of LMWH induced thrombocytopenia 33. Similarly, Leyvraz et al. report that none of 174 patients treated with LMWH developed HIT 79. Other observations are consistent with LMWH being less immunogenic. Amiral et al. 56 detected anti-PF4-heparin antibodies in 17% of patients receiving UH while only 8% of patients on LMWH developed antibodies. Interestingly there was no thrombocytopenia in either group of patients. Other reports indicate that platelets are activated less by LMWHs than UH 34,80 and LMWH has a lower affinity for PF4 81. Nevertheless case reports indicate that HIT can occur with LMWHs 82-85. It should be stressed that it is often inappropriate to use LMWH to treat HIT caused by UH 33,86,

22 although it may be successful when cross-reactivity has been excluded in a laboratory assay 87,88.

The LMW heparinoid, Orgaran (danaparoid sodium, Org 10172), shows an even lower propensity to cause HIT. To date there have been no reports in Medline or Current Contents of thrombocytopenia solely caused by Orgaran. One of the closest reports is that of a man who developed HIT while receiving LMWH without in vitro cross- reactivity with Orgaran 89. He initially appeared to tolerate Orgaran but after 4 days became severely thrombocytopenic and was positive in a platelet aggregation test with Orgaran 89. However, this is a rare case and I and others have observed that only 0-20% (best estimate ≈10%) of plasmas from HIT patients will cross-react with Orgaran to activate platelets in vitro 59,90-95. This has lead to its use in the treatment of HIT 54,92.

Thrombosis Bleeding is rarely a complication of HIT 33 despite the occurrence of marked thrombocytopenia. Laster et al. 58 report that out of 169 HIT patients, only 8 suffered haemorrhages attributed to the thrombocytopenia. However, 7 of these 8 died as a result. Various case reports do describe bleeding from venipuncture sites 15,21,96 as well as more serious haemorrhage (eg. intracerebral) 16,65,97,98. Paradoxically, the most common complication of HIT is thrombosis.

While patients with HIT are at increased risk of thrombosis, the frequency of this complication is uncertain. Arterial and venous thrombosis are commonly reported in case studies 16,20,59,60,99. However, early prospective studies found no cases of thrombosis associated with HIT 24,26,30. More recent and larger studies report a higher frequency of thrombotic events. In a randomised clinical trial 9/332 patients receiving unfractionated heparin developed HIT and eight of these nine experienced a thrombotic event 33. However, as the authors point out, these patients received heparin for prophylaxis after surgery and were specifically monitored for signs of venous thrombosis because they were presumed to be at higher risk. In a retrospective study, the same group observed that about half the HIT patients who did not have signs of thrombosis at diagnosis subsequently developed thrombosis over the next 30 days 100. Laster et al. 58 reports that the rate of complications (largely thrombotic) in HIT patients

23 dropped from 61% to 22% with improved clinical surveillance. Boshkov et al. 57 reviewed the clinical data of 53 patients with HIT (confirmed by 14C-serotonin release assay) and observed 68% suffered thrombosis.

Arterial thrombosis is perhaps the classic thrombotic event in patients with HIT and results in serious complications such as myocardial infarct, limb gangrene, stroke, renal failure and multiple organ failure 28,35,98,101. Bell 62 describes thrombosis mainly in the arterial side, and rarely in the venous side, of the circulation. Postmortem / embolectomy examination typically reveals that arteries are occluded by platelet and fibrin rich, but erythrocyte-poor, thrombi 35,53,88. This lead to the, now rarely used, term “white clot syndrome” 52. Recent experience suggests that thrombosis on the venous side of the circulation may, in fact, be more common than arterial thrombosis in some settings 33,44,100 and most commonly presents as proximal DVT and pulmonary embolism 54. Other reports indicate roughly equal numbers of patients with arterial or venous thrombi 60,101 as well as patients with both 52,88. Rarely, skin necrosis at the site of heparin injection is observed and has been attributed to thrombi in microcirculation 57,102.

The discrepancies between different authors as to the most common site of thrombosis associated with HIT may be due to different investigators studying different patient populations that are heparinised for different reasons. For example, Boshkov et al. 57 reviewed the clinical data of 36 patients who suffered thrombosis attributed to HIT and observed that there was a strong association between the site of thrombosis and the reason for which heparin was administered 57. Specifically, patients who developed HIT while receiving heparin to prevent DVT or pulmonary embolism after surgery were more likely to suffer venous thrombi. This contrasted with cardiovascular patients who were heparinised following myocardial infarction or angioplasty and developed HIT. They were more likely to experience arterial thrombi. The authors concluded that thrombosis was more likely to occur at a site of pre-existing pathology 57.

Makhoul et al. 59 describe 25 patients (about 0.1% of all the patients who received heparin) who developed thrombosis as a consequence of HIT. They observed that patients who had a procedure requiring a catheter in the femoral artery or vein were

24 more likely to experience thrombosis in the same leg that received the catheter. They propose that endothelial damage may increase the susceptibility to thrombosis.

The ability to predict whether a patient with HIT will develop a thrombotic complication would enable specific patients to be selected for more aggressive anti-thrombotic therapy and probably also improve our understanding of the pathophysiology of this disease. Unfortunately, while it is clear that HIT patients in general are at increased risk of thrombosis there are no strong predictive factors for thrombosis within the HIT population. Most authors contend that the risk of thrombosis is not related to the severity of the thrombocytopenia 57,58,88 or the strength of the platelet aggregation reaction (measured by lag time, or time until maximum aggregation) 103. However, there is a recent report that suggests that lower nadir platelet counts are associated with increased risk of thrombosis but the predictive value of this is poor for an individual patient 104. The concentration of the coagulation factor inhibitors AT-III, heparin cofactor II, and protein C appear to be low during the period of thrombocytopenia regardless of whether the individual suffers thrombosis or not 57. There is, however, some evidence that larger doses of heparin may be associated with thrombosis complicating HIT 58.

It is also worth noting that failure to stop heparin does not invariably cause thrombosis. For example, Laster et al. 58 describe 5 HIT patients who continued to receive heparin after diagnosis of HIT was confirmed by aggregometry. Three of these (two receiving heparin flushes and one dialysis patient) had no complications. However, one patient receiving a heparin flush experienced a fatal haemorrhage and another an arterial thrombosis while receiving heparin with parenteral nutrition. This illustrates why it is important to understand this disease and, if not prevent it, be able to predict which patients are at higher risk of thrombosis.

Clinical diagnosis

The early diagnosis of HIT and the cessation of heparin is critical to reduce the morbidity and mortality caused by this disease. At the same time, unnecessary withdrawal of heparin may also place the patient at risk of thrombosis. Unfortunately, the differential diagnosis of HIT is often complex because there are few symptoms that

25 are exclusive to HIT and many of the possible indicators may be absent. A combination of clinical symptoms and laboratory findings are used to support a diagnosis of HIT. For an overview, Hacket et al. 105 provide a good description of the general diagnostic approach to all drug-induced immune thrombocytopenias.

A history of recent heparin exposure is probably the only absolute requirement for a diagnosis of HIT, but in rare cases HIT may occur with very low doses of heparin that may not be initially recognised as heparin administration 74. The development of thrombocytopenia (see Thrombocytopenia, p.21) concurrent with heparin should alert the physician to the possibility of HIT, particularly if there is a rapidly falling platelet count 46. However, there are reports of HIT, identified by positive HIPAA and thrombotic complications, in patients receiving heparin but with normal platelet counts 63. In Type II HIT the onset of thrombocytopenia would not be expected until after about 5 days of heparin therapy 30,54,101 but thrombocytopenia may occur earlier if there has been previous heparin exposure 16,27,30,35. This susceptibility may last months 16,65. It is important to recognise that many other conditions may reduce the platelet count and these must be ruled out as a cause of the thrombocytopenia before it can be attributed to heparin 62. Platelet counts are inherently variable so thrombocytopenia should be indicated by at least two consecutive platelets counts 33. A clinical suspicion of HIT should also be raised if a patient develops new thrombosis or requires increasing doses of heparin 46,65,98.

When investigating a possible case of HIT, considerable effort is aimed at demonstrating that heparin is having a deleterious effect on platelets. This helps distinguish the more serious Type II HIT from the benign Type I and helps to rule out other causes of the thrombocytopenia/thrombosis. The return to normal platelet levels after cessation of heparin is good evidence that the thrombocytopenia can be attributed to heparin. This would typically be expected to take around 5 days 20,24,29,58.

The most conclusive indication that heparin is causing the thrombocytopenia is if thrombocytopenia/thrombosis returns when the patient is rechallenged with heparin 10,12,14,16,21,35,98,106,107, however, this does not always occur 57,58, particularly if rechallenge occurs months later 16. It must be emphasised that rechallenge with heparin is no longer clinically responsible and is ethically unacceptable due to the known risk of

26 thrombosis. This is one of the reasons why in vitro assays, which measure the activation of normal platelets by HIT antibodies, are used to aid in the diagnosis of HIT. These assays, such as platelet aggregometry, 14C-serotonin release assay and HIPAA, are discussed later (Laboratory diagnosis of HIT, p.46). A positive result in a well controlled in vitro assay is considered good evidence of Type II HIT. However, a negative result does not conclusively prove that a patient does not have HIT 108 and diagnosis should be made on the overall clinical picture 27.

Greinacher et al. 91 used a scoring system that weighted the symptoms of heparinised patients to determine the likelihood of HIT. Other authors have suggested the criteria that are presented in Table 1. Most or all of these conditions would have to be fulfilled to be confident of a positive diagnosis. However, as Kappers-Klunne et al. point out, there are no generally accepted criteria for the diagnosis of HIT 46.

Table 1. Criteria for the diagnosis of HIT 1. Normal platelet count before heparin 46,94 2. Thrombocytopenia while receiving heparin (usually for a minimum of 5 days) 27,29,101 • specifically platelets < 140x109 / l 94 • specifically platelets < 100x109 / l 20,62 • specifically platelets < 60x109 / l or < 100x109 / l if fall is >50% or if fall is >30% and there is concomitant acute thrombosis 46 • specifically platelets < 100x109 / l or fall > 50% 54 • Indicated by at least two consecutive counts 33,62 • Confirmed by peripheral blood film 28,61,62 3. Exclude other causes of thrombocytopenia 20,27,29,62,94,101 4. Positive laboratory assay 27,94,101 5. Resolution of thrombocytopenia after heparin withdrawal 20,27,29,62,101

Normal platelet physiology

Before discussing the mechanism by which heparin causes thrombocytopenia it is worthwhile summarising the normal physiology of platelets. Platelets are small, discoid 27 (1.5-2.5 µm x 0.5-0.9 µm 109,110) anucleate blood cells that play a key role in haemostasis. Platelets are derived from megakaryocytes, which are huge (25-50 µm 111) multinucleated bone-marrow cells. Normal platelet counts are in the range 150-350x109/l 112 with an average of about 250x109/l 113. It is estimated that 35,000 platelets/µl of plasma are produced each day 113 and the average platelet survival is approximately 10 days 113,114.

The role of platelets in thrombus formation can be summarised as follows: The endothelium, which lines blood vessels, prevents platelet adhesion to the vessel walls. Damage to a blood vessel exposes constituents of the subendothelium such as collagen, von Willebrand factor (vWF) and fibronectin. Platelets adhere and spread on the exposed subendothelium. Platelets bind to collagen fibrils largely through GPIa/IIa receptors on their surface 110 and to vWF through the GPIb/IX/V complex 110,115.

Adhesion triggers the release of thromboxane A2 and ADP. These agonists act synergistically to activate nearby platelets and recruit them to the site of injury. The platelet also undergoes a remarkable shape change from discoid to a sphere covered with spines 1-2µm long 109,110. The cellular membrane of the activated platelet changes to include more phosphatidylserine, which facilitates the plasma coagulation cascade 116. This generates thrombin, which not only catalyses fibrin formation but is a potent platelet agonist in its own right.

The fibrinogen receptor GPIIb/IIIa is critical to platelet aggregation. This glycoprotein is the most common receptor on the platelet surface. Spaced about 20 nm apart there are 40,000-80,000 receptors per platelet, 110. GPIIb/IIIa on non-activated platelets will bind fibrinogen provided the fibrinogen is immobilised at high density on a surface 110. However, activated platelets display modified GPIIb/IIIa that will bind soluble fibrinogen and when fibrinogen links adjacent platelets aggregation results. Divalent cations are essential for this process. vWF can substitute for fibrinogen and cross-link GPIIb/IIIa, particularly in situations where there is high shear forces or a lack of fibrinogen 117. However, the fibrinogen contained within the α-granules is usually sufficient to support platelet aggregation provided the agonist is strong enough to induce degranulation 117. Clinically monoclonal Fab fragments against GPIIb/IIIa (abciximab) are now being used as an anti-platelet agent.

28 Platelet activation coincides with a spike of free intraplatelet Ca2+, lasting a couple of 110,118 2+ seconds, that rises from ≈0.3µM to 1-5µM . Ca , inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG) play major roles in the signal transduction within the platelet 118. Their release is caused by activation of G-proteins by agonists and this triggers phospholipase C to cleave phospatidylinositol bisphosphate (PIP2) into the 119 2+ second messengers IP3 and DAG . IP3 mobilises Ca from the dense tubular system of the platelet and there is also an influx of Ca2+ from the exracellular medium 117,118.

Platelet activation triggers the release of platelet-granule contents 110,117. Serotonin, ADP and Ca2+ are released from the dense (amine) granules and potentiate vasoconstriction, further platelet aggregation and plasma coagulation. A range of proteins are released from the α-granules. Some of these, such as PF4 and histidine-rich glycoprotein, antagonise the anticoagulant action of heparin. Others, such as fibrinogen, vWF and fibronectin, promote platelet aggregation. Plasma factors from the α-granules may promote (eg Factor V, Factor XI) or inhibit (protein S) the coagulation cascade. It is interesting to note that α-granules contain proteins derived from the plasma including IgG 110,120,121. Very strong platelet activation releases the contents of the lysosomal granules, which contain enzymes that may contribute to vascular damage.

A key characteristic of platelet activation is that a relatively small stimulus to a limited number of platelets can be amplified by positive feed-back to incorporate many platelets and fibrin into a haemostatic plug.

Pathophysiology of HIT

An immune basis for HIT As the potentially serious consequences of HIT became recognised in the 1970’s, researchers began to suggest that the mechanism may be immunological 12,14,16,20,60,65,122. This was based on the observation that thrombocytopenia often took a week or more to develop after the initiation of heparin therapy and the fact that HIT was likely to occur within a few days upon re-challenge 12,16,65.

29 A HIT antibody Platelet aggregometry has provided a tool to investigate the mechanism of HIT. When platelet rich plasma from a normal donor is mixed with HIT patient plasma and heparin the platelets usually aggregate 14,17,19,96. The aggregation is not passive agglutination because EDTA 96 and metabolic inhibitors 123 prevented aggregation. This aggregation is presumed to be a model for events in vivo. However, it should be noted that it is very difficult to obtain direct evidence to prove this assumption.

A number of authors isolated the pro-aggregatory/anti-platelet factor in the HIT plasma to the immunoglobulin fraction 17,19,20,95,96, usually an IgG 12,16,60,124-126 but sometimes IgM 16,18. Patients with HIT also appear to have elevated platelet associated IgG 30,126- 129. However, this can occur in other situations 30,120,127 and less than 1% of total platelet IgG is associated with the cell surface 120 so it is unclear what relevance of platelet- associated IgG has in understanding the mechanism of HIT.

It was concluded that HIT patients were producing an antibody that in association with heparin, induced platelet aggregation. However, the identity of this antibody and its antigen remained controversial.

Role of the platelet FcγγγRII in HIT

Of particular relevance to the mechanism of HIT is the ability of IgG to activate platelets via the platelet Fc receptor (FcγRII, CD32). There are about 600-1,500 FcγRII receptors per platelet and expression increases upon activation 110,130. Crosslinking the FcγRII sends a powerful activation stimulus to the platelet. Factors, such as aggregated IgG 131- 134, that cross-link FcγRII, cause platelet aggregation. Many monoclonal antibodies against platelet antigens (eg. anti-GPIIb/IIIa) induce platelet activation but this does not 135-137 occur with the F(ab’)2 portions of the monoclonal IgG . It is thought that the anti- platelet monoclonal antibodies bind to the platelet by their Fab portion and then the Fc region binds and clusters FcγRII on adjacent platelets and induces activation. Furthermore, the FcγRII can be specifically blocked by a monoclonal antibody called “IV.3” and this prevents an anti-platelet monoclonal IgG from triggering platelet activation 135-137.

30 Similarly, platelet aggregation or serotonin release caused by HIT antibody plus heparin is also inhibited by IV.3 138,139 or high concentrations of rabbit IgG 132 which also blocks the FcγRII. Likewise, F(ab’)2 prepared from IgG of HIT patients does not activate platelets 138,140. It is now widely accepted that FcγRII is integral to the activation of platelets by HIT antibody plus heparin in vitro. Whether this is also the case in vivo is difficult to determine but it seems a reasonable supposition. Interestingly, Chong et al. reported that HIT patients may express elevated FcγRII on their platelets during the acute phase of their disease 41.

Fc-receptor polymorphism

The importance of FcγRII in platelet activation induced by HIT antibody plus heparin has lead to investigations into whether a polymorphism in FcγRII may alter the susceptibility to HIT. Of particular interest has been an allelic polymorphism at amino acid 131 of FcγRII. This can be either homozygous Arg/Arg, His/His or, more commonly, heterozygous Arg/His 47,141,142. Burgess et al. observed that none of the 19 HIT patients investigated were homozygous Arg/Arg and concluded that “the presence of the FcγRIIHis131 allele predisposes to HIT” 141.

Brandt et al. 142 came to the same conclusion but with slightly different data. They did observe HIT patients with Arg/Arg phenotype but also observed a much higher frequency of His/His phenotype in the HIT population compared to non-HIT 142. Brandt et al. also investigated the effect of this polymorphism on the sensitivity of platelets to aggregation by HIT antibody plus heparin. Surprisingly, they observed that normal platelets from none of 9 His/His donors aggregated in the presence of HIT plasma plus heparin 142. One would have expected that if the FcγRIIHis131 allele is associated with HIT then such platelets would be the most sensitive to HIT antibody induced aggregation. In fact, two years later Denomme et al. 143 reported just this finding, completely at odds with that of Brandt et al. 142. Denomme et al. observed that normal His/His platelets were more sensitive than His/Arg, which were more sensitive than Arg/Arg, to activation by HIT serum containing anti-PF4-heparin IgG1 143. They also observed that the frequency of the His allele (homo- or heterozygous) occurred in 56.5% of HIT patients but only in 47.1% of control patients 143. While this difference was statistically significant it is not substantial and it can only be a partial explanation for the 31 occurrence of thrombocytopenia/thrombosis in patients with anti-PF4-heparin IgG. To further add to the debate, Arepally et al. have reported no difference between HIT and healthy controls in the frequency of His or Arg alleles 47.

Heparin is not the HIT antigen The obvious target antigen for the HIT antibodies was heparin itself. This was reinforced by observations that high concentrations of heparin (10-100 U/ml) inhibited the antibody induced platelet aggregation 60,144-146. This was presumed to be due to the excess heparin binding all the antibody and preventing antibody interaction with the platelets, akin to a prozone effect 129. Normally heparin is considered a poor immunogen 14,16,65 and generation of anti-heparin antibodies required rabbits to be immunised with a methylated-BSA-heparin precipitate 147. Babcock et al. 60 suspected that heparin concentrations greater than 10 U/ml inhibited platelet aggregation by a non- specific mechanism.

A number of groups looked for specific antibody binding to heparin but virtually all failed to find any difference between HIT and normal individuals. Sandler et al. 125 observed that heparin-sepharose could not adsorb the platelet-aggregating IgG from HIT serum.

Green et al. 17 used three techniques in an attempt to detect binding of HIT antibody to heparin alone. First they observed that when plasma was mixed with 35S-heparin then immunoglobulins precipitated, no more radioactivity was precipitated with HIT plasma than with normal plasma. Second, a solid phase RIA demonstrated that immobilised HIT antibody bound no more heparin than Normal antibody. Finally, column gel chromatography showed that when 35S-heparin was mixed with patient plasma then applied to a Sephadex-G50 column, no peak of 35S-heparin eluted with the immunoglobulin fraction.

Kelton et al. 148 performed further elegant experiments. They found no difference in the binding of 3H-heparin to HIT or Normal IgG that had been adsorbed to Protein A- sepharose. They used Ouchterlony diffusion analysis but observed no precipitin lines between heparin and HIT IgG, despite testing at different temperatures and ionic strength. They placed HIT IgG in dialysis bags and observed that no more 125I-heparin 32 accumulated in the HIT IgG bag than the Normal IgG bag. Their calculations indicate that this technique was sensitive enough to detect even very low affinity antibodies. Finally, precipitation of HIT IgG with ammonium sulfate precipitated the same amount of 125I-heparin as Normal IgG.

Greinacher et al. 95 report that IgG from HIT sera did not bind specifically to acrylic beads coated with either heparin or dextran sulfate while a wide range of sulfated oligosaccharides were able to induced platelet aggregation in conjunction with HIT sera 95. They concluded that the HIT antibody is not heparin specific and that the degree of sulfation is the most important factor in determining if a oligosaccharide triggers platelet activation with HIT sera 95.

One early report claims to have observed heparin associated more with partially purified IgG from HIT patients than IgG from normal individuals 126. More recently, Adams et al. 149 have claimed to demonstrate that HIT IgG will bind to heparin alone. Deficiencies in this report are discussed later (Dissenting views, p.45) and it does not provide enough evidence to reject the findings of others that heparin does not bind specifically to HIT antibody.

It was recognised that heparin preparations contain additives/contaminants that may be the HIT antigen or directly thromboplastic 15,55,62,95 but none have been identified as a cause of HIT. It is clear that heparin, and not a contaminant, is responsible for platelet activation in aggregometry or 14C-serotonin release assays because its effect can be neutralised by protamine 125 and platelet activation is independent of the source of heparin 59,148 which can be substituted for by synthetic heparin 150, dextran sulfate 151 or even DNA 151.

Platelet HIT antigens It became suspected that HIT antibodies were a “heparin-dependent platelet membrane antibody” 98 and Gruel et al. 152 demonstrated that HIT antibodies mixed with heparin bound to platelets fixed onto microtitre wells. However, early attempts to identify such target antigen(s) were inconclusive. Howe and Lynch 153,154 observed that HIT sera contained antibodies that bound to platelets in the presence of heparin. They detected three proteins (180, 124 and 82 kD) by western blot that appeared to bind HIT antibody 33 and suggested that they may correspond to thrombospondin, a thrombospondin degradation product and glycoprotein V respectively 154. However, Chong 29 says that the molecular weight of thrombospondin, under the non-reducing conditions that were used, would have been about 450 kD. He has also shown that platelets congenitally deficient in glycoprotein V aggregate normally with HIT plasma 29,123 so glycoprotein V is unlikely to be involved.

Wolf and Wick purported to have identified glycoprotein Ib and another 207 kD heparin-binding protein as platelet membrane proteins that bound HIT antibodies 155. Adelman et al. observed that platelet aggregation can be inhibited by a monoclonal antibody (6D1) directed against glycoprotein Ib 140. However, Chong has shown that platelets from Bernard-Soulier patients (which lack glycoprotein Ib) are aggregated by HIT antibodies 123. Both he 29 and Adelman et al. 140 recognise that the platelet FcγRII is closely associated with glycoprotein Ib. Chong 29 contends that the unknown protein (207 kD non-reduced, 57 kD reduced) observed by Wolf and Wick 155 is in fact the platelet Fc receptor, which is reported to be 255 kD under non-reducing conditions and 50 kD reduced 156. He argues that aggregation was inhibited when mAb 6D1 bound to glycoprotein Ib and sterically hindered the interaction between the neighbouring FcγRII and HIT IgG 139.

Pfueller et al. describe a patient who developed thrombocytopenia while on heparin but thrombocytopenia and thrombosis remained after heparin withdrawal. A heparin- independent platelet aggregating IgG was isolated from the plasma and appeared to recognise glycoprotein IV (=GPIIIb) 157. It is impossible to know if the presence of this antibody was caused by heparin or was simply coincidental.

HIT antibodies against PF4-heparin

PF4 is a protein released from the α-granules of platelets upon platelet activation (see section starting p.64). It is known to neutralise heparin, which it binds with high affinity. In 1992, Amiral et al. 158 reported that plasma from most HIT patients, but not normal individuals, contained antibodies that recognised a complex formed between heparin and platelet factor 4 (PF4). This was confirmed by other groups 43,45,148,159. These observations were generally made using an ELISA, where a mixture of PF4 and heparin

34 was coated onto microtitre wells. Diluted plasma was incubated in the wells and the binding of antibody detected with a peroxidase conjugated secondary antibody and colorimetric substrate. No HIT antibody binding was observed to heparin complexed to other heparin binding or platelet proteins 158,159.

There is an optimum ratio of approximately 0.5-1 U heparin per 20µg PF4 in the solution coating the microtitre wells 43,45,159. This ratio corresponds to approximately one molecule of heparin to two tetramers of PF4 43,45. However, more PF4 may be required with expired batches of heparin to overcome the inhibitory effect of small heparin fragments 45. A range of polyanionic polysaccharides can be substituted for heparin 159 and they each have their own optimum PF4:polyanion ratio 159. The chain length of heparin is also important. A minimum chain length of around 10 saccharide units is required for HIT antibody binding to PF4-heparin 45,159. It is not surprising therefore that PF4 complexed to the pentasaccharide that binds ATIII, is not recognised by HIT antibodies 150,160. Low molecular weight heparins have a broader optimum range than unfractionated heparin and are recognised by HIT antibodies in the PF4-heparin ELISA 159. There is an inverse correlation between heparin chain length and the amount of heparin required for antibody recognition 159. Also, the more highly sulfated polysaccharides are the most efficient at promoting HIT antibody binding to PF4 159.

It appears that the complexes of PF4-heparin that coat the ELISA wells are multimolecular. That is they consist of many PF4 and heparin molecules crosslinking each other. Greinacher et al. showed that high concentrations of heparin would release PF4 from wells coated with PF4-heparin 43 presumably by disrupting multimolecular complexes of PF4-heparin that were irreversibly bound to the well at only a few points. Little PF4 was released by heparin from wells coated with PF4 alone 43.

Observations that Grey’s platelets, devoid of α-granules, were not aggregated by HIT plasma and heparin suggested that the α-granules may be important 161. Horne and Alkins elegantly demonstrated that, when supplemented with exogenous PF4, Grey platelets could be activated by HIT IgG plus heparin 162. This confirms that Grey platelets are unresponsive because they lack PF4 and not because of some other defect.

35 Clearly there is a high correlation between HIT and the presence of anti-PF4-heparin antibodies but this is not absolute. In prospective studies, where patients receiving heparin have been tested for anti-PF4-heparin antibodies, it has been observed that anti- PF4-heparin antibodies develop without thrombocytopenia 56,163,164. Amiral et al. observed 19/109 (17%) of patients receiving unfractionated heparin and 8/100 (8%) of those receiving LMWH developed anti-PF4-heparin antibodies but the platelet counts in all patients remained above 150x109 /l 56. The most common isotypes were IgM > IgA > IgG 56. Similarly, Trossaërt et al. 164 observed that 14/51 (27%) of cardiopulmonary bypass patients had developed anti-PF4-heparin antibodies when tested on the 8th day of heparin therapy. Importantly, preoperative samples were negative. In this case most of the anti-PF4-heparin antibodies were of the IgG class 164.

Not all HIT patients have antibodies against PF4-heparin. Occasional patients have been reported to be negative in a PF4-heparin ELISA but positive in a functional assay 43,158,159. It is estimated that the PF4-heparin ELISA falsely reports about 10% 163 or 25% 91 of HIT patients as negative. This suggests that other platelet antigens may be involved in some cases of HIT. Interleukin-8 and neutrophil activating peptide-2 have been identified a alternative candidate antigens (see HIT antibodies against IL-8, NAP-2, p.38).

Amiral et al. have twice isotyped the anti-PF4-heparin antibodies in the plasma of HIT patients 159,165. IgG antibodies, with or without IgA or IgM, were present in 13/15 (87%) 159 and 26/38 (68%) 165 of cases. The remaining patients produced only IgA and/or IgM antibodies, without IgG. While most of these IgA/IgM only plasmas were negative in a platelet aggregation assay the authors are adamant that, based on clinical observations, type II HIT is the correct diagnosis 165. Amiral et al. suggest that anti-PF4- heparin IgA and IgM may also be involved in the pathogenesis of HIT without the involvement of the platelet FcγRII. However, they did not consider the possibility that patients may have antibodies against other platelet proteins, such as NAP-2 or IL-8 166, which may have caused the HIT.

36 Are anti-PF4-heparin antibodies pathogenic? HIT is associated with antibodies directed against complexes of PF4 plus heparin, so there has been speculation that the anti-PF4-heparin antibodies cause the platelet activation and thrombocytopenia. However, direct evidence for this has been scanty. Greinacher et al. 43 purified HIT antibodies by affinity for endothelial cells in the presence of heparin. When eluted, the material that bound to the endothelial cells usually contained anti-PF4-heparin antibodies as detected by ELISA. This eluate also activated platelets in a serotonin release assay. Greinacher et al. concluded that PF4 complexed to heparin is the antigen involved in HIT 43. However, endothelial cells posses a wide range of different antigens including many that are common to platelets, for example ICAM-2 167,168, PECAM-1 169, P-selectin 170 and p24/CD9 171-173. Monoclonal antibodies against p24/CD9 can even cause platelet activation 174. Thus, in theory, Greinacher et al. 43 may have co-purified anti-PF4-heparin along with a different antibody responsible for the activation of platelets in the serotonin release assay. Clearly isolation of anti-PF4-heparin antibodies in a more purified system would help resolve this issue. The platelet aggregating ability of HIT IgG, affinity purified on a heparin- PF4-agarose column, is presented in Chapter 4. Even so there is still no direct proof that the antibody responsible for platelet activation in SRA or aggregometry is responsible for HIT, although it is very likely.

Recently, Blank et al. 175 have attempted to provided in vivo evidence that anti-PF4- heparin antibodies are pathogenic and cause thrombocytopenia. They immunised mice with total IgG derived from HIT patients. The immunised mice developed anti-idiotype antibodies against the human HIT antibody (Ab2) and after a few months anti-anti- idiotype antibodies (Ab3) against the Ab2. The Ab3 mimicked the effect of the original human HIT antibody in that they bound to PF4-heparin in ELISA and the injection of 175 heparin into mice with HIT Ab3 caused rapid thrombocytopenia . Unfortunately, this experiment still does not prove that it is the anti-PF4-heparin antibodies that cause thrombocytopenia because the immunising human IgG (purified on protein A agarose) may have contained other pathogenic antibodies to which mouse Ab3 developed.

37 HIT antibodies against IL-8, NAP-2 The observation that not all HIT plasma contain antibodies against PF4-heparin 91,163 suggests that other antigens may be involved. Amiral et al. have identified interleukin-8 (IL-8) and neutrophil-activating peptide-2 (NAP-2) as possible, rare, HIT antigens 166. They investigated 87 patients with HIT (confirmed by heparin-dependent aggregometry and/or anti-PF4-heparin ELISA). 15 of these patients were negative in anti-PF4-heparin ELISA despite being positive by aggregometry. Six of these 15 possessed antibodies that bound to microtitre wells coated with IL-8 while a further three of them bound to NAP-2 coated wells 166. Heparin did not enhance the binding of these antibodies to IL-8 or NAP-2. These patients appeared to fall into distinct populations in that their antibodies recognised either PF4-heparin (72/87), or IL-8 (6/87), or NAP-2 (3/87), or an unknown antigen (6/87), but not any combination of these 166.

A degree of caution should be exercised in interpreting these results, particularly the significance of the IL-8 results. Amiral et al. recognise that substantial levels of IL-8 antibodies have been observed in patients with malignancy, infection or after surgery 166. While Trossaërt et al. observed anti-IL-8 antibodies in 2/51 patients before they underwent cardiopulmonary bypass and received heparin 164. A subsequent report has also indicated that anti-IL-8 and anti-PF4-heparin antibodies can occur concomitantly and that anti-NAP-2 antibodies have been detected in an individual who was “unlikely to have HIT” 176.

Epitopes on PF4

To date, no studies have been performed to identify HIT epitopes on NAP-2 or IL-8 but some progress has been made with the PF4-heparin antigen.

Amiral et al. 159 observed that PF4 must be an intact tetramer for efficient binding of HIT antibody and reduction of disulfide bonds abrogates antibody binding 48,159. However, it is not clear to what degree this just reflects the lower affinity that reduced PF4 has for heparin 177,178. Horsewood et al. 179 observed that a subset (5/24) of HIT sera could recognise PF4 that had been reduced with dithiothreitol then bound to ELISA wells in the presence of heparin. They then investigated different peptide fragments of PF4. The same HIT sera bound to a peptide spanning the sequence from cys-52 to the

38 C-terminal ser-70. However, a peptide only one amino acid shorter (leu-53 to ser-70) was not recognised by these sera. None of the other peptides that they tested which spanned the N-terminal or middle region of PF4 reacted with any of the HIT sera 179. Horsewood et al. conclude that the 19 C-terminal amino acids are required to form the epitope that was recognised by the subset of HIT sera. The ability of the PF4 peptides to bind heparin appeared to correlate with their ability to bind the HIT sera. This suggests that, in this subset of patients, the heparin molecule forms part of the epitope to which the HIT antibodies bind 179. The authors propose that the substantial majority of HIT sera that did not bind reduced PF4 must recognise an epitope formed by non-adjacent amino acids which is produced when heparin induces a conformational change in PF4 179.

The study by Horsewood et al. 179 raises the likelihood that there is in fact more than one epitope on PF4-heparin that can be recognised by HIT antibodies. This appears to have been confirmed by Suh et al. 48 who observed that antibodies from four HIT patients recognised two or three different epitopes on PF4. Suh et al. also made an interesting observation that HIT antibodies only bound to PF4 immobilised on heparin- sepharose when the heparin was end-linked to the sepharose 48. When heparin was linked at many points along its length to the sepharose, the HIT antibodies did not bid to a PF4-heparin-sepharose column. This was interpreted as providing evidence that heparin must wrap around the PF4 tetramer to create the HIT epitope.

Recently, Ziporen et al. 180 have conducted a series of experiments, with PF4 mutants, in an attempt to identify HIT epitopes. Point mutations were induced in PF4 to individually replace each of the C-terminal lysines with alanine. This reduced but did not abolish heparin binding. Only in PF4 with substituted lys-61 was there a partial drop in recognition by HIT antibody compared to normal PF4. They concluded that while the lysines were implicated in heparin binding they were not required for HIT antibody binding to PF4-heparin 180. Secondly, they observed that polyclonal antibodies raised against the last 23 C-terminal amino acids of PF4 did not block HIT antibody binding. However, I would argue that HIT antibodies may still recognise non-contiguous epitopes in this region. Finally, they discovered that replacing the amino acids between cys-36 and cys-52 with corresponding sequences from NAP-2 abolished the binding of many HIT antibodies. The mutation of Pro-37 appeared to be largely responsible for this 39 effect 180. The location of pro-37 on the surface of the PF4 tetramer, but away from the putative heparin binding region, is illustrated in Figure 6 (p.76).

Role of T cells in HIT

Recently, Bacsi et al. 181 have investigated the role of helper T cells in the development of HIT antibodies against PF4-heparin. They isolated peripheral blood mononuclear cells (PBMC) from two HIT patients. When these PBMC were cultured with PF4- heparin they observed an expansion of T cells expressing the V beta 5.1 subfamily of T-cell receptor (TCR). This did not occur when HIT PBMC were cultured with PF4 alone or heparin alone. Nor were any T-cell clones preferentially expanded when Normal PBMC were incubated with PF4-heparin. This provides the first direct evidence to support the assumption that helper T cells are involved in the generation of anti-PF4- heparin antibodies in HIT. Interestingly, Bacsi et al. also sequenced the TCR mRNA transcripts from the HIT T cells that were preferentially expanded by PF4-heparin. They observed that the TCR from both patients shared a common tetrapeptide that probably recognised part of the processed PF4 molecule 181.

Mechanism of thrombosis Understanding the association of thrombosis with HIT is a key goal of HIT researchers for the following reasons: Thrombosis is the most serious complication of HIT and without it HIT would be a fairly benign side-effect of heparin therapy. There are no factors that clearly predict which HIT patients will thrombose. Lastly, thrombosis associated with thrombocytopenia is counterintuitive and explanations for it should aid in its prevention.

Macroscopic platelet-rich thrombi (“white clots”) causing arterial thrombosis in HIT patients are presumably due to in vivo platelet activation. This is supported by in vitro experiments that demonstrate platelet aggregation by HIT plasma and heparin 20,24,65,127. However, only about half of HIT patients whose plasma produces platelet aggregation in vitro actually develop thrombosis 57,58,100 and venous thrombosis, without “white clots”, is probably at least as common as arterial thrombosis 33,44,60,101.

40 Additional mechanisms have been proposed to account for the association of thrombosis with HIT. PF4, released during platelet activation, may neutralise heparin and return the patient to a pre-existing pro-thrombotic state 13,35,65,67. There is some evidence that HIT correlates with a pro-coagulant environment. Boshkov et al. 57 report that at time of thrombocytopenia there was often reduced levels of antithrombin III, heparin-cofactor II and protein C, relative to levels 2 days after heparin was stopped. However, there was no difference in levels of these coagulation inhibitors between HIT patients who developed thrombosis and those who did not 57. It is claimed that platelet activation can activate the intrinsic pathway of coagulation 65,67. Warkentin et al. observed that platelets, activated by HIT antibody and heparin, release procoagulant microparticles 182,183. This did not occur with sera from patients with quinidine/quinine induced thrombocytopenia, who tend to bleed rather than thrombose. The platelet microparticles were procoagulant in that they shortened the Russel viper venom clotting time of normal plasma and they appeared to enhance prothrombinase activity, which would increase thrombin production 182. Interestingly, the plasma of HIT patients contained higher levels of platelet microparticles than patients with other causes of thrombocytopenia. However, plasma coagulation tests reveal that prothrombin times, activated partial thromboplastin times and fibrinogen levels of HIT plasma are usually within the normal range after neutralisation of the heparin 53,98.

Warkentin and others have also suggested that acquired APC resistance, as a result of warfarin therapy, may place HIT patients at higher risk of thrombosis during the early stages of warfarinisation 184,185. Given the relatively high incidence (5%) of the Factor V Leiden mutation in Caucasian populations 186 it is possible that genetic APC resistance predisposes to thrombosis in HIT patients. In an abstract, Pötzsch et al. reported that 24/38 HIT patients who developed venous thrombosis were APC resistant while none of the 18 HIT patients with thrombocytopenia alone were APC resistant 185. However, in a larger study, Lee et al. observed that there was no significant association between the heterozygous factor V Leiden mutation and the risk of thrombosis in HIT patients 187.

Damage to the endothelium may be an important trigger for thrombosis. It has been suggested that HIT antibodies may bind to PF4-GAG complexes on the endothelial cell surface, triggering immune-mediated damage and predisposing to thrombosis 43,45,74. Also physical injury to the endothelium caused by a surgical procedure may locally 41 increase the susceptibility to thrombosis 59. Recently, Kwaan and Sakurai 188 examined, histologically, tissue from HIT patients requiring limb amputation. They observed many small arteries were occluded by platelet aggregates. These thrombi were surrounded or infiltrated by proliferating endothelial cells which had bound IgG, IgA and IgM 188. They argue that antibody mediated endothelial damage promotes platelet activation and the endothelial cell hyperplasia. I would suggest a related possibility is that areas of angiogenesis may be targeted by the HIT antibodies because proliferating endothelial cells preferentially bind PF4 189,190.

A paradigm for HIT Having discussed the evidence for various mechanisms by which heparin may induce thrombocytopenia it is now appropriate to present a model that incorporates this evidence. Many aspects of this model are supported only by indirect evidence but it provides a paradigm in which theories can be tested and refined as experimental techniques advance. This model is influenced by descriptions from Warkentin et al. 54, Greinacher 44, Kelton 148 and Visentin et al. 45.

1. The initiation of heparin therapy is associated with elevated concentrations of plasma PF4. Many factors are likely to contribute to this: Heparin releases PF4 from endothelial cell proteoglycans 191,192. Platelets are weakly activated by heparin 34 and heparin enhances the effect of low concentrations of platelet agonists (see Effect of heparin on platelets, p.63). PF4 on the platelet membrane also increases with platelet activation 193. The consequence of these effects is presumed to be the formation of PF4-heparin complexes on the platelet surface or in the plasma. Then the patient’s immune system interprets the PF4-heparin as foreign antigen and, over 1-2 weeks, produces HIT antibodies in response.

2. The next stage is somewhat controversial, in that there are two schools of thought. If PF4-heparin complexes remain present then they could either stay in solution or bind to platelets. Antibody could potentially bind to PF4-heparin in either state. Some researchers 44,45,54,146,194 contend that HIT IgG forms immune complexes in the fluid- phase with PF4-heparin and then the Fc portion of these complexes activates platelets through the FcγRII. This model is illustrated in the Fluid-phase Antigen Model in

42 Figure 1. Other groups 43,99,148,159,195 favour a model where PF4-heparin collects on the platelet surface and then binds antibody. IgG coated platelets activate adjacent platelets by crosslinking their FcγRII (Platelet-bound Antigen Model in Figure 1).

3. Activated platelets may form a large thrombus or microaggregates that are removed by the spleen or reticuloendothelial system. The thrombocytopenia in HIT is attributed to increased platelet consumption/ injury 124,126 and shortened platelet survival 18 rather than a failure to produce platelets 35.

4. Thrombotic complications may result from aggregated platelets forming arterial or venous emboli 33,35,53,57,58,88, from activated platelet microparticles promoting the plasma coagulation cascade 182,183, or from PF4 bound to endothelial GAGs being targeted by HIT antibody, which may result in endothelial injury and a pro-coagulant site 43,45,74.

43 Fluid-phase antigen model

Platelet

Platelet-bound antigen model

Platelet

PF4 HIT IgG Heparin FcγRII

Figure 1 - Models of platelet activation by HIT IgG Fluid-phase antigen model: HIT IgG binds to complexes of PF4-heparin in solution. These immune complexes then bind and activate platelets when the Fc portion of the IgG binds to the FcγRII. This model is based on descriptions by Greinacher 44, Warkentin et al. 54,183, Visentin et al. 45, and Warner and Kelton 194. Platelet-bound antigen model: Complexes of PF4-heparin form on the platelet surface. The Fab portion of HIT IgG binds to this immobilised antigen. Only then does the Fc portion of the platelet-bound IgG cross-link FcγRII and trigger platelet activation. This model is based on descriptions by Amiral et al. 159, Greinacher et al. 134, Kelton et al. 148, Jackson et al. 99, and Horne and Alkins 195. Note that blocking the FcγRII with the monoclonal antibody IV.3 will prevent HIT IgG from binding platelets in the fluid-phase antigen model but not the platelet-bound antigen model. However, IV.3 will prevent platelet activation in both models.

44 Dissenting views Not all researchers accept that antibodies in patients with HIT are directed against PF4- heparin. Adams et al. 149 have recently reported experiments claiming to demonstrate heparin specific antibodies in plasma samples from HIT patients. They observed that plasma from HIT patients formed a precipitin line with heparin in an immunodiffusion assay, whereas normal plasma did not. They also report that antibodies in HIT plasma bound to heparin-sepharose alone when incubated for 12 hrs 149. However, it could be argued that the plasma contained PF4 which complexed to heparin, enabling antibody binding and precipitation. Indeed, no assessment of PF4 in the plasma was performed and Greinacher et al. 95 have reported that HIT antibody does not bind to heparin-coated acrylic beads.

A better controlled experiment by Adams et al. involved the use of protein A purified IgG from HIT and normal individuals. These samples were mixed with heparin sepharose and those proteins that bound were separated on SDS-PAGE. A 50 kD band, that reacted with fluorescent-labelled anti-IgG, was present in the HIT IgG but not the Normal IgG, although the Normal data was not shown 149. It seems unlikely that PF4 would have bound to protein A and thus be present in the IgG samples. Unfortunately, if PF4 was present it would have produced bands just off the end of the published SDS- PAGE gel. Adams et al.’s conclusion that a significant number of HIT patients posses heparin specific antibodies conflicts with those of others 17,95,148 who failed to demonstrate any heparin binding antibodies in HIT plasma.

The role of heparin in the initial immunisation of HIT patients has also recently been questioned. Suzuki et al. 196 report 5 cardiac patients who supposedly developed Type II HIT, 1-9 hours after starting heparin without any prior exposure to the drug. The definition of thrombocytopenia in these patients was a fall in platelet levels of just 30x109/l. Only one of the patients had less than 150x109 platelets/l at the time that they were considered thrombocytopenic and the counts do not appear to have been confirmed by either a blood smear or even a repeated test. The authors claim that the “HIT” patients were positive in platelet aggregometry and anti-PF4-heparin ELISA. However, they did not rule out the presence of heparin-independent platelet-aggregating factors in the patient plasma. The detection of anti-PF4-heparin antibody in the plasma is interesting, but its significance uncertain, because it is not clear if cardiac patients who 45 did not develop “thrombocytopenia” with heparin may also have been positive in the ELISA. The authors propose that the disease of some patients promotes platelet activation and release of PF4. This PF4 binds to endogenous glycosaminoglycans and immunises the patient before heparinisation. When heparin is administered the antibodies are proposed to further activate platelets and produce thrombocytopenia. The authors suggest that anti-PF4-heparin antibodies should be measured prior to heparin therapy. Given the uncertainties in this paper, independent verification of these observations is required before they are taken too seriously.

Laboratory diagnosis of HIT

In vitro laboratory assays can play an important role in the diagnosis of HIT. This is because many of the symptoms, particularly thrombocytopenia and thrombosis, may be caused by other factors, such as sepsis, neoplasia and other drugs 29,33,62. Laboratory assays aim to demonstrate the presence of HIT antibodies in patient serum/plasma and can be divided into two groups: 1. Functional assays that observe the activation of normal platelets by HIT antibodies in conjunction with heparin. 2. Immunoassays that detect the specific binding of HIT antibodies to purified antigen (eg. PF4-heparin).

Elevated platelet associated IgG, which has also been observed in HIT 74,126-129,153, is not diagnostically useful because it is common in other disorders 127,197

Platelet aggregation Platelet aggregometry is one of the oldest functional assays used to detect HIT antibodies 20,24,65,127,198. It involves stirring platelet rich plasma with test plasma at 37°C in a specially designed instrument (platelet aggregometer). Test serum should be heat inactivated (56°C, 30 min) to destroy residual thrombin 20,182. The addition of heparin (around 0.1-0.5 U/ml) triggers platelet aggregation if the test plasma contains HIT antibody. Aggregation is usually monitored by the corresponding increase in light transmittance through the platelet suspension. A typical platelet aggregation profile, induced by HIT plasma and heparin, is shown in Figure 2. Characteristic of this profile is a significant lag period before accelerating aggregation 65. A test plasma is usually considered positive if it produces at least a 20% increase in light transmission within 15-20 min of adding heparin 88,101,127. Many HIT plasma will reliably produce 100% 46 aggregation (personal observation). Significantly the source of heparin does not matter. Once a patient has developed HIT, antibodies in their plasma will usually aggregate platelets in the presence of bovine heparin, porcine heparin, or even LMWH 14,17,19,21,59. Plasma may remain positive in an aggregation assay for a period from 1 week to greater than 2 years after a patient stops heparin 58.

100%

80%

60% 0.5U/ml Heparin 40%

Platelet Aggregation 20%

0% -5 0 5 10 Time After Heparin (min)

Figure 2 - Typical platelet aggregation profile caused by HIT plasma and heparin Platelet rich plasma and HIT plasma are mixed in an aggregometer then heparin is added. Aggregation is measured by the increase in light transmittance.

The attraction of platelet aggregometry has been that it is relatively simple, it has high specificity, and uses concentrations of components that are achievable in vivo 127. However, the reported sensitivity of platelet aggregometry differs among researchers 58,127. This has been attributed to variability in donor platelets 27,129,199 but does not correlate with HLA or ABO type of the donor 199. Some authors have recommended utilising a panel of platelet rich plasma 200,201 to improve the sensitivity, while others suggest selecting donors who are known to produce a more sensitive assay 28,145. It is worth noting that platelet donors should be selected on the basis of their sensitivity to HIT plasma rather than on sensitivity to aggregation induced by monoclonal anti-platelet antibodies, which show no correlation with sensitivity to HIT antibodies 131.

47 Occasional case reports indicate that heparin dependent platelet aggregation only occurs with the patient’s own platelets but the interpretation of such observations is difficult because platelets from patients with many other conditions, such as burns 202, severe vascular disease 203 and prosthetic heart valves 204 will aggregate upon mixing with heparin. Thus, ill patients may have hyperaggregable platelets regardless of whether they have HIT or not. This illustrates the point that care must be taken to include appropriate controls for the aggregation assay, or any other functional assay. Specifically, aggregation should not occur when saline is added instead of heparin or when non-HIT plasma is used. It is obviously also important that the platelets are viable and sensitive. One of the simplest ways to demonstrate this is to include a weakly positive HIT plasma as a positive control 131.

14C-Serotonin Release Assay The two point 14C-serotonin release assay (SRA) is considered the gold standard for the laboratory diagnosis of HIT 28,205,206 although others argue that none of the available assays are 100% sensitive or specific 207. Washed normal platelets are incubated with 14C-serotonin which is taken up into the dense granules. These labelled platelets are incubated with test plasma and heparin for 1 hr at 22°C. The presence of HIT antibodies causes platelet activation and degranulation which is quantitated by the release of 14C-serotonin. In order to improve the specificity of this assay, Sheridan et al. 144 developed a two point test. This involves measuring serotonin release at both a high (100 U/ml) and low (0.1 U/ml) heparin concentration. A test plasma is deemed positive if more than 20% of the 14C-serotonin is released at the low heparin concentration but not the high concentration 144,208.

The attraction of this technique is its high specificity and sensitivity 29,33. The disadvantages of the 14C-serotonin release assay are that it uses radioactivity, requires fresh platelets and some specific expertise is needed for the washing and handling of platelets. In practice this technique is limited to specialised haematology laboratories.

Variations of functional assays eg. HIPAA, Platelet Microparticles A number of variations of functional assays have been developed. The heparin induced platelet aggregation (HIPA) assay was developed by Greinacher et al. 93. This assay is 48 essentially the same as a 14C-serotonin release assay except that platelets are not loaded with 14C-serotonin, rather platelet activation is determined semi-quantitatively by visual inspection of platelet aggregation in the microtitre wells. Greinacher et al. contend that their assay is as sensitive as the SRA 93. However, Kappers-Klune et al. 46 disagree and concluded that no predictive value of HIPAA could be determined in their hands.

Lee et al. 209 have developed an assay for HIT based on the observation that platelets, activated by HIT plasma plus heparin, release platelet microparticles and these can be quantitated by flow cytometry 182. They mixed platelets, test sera and heparin for 1 hr as in the SRA. Samples of this reaction were labelled with a fluoresceinated anti-GPIbα monoclonal antibody. The microparticles were distinguished from platelets on the basis of their flow cytometric profile. They estimated that the overall agreement between the SRA and microparticle release assay was 96% 209 and the microparticle technique had the advantage of not requiring radioactivity.

ELISA In 1992 Amiral et al. reported that HIT plasma contained antibodies that recognised complexes of PF4-heparin and developed an ELISA to detect these antibodies 158,159. Microtitre wells are coated with a mixture of PF4 (20µg/ml) and heparin (≈0.5 U/ml). Diluted test plasma is incubated with these wells and the binding of anti-PF4-heparin antibodies detected by a secondary antibody then a colorimetric substrate.

This technique is still fairly new and an accepted criteria for a positive result have not been established. Amiral et al. defined absorbances above OD=0.5 as positive and those between 0.5 and 0.25 as uncertain. When I used a commercial ELISA kit, based on Amiral’s publications, I observed that the plasma from many normal individuals resulted in absorbances well above OD=0.5 90. This highlights two important disadvantages of the ELISA. Firstly, different plasmas display wide variation in the degree of non-specific binding and this results in considerable scatter of Normal results. Secondly, the final absorbance that is obtained is influenced by many factors other than the concentration of HIT antibody. For example, density of PF4-heparin, degree of blocking, strength/affinity/concentration of secondary antibody, nature of the substrate and the time that colour is allowed to develop. I would contend that laboratories who

49 use ELISA for the diagnosis of HIT need to determine their own Normal range and the criteria for a positive result. The key advantages of the ELISA technique over functional assays are that it does not require live platelets and can be performed in virtually any diagnostic laboratory using standard techniques.

The clinical relevance of anti-PF4-heparin antibodies measured in ELISA is uncertain 46 and the rate of false positives is quite high. Illustrating this, 14/51 (27%) of patients undergoing cardiopulmonary bypass developed anti-PF4-heparin antibodies by ELISA 164 without signs of thrombocytopenia. Two of these patients were also positive by aggregometry so may possibly have had HIT without thrombocytopenia. Some of the cardiopulmonary bypass patients appeared to have anti-PF4-heparin antibodies by ELISA even before bypass 164, although the authors do not comment as to whether this may be due to previous heparinisation. Similarly, Amiral et al. report 19/109 (17%) of patients receiving UH and 8/100 (8%) of patients receiving LMWH developed anti-PF4- heparin antibodies (mostly IgM and IgA) by ELISA but none of these developed thrombocytopenia 56.

Comparisons of HIT assays Look et al. compared aggregometry with ELISA and concluded that aggregometry was more sensitive than ELISA 205. They attributed this to performing aggregometry with specific donor platelets that were known to react with HIT plasma and to investigating aggregation at 0.1, 0.4 and 1 U/ml heparin. Furthermore, they reported only 52% agreement between the two assays 205. Unfortunately, this study is difficult to interpret because no indication of specificity is provided. In a similar study, Nguyên et al. 210 observed 84% agreement between aggregometry and ELISA but ELISA detected more positive samples than platelet aggregation. Neither ELISA nor aggregometry reliably predicted thrombotic risk 205,210.

Greinacher et al. 91 compared platelet aggregometry, HIPAA, and ELISA and concluded that aggregometry was less sensitive than HIPAA or ELISA which are about equal. There is general agreement that the anti-PF4-heparin ELISA and functional assays recognise different, but overlapping, groups of patients and that the two types of assay may complement each other 91,163,205

50 Prevention of HIT

Prevention through early diagnosis While HIT can really only be prevented by not using heparin, early diagnosis of HIT and the cessation of heparin may reduce the risk of associated thrombosis. Regular platelet counts are the key to monitoring heparinised patients for the development of HIT. Generally, it is recommended that platelet counts be performed every 3 days 78 or daily if levels have declined substantially but are still above 100x109/l 62. Laster et al. 58 initiated daily platelet counts and observed that the risk of thrombosis (and rarely haemorrhagic complications) fell from 61% of HIT patients in 1983 to 22.5% in 1986 58. This was attributed to the increased platelet monitoring which led to early diagnosis and immediate cessation of heparin. They also describe a procedure for responding to low platelet counts 58. When the count falls below 100x109/l heparin is stopped and a platelet aggregation test performed. If this is negative then it is repeated 3 days later, Only if the aggregometry result remains negative, is heparin reinstated if required. It is important to note that the possibility of HIT should be considered if a heparinised patient develops new thrombosis or haemorrhage even if platelet counts are within the normal range 46,63.

Recently, Wallis et al. 104 compared the efficacy of ceasing heparin early (< 48 hrs) and late (> 48 hrs) after the onset of thrombocytopenia. Interestingly, they observed no significant difference in the rate of morbidity or mortality between the two groups of patients. If anything, the rate of thrombosis was slightly higher in the group who stopped heparin early. The authors conclude that other anti-thrombotic strategies, in addition to heparin cessation, should be considered in HIT patients 104.

Prevention by using alternative anticoagulants The use of LMWH and LMW heparinoid (danaparoid=Orgaran) has been associated with a significantly lower risk of HIT than unfractionated heparin 33,79,211. There are a few case reports of LMWH induced thrombocytopenia 82-85 but to date there do not appear to be any instances of Orgaran directly causing this complication. There appears to be a trend towards using LMWH in place of unfractionated heparin, where possible,

51 because LMWH requires less monitoring 79,211,212. Hopefully there will be a corresponding decrease in the rate of HIT.

Another approach that may reduce the risk of HIT is to minimise the time that a patient receives heparin. It has been suggested that oral anticoagulants (vitamin K antagonists eg Warfarin) and heparin can be initiated concurrently but heparin terminated after about 3 days, once the vitamin K antagonists have become effective. This should provide immediate anticoagulant cover but the duration of heparin is too short for HIT to develop 30.

Treatment of HIT

The key treatment of HIT is to immediately stop heparin 29,39,67,88 and, if necessary, institute an alternative anticoagulant that does not cross-react with the patients antibodies. Plasmapheresis has also been used in an attempt to reduce the concentration of pathogenic antibodies. Recently Robinson and Lewis 213 have compared the efficacy of plasmapheresis in what appears to be a retrospective study. They concluded that plasmapheresis within 4 days of the onset of thrombocytopenia was associated with reduced mortality from HIT, while patients who were plasmapheresed later than 4 days, or not at all, had a higher 30 day mortality rate. However, the authors also note that the patients that benefited from early plasmapheresis also had heparin stopped sooner after thrombocytopenia than the other patients.

Failure to stop heparin runs the risk of serious and life threatening thrombosis and rarely haemorrhage 58. Once heparin has been withdrawn the platelet counts recover in 5- 7 days 20,24,29,58 and presumably the risk of thrombosis due to the HIT antibody is diminished. However, the patient may still require anticoagulant cover and there is no clear guideline as to the most appropriate drug to use. Early approaches to alternative anticoagulation, such as Dextran 29,62 or Aspirin 29,39,59, have been superseded or found ineffective. Some others, like, hirudin and argatroban are still largely experimental in the treatment of HIT.

52 Treatment with LMWH LMWH has been used as an alternative to unfractionated heparin. Leroy et al. 88 treated 34 cases of HIT with LMWH (CY 216). All but three survived. Retrospective analysis of some patient plasma indicated that the fatalities were associated with a positive aggregation assay using the LMWH. However, some patients cross-reacted with the LMWH and survived. The authors do not clarify whether the non-fatal thromboses, that they observed, occurred in patients whose plasma cross-reacted with the LMWH. Nevertheless, they conclude that it is “imperative to perform indirect aggregation tests with LMW heparin before prescribing it to patients with [HIT]” and LMWH should only be used if such tests are negative 88. Similar sentiments have been expressed by others following unsuccessful administration of LMWH that was subsequently found to support platelet aggregation in vitro 214,215. While there are a number of reports that indicate the successful use of LMWH once cross-reactivity has been excluded by in vitro assays 87,216-218 LMWH remains a poor alternative to unfractionated heparin in HIT because the rate of in vitro cross-reactivity is generally high (see Table 2, p.55).

Treatment with Oral Anticoagulants Warfarin (or another vitamin K antagonist/oral anticoagulant) was the treatment of choice for HIT in the 1980’s. Chong 27,29,129 recommended that, upon the diagnosis of HIT, heparin is stopped immediately and warfarin therapy initiated. To provide anticoagulation for the few days that warfarin takes to be effective, the use of dextran or anti-platelet agents (aspirin, dipyridamole) were suggested 27,29,129. This approach has been supported by others 101. Bell 62 and King 30 also suggested warfarin but advocated continuing heparin until warfarin became effective. The use of warfarin is no longer considered such a good treatment for HIT. Warkentin et al. 183,184 and Pötzsch et al. 185 have drawn attention to a potentially serious effect of warfarin. That is, warfarin not only inhibits the vitamin K dependent pro-coagulant factors but also inhibits the anti- coagulant action of the protein C pathway. They observed that venous limb gangrene was associated with the use of warfarin in HIT patients 184. The mechanism for this was proposed to be an imbalance between pro-coagulant and anti-coagulant pathways of haemostasis. Specifically, the HIT patients that developed venous limb gangrene had signs of elevated thrombin generation, which was attributed to platelet-derived

53 microparticles released following platelet activation 183. Concurrently, the warfarin decreased the protein C activity of treated individuals. Thus some of the HIT patients were producing excess thrombin without a counterbalancing increase in protein C activity and it appears to be these individuals who suffered the venous limb gangrene. Interestingly, these patients still gave the impression of being well anticoagulated because their INRs were significantly longer than warfarinised patients who did not develop venous limb gangrene.

Thus warfarin may be contraindicated and “potentially dangerous” 183 in patients with HIT, at least until the thrombocytopenia has resolved and parenteral anticoagulation is established185.

Treatment with Orgaran Currently, either Orgaran (danaparoid sodium, Org 10172, LMW heparinoid) or ancrod is the treatment of choice for HIT 54,108. Orgaran is a mixture of heparan sulfate (84%), dermatan sulfate (12%) and chondroitin sulfate (4%) 219. The main anticoagulant effect of Orgaran comes from its anti-Factor Xa activity rather than its minimal anti-thrombin action 219. The use of Orgaran in treating HIT is supported by its low rate of cross- reactivity in in vitro assays. The best estimate appears to be that about 10% of HIT patients possess antibodies that cause platelet activation in the presence of Orgaran 59,90- 92,94. Table 2 (p.55) illustrates that this compares favourably with the 90% cross- reactivity rate of LMWH. The clinical experience with Orgaran is also positive. The largest collection of patient data concerning the treatment of HIT with Orgaran was compiled by Magnani 92 who is from the company that developed the drug. He compiled the clinical outcomes of 230 HIT patients, from around the world, that were treated with Orgaran and reported only 15 (7%) treatment failures 92. The conclusion that Orgaran is a suitable alternative to heparin in HIT patients, who show no evidence of in vitro cross- reactivity, is supported by other authors 94,129,220-222. Warkentin and Barkin 223 have recently summarised the clinical use of Orgaran to treat HIT.

54 Table 2 - In vitro cross-reactivity of HIT antibodies with heparin-like anticoagulants Investigators Fragmin Clexane Danaparoid Method Chee et al. 224 40/45 (89%) 34/41 (83%) 3/45 (7%) Aggregation Slocum et al. 217 43/126 (34%) Aggregation Ramakrishna et al. 216 6/15 (40%) 2/15 (13%) Aggregation Kikta et al. 225 13/51 (25%) 10/51 (20%) Aggregation Ortel et al. 221 0/5 (0%) Aggregation Greinacher et al. 95 40/40 (100%) 20/20 (100%) 0/40 (0%) HIPA Greinacher et al. 93 14/14 (100%) 14/14 0/14 (0%) SRA Greinacher et al. 91 69/70 (99%) 69/70 (99%) 7/70 (10%) HIPA Harbrecht 226 6/6 (100%) 6/6 (100%) 0/6 (0%) Aggregation Chong et al. 94 3/17 (18%) Aggregation Goual-Heilmann 1/5 (20%) Aggregation et al. 215 Borg et al. 227 ≈28/33 (85%) ≈28/33 (85%) Aggregation Makhoul et al. 59 12/14 (86%) 0/13 (0%) Aggregation Amiral et al. 160 0/49 HIT plasmas reacted with PF4 mixed ELISA with the ATIII-binding pentasaccharide Arepally et al. 163 6/12 (50%) and 4/12 (33%) HIT plasmas ELISA cross-reacted with two different LMWHs Newman et al. 90 23/26 (88%) 23/26 (88%) 13/26 (50%) Fluid-phase EIA

This table summarises studies that have investigated cross-reactivity of heparin dependent antibodies with LMWH (Dalteparin and Enoxaparin) and Danaparoid. Only publications with more than four cases have been included and data compiled by Magnani 92 has been excluded because many of the individuals may be reported in other publications.

Treatment with Ancrod Ancrod has been used to provide rapid (within 12 hr) anticoagulation in patients with HIT 228. Ancrod is a serine protease derived from the venom of the Malayan pit viper that cleaves fibrinopeptide A in fibrinogen 229,230. In contrast thrombin cleaves fibrinopeptide A and fibrinopeptide B from fibrinogen and activates fibrin stabilising

55 factors. Thus, ancrod promotes the formation of an unstable and non-cross-linked fibrin matrix that is readily degraded before a thrombus can form 228,229. This leads to rapid depletion of fibrinogen so reduces fibrin clot formation and inhibits platelet aggregation, which is dependent upon binding of fibrinogen to the GPIIb-IIIa complex on the platelet membrane 110,117. The successful use of ancrod to treat HIT has been described in small studies 228,231. Demers et al. 231 used ancrod to provide short term anticoagulation until warfarin, which was administered concurrently, became effective. Cole et al. 228 report treating HIT patients with ancrod for longer periods (up to 20 days) before they were switched over to warfarin. Lathan and Staggers 229 successfully used ancrod alone to anticoagulate a man with a history of HIT who required a coronary artery bypass. In the opinion of some, ancrod is an even better choice than Orgaran for anticoagulation of HIT patients 232. However, Warkentin and Barkin 223 point out that it has the disadvantage of requiring slow administration and may potentiate the early pro- thrombotic risk of warfarin.

56 Heparin

Heparin was discovered and partly purified by McLean around 1916 233,234. He initially called it heparphosphatid because it was purified from liver and assumed to be lipid. Howell and Holt 235 proposed the name "Heparin", and characterised it a couple of years later. While the structure and the mode of action of heparin (thought to activate a “pro- antithrombin”) that they proposed do not conform to the current understanding, they clearly demonstrated the potent anticoagulant effect of heparin 235. The fascinating early history of the discovery and development of heparin is further described by Best 236. We now know that heparin is a glycosaminoglycan that exerts it anticoagulant effect by accelerating the inactivation of thrombin (see Mode of anticoagulant action, p.61). The benefits of post-operative heparin as prophylaxis against thromboembolic complication was demonstrated by Crafoord and Jorpes 237,238. Over its 60 year history of clinical use, heparin has proven to be generally safe and effective in preventing thrombosis 239-241.

Clinical use of heparin

Heparin is used in a wide range of situations where anticoagulation is required. Low doses of heparin (5,000 IU by deep subcutaneous injection every 8-12 hrs 242,243) are effective for the prophylaxis of venous thrombosis following surgery, trauma or prolonged immobilisation 243,244. Higher doses of heparin (20,000-40,000 IU per day 242) are used therapeutically in patients with thrombosis such as DVT, pulmonary embolism and myocardial infarction 243,244. The dose of heparin is usually adjusted so that the patient’s plasma takes 1.5-2.5 times as long to clot as normal plasma in an activated partial thromboplastin time test 243,245,246. This corresponds to a plasma heparin concentration of about 0.2-0.4 U/ml 243. Other applications of heparin include mini doses to flush indwelling catheters and very large doses to maintain extracorporeal circulation during dialysis or cardiopulmonary bypass 244. While all dosages of heparin have been associated with HIT, heparin retains some advantages over alternative anticoagulants. It is inexpensive, acts immediately, its action is reversible with antidotes, and it is generally safe and well tolerated.

57 Other side effects

Obviously heparin can induce thrombocytopenia but haemorrhage (without thrombocytopenia) is the most common adverse effect of heparin therapy 243,244. The risk of bleeding is greater with higher doses of heparin 244,247 and when heparin is administered periodically rather than by continuous infusion 248. However, other factors, such as co-morbid conditions, old age and PT or APTT clotting times that are more than 2-3 time that of control plasma, are at least as important predictors of haemorrhage 247- 249. In addition, concomitant administration of other anticoagulants, anti-platelet or thrombolytic drugs may also increase the risk of bleeding 242,247,248,250. Bleeding is treated by stopping heparin or reducing its dose 245. If necessary heparin can be rapidly neutralised by an injection of protamine sulfate 245.

Long-term administration of heparin has been associated with osteoporosis 244,251,252.

Structure and synthesis of heparin

Heparin is a heterogeneous mixture of highly sulfated glycosaminoglycans of different chain lengths and monosaccharide sequences. The molecular weight of the heparin chains varies from 1.8 kD to 30 kD with a mean of about 15 kD (≈50 monosaccharide units) 243,244. However, a minimum chain length around 18 monosaccharide units (5.4 kD) is required for maximum anticoagulant activity. The sequence of linear (ie unbranched) monosaccharide units consists of alternating glucosamine and hexuronic acid moieties. The hexuronic acid units may be either iduronic acid or, less frequently, glucuronic acid (Figure 3).

58 6 COO- 5 O O O - 4 1 COO OH OH O OH O 2 O 3 N OH OH Glucosam ine Iduronic acid Glucuronic acid

Hexuronic acids Figure 3 - Fundamental sugar moieties of heparin Heparin consists of alternating glucosamine and hexuronic acids that have been modified by sulfation and acetylation. The numbers on the glucosamine structure indicates the convention for numbering carbon atoms in these structures. Note that the iduronic and glucuronic acids differ only in the orientation of the C6 carboxy group around the C5 carbon.

The structure of a hypothetical heparin chain is shown in Figure 4. In heparin, the glucosamine units are usually sulfated but small amounts of N-acetyl-glucosamine and plain glucosamine units may be present 253,254. A specific pentasaccharide sequence is important for the heparin’s anticoagulant activity because it is this site that binds antithrombin III. However, this site is present in only about 1/3 to 1/2 of heparin molecules 255,256.

59 - - - H2COH COO H COSO 2 3 H2COSO3 O O O O O O - COO COO- O OH O OH O OH OH OH OH O O O

- - OSO NAc OH HNSO OH - 3 3 HNSO3 2-O-sulfo- N-acetyl- Glucuronic N-sulfo- Iduronic N-sulfo- iduronic glucosam ine acid 6-O-sulfo- acid 6-O-sulfo- acid glucosam ine glucosam ine

- - - - H2COSO3 COO H COSO 2 3 H2COSO3 O O O O O O COO- - - COO O OH OH O OSO O OH OH 3 OH O O O

- - - - OSO3 NAc OH HNSO OSO 3 3 HNSO3 2-O-sulfo- N-acetyl- Glucuronic N-sulfo- 2-O-sulfo- N-sulfo- iduronic 6-O-sulfo- acid 3-O-sulfo- iduronic 6-O-sulfo- acid glucosam ine 6-O-sulfo- acid glucosam ine glucosam ine

Antithrom bin III Binding Site Figure 4 - Structure of heparin Representation of a hypothetical heparin chain, including the specific antithrombin III binding site. Based on descriptions by Lindahl and Kjellén 253,257.

Each hexose (= monosaccharide) unit has three carbons that have the potential to be sulfated. these are C-2, C-3, and C-6. However, not all sites that can be sulfated actually become so. Many of the reactions in heparin synthesis fail to go to completion. In fact, it is the incomplete nature of these reactions that results in the quasi-random sequence of monosaccharide units. The synthesis of heparin is outlined by Lindahl and Kjellén 253 and best understood by observing the different hexose structures in Figure 3 and Figure 4.

A uniform chain of alternating D-glucuronic acid and N-acetyl-D-glucosamine units is constructed on a protein core from uridine-diphosphate-glucuronic acid and uridine- diphosphate-N-acetyl-D-glucosamine within mast cells. Deacetylation of the N-acetyl- D-glucosamine units results in an NH3+ group which is quickly and completely sulfated. A C-5 epimerisation reaction then converts most of the glucuronic acid units into iduronic acid. Finally O-sulfation of the C-2, C-6, and C-3 carbon atoms takes place 253.

The initial deacetylation/sulfation of N-acetyl-D-glucosamine is critical to the subsequent modification of the chain. For a glucuronic acid unit to be converted into

60 iduronic acid it must be flanked by N-sulfo-D-glucosamine on one side (through C-4) and by N-acetyl-D-glucosamine on the other (through C-1) 258. The higher iduronic : glucuronic acid ratio present in heparin, compared to heparan sulfate, is due to a greater degree of N-deacetylation/sulfation, resulting in greater C-5 epimerisation activity 253. Furthermore, C-6 and C-2 sulfation is enhanced by proximity of an N-sulfated D-glucosamine unit 259 and this provides an explanation for the greater degree of sulfation present in heparin vs heparan sulfate.

Mode of anticoagulant action

The anticoagulant activity of heparin comes largely from its ability to bind antithrombin III (ATIII) and thus catalyse the inactivation of serine protease clotting factors by ATIII. Heparin binds ATIII with moderately high affinity, around 100 nM 260-262. This binding of ATIII to a specific pentasaccharide sequence on heparin appears to induce a conformational change in ATIII 260,261,263. If a given heparin chain contains the ATIII binding site and has a minimum length of 18 monosaccharide units 81,264 (Mr>5,400) then it can serve as a surface to bring together thrombin and ATIII in a trimolecular complex 265. ATIII then irreversibly destroys the activity of thrombin by forming a covalent bond with the clotting factor 266. This reduces the affinity of ATIII for heparin and releases the heparin to catalyse further reactions 267.

ATIII, when bound to heparin, will also inactivate factor Xa, but this activity is not affected by the heparin chain length 81,264. Inhibition of other coagulation factors, such as IXa 268, XIa 269, XIIa 270 and VIIa 271, may also play a role in the action of heparins.

LMWH has relatively more anti-Xa activity for its anti-thrombin activity compared to unfractionated heparin 272,273. However, the exact mechanism of LMWH anticoagulant activity is uncertain 274. Interestingly, Hemker and Béguin 275 assert that the predominant effect of LMWH is also through inhibition of thrombin. They argue that the concentration of Factor Va, and not Factor Xa, limits the production of thrombin when there is ample phospholipid. In the absence of added phospholipid, coagulation is dependent upon traces of thrombin activating platelets and the low anti-thrombin activity of LMWH is able to inhibit this 275.

61 Thrombin can also be inactivated by a complex of heparin and heparin-cofactor-II (HCII). This reaction does not require the specific ATIII binding site 276, instead it is dependent upon the overall negative charge of heparin 277. However, the inhibition of thrombin by HCII requires a higher concentration of heparin and a greater chain length (minimum of 26 monosaccharide units) than that required by ATIII 277.

Pharmacokinetics of heparin

Heparin is given by intravenous injection because it is degraded in the gastrointestinal tract. 278. Intramuscular injection runs the risk of large haematomas. The fate of heparin in vivo is varied.

Heparin will bind to a wide range of proteins, often modulating the activity of the protein 279. Of specific interest here, are plasma proteins that can bind to heparin and suppress its anticoagulant activity 280. Examples of such proteins are vitronectin 281,282, fibronectin 282,283, histidine-rich glycoprotein 282,284,285 and PF4 81,286,287. Zammit et al. comprehensively studied the relative binding of the protein constituents of whole plasma and concluded that histidine-rich glycoprotein is the only plasma protein likely to substantially inhibit heparin activity by competition with ATIII 282. Many other proteins bound heparin but they were unlikely to influence coagulation.

The in vivo clearance of a single bolus of heparin displays complex kinetics. Bjornsson et al. 288 observed that moderate doses of heparin (25-75 U/kg) were cleared with first order kinetics with the half-life increasing with dosage. In real patients, being treated for venous thromboembolism, the half-life of heparin activity is about 1-1.5 hr 289 but this varies widely between individuals and is dependent upon the method used to assay the heparin 288,289.

De Swart et al. 290 investigated the kinetics of high dose heparin (75-250 U/kg) and paid particular attention to the early stages of clearance. They observed that in the first half hour there was an initial rapid drop in heparin activity, which they attributed to adsorption onto the endothelium. This was followed by heparin clearance, which at 75 U/kg was nearly first order but at higher heparin doses a component of zero order kinetics became more apparent 290. Thus heparin is eliminated with a combination of saturable (first order) and linear (zero order) kinetics. Renal clearance, with zero order 62 kinetics, probably accounts for the slower removal of heparin 290 but this mechanism plays little role in the clearance of unfractionated heparin except at high doses 246,290,291. However, it is probably the main method of removal of LMWH 273 which is slower than for UH 292.

The experiments just described do not provide the whole picture of the catabolism of heparin because they concentrate on measuring heparin activity rather than quantitating and characterising the heparin molecules. Dawes and Pepper 293 injected 1000 U of iodinated heparin into volunteers and observed that the radioactivity in the plasma declined rapidly as it was taken up by the liver and spleen. However, plasma radioactivity rose to a peak about 3 hrs after injection as the sequestered heparin was returned to the plasma. Importantly, they observed that this peak in radioactivity was not associated with a rise in anticoagulant activity. In fact, they demonstrated that, while the heparin in the plasma after 3 hrs was of full molecular weight, it was largely desulfated and did not bind ATIII 293. They conclude that first steps in heparin catabolism are N- and O-desulfation that occur in the reticuloendothelial system of the spleen and liver and that this destroys biological activity. Evidence that endothelial cell do indeed play a role in heparin catabolism comes from observations that heparin binds to endothelial cells 294-297, is internalised 294,295,297,298, degraded 294,297 and can be released back into the exracellular environment 298.

Effect of heparin on platelets

Considering that heparin is an anticoagulant it seems somewhat paradoxical that heparin is also a weak platelet agonist. High heparin concentrations (50µg/ml) can cause spontaneous platelet aggregation in vitro 299 but detecting aggregation produced by lower concentrations (10µg/ml) requires sensitive techniques such as aggregometry with an expanded scale 34 or electronic particle size analysis 40. More noticeable, is the potentiating effect that heparin has on aggregation induced by other agonists. Heparin (at near pharmacological concentrations) increases the degree of aggregation and/or lowers the concentration of agonist required for aggregation induced by ADP 34,37,38,80,300-302, adrenaline 34,37,38,80,300-302, arachidonate 37,38, Staphylococcus aureus 38 and aggregated IgG 38,131-134. This effect has also been observed in platelets

63 collected from individuals following injection of heparin 300,301. In contrast to unfractionated heparin, LMWH does not potentiate the effect of platelet agonists 34,80.

The effect of heparin on collagen induced platelet aggregation is less clear. Some reports indicate potentiation 38,80 but others found no effect 300 or even inhibition 303. The data of Mohammad et al. 37 suggest that high (18 U/ml) heparin concentrations may inhibit the effect of collagen while low concentrations (1.2 U/ml) may enhance it. Ristocetin is one of the few platelet agonists that is opposed by heparin 301 and this effect may be due to heparin impairing the ability of vWF to bind platelets 304.

The proaggregatory effects of heparin may well be clinically significant because the hyperaggregable platelets from patients with anorexia nervosa or severe peripheral vascular disease are activated by heparin in vitro 80,203. Lecrubier et al. observed that spontaneous platelet aggregation may be more common in patients with prosthetic heart valves who were treated with heparin than those who were not 204. Heparin may promote 305,306 platelet aggregation because it antagonises the anti-aggregatory effects of PGI2 , 307 306 PGE1 and PGD2 , perhaps by inhibiting adenylate cyclase which is normally 307 activated by PGE1 . There is also evidence that heparin promotes platelet aggregation by generating thromboxane 299.

PF4

Platelet factor 4 (PF4) is a protein released from the alpha granules of platelets upon platelet activation 308-310. In the absence of its proteoglycan carrier, high concentrations of PF4 are only soluble at high ionic strength 311,312. PF4 has high affinity for heparin and neutralises the anticoagulant activity of heparin 81,313. The tissue localisation of PF4 is largely restricted to platelets, megakaryocytes and endothelial cells 314 but McLaren et al. have reported PF4 antigen in the granules of mast cells and perhaps basophils but not leucocytes 315. A major and definitive role for PF4 has not been identified. Rather, a variety of effects on various physiological functions, such as, angiogenesis, platelet production, coagulation and immunoregulation have been observed and PF4 is now considered to be involved in the mechanism of HIT.

64 PF4 in the Plasma

The level of circulating PF4 in the plasma is generally low, although estimates vary. Mean values ± SD of 13.9±6.1 ng/ml 316, 13.9 ± 6.1 ng/ml 191, 18.1±6.6 ng/ml 192, 13±5.5 ng/ml 317, have been measured. The true value may in fact be lower. Handin et al. report 16±4 ng/ml but believed that much of this was due to residual platelets 318. A couple of laboratories detected only 1.8±1 ng/ml 319 or 3.4±1.8 ng/ml 320. Others report very high levels like 720 ng/ml 309 but Kaplan et al. 321 contend that this was probably due to platelet contamination.

It has been suggested that cardiovascular disease and other conditions that cause platelet activation may raise the circulating PF4 concentration 317-319. However, Kaplan and Owen contend that elevated PF4 is a sign of in vitro release and that measuring βTG levels (or βTG : PF4 ratio) gives a better indication of the in vivo platelet state 322. They report that the only condition to genuinely increase circulating PF4 is heparinisation. The in vivo interaction between PF4 and heparin is reviewed in detail later (see Interaction between PF4 and heparin, p.74).

Plasma Kinetics of PF4 Animal studies indicate that injected human PF4 is cleared from the circulation with biphasic kinetics 177,189,192,323-325. Initially, a period of rapid clearance dominates but this is followed by a period of slower elimination. Estimates of the half lives of the initial & later clearances respectively include 2.1 & 70 min (monkeys)324, 0.75 & 20 min (rabbits)325, 6.2 & 91 min (rats)323, 2 & 41 min (hamster)189, 1 & 18 min (rabbits)177, 1.2 & 17.1 min (rats)192. Injecting high doses of PF4 does not alter theses kinetics 189,325 but results in circulatory problems due to microthrombi before all the binding sites are saturated 189. The addition of heparin slows the removal of PF4 from the plasma by eliminating the short half-life component 177,189,192,325. Interestingly, the inclusion of heparin with PF4 increases the amount of intact PF4 appearing in the urine by two orders of magnitude 325.

The early fast phase of clearance has been attributed to adsorption of PF4 to the microvasculature 192,324 and is supported by observations that PF4 binds to endothelial cells in vitro 326,327. However, Rucinski et al. observed that within 5 min of the injection

65 of 125I-PF4 into rabbits about 40% of the radioactivity was localised in the liver 325,328. The fact that a subsequent injection of heparin could remobilise part of the hepatic radioactivity suggests that the PF4 was bound to the endothelium within the liver 325. Over time, however, the liver catabolises the PF4 that it takes up and releases low molecular weight degradation products back into the circulation 325. Experiments with cultured hepatocytes confirms their ability to internalise and degrade PF4 328 but this is also a characteristic of cultured endothelial cells 327.

Physiological role of PF4

PF4 in Inflammation and Immune regulation PF4 displays a variety of proinflammatory and immunoregulatory activities. Deuel et al. first showed that high concentrations of PF4 (1-5 µg/ml) were chemotactic for neutrophils and monocytes 329. This was subsequently confirmed by others 330,331 and increased adherence of PF4-stimulated neutrophils to plastic and endothelial cells was observed 331. PF4 appears to prime neutrophils by a different mechanism to GM-CSF 332 and both N-terminus and C-terminus regions are important for this activity 333. However, it is not clear whether the high PF4 concentrations are physiologically relevant and Walz et al. observed that the neutrophil chemotactic potency of PF4 was negligible compared to NAP-2 334. Interestingly, peptide fragments of PF4 tend to be more potent neutrophil activators 333 and monocyte chemoattractants 335 than the whole protein.

Another curious effect of PF4 is its ability to reverse immunosuppression. Katz et al. studied a model of immunosuppression where the injection of syngeneic lymphoma cells or concanavalin A suppresses the immune response to sheep red blood cells 336,337. They observed that the injection of releasate from mouse or human platelets could more than overcome the effect of the immunosuppressive agents 336,337. PF4 was identified as the active component in the platelet releasate on the basis of its characteristic high affinity for heparin. It probably acts by binding and inhibiting activated peripheral T suppressor cells 337. Subsequently, a peptide consisting of the last 13 C-terminal amino acids of PF4 was shown to have the same activity, which was not dependent upon the integrity of the four C-terminal lysines that are involved in heparin binding 338.

66 Interestingly, the anti-immunosuppressive effect of PF4 was dependent upon an enzyme released from platelets along with PF4 because immediate treatment of platelet releasate with a serine protease inhibitor resulted in the releasate having no effect on immunosuppression. Whereas addition of the protease inhibitor after the platelet releasate had been incubated by itself for 60 min still allowed the PF4 to reverse the immunosuppression 336. Incidentally, PF4 that is purified from a recombinant source 339 or outdated platelets 336 by affinity for heparin sepharose possesses anti- immunosuppressive activity without the need for protease.

PF4 and coagulation The ability of PF4 to antagonise the anticoagulant activity of heparin in vitro has, not surprisingly, lead to speculation that PF4 is procoagulant in vivo 81,325. Evidence supporting this comes from studies by Busch and Whyte 340 who perfused a mixture of thrombin and ATIII through the microcirculation of a rat heart or through a column of microcarrier beads coated with bovine endothelial cells. In each case the heparin-like glycosaminoglycans on the endothelial cells accelerated the inactivation of thrombin by ATIII. However, the inclusion of PF4 (10µg/ml) in the perfusate either partially or completely abolished this effect 340. Marcum et al. observed a similar effect of PF4 in rat hind limbs that were perfused with thrombin and ATIII 341.

However, there is evidence that PF4 can also have anticoagulant activity. Specifically PF4 can inhibit the activation of factor XII (Hageman factor) by dextran sulfate 342 and lengthen the plasma clotting time. The addition of PF4 to plasma does not alter the kaolin clotting time 342 but pretreatment of kaolin or glass with PF4 appears to reduce the ability of these substances to activate factor XII 343.

PF4 also displays an anticoagulant effect by increasing the production of activated protein C 344. It appears to do this by binding to an anionic domain on protein C, which induces a conformational change 345. PF4 also binds to a glycosaminoglycan domain on thrombomodulin and perhaps neutralises some of its negative charge 345. The effect of PF4 is to markedly increases the affinity of a thrombin-thrombomodulin complex for protein C and thus accelerate, 25 fold, the production of activated protein C by the thrombin-thrombomodulin complex 344. It is noted that this effect of PF4 only occurs at

67 high concentrations (3-10 µg/ml) and so may only be physiologically relevant in localised areas of platelet activation 345.

Regulation of Platelet production by PF4 Han et al. observed that PF4 inhibited megakaryocyte colony formation and maturation when added to bone marrow cell cultures at concentrations above 2.5 µg/ml 346,347. As little as 30 min exposure to 5 µg/ml PF4 inhibited megakaryocyte colony formation 346. They argue that these concentrations of PF4 may be achievable in the marrow sinusoid, which does not exchange freely with the plasma 346. Others 348,349 required substantially higher concentrations of PF4 (≥10 µg/ml) to detect significant inhibition of megakaryocyte maturation 348 and observed that PF4 is 100-1000 times less potent than NAP-2 in inhibiting megakaryocytopoiesis 348. Han et al. also make an important distinction on the effect of PF4. Namely, while they observed that PF4 inhibits the proliferation of normal haematopoietic cells they also found that PF4 simultaneously supports the viability of these cells, particularly in the presence of chemotherapeutic drugs 350.

Lebeurier et al. contend that a sequence of amino acids between from Asn-47 to Pro-58 is important for the inhibitory effect of PF4 on megakaryocytopoiesis because this peptide has a similar effect to native PF4 351. Importantly, they also demonstrated that injection of 1 µg PF4 (or 5 µg of the peptide) per day into mice for four days reduced the ability of the bone marrow to subsequently form megakaryocyte colonies 351.

Lecomte-Raclet et al. report that the core of PF4 contains a peptide sequence (p34-58) that has 60-300 fold greater potency as an inhibitor of haematopoiesis than native PF4 352. It is not clear if this serves a physiological function because it is inaccessible in the whole molecule 352. However, they propose that it may be unmasked when PF4 binds to heparin-like molecules on the surface of haematopoietic progenitor cells 352.

Platelet associated PF4 When released from platelets, PF4 is complexed to a high molecular weight chondroitin sulfate proteoglycan carrier 312,353,354 that heparin 287 or salt 312 will readily displace. Release of PF4 is particularly associated with the second phase of platelet

68 aggregation 308. The total amount of PF4 in platelets is reported to be variously 20-30 µg 317, 7.7 µg 318 and 9-35 µg (mean 18±7 µg) 355 per 109 platelets.

Platelet activation also results in increased expression of PF4 on the surface 356. Binding of extracellular 125I-PF4 has been demonstrated and reaches equilibrium in 15-30 min 193. 1 µg/ml PF4 saturates the ≈2700 binding sites per unstimulated platelet with a Kd of 27 nM 193. While the expression of PF4 on the platelet surface increases with thrombin stimulation, thrombin does not increase the number of PF4 receptors. Instead it is presumed that PF4, released by thrombin, binds to existing receptors 193.

Endothelial associated PF4 Busch et al. 326 demonstrated that PF4 will bind to cultured endothelial cells in a specific and saturable manner. Binding was presumed to take place through glycosaminoglycans on the endothelial surface and a range of glycosaminoglycans in solution competed with endothelial cells for PF4. Rybak et al. observed that binding of PF4 to endothelial cells reaches equilibrium in about 20 min 327. Approximately 10 µM (300 µg/ml) PF4 was required to saturate the binding sites that had a Kd ≈ 3 µM for PF4 327. The Bmax of this binding was ≈64 pmol/105 cells 327. Bound PF4 was internalised and degraded by the endothelial cells 327. The PF4 that binds endothelial cells may also antagonise the anticoagulant effect of heparin-like molecules 340,341 (see PF4 and coagulation, p.67).

Interestingly, the binding of PF4 to the endothelial cells in vivo is not identical to that in vitro. Hansell et al. 189 investigated the binding of injected PF4 to the microvasculature of hamster cheek pouches. They observed that binding of fluoresceinated PF4 (≈1.5 mg/kg body weight) was localised to short sections of the venules and arterioles. When animals were pretreated with extra unlabelled PF4 at 80 mg/kg of body weight there was no decrease in the number of sites binding the FITC-PF4. Instead, microthrombi, labelled with PF4, and leucocytes appeared at these sites. One can speculate that at these sites the binding of PF4 neutralised the anticoagulant/antithrombotic activity of the endothelial glycosaminoglycans and thus resulted in localised platelet activation. In contrast to the sparse and uneven binding in vivo, FITC-PF4 was internalised by more than 80% of cultured sub-confluent

69 endothelial cells in vitro. Hansell et al. contend that PF4 only binds to proliferating endothelial cells in vitro 189. Subsequently, the same group has elegantly demonstrated that PF4 will preferentially bind to the endothelium of newly formed blood vessels in the area of an angiogenic stimulus 190. They speculate that this may target the anti- angiogenic effects of PF4 to areas of angiogenesis.

PF4 and Angiogenesis The inhibition of angiogenesis by PF4 has been investigated using a chicken chorioallantoic membrane assay 357-359. Maione et al. 358 observed that whole PF4 protein at 1-6.5 nmol per disk produced a dose dependent inhibition of vascularisation. Peptide fragments indicated that this activity was associated with the last 13, C-terminal, amino acids of PF4 358. Recombinant PF4 was also observed to reversibly inhibit the proliferation 358 and migration 360 of cultured human umbilical vein endothelial cells but not fibroblasts or keratinocytes 358.

The same group then demonstrated that the growth of tumour cells, which had been injected subcutaneously into mice, could be inhibited by injecting recombinant PF4 (50 µg/day) into the tumour 360,361. This effect was also observed in athymic nude mice, which excludes a role for T cells and makes it unlikely that PF4 was just promoting an immune response against the tumour cells. They argued that this effect was due to the inhibition of angiogenesis because PF4 was not toxic to the tumour cells in vitro 360.

Structure of PF4

PF4 is classed as a CXC (or α) chemokine. Other members of this family include IL-8, βTG, and NAP-2 314,362. Under physiological conditions, PF4 exists as a tetramer of identical subunits 193,363,364. Each subunit of PF4 consists of a known 70 amino acid sequence (7.8 kD) 365-367. The PF4 gene has been cloned 330,359,368,369but there is some evidence that there is an alternate gene with three amino acid substitutions near the C-terminus of the protein 370.

On SDS PAGE, without reducing agents, PF4 tetramers dissociate into monomers that run as a single band at 7.8 kD359, 8 kD 330, 9 kD 179,339, 9.6 kD 371, 10 kD 372, or 11.6 kD 311. The pI is reported to be 7.6 371. Two intra-chain disulfide bonds exist

70 between the four cys amino acids, and there is no free cysteine present in the subunits 366 and no covalent bonds between subunits. The carboxy terminal region has two pairs of lysine residues that are separated by a pair of isoleucine amino acids 365,366. This region appears important for heparin binding (see In vitro molecular interactions, p.74).

X-ray crystallography and NMR studies have largely determined the three dimensional structure of the tetramer. St.Charles et al. 373 investigated the crystal structure of bovine PF4 while Zhang et al. 374 that of human PF4. They both demonstrated that the PF4 tetramer was not simply a tetrahedron (triangular pyramid) formed by four spherical subunits as claimed by Cowan et al. 375. Instead, they described it as a pair of dimers. They suggested that the dimers may consist of a “floor” of anti-parallel β-sheet, on top of which lies two α-helices 373,374. Each monomer contributes one α-helix and to half of the β-sheet floor (Figure 5-i). Interestingly, these dimers are very similar to those that comprise the whole IL-8 molecule 362. The PF4 tetramer appears to comprise of two dimers joined by the β-sheets lying back to back and forming a “β-sheet bilayer” 373,374 (Figure 5-ii). This results in pairs of α-helices on opposite sides of the PF4 tetramer. Another way of visualising the three dimensional structure is to imagine that the dimers form half a sphere, with the β-sheets at the equator and the α-helices forming the poles. Together, the two half-sphere dimers form a roughly spherical PF4 tetramer (Figure 5-iii).

In order to discuss the interaction between PF4 subunits each subunit must be named. St.Charles et al. labelled the first subunit ‘A’, “its partner in the extended β-sheet as ‘B’, its nearest neighbour in the opposite β-sheet as ‘C’ and its farthest neighbour ‘D’ ”. This is illustrated by the colours in Figure 5-ii .

The subunits of PF4 are held together by non-covalent bonds. The dimer is maintained by hydrophobic and hydrogen bonding between monomers, while ionic bonds between dimers are predominantly responsible for tetramer integrity 373,374. However, the N-terminal regions of A and C subunits (similarly B and D) lie anti-parallel to each other, across the plane of the β-sheets, and hydrogen bonding between N-terminal residues also contributes to tetramer stability 374.

71 Mayo and Chen 363 have used NMR to investigate the association of PF4 subunits in solution. They concluded that the formation of tetramers results from the coalescing of dimers but under most conditions the tetramer state was heavily favoured. However, acidic conditions cause the tetramer to partly dissociate into dimers and monomers 363. This feature was used to investigate the nature of the dimer intermediate. Mayo and Chen 363 suspect that the dimer comprises A and C (or B and D) subunits. This conflicts somewhat with the models presented by others where A and B (C and D) subunits are suggested 373,374 and would mean that the PF4 dimer does not resemble the IL-8 dimer.

72 (i) (ii)

(iii)

Figure 5 - Tertiary and quaternary structure of PF4 (i) Representation of a PF4 dimer, viewed looking down on the β-sheet “floor” (dark colours) with an α-helix (light colours) of each subunit lying on top. (ii) Representation of a PF4 tetramer consisting of two dimers viewed from the front. The two β-sheets are seen side-on and lie back-to-back forming the equator of the tetramer. The pairs of α−helices are shown on the top and bottom of the tetramer. (iii) A space filling model of the PF4 tetramer showing its roughly spherical shape. In all models the different colours represent subunits. Blue is subunit A, red is B, green is C and yellow is D. These pictures were developed from the data supplied by Zhang et al. 374 to the Brookhaven Protein Databank (www.rcsb.org, Brookhaven Code 1RHP). The images were generated using the RasMol version 2.6 computer program which is written by Roger Sayle (Glaxo Welcome) and is available at www.umas.edu/microbio/rasmol.

73 Interaction between PF4 and heparin

In vitro molecular interactions

Heparin binds quickly 371, and with high affinity, to PF4. Estimates of the Kd are 30 nM 376,377 and 4 nM 345. The fact that 1.4 M NaCl is required to break this electrostatic bond 177 gives an indication of how strong it is. The disulfide bonds within PF4 are important for heparin binding because PF4 reduced with β-mercaptoethanol is eluted from immobilised heparin at only 0.5 M NaCl 177. Reduction of disulfide bonds decreases the affinity for heparin 20 fold 178 and results in PF4 dimers rather than tetramers 178. Interestingly, heating PF4 to 100°C does not diminish the affinity for heparin 177.

PF4 neutralises the anti-IIa (thrombin) and anti-Xa activity of unfractionated heparin 286,313 but is only able to partly neutralise the anti-Xa activity of LMWH 286. The minimum size of heparin required for PF4 binding lies somewhere between 8 and 16 saccharide units 313. PF4 is unable to properly bind and neutralise the anti-Xa activity of an octasaccharide that contains ATIII binding site 313.

The binding of PF4 to heparin appears to be quite different to that of ATIII. While ATIII binds to a specific saccharide sequence, present on a minority of heparin molecules, PF4 will interact with binding sites present on all heparin chains 378-380. The heparin binding capacity of PF4 has been variously reported as ≈27 units 365 or 100µg 313 of heparin per milligram of PF4. While the stoichiometry of this interaction is reported to be 1:1 381, Bock et al. 379 observed that at low (limiting) concentrations of heparin a high molecular weight multimolecular complex formed with PF4. Only when excess heparin was added did this complex break down into complexes consisting of one heparin molecule and one PF4 tetramer. The multimolecular complex could be reformed by addition of excess PF4 379. Formation of the multimolecular complex was favoured by acidic pH and by low ionic strength. In addition, high molecular weight heparins (> 15,000) more readily formed multimolecular complexes with PF4 379.

Ibel et al. 382 proposed a model where by heparin wrapped around the PF4 tetramer. They conducted experiments in an excess of heparin, thus enforcing the 1:1 stoichiometry of the PF4-heparin complex. Using neutron scattering, they observed that

74 the heparin molecule was further from the centre of the complex than PF4. Thus they concluded that heparin surrounded the PF4 tetramer, rather than being incorporated into it. The most likely interaction was hypothesised to be a complex where by the negatively charged sulfate groups on the heparin interacted with a positively charged patch present on the surface of each of the four subunits of PF4.

X-ray crystallography of bovine PF4 revealed “a belt of positively charged residues around the PF4 tetramer”, partly due to four lysine residues present on the surface of an alpha-helix in each monomer 373. Stuckey et al. 383 proposed that heparin either bound to this region or was located in a shallow hydrophobic crevice formed between the two alpha-helices of each dimer. Talpas and Lee 384 observed that binding of heparin to human PF4 perturbs the 1H NMR resonance of a histidine residue which lies within the belt of positive charge to a greater degree than one which does not. This has been considered strong evidence for electrostatic interactions between the positive charge belt of PF4 and heparin. Carboxypeptidase digestion of PF4 to remove the C-terminal lysines markedly reduces affinity for heparin 376. Site directed metagenesis studies indicate that changing any of these lysines to alanine decreases, but does not abolish heparin binding 180,385. Substituting all four lysines simultaneously further reduced the affinity for heparin, but not as much as replacing arginines, within the positively charged belt, with glutamine 385. Consequently, Mayo et al. 385 argue that arginines are more important than lysines for heparin binding. This appears to conflict with earlier reports where chemical modification of lysines reduced heparin binding but modification of arginine residues had no effect 371. The location of amino acids that are purported to have a role in heparin binding are shown on a space-filling model of PF4 in Figure 6.

75 Figure 6 - Space-filling model of human PF4 showing putative heparin- binding and HIT antibody-binding sites The surface of the PF4 tetramer exhibits a belt of positive charge which is formed by lysines-61, 62, 65 and 66 (red), arginines-20, 22 and 49 (orange), histidine-23 and lysines- 31 and 46 (yellow) 374,385. It is likely that heparin binds this region although the relative importance of lysine and arginine residues is debated. Ziporen et al. claim that some HIT antibodies recognise an epitope around proline-37 (blue) and they have published a similar illustration 180. This Figure was generated with the same software and data as Figure 5.

In vivo release of PF4 by heparin

The high affinity that PF4 has for heparin results in interesting interactions in vivo. Dawes et al. 191 observed that a single intravenous injection of heparin resulted in an immediate (within 2 min) increase in plasma PF4 levels and this has been confirmed by others 192,320. The PF4 peak coincided with the plasma heparin concentration and the levels of both reactants declined together with a half life of 15-20 min191. By 90 min the PF4 concentration was almost back to normal. Importantly, a repeat dose of heparin at this time did not trigger an increase in PF4. It took one 320 or two 191 days for the amount

76 of PF4 released with a second dose of heparin to be just half that of the first dose. Only after 6 days were individuals fully responsive again 191. The most likely source of the released PF4 is the vascular endothelium because heparin by itself does not usually stimulate the release of PF4 from platelets 191.

Other drug-induced and immune thrombocytopenias

Heparin is not alone in its ability to cause thrombocytopenia. A wide range of drugs may induce thrombocytopenia by a variety of mechanisms 129. Chemotherapy may disrupt haematopoiesis and thus suppress platelet production along with other blood cells. Ristocetin, which was used as an antibiotic, directly aggregates platelets 386 resulting in platelet consumption exceeding production. Many drugs cause thrombocytopenia through an immune mechanism 105. Quinine/quinidine, heparin and gold drugs are the most common causes of drug-induced immune thrombocytopenias 121. Antibody mediated thrombocytopenia can also arise spontaneously. Idiopathic thrombocytopenic purpura (ITP) and the antiphospholipid syndrome (APS) are both autoimmune conditions where patients posses antibodies directed against platelet proteins, without requiring a foreign compound. An overview of antibody associated thrombocytopenias and their pathophysiology helps to put HIT in perspective and can provide some insights into the mechanisms involved in HIT.

Quinine/quinidine induced thrombocytopenia (QIT)

QIT usually presents as a sudden and severe thrombocytopenia (less than 10x109 platelets/l) 121,129. Patients report warm flushes and chills within minutes of ingesting the drug. Within ≈12 hrs petechiae and purpura develop, followed by haemorrhage in the gastrointestinal and urinary tracts in severe cases 121. Thus, bleeding is the dominant symptom, and in this sense, QIT is a more typical thrombocytopenia than HIT where thrombosis is the chief concern. However, like HIT, the cause of the thrombocytopenia in QIT appears to be decreased platelet survival 387. The disease is usually self limiting, provided the drug is withdrawn. Symptoms abate over 3-4 days and platelet counts normalise within a week 121. Rarely, serious intracerebral and intrapulmonary haemorrhage can occur 121.

77 Antibodies in patients with QIT appear to recognise platelet antigens in association with the drug. The two main antigens that have been recognised are the GPIb-IX complex (more common) and the GPIIb-IIIa complex 388,389. Many patients recognise epitopes on both complexes 390. Recently, Burgess et al. 391 have demonstrated that there is even patient heterogeneity in the subunit of GPIb-IX that antibodies bind. In QIT the Fab region of the antibody binds to antigen immobilised on the platelet surface 392,393. In this thesis I will present data demonstrating that HIT antibodies also bind to antigen immobilised on the platelet surface.

Idiopathic/Immune thrombocytopenic purpura (ITP)

George et al. 121 describe ITP as “an acquired disease of children and adults characterised by a low platelet count, an essentially normal bone marrow, and absence of evidence for other disease”. The disease in children is often associated with a viral or other infection and usually resolves spontaneously. In contrast, adult ITP is chronic and is considered distinct from the childhood condition. The symptoms of chronic ITP are similar to those of QIT except that there is no history of quinine/quinidine. Platelet counts are less than 20x109/l, purpura is common but the occurrence of severe haemorrhage is probably less than 5% 121. Interestingly, in early experiments Harrington et al. observed that infusion of ITP plasma into a normal individual induced thrombocytopenia 394. It is now thought that anti-platelet antibodies are the likely cause. The platelet antigens most commonly associated with ITP are GPIIb-IIIa or GPIb- IX 121,395,396 but curiously, similar autoantigens have also been found in individuals with non-immune thrombocytopenia 397. Antibodies from patients with ITP do not appear to recognise PF4-heparin 163. The spleen appears to be the major site of platelet destruction that causes thrombocytopenia in ITP. For this reason, and the fact that the spleen produces antibody, splenectomy is an effective treatment 121.

Insights into the HIT antigen offered by the antiphospholipid syndrome

The symptoms, pathogenesis and controversies of the antiphospholipid syndrome (APS) share many similarities with HIT 398. A full appraisal of the APS literature is beyond the scope of this review so instead those characteristic particularly relevant to HIT will be highlighted.

78 APS is associated with thrombosis, thrombocytopenia and recurrent spontaneous abortion 399,400. Initially it was believed that patients possessed antibodies, designated antiphospholipid antibodies (aPL), to negatively charged or neutral phospholipids. Subsequently it was discovered that antibody binding to phospholipids was dependent 401-403 upon plasma proteins such as β2-glycoprotein I (β2GPI) and prothrombin . It has now been suggested that the antibodies in patients serum/plasma are directed against modified β2GPI or prothrombin that arises as a result of interaction with phospholipid.

This is very similar to HIT where PF4 corresponds to β2GPI/prothrombin and heparin is analogous to phospholipid. However, in both HIT and APS the antibodies do not invariably cause thrombocytopenia, thrombosis or other pathology. This highlights the uncertainty of whether these antibodies are truly pathogenic or just associated with the disease. For the record, Arepally et al. report that none of 15 plasmas with anticardiolipin antibodies bound to PF4-heparin in ELISA 163.

A number of mechanisms have been proposed to explain the apparent co-dependence of phospholipid and β2GPI/prothrombin for binding aPL. These theories may be equally applicable to the binding of HIT antibodies to PF4 and heparin. None of these mechanisms need be mutually exclusive either within or across individuals. The binding of phospholipid and β2GPI may bring together regions from each molecule and thus 401 form a new antigen, directly contributed to by both phospholipid and β2GPI . In such a case, it would be unlikely that either molecule could be substituted by a range of substantially different molecules. Another hypothesis proposes that a new antigenic site is exposed or formed within β2GPI as a result of binding phospholipid. This is supported 404 by observations that aPL bind to β2GPI on γ-irradiated polystyrene and polyvinylchloride 405 microtitre plates in the absence of phospholipid. Both microtitre plate surfaces are more hydrophilic, anionic, and carboxylated than conventional polystyrene plates. The authors argue that aPL are directed against a cryptic epitope on

β2GPI that is exposed or formed when β2GPI undergoes conformational change during adsorption to a suitable surface. A third mechanism, suggested by Roubey et al. 406, proposes that aPL have low binding affinity for β2GPI molecules free in solution.

However, when β2GPI binds at sufficiently high density to a microtitre plate or phospholipid membrane the bivalent nature of immunoglobulins stabilises the binding

79 of the “antiphospholipid” antibody. When both Fab regions of the antibody bind to joined β2GPI molecules the affinity of the antibody is greatly enhanced.

Platelet activation by HIT antibody is highly Fc dependent but the mechanism of activation by aPL antibodies is not so clear cut. Lin and Wang 407 observed that anticardiolipin antibodies raised in rabbits caused platelet activation but this was not inhibited by blocking the platelet Fc receptor. In contrast Arvieux et al. 408 report that platelet activation by mAbs against β2GPI was suppressed by blocking the Fc receptor. Interestingly, platelet activation by these antibodies required subthreshold levels of weak platelet agonists. Similarly, Shi et al. 409 observed that aPL antibodies only bound to activated platelets.

Key Questions

Considerable advances have been made in the last 10-20 years in understanding HIT. These include, recognising the distinction between immune and non-immune forms, demonstrating the importance of the platelet FcγRII, discovering a strong association between anti-PF4-heparin antibodies and HIT and the use of alternative heparin-like anticoagulants to treat HIT. However, a number of key questions about HIT remain to be answered or further investigated:

Why does HIT only occur in a few percent of patients?

The vast majority of patients who receive heparin do not develop HIT and there are no clear predictive factors that can help identify susceptible individuals (see Incidence of HIT, p.21). Furthermore, about 20% of heparinised patients produce anti-PF4-heparin antibodies but most of these do not develop thrombocytopenia 56,164. Of the patients that do develop HIT, it is unclear why thrombosis only occurs in a subset of them (see Thrombosis, p.23). Resolving these issues should drastically reduce the morbidity and mortality associated with this disease. This thesis investigates aspects of the pathophysiology of HIT and provides data that brings us closer to understanding this disease.

80 How does the HIT antibody bind and activate platelets?

IgG from HIT plasma (plus heparin) can clearly cause platelet activation but there are conflicting models of the mechanism. Some researchers propose that HIT IgG binds soluble antigen to form an immune complex and then activate platelets by cross-linking the platelet Fc receptor (Figure 1- Fluid-phase antigen model, p.44). Others support a model where antigen forms on the platelet surface, binds HIT antibody and only then triggers platelet activation through the Fc receptor (Figure 1- Platelet-bound antigen model, p.44). Data is presented in this thesis that supports the second model and should largely resolve this issue.

What is the best way to diagnose HIT?

The diagnosis of HIT is problematic. The clinical impression is supported by laboratory assays such as platelet aggregometry, 14C-serotonin release assay and ELISA. Agreement between these assays is usually only moderate (see Comparisons of HIT assays, p.50) and a negative test result does not reliably exclude the possibility of HIT 108. A modified immunoassay, which has some advantages over the ELISA, is presented in this thesis but there remains the need for diagnostic tools with greater sensitivity and specificity.

What is the best way to treat HIT?

It is generally accepted that prompt cessation of heparin is desired in HIT but there is debate as to which is the best anticoagulant to replace heparin. I and others have demonstrated that LMWH have a very high rate of cross-reactivity with the HIT antibodies in vitro, which makes them unsuitable (see Treatment with LMWH, p.53). Recently, it has been shown that warfarin probably increases the risk of thrombosis (see Treatment with Oral Anticoagulants, p.53). Various other anticoagulants have been used successfully with danaparoid being one of the most popular. I show that while danaparoid cross-reacts weakly in about half of HIT patients by an immunoassay this is not clinically significant because a number of such patients recovered while receiving this drug.

81 Are the anti-PF4-heparin antibodies pathogenic?

It is clear that antibodies against complexes of PF4 and heparin are associated with HIT but it remains to be proved that these antibodies are the cause of the pathology (see Are anti-PF4-heparin antibodies pathogenic?, p.37). I present evidence that, under conditions that are as physiological as possible, affinity-purified anti-PF4-heparin IgG (plus heparin) can cause platelet activation in vitro.

What is the HIT epitope(s) ?

If we suppose that HIT antibodies against PF4-heparin are in fact pathogenic then it is of interest to identify the specific epitope(s) that are recognised. I present evidence that in many cases the antibodies recognise epitopes solely on PF4 and not epitopes composed of part of a PF4 molecule and part heparin. Various groups have localised different regions of PF4 that may be involved in binding a subset of HIT antibodies (see Epitopes on PF4, p.38) but it is clear that more research is required in this area. I have taken the approach that a first step in understanding the binding of an antibody is quantitating its avidity. Using my technique to affinity purify HIT anti-PF4-heparin IgG I have been able to demonstrate that these antibodies have surprisingly high avidity.

82 Chapter 2 - IgG binding to PF4-heparin complexes in the fluid phase and cross-reactivity with low molecular weight heparin and heparinoid.

Introduction

About 2-5% of patients receiving heparin develop immune thrombocytopenia 28. Heparin-induced thrombocytopenia (HIT) may be complicated by limb- and life- threatening thrombosis. Early diagnosis of HIT is of clinical benefit and may be life- saving. Currently, diagnosis is usually made on a clinical basis and is subsequently confirmed by a positive laboratory test. The laboratory tests are commonly functional assays that measure either the release of 14C-serotonin or platelet aggregation in the presence of heparin and patient plasma 144,145. More recently, an ELISA has been developed that measures antibodies which bind to a complex of platelet factor 4 (PF4) and heparin immobilised on a microtitre plate 158. A PF4-heparin complex is now widely believed to be the major target antigen for the antibodies in HIT 43,45,159.

Low molecular weight heparins (LMWH) and the heparinoid, danaparoid, have been used as alternative anticoagulants in patients who have developed HIT 59,215,216,218,369. A number of reports indicate that patients whose antibodies cross-react with LMWH in a functional assay should not be given these anticoagulants 88,214,215,369,410. However, there is little data concerning cross-reactivity, measured in an immunoassay, of HIT anti-PF4- heparin antibodies with PF4 complexed to LMWH or heparinoid. The clinical significance of any cross-reactivity in an immunoassay is also unclear. This is important because some patients have been found by ELISA to have anti-PF4-heparin antibodies without thrombocytopenia or thrombosis 56,159,163,411. Studies are clearly needed to address these issues.

Two broad theories have been developed that incorporate a role for PF4-heparin complexes and anti-PF4-heparin antibodies in the pathophysiology of HIT. The first, proposes that heparin administration induces the release of PF4 from endothelial cells and elevates plasma PF4 concentrations. The PF4 forms complexes with heparin which bind to the surface of platelets. The Fab portion of the anti-PF4-heparin antibodies then

83 bind the platelet-bound antigenic complexes and induce platelet activation 159,195. The anti-PF4-heparin ELISA, developed by Amiral et al. 158,159 simulates this model. In the ELISA, the target antigen is a solid-phase complex of PF4-heparin coated onto microtitre wells and is akin to a platelet bound complex. The second hypothesis also proposes that heparin elevates circulating PF4 such that PF4-heparin complexes are formed. IgG antibodies bind these complexes in the fluid phase and then the PF4- heparin-IgG complexes activate platelets via the platelet Fcγ receptors II 45. These theories are investigated in detail in Chapter 4 but there has been no explicit demonstration that HIT antibodies will bind to soluble PF4-heparin.

I developed a novel fluid phase enzyme-immunoassay (EIA), simulating the second model for HIT, where complexes of PF4-heparin are reacted in solution with HIT antibodies. In this study, the anti-PF4-heparin ELISA is compared with the fluid phase EIA from a diagnostic point of view. I also investigate the cross-reactivity of HIT antibodies with other heparin-like compounds and, importantly, correlated the test results with clinical outcomes of the patients who were treated with the heparinoid, danaparoid.

Methods

Patients Serum and plasma (citrated or ACD) samples were collected from patients with clinically suspected HIT, ie. patients who developed thrombocytopenia while receiving unfractionated heparin and in whom other causes of thrombocytopenia were excluded clinically. However, only individuals confirmed to have HIT by either platelet aggregation or serotonin release assay were included in this study. Control serum and plasma samples were collected from healthy volunteers (“Normals”) and patients with thrombocytopenia due to causes other than heparin (“Non-HIT thrombocytopenic controls”). This protocol was approved by the Eastern Area Health Service ethics committee and informed consent was obtained from the patients whenever appropriate.

84 Purification of PF4 PF4 was purified from 1800 ml of platelet poor plasma derived from expired platelet packs that were kindly provided by the Australian Red Cross Blood Transfusion Service (Sydney, NSW). Levine and Whol 311 have also purified PF4 from the plasma, rather than the cellular component, of platelet concentrates. PF4 was extracted by its affinity for heparin-sepharose that was thoroughly washed with 2 M NaCl to remove any heparin that may not have been covalently bound. 52 g NaCl was dissolved in the platelet poor plasma to make it 0.5 M NaCl. This plasma was mixed at 4°C overnight with 10 ml heparin-sepharose beads (≈2 mg heparin/ml of sepharose, Pharmacia, Sweden). After washing the sepharose with buffer containing 0.8 M NaCl, 20 mM HEPES pH 7.5, and 5 mM EDTA, a crude PF4 extract was eluted with a similar buffer that contained 2 M NaCl. To remove contaminants, the crude PF4 extract was diluted 1:3 with water and applied to a 7 ml column of S-sepharose (Pharmacia) at 1 ml/min. PF4 became bound to the S-sepharose while the contaminants flowed through. The column was washed with 30 ml of 0.5 M NaCl, 20 mM HEPES pH 7.5, 5 mM EDTA. The PF4 was eluted from the S-sepharose under gravity by repeatedly adding 1 ml of 1.2 M NaCl, 20 mM HEPES pH 7.5, 5 mM EDTA and collecting 1 ml fractions. The concentration of PF4 was determined using BCA protein assay reagents (Pierce, U.S.A.) as reported by others 372. Fractions containing the protein peak were pooled and assayed again to obtain a final PF4 concentration. The purified PF4 ran as a single band on SDS- PAGE (Figure 8, p.91).

Biotinylation of PF4

PF4 was biotinylated through tyrosine and histidine groups 412 with heparin binding sites protected. 4 mg of crude PF4 was coated onto 3 ml (bead volume) of heparin-sepharose in 14 ml 200 mM NaCl, 20 mM HEPES pH 7.4. 400 µl of diazobiotin (Boehringer Mannheim Biochemica, Germany, 10 mg/ml in dimethylsulfoxide) was added to the PF4-heparin-sepharose suspension and incubated for 1 hr at room temperature. Sepharose was washed with 0.8 M NaCl, 20 mM HEPES pH 7.5, 5 mM EDTA and then biotinylated PF4 was eluted with a solution of 2 M NaCl, 20 mM HEPES pH 7.5, 5 mM EDTA. Biotinylated PF4 was purified on S-sepharose column as above.

85 SDS-PAGE SDS-PAGE was performed either using freshly prepared 15% continuous gels or using precast 4-15% gradient gels (Bio-Rad Laboratories, USA). The 15% continuous gels were prepared as follows: Glass plates were thoroughly cleaned then assembled, separated with 1.5 mm spacers. A 15% resolving gel was prepared by mixing 5.5 ml water, 5.6 ml of 40% acrylamide/bis 37.5:1 (Bio-Rad Laboratories), 3.8 ml of 1.5 M Tris pH 8.8, 150 µl 10% SDS then adding 75 µl of 10% ammonium persulfate (freshly prepared, ICN Biomedicals, USA) and 6 µl TEMED (N,N,N’,N’- tetramethylethylenediamide, Progen Industries, Australia). This solution was poured between the gel plates, covered with a layer of water and allowed to polymerise for 30 min. A 5% stacking gel was prepared by mixing 4.4 ml water, 750 µl 40% bis:acrylamide, 760 µl 1.0 M Tris pH 6.8, 60µl 10% SDS then adding 60 µl of 10% ammonium persulfate and 6 µl TEMED. The water on top of the polymerised resolving gel was removed and replaced with the stacking gel solution. A 10 well comb was inserted and the stacking gel polymerised for 30 min.

Protein samples were prepared by mixing with an equal volume of 2x sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol and bromophenol blue to colour). If reducing conditions were required then 4% β-mercaptoethanol was freshly added to the 2x sample buffer. Samples were electrophoresed at 100 V for 1-1.5 hr until the bromophenol blue dye-front reached the end of the gel. A 10 kD protein ladder (Gibco BRL, USA) was used for molecular weight standards. Gels were either transferred to a PVDF membrane or stained with coomassie blue or colloidal coomassie blue.

Stained gels were photographed using a Kodak DC 40 digital camera. The image was processed by the associated Kodak Digital Science 1D software (version 1.6) using the “coomassie blue” setting and saved as a TIFF file. No further manipulation of these images has been performed except to crop irrelevant areas from the edge of the image.

Coomassie blue stain Polyacrylamide gels were stained for 1 hr with agitation in a solution containing 0.02% coomassie brilliant blue R-250 (Sigma), 50% methanol and 10% acetic acid then

86 destained overnight in the same solution but without the coomassie blue. Gels were rehydrated in water for at least 3 hrs before being photographed.

Colloidal coomassie blue stain

A stock solution of stain was prepared by dissolving 11.75 ml H3PO4 (85%, BDH, UK) in 350 ml H2O and adding a suspension of 0.5 g G250 Coomassie Brilliant Blue (Bio-

Rad ) in 50 ml H2O while stirring. The mixing was maintained until 50 g (NH4)2SO4, which was added slowly, had dissolved. Just before use, 40 ml of stock stain solution was mixed with 10 ml methanol. This was then gently agitated with the gel overnight in a sealed container. Destaining was performed with water over the next night then the gel was photographed.

Electrotransfer to membranes from SDS-PAGE gels Polyacrylamide gels were soaked for 20 min in electrotransfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) to remove SDS. A polyvinylidene difluoride (PVDF) membrane (PolyScreen®, DuPont, USA) was laid on top of the washed gel and this was sandwiched between filter-paper and fibre pads to ensure even electrotransfer. The gel and membrane were placed in an electrotransfer apparatus with the membrane on the anode side of the gel. Electrotransfer was performed at 100 V for 1 hr in the electrotransfer buffer.

Fluid-Phase Enzyme Immunoassay (EIA) The fluid phase EIA for anti-PF4-heparin IgG is based on the Protein A antibody- capture ELISA assay reported by Nagi et al. 413 and is illustrated in Figure 7. PF4 (20 µg/ml), of which 5% was biotinylated, was mixed with an optimal concentration of heparin (0.55-0.88 U/ml, Fisons, Australia) in a solution of phosphate buffered saline and 0.05% tween 20 (PBS-tween). This antigen mixture (200 µl) was incubated for 1 hr with 200 µl of serum/plasma, diluted 1/50 or 1/10 in 1% bovine serum albumin in PBS- tween (1%BSA). Subsequently, 190 µl samples of this antigen-antibody mixture were incubated in duplicate microcentrifuge tubes containing 10 µl of Protein G Sepharose (binding capacity ≈17 mg IgG/ml, Pharmacia, Sweden) which had been blocked with 1%BSA. The sepharose beads, containing biotin-PF4-heparin-antibody complex were

87 separated from unbound antigen by centrifugation and washed three times with PBS- tween. The amount of biotin-PF4-heparin-antibody complex immobilised to the beads was determined by incubating the sepharose pellet for 30 min with streptavidin conjugated to horse radish peroxidase (Amersham, England), diluted 1/5000 in 1%BSA. After another three washes, 400 µl of TMB peroxidase substrate (Kirkegaard & Perry

Laboratories Inc., USA) was added to the tubes for 15 min and then 50 µl 0.6 M H2SO4 was added to halt colour development. 200 µl of supernatant was transferred to microtitre wells and the absorbance at 450 nm was measured in a microtitre plate reader (Molecular Devices, USA).

Colour

Substrate

Wash

G G

12 3

Legend

Biotinylated PF4- IgG in HIT plasma heparin complex

Protein G Streptavidin- G sepharose peroxidase

Figure 7 - Schematic representation of the fluid-phase assay. (1) HIT plasma was incubated with biotinylated PF4-heparin. (2) All IgG was precipitated by the addition of protein G sepharose. (3) Streptavidin-peroxidase was used to detect the biotin-PF4-heparin that coprecipitated with the IgG. 88 The Normal range for the fluid-phase assay was defined as being within three standard deviations (SD) of the mean of the logarithm transformed absorbance data from 26 Normal individuals. A positive result was defined as one that was greater than the mean plus 3 SD and values below that were considered negative. The logarithm transformation was used to make the distribution of the data approximate that of a normal curve. The cut-off points are graphed after reverse transformation back into the original absorbance units. Kappers-Klunne et al. similarly used a Normal range of 3 SD in an ELISA 46.

In some experiments heparin was substituted with either dalteparin (Fragmin, Pharmacia AB, Sweden), enoxaparin (Clexane, Rhone-Poulenc Rorer, France) or danaparoid (Orgaran, Organon, The Netherlands). As with heparin, the optimal concentration of each anticoagulant was determined with PF4 concentration at 20 µg/ml (Figure 11). The optimum concentrations used were, for enoxaparin 7.5 µg/ml (about 0.75 anti-Xa U/ml), for dalteparin 0.5 anti-Xa U/ml and for danaparoid 0.56 anti-Xa U/ml (40 µg/ml). Only samples previously shown to have anti-PF4-heparin antibodies were assayed for cross- reactivity. The screening of HIT plasmas was performed with assistance from Ms Rebecca Swanson.

Heparin-PF4 Antibody ELISA Heparin-PF4 Antibody ELISA kits (Stago Diagnostica, France) were a generous gift from Dr J. Amiral (Serbio Research Laboratory, France). The assay was performed according to the manufacturer’s instructions. PF4-heparin coated microtitre wells were incubated in duplicate with 1/101 dilutions of serum/plasma for 1 hr, washed, then incubated with a peroxidase conjugated secondary antibody directed against human IgG, IgM and IgA. Following another wash, peroxidase substrate solution was added for

5 min before the colour development was stopped by the addition of H2SO4. Abs490 was measure using a microtitre plate reader. Some of the ELISAs were kindly performed by Ms Rebecca Swanson.

89 Functional Assays Functional assays were kindly performed by Ms Sue Evans (Prince of Wales Hospital) as previously described 139,144. Briefly, the 14C-serotonin release assay involved incubating (1 hr, 22°C) 75 µl 14C-Serotonin labelled platelets (300x109/l) with 20 µl heat inactivated test serum and 5 µl heparin (final concentrations 0.1 U/ml and 100 U/ml). The amount of 14C released into the supernatant indicated the degree of platelet activation and was determined using a β-counter. Sera were considered positive if more that 20% of the 14C-serotonin label was released at the low, but not high, heparin concentration. Most patients were also tested for cross-reactivity with dalteparin (0.5 anti-Xa U/ml) and danaparoid (0.1, 0.5 anti-Xa U/ml). 20% release of 14C-serotonin at any of these concentrations was considered positive cross-reactivity.

Platelet aggregometry was performed by mixing, at 37°C in an aggregometer, 340 µl platelet rich plasma with 150 µl test serum and then adding 10µl heparin (final concentrations 0.5 U/ml and 100 U/ml). The degree of aggregation was determined by the increase in light transmittance. Sera were considered positive if there was more than 20% platelet aggregation at the low, but not the high, heparin concentration. Most patients were also tested for cross-reactivity with dalteparin and danaparoid at final concentrations of 0.1, 0.5 and 1 anti-Xa U/ml. Greater than 20% aggregation at any of these concentrations was considered a positive cross-reaction.

The cross-reactivity tests with LMWH and heparinoid were performed with both the EIA and the functional tests (14C-serotonin release or platelet aggregometry) by operators who were “blinded” to the clinical outcomes of the HIT patients. The EIA tests were all performed retrospectively but the functional tests were performed in part retrospectively and in part prospectively. Patients A and B, who showed cross-reacting antibodies with danaparoid detected by functional assay, were patients from other hospitals. Their sera were referred to the Prince of Wales Hospital Haematology laboratory for HIT antibody testing after the treating physicians had commenced or completed danaparoid treatment.

90 Results

PF4 Purification PF4 was purified from the plasma of expired platelet packs in a two step procedure. First, PF4 was purified by affinity for heparin-sepharose. The principal contaminants of this PF4 preparation had molecular weights of 57 kD and 180 kD while PF4 ran at 10 kD (Figure 8). In the second step, the crude PF4 preparation was applied to a S-sepharose column. The contaminants flowed through but PF4 bound and was subsequently eluted in a pure form (Figure 8). This technique produced 3-5 ml of PF4 at 2-3 mg/ml and a total yield of about 10 mg PF4, which corresponds to about 5 µg PF4 per ml of the original plasma.

The identity of the PF4 was confirmed immunologically by ELISA. Microtitre wells that were coated with PF4, bound rabbit anti-PF4 antibodies from two different sources but did not substantially bind non-immune rabbit antibodies (Figure 9).

12 3 M

180 kD 200 kD

100 kD ATIII 57 kD 50 kD

PF4 10 kD 10 kD Figure 8 - SDS-PAGE demonstrating purification of PF4 Protein solution from the purification of PF4 was electrophoresed through 4-15% polyacrylamide gel under non-reducing conditions. Lane 1 contains 10 µl crude PF4 that was eluted from heparin-sepharose after incubation with plasma from platelet packs. The crude PF4 was applied to a S-sepharose column and the flow-through (Lane 2, 10 µl) contained the contaminants, one of which is presumed to be ATIII. Pure PF4 (Lane 3, 1 µl) was eluted from the S-sepharose. Molecular weight markers (200 kD and 120-10 kD at 10 kD intervals) were run in Lane M.

91 4

3.5

3

2.5

2

1.5

Absorbance (650 nm) 1

0.5

0 0.0001 0.001 0.01 0.1 1 Concentration of rabbit serum (%)

Figure 9 - Antibody confirmation of PF4 ELISA was used to confirm the identity of the purified PF4 by the binding of two different anti-PF4 antibodies. Covalink microtitre wells (Nunc) were coated overnight with PF4

(20 µg/ml in 50 mM PO4 buffer pH 7.5). Wells were blocked for 2 hr with 1% BSA then incubated for 1 hr with dilutions of immune and non-immune rabbit serum in 1% BSA. After five washes with PBS-tween, wells were incubated for another hour with a 1/500 dilution of HRP conjugated swine anti-rabbit-immunoglobulin (Dako, Denmark). Wells were washed five more times then 170 µl of TMB peroxidase substrate was added for 6 min before the absorbance at 650 nm was measured. The binding of two different rabbit anti-PF4 sera, generously provided by Prof. J. Dawes ( ) and Prof. C.N. Chesterman ( ), to microtitre wells coated with PF4 was investigated. As controls, the binding of non-immune rabbit serum ( ) to PF4 coated wells and anti-PF4 serum to uncoated wells ( ) was also determined.

Biotinylation of PF4 PF4 was successfully biotinylated with diazobiotin. Figure 10 indicates that when the biotinylated PF4 was run on SDS-PAGE and transferred to a PVDF membrane a single band was observed when stained with coomassie blue or probed with streptavidin.

92 12M

Origin

37.5kD 29.2kD 17.8kD

PF4 9kD

Figure 10 - Successful biotinylation of PF4 4 µg of biotinylated PF4 was electrophoresed through a 15% SDS-PAGE gel and proteins were electrophoretically transferred to a PVDF membrane. The membrane was cut in half and the left side (Lane 1) stained with coomassie blue. The right side of the membrane was probed to detect biotinylated proteins (Lane 2) as follows: The membrane was wetted in 90% ethanol and washed with water. After blocking for 45 min with 1% BSA in PBS, the membrane was incubated for 45 min with 1/2000 streptavidin-HRP conjugate (Amersham, England) dissolved in a solution containing 1% BSA, 0.01% tween-20 and PBS. The membrane was washed four times for 5 min in PBS with 0.01% tween-20 then HRP was detected by incubating 4CN substrate (DuPont) for 3 min. Lane M shows Kaleidoscope polypeptide molecular weight standards (Bio-Rad Laboratories).

Fluid phase EIA HIT antibodies bound to PF4-heparin complexes in the fluid-phase EIA. The strength of binding was strongly dependent upon the heparin concentration. Figure 11 illustrates that HIT IgG only bound to PF4 in the presence of a narrow range of heparin concentrations. In this example the optimum concentration was ≈0.9 U/ml, although it was lower with different batches of PF4 and heparin. The fluid-phase assay can also be used to measure antibodies against PF4 complexed to LMWH or heparinoid so various concentrations of these anticoagulants are also shown in Figure 11.

93 2

1.5

1

0.5 Binding of HIT Antibody (Abs 450 nm)

0 0.01 0.1 1 10 Anticoagulant Concentration (U/ml)

Figure 11 - Optimisation of heparin/LMWH/heparinoid concentration in fluid-phase EIA

Various concentrations of heparin ( ), dalteparin ( ), enoxaparin ( ) or danaparoid ( ) were used in the fluid-phase EIA with a PF4 concentration of 20 µg/ml. Each line represents the mean absorbance from two different HIT patients.

Twenty eight patients with confirmed HIT were investigated using the fluid phase EIA for antibody binding to PF4-heparin. Initially plasma samples were tested at 1 in 50 dilution. Negative samples were retested at 1 in 10 dilution, as were all 26 Normal healthy volunteers and 30 control patients with non-HIT thrombocytopenia (Figure 12). The higher antibody concentration did not substantially increase the binding of the Normal or non-HIT control IgG. The upper limits of the normal range was OD 0.22 and 0.28 when Normal plasmas were tested at 1/50 and 1/10 respectively. Anti-PF4-heparin IgG was detected in plasma from 26/28 (93%) of the known HIT patients tested but in none of the Normal or non-HIT thrombocytopenic controls (Figure 12).

94 2

1.8

1.6

1.4

1.2

1

0.8 Absorbance (450nm)

0.6

0.4

0.2

0 Normals HIT Non-HIT

Figure 12 - Fluid-phase EIA for HIT antibodies HIT patients, healthy individuals (Normals), or patients with thrombocytopenia due to causes other than heparin (Non-HIT) were tested for antibodies to PF4-heparin in the fluid-phase assay. Serum/plasma samples were assessed at a dilution of 1/50 ( ), or at 1/10 ( ) because they were negative at 1/50. Samples were considered positive if, using log transformed data, their absorbances were greater than three standard deviations above the mean of the Normals at 1/10 (dashed line).

Heparin-PF4 Antibody ELISA I also investigated anti-PF4-heparin antibodies using a commercial ELISA kit. The manufacturer’s instructions state that absorbances greater than 0.5 Units should be regarded as clearly positive. Using this criteria, 7/32 of the normal samples would be positive. This rate of false positives was unacceptable so I applied the same criterion as that used for the fluid phase assay to determine a positive result. This resulted in 24/28 95 (86%) of the HIT plasmas being detected as positive, while all the Normals were negative (Figure 13).

4.5

4

3.5

3

2.5

2 Absorbance (490nm) 1.5

1

0.5

0 Normals HIT

Figure 13 - ELISA for HIT antibodies HIT patients and Normal controls were assayed according to the instructions with the anti- PF4-heparin ELISA kit. Samples were considered positive if, using log transformed data, their absorbances were greater than three standard deviations above the mean of the Normals (dashed line).

Effect of heparins on the coating of PF4 to microtitre wells Preliminary experiments indicated that heparin promoted the coating of microtitre wells with PF4, so the effect of different heparin-like anticoagulants on PF4 coating was investigated. Figure 14 indicates that microtitre wells coated with 2µg PF4-dalteparin bound up to 0.8 µg PF4 per well, which was four times as much as wells coated with

96 PF4-heparin. Coating with PF4-danaparoid resulted in only 0.09 µg PF4 per well, which was barely above the 0.08 µg PF4 per well observed with PF4 alone.

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

Amount of PF4 bound (µg/well) 0.1

0.0 00.511.52 Concentration of Anticoagulant (U/ml)

Figure 14 - Effect of different heparin-like anticoagulants on the binding of PF4 to microtitre wells. Covalink BreakApart microtitre wells (Nunc) were coated overnight with 2 µg of 125I-PF4 (see Iodination of Proteins, p.114) mixed with various concentrations of heparin ( ), dalteparin ( ) or danaparoid ( ) in 100 µl. After washing, bound radioactivity was determined in a γ-counter.

Cross-reactivity The fluid phase EIA for anti-PF4-heparin IgG permits the substitution of heparin with other anticoagulants. I investigated whether the antibodies in patients with HIT cross- reacted with dalteparin, enoxaparin or danaparoid. Figure 15 illustrates that 23/26 (88%) HIT sera/plasmas cross-reacted with PF4-dalteparin or PF4-enoxaparin. A lesser number, 13/26 (50%), reacted with PF4-danaparoid. These antibody reactions with danaparoid were also generally weaker than that with the LMWHs, dalteparin and enoxaparin (Figure 15). Cross-reactivity tests with LMWH and heparinoid were also determined using functional assays (14C-serotonin release and/or platelet aggregometry).

97 Consistent with the EIA results, the functional assays showed 21/23 (91%) of patient plasmas/sera also cross-reacted with the LMWH, dalteparin (Figure 15). In contrast, only 4/24 (17%) of the HIT samples tested cross-reacted with the heparinoid, danaparoid, using the functional assays. Figure 16 indicates that there is a high degree of correlation between the levels of anti-PF4-heparin antibodies and cross-reacting antibodies binding to PF4 complexed to the heparin-like anticoagulants.

2.5

2

1.5

1 Binding of HIT IgG (Abs 450nm)

0.5

0 PF4 alone PF4-heparin PF4- PF4- PF4- dalteparin enoxaparin danaparoid

Figure 15 - Cross-reactivity of HIT antibodies with PF4 complexed to heparin-like anticoagulants The fluid phase EIA was used to assess the degree to which IgG present in HIT sera/plasma bound to PF4 alone, PF4-heparin, PF4-dalteparin (Fragmin), PF4- enoxaparin (Clexane), or PF4-danaparoid (Orgaran). The positive cut-off (dashed line) is three standard deviations above the mean (log transformed) absorbance of the Normal samples using PF4-heparin. The binding of normal antibodies is indicated by triangles ( ). Circles indicate HIT samples which have either been shown to be positive ( ), negative ( ) or not tested ( ) in a functional assay with the corresponding drug.

98 ) 2.5

2

1.5

1

0.5 anti-PF4-dalteparin (Abs 450nm 0

) 2.5

2

1.5

1

0.5 anti-PF4-enoxaparin (Abs 450nm 0

) 0.7

0.6

0.5

0.4

0.3

0.2

0.1

anti-PF4-danaparoid (Abs 450nm 0 00.511.52 anti-PF4-heparin (Abs 450nm)

Figure 16 - Correlation of HIT antibody cross-reactivity Correlation of anti-PF4-heparin antibody levels with their antibody binding to PF4- LMWH/heparinoid complexes in the fluid phase EIA. Absorbances, derived from Figure 15, are shown with the linear regression line fitted. Spearman’s rank order correlation coefficients for the cross-reactions with heparin are: dalteparin = 0.84, enoxaparin = 0.87, danaparoid = 0.77.

99 Clinical outcomes of HIT patients treated with danaparoid The clinical outcomes of HIT patients, who received danaparoid after heparin withdrawal, were kindly traced by Prof. B.H. Chong, Dr S. Wright, Dr A.E. Blackwell and Dr P. Warburton. Inspection of hospital clinical notes and other records indicated that 21 patients (14 from the group analysed in Figure 15, plus an additional 7, which were not investigated elsewhere in this study) received danaparoid for 3 or more days. Plasmas from 11 of these were negative for antibodies cross-reacting with danaparoid in both the fluid phase immunoassay and a functional assay. Another 8 were also negative in a functional assay but positive for anti-PF4-danaparoid antibodies in the fluid-phase EIA. All of these patients who were negative in a functional assay, regardless of the fluid-phase immunoassay, recovered with a resolution of thrombocytopenia and thrombosis (Figure 17a and b).

The remaining two patients, Patients A and B, had antibodies that cross-reacted with danaparoid in both the fluid-phase EIA and a functional assay. In Patient A (Figure 17c) her left deep vein thrombosis (DVT) and pulmonary embolism improved clinically and the thrombocytopenia resolved while she was treated with danaparoid for three days. In contrast, the severe thrombocytopenia in Patient B (Figure 17d) persisted and his DVT became worse while he was treated with danaparoid for 15 days. After cessation of danaparoid his DVT became even more severe and extensive. At the time no cross- reactivity data was available. The platelet counts started to increase before cessation of danaparoid but peaked 3 days later and subsequently dropped. The reason of the second decline in his platelet counts was uncertain and might be attributed to progression of his extensive thrombosis. He was on warfarin throughout his hospitalisation and developed venous gangrene of his lower limb. In hind sight, this might be related to warfarin therapy 184,185. He was treated with several other antithrombotic agents including urokinase, anti-glycoprotein IIb-IIIa antibody (ReoPro), tissue plasminogen activator and prostaglandin E1. The patient eventually died as a result of his unabating thromboembolism.

100 300 (a) (b) (c) (d) / l) 9

200

Platelet Count (10 100

0 0102030 0102030 0102030 0102030 Days Days Days Days

Figure 17 - Serial platelet counts of representative HIT patients treated with danaparoid (Orgaran). Solid black bar shows duration of heparin administration, striped bar indicates danaparoid therapy, open bar shows warfarin therapy. The zero time point indicates the start of available records. (a) Typical profile of the 11 patients who were negative in both fluid- phase EIA and functional assay. (b) Typical profile of the 8 patients who were positive in the fluid-phase EIA but negative in a functional assay. (c) Profile of Patient A, one of the two patients who were positive in both types of assay. She recovered during a short course of danaparoid. (d) Profile of Patient B, the other patient positive in both assays. He was admitted to a small regional hospital with normal platelet count. Thrombocytopenia developed while receiving heparin. Detailed records became available once he was admitted to a major hospital with HIT. Despite treatment with many antithrombotic agents, he eventually died following major thrombosis.

Discussion

A convenient and affordable source of pure PF4 was required for this project. The technique that I developed used plasma from expired platelet packs that had been stored at 4 °C. This avoided the need to keep platelets viable. The first step of purification isolated proteins that remained bound to heparin-sepharose after washing with 0.8 M NaCl. This crude PF4 preparation was remarkably pure but did contain two contaminants, the principal one having a molecular weight of 57 kD. This contaminant 101 is most likely to be antithrombin III (ATIII) which has a published molecular weight of 58 kD 414 and runs at 55-57.5 kD on reducing SDS-PAGE 282. The strategy for removing this contaminant was based on the presumption that the ATIII contaminant was present because it bound heparin-sepharose through the specific pentasaccharide sequence. In contrast PF4 is thought to bind heparin through ionic interactions. Thus PF4 was predicted to bind to negatively charged S-sepharose whereas ATIII would not bind because S-sepharose lacks the required pentasaccharide sequence. Indeed, Figure 8 (p.91) shows that the 57 kD contaminant passed through the S-sepharose column but PF4 bound and was then eluted as a pure preparation. The identity of the minor contaminant (180 kD under non-reducing conditions) in the crude PF4 preparation is uncertain. Zammit et al. 282 identified plasma proteins that bind heparin and observed that complement factor H ran at ≈170 kD on a reducing SDS-PAGE gel. Considering that factor H is a single polypeptide chain 415 this would seem to be a reasonable candidate for my 180 kD contaminant. However, Carron et al. 416 have reported that factor H, surprisingly, runs at 140 kD under non-reducing conditions and 170 kD under reducing conditions. Thus, the 180 kD contaminant that I identified under non-reducing conditions is unlikely to be factor H. Instead it is probably just IgG, which runs at ≈180 kD with these gels and markers (see Figure 31, p.130). Regardless of its identity, this contaminant did not bind to S-sepharose and so was removed by the same process as ATIII (Figure 8). Subsequently I have discovered that Medici et al. 372 similarly used a cation exchange column to remove ATIII (confirmed immunochemically) from a crude PF4 preparation that had been eluted from heparin-sepharose. Levine et al. 311 have also purified PF4 from the plasma of platelet concentrates rather than the platelets themselves.

The PF4 that I purified had a apparent molecular weight of 10 kD on SDS-PAGE, which is somewhat different from the theoretical 7.8 kD for PF4 365. However, the reported molecular weight of PF4 by SDS-PAGE ranges from 7.8 kD to 11.6 kD 179,311,330,339,359,371,372. Confirmation that this protein is in fact PF4 comes from the observations that it was recognised by two different rabbit anti-PF4 antisera (Figure 9, p.92) and that it was eluted from heparin-sepharose only at high salt concentrations.

102 PF4 was biotinylated using diazobiotin, which reacts with tyrosine and histidine groups 412, rather than the more common NHS-biotin which reacts with primary amines 417. This approach was used in order to retain the heparin binding capability of PF4 that could have been lost by biotinylation of the ε-amines in lysine. For the same reason, PF4 was mixed with heparin-sepharose before diazobiotinylation in an attempt to further protect any heparin binding sites from modification. After biotinylation any PF4 that had lost affinity for heparin was washed away from the heparin-sepharose before viable PF4 was eluted. Figure 10 (p.93) indicates that diazobiotinylation was successful.

I developed a fluid phase enzyme immunoassay for the detection of the antibodies in HIT. This assay measures the binding of IgG from patient plasma to complexes of PF4- heparin in solution. Plasma were deemed to contain HIT antibodies if they fell outside the Normal range. The upper limit of the normal range was defined as 3 SD above the mean of the Normal individuals and was calculated using logarithm transformed data to make the distribution more Gaussian.

The fluid phase assay is both sensitive and specific because it detected anti-PF4-heparin IgG in the plasma of 93% of HIT patients while all the Normal and non-HIT thrombocytopenic controls were negative. In comparison, a commercial ELISA kit for the detection of anti-PF4-heparin antibodies produced a high background and an unacceptable rate of false positives when the criterion for diagnosis provided with the kit (Abs490>0.5) was followed. I overcame this by using the same criterion as that for the fluid-phase EIA (mean of the Normals + 3 SD). This made the ELISA specific (no false positives) and of similar sensitivity (86%) to the fluid-phase EIA. It is unclear why the ELISA produced absorbance values that were so much greater than that anticipated by the manufacturer but this was a consistent finding across kits from two different batches.

It is interesting to note that the variation among the Normal individuals is considerably greater in the ELISA (Figure 13) compare to the fluid-phase assay (Figure 12). This is reflected in their coefficients of variation (based on log transformed data) of 51% and 12% respectively. This is probably due to non-specific binding of some antibodies to the ELISA wells. The fluid-phase EIA avoids this by precipitating all IgG then detecting the antigen which is specifically bound to the IgG (see Figure 7, p.88). As a result low

103 levels of HIT antibodies can be detected by increasing the concentration of plasma without increasing non-specific binding.

The fluid-phase EIA only detects antibodies of IgG class. This may account for the failure of the fluid-phase EIA to detect anti-PF4-heparin antibodies in two samples with confirmed HIT. These patients may have had exclusively IgA or IgM antibodies, except that they were also negative by ELISA, which is sensitive to IgA and IgM. Recently HIT associated with antibodies against IL-8 or neutrophil-activating peptide-2 have been described 166 and this may be a better explanation for the lack of anti-PF4-heparin antibodies in these patients. Nevertheless, the fluid phase assay compares favourably with the ELISA kit. It is marginally more sensitive than the ELISA and less susceptible to background or non-specific binding, even at high plasma concentrations.

The fluid-phase assay demonstrates that HIT antibodies will bind to solubilised PF4- heparin complexes and not just to PF4-heparin immobilised on microtitre wells. This is important because it demonstrates that immune complexes of HIT antibody and PF4- heparin can form in solution. This is consistent with the theory that in vivo such immune complexes form and then bind to platelets via Fcγ receptors II but it does not prove the theory. The binding of HIT IgG to platelets is investigated in detail in Chapter 4.

The fluid-phase EIA also has the advantage that the amount of antigen (PF4-heparin) available for binding to the antibody is known to be the amount added to the tube. In contrast the amount of PF4-heparin adhering to an ELISA well varies with the heparin concentration and the nature of the heparin(oid) compound. I observed that in the presence of an optimal concentration of LMWH four times as much PF4 bound to microtitre wells compared to unfractionated heparin, which in turn was twice as much as that with danaparoid (Figure 14). For this reason, the ELISA is not well suited to investigating differences in HIT antibody binding to PF4 complexed with different heparin-like anticoagulants because any variation in antibody binding may simply reflect the amount of antigen available and not the avidity of the antibody for the antigen. Thus, the fluid-phase EIA is more appropriate than ELISA for comparing reactivity of anti- PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants.

104 Using the fluid-phase EIA, I found 88% of the patient sera cross-reacted with the LMWHs, enoxaparin and dalteparin. This cross-reaction rate is comparable with that observed when using aggregation or 14C-serotonin release assays 93,95,217,224. Not only was the number of individuals reacting with heparin and LMWH similar but the intensity of the reaction was also analogous. This indicates that LMWH-PF4 is potentially as good an antigen for HIT antibodies as heparin-PF4. In contrast, danaparoid-PF4 binds considerably less frequently and intensely than heparin-PF4 to IgG from HIT patients (Figure 15). This lower affinity is probably a function of danaparoid-PF4 complexes being less stable than heparin-PF4 complexes. The glycosaminoglycans in danaparoid have less negatively charged sulfate groups than heparin so would display weaker binding to the positive charges on PF4. Similar results have been observed using the serotonin release assay and PF4-heparin ELISA where decreases in sulfation of oligosaccharides results in decreased detection of HIT antibodies 134,159. The 50% rate of cross-reactivity of the antibodies with danaparoid- PF4 by EIA is considerably higher than the 0-20% observed by others who used platelet aggregometry or 14C-serotonin release assays 93,216,224,225. The reason for the divergent results is unclear. It is possible that aggregometry and 14C-serotonin release assays are less sensitive and were able to detect only high affinity antibodies or antibodies that cause platelet activation. Following the publication of this data 90 a number of the conclusions have been confirmed by Wang et al. 418,419. They developed an assay that also detected the binding of HIT IgG to soluble complexes of PF4-heparin. Their assay was similarly sensitive and specific and showed high cross-reactivity with LMWH 418.

I observed a high correlation between antibody binding to PF4-heparin and to PF4 complexed with alternative anticoagulants (Figure 16). This suggests that the antibodies are genuinely cross-reacting rather than being different immunoglobulin molecules directed against distinct epitopes. Substantial correlation was even observed with the weak binding to PF4-danaparoid, indicating that individuals with strong binding to PF4- heparin are likely to have the strongest binding to PF4-danaparoid.

HIT antibodies that only cross-react with danaparoid in an immunoassay may not be clinically significant. On retrospective analysis, 21 patients with HIT were treated with danaparoid after heparin withdrawal. In 8 of these patients, thrombocytopenia and

105 thrombosis recovered despite their plasma/serum showing cross-reactivity with PF4- danaparoid by the EIA, although there was no cross-reactivity by the functional tests.

In contrast, the clinical significance of the HIT antibodies with danaparoid cross- reactivity detected by functional assays is unclear. Two HIT patients (Patients A and B) possessed antibodies that cross-reacted with danaparoid in a functional assay. In Patient A, the thrombocytopenia resolved and the thrombosis clinically recovered with danaparoid therapy. Wang et al. 418 report a similar case where a patient cross-reacted with danaparoid in a HIPA assay but thrombocytopenia resolved while receiving danaparoid.

However, danaparoid is not always tolerated so well. Patient B’s platelet counts remained markedly low during danaparoid therapy. His thrombosis became even worse after withdrawal of danaparoid and then progressed unabated, leading to devastating consequences and finally his death despite treatment with several potent anticoagulant, fibrinolytic and anti-platelet agents. Similarly, the thrombocytopenia of a patient reported by Tardy/Poncet et al. persisted during danaparoid therapy and recovered only after cessation of the offending drug. In that patient cross-reactivity of the HIT antibody with danaparoid, detected by platelet aggregometry, developed during therapy 420. In summary, it would appear from current data that danaparoid treatment of patients with cross-reacting antibodies detected only by immunoassay, such as the fluid-phase EIA, is probably safe. It is not yet possible to predict the clinical outcome of patients, with cross-reacting antibodies detected by functional assays, who are treated with the heparinoid. An approach, suggested by Hill et al. 421 is to use doses of danaparoid below that which facilitates platelet aggregation in vitro. Nevertheless, any patients who demonstrate cross-reactivity with danaparoid in vitro require particularly close monitoring if treated with danaparoid.

106 Chapter 3 - Further characterisation of antibody and antigen in HIT

Introduction

Type II heparin-induced thrombocytopenia (HIT) with thrombosis is a serious side- effect of heparin therapy that occurs in 1-3% of patients treated with heparin 28,33,46,54,66,86. Patients produce antibodies that recognise a complex of platelet factor 4 (PF4) and heparin 43,45,158,159. IgG, bound to the PF4-heparin complex, is thought to activate platelets via Fc receptors on the platelets 45,138,139. Activated platelets may cause thrombosis or may be cleared by the reticuloendothelial system resulting in thrombocytopenia. The exact nature of the epitope(s) recognised by the HIT antibodies has not been elucidated. Suh et al. 48 observed that different patients appear to have antibodies that recognise different epitopes on PF4-heparin. Ziporen et al. 180 concur, but suggest that a region just after the third cysteine of PF4 is important for the binding of many HIT sera.

Understanding the role heparin plays in forming the epitope to which HIT antibodies bind is important in explaining the pathogenesis of HIT. Antibody binding to PF4 is optimum in a narrow range of heparin concentrations. This optimal PF4:heparin ratio corresponds to concentrations at which a multimolecular complex of PF4 and heparin is formed 43,159,379. Some HIT antibodies also bind weakly to PF4 alone in ELISA 43,45,159, although this is not always observed 179. It is not clear whether binding to PF4 alone indicates that some antibodies recognise native PF4 or if immobilisation of PF4 on the ELISA wells antigenically modifies PF4 to facilitate antibody binding. Furthermore, antibodies to PF4 alone may be the same immunoglobulin molecules as those that recognise PF4-heparin. Alternatively, there may be two distinct antibody populations, one recognising PF4-heparin and another PF4 alone. Discussion in recent reports indicate that it is still unclear whether HIT antibodies bind to epitopes consisting of polysaccharide plus PF4 or if the binding of heparin induces a change in PF4 that perhaps exposes a neoepitope 48,179.

I addressed issues of epitope specificity and the role of heparin by investigating HIT antibody binding to native PF4 in the fluid-phase and by specifically depleting HIT 107 plasma of antibodies with agarose beads coated with PF4-heparin and PF4 alone. I also developed a technique to affinity purify HIT anti-PF4-heparin IgG. This has enabled me to estimate, for the first time, the functional binding affinity of this IgG for PF4 and PF4-heparin. This information not only supports my other conclusions but provides fundamental data on the nature of HIT antibodies that may help understand this disease.

Methods

Patients Plasma was collected from 14 patients with confirmed HIT (thrombocytopenia while receiving heparin, positive 14C-serotonin release or platelet aggregation assay, exclusion of other causes of thrombocytopenia, and recovery of platelets upon heparin withdrawal). Only plasmas containing anti-PF4-heparin IgG were included. The plasmas of 12 healthy volunteers were used as normal controls.

Two HIT patients (“Patient 1” and “Patient 2”) underwent plasmapheresis, yielding sufficient plasma to affinity purify HIT IgG. Patient 1 was a 72 year-old woman treated with heparin (i.v. 36,000 U/day) after developing pneumonia, arterial fibrillation and cardiac and renal failure. She became thrombocytopenic after 6 days and was positive in a 14C-serotonin release assay. Her condition transiently improved after plasmapheresis but she subsequently deteriorated and died. Her history was kindly provided by Prof. B.H. Chong.

Patient 2 was a 67 year-old woman requiring cardiac bypass surgery who received heparin (s.c. 5000 U twice daily) for a week before thrombocytopenia and a positive platelet aggregation test prompted cessation of heparin. She made an uneventful recovery after plasmapheresis and cardiac surgery. Her history was kindly provided by Dr J.V. Lloyd.

ELISA PF4 was purified from platelet poor plasma derived from expired platelet packs by affinity for heparin-sepharose and S-sepharose as previously described (see Purification of PF4, p.85). The ELISA was based on that developed by Amiral et al. 158,159.

108 Polystyrene microtitre wells (Covalink, Nunc, Kamstrup, Denmark) were coated overnight with 100 µl of either PF4 (20 µg/ml) ± porcine heparin (optimum at 0.7 IU/ml, Fisons, Australia) or β-thromboglobulin, (βTG, 20 µg/ml, a generous gift from Dr M. Yang) or bovine serum albumin (BSA, 20 µg/ml, Sigma, U.S.A.). Wells were washed with phosphate buffered saline containing 0.05% Tween-20 (PBS-tween) then blocked for 1 hr with 1% BSA. HIT or normal plasma were diluted 1/1000 in 1% BSA and incubated in duplicate wells for 1 hr. Wells were washed then incubated for 1 hr with 1/5000 peroxidase-conjugated anti-human-IgG antibody (Pierce, U.S.A.). After washing, 170 µl of peroxidase substrate (3, 3’, 5, 5’-tetramethylbenzidine, Kirkegaard & Perry Laboratories Inc., U.S.A.) was incubated in the wells for 15 min and absorbance at 650 nm was measured in a microtitre plate reader. Absorbance values from duplicate wells were averaged. In Figure 21 the wells were pretreated with 100 µl sulfoMBS (m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester, 125 µg/ml, Pierce) for 3 hr before coating with PF4±heparin. In subsequent experiments this treatment was omitted because it was found to have no effect on IgG binding to PF4±heparin (see Effect of sulfoMBS activation of Covalink, p.118). I used a twenty fold higher dilution of plasma than the 1:50 used by others 43,159 because my secondary antibody and peroxidase substrate was particularly sensitive.

Fluid-phase Enzyme Immunoassay The fluid-phase EIA was performed as previously described (see Fluid-Phase Enzyme Immunoassay (EIA), p.87). Briefly, biotinylated PF4±heparin was incubated with diluted HIT plasma then all IgG precipitated by protein G sepharose. The amount of biotinylated PF4 that coprecipitated with the IgG was determined using streptavidin peroxidase and colorimetric substrate.

Depletion of HIT antibodies PF4-agarose was prepared by incubating 3 ml PF4 (3 mg/ml) with 0.6 ml N-hydroxysuccinimide-activated agarose beads (AffiGel 10, BioRad Laboratories, U.S.A.). After 5 hr another 0.6 ml of beads was added to the supernatant and incubated overnight. Any remaining N-hydroxysuccinimide ester was quenched by resuspending the agarose in 3 ml 0.1 M Tris pH 7.5 for 1 hr. The PF4-agarose preparations were

109 pooled and washed to remove any PF4 that was not covalently bound. A BCA protein assay (Pierce) indicated 2.2 µg PF4 per microlitre of washed agarose beads. Plain agarose was prepared similarly but without PF4. Heparin-PF4-agarose was prepared by mixing 8 IU heparin per millilitre of PF4-agarose beads for 30 min. The agarose beads were blocked with normal IgG and washed with 1% BSA.

HIT plasma (100 µl of a 1/20 dilution) was specifically depleted of antibodies by incubating for 1 hr with 0-40 µl of agarose beads coated with PF4±heparin. Depleted plasma was assayed in the fluid-phase EIA and solid-phase ELISA. Samples were diluted so that there was a linear relationship between the resulting absorbance and antibody concentration. The antibody concentration, relative to the undepleted sample, was calculated from a linear regression line obtained by plotting known dilutions of undepleted sample against corresponding absorbances.

Total IgG and anti-adenovirus IgG assays Determination of total IgG and anti-adenovirus IgG were used to quantitate the degree of non-specific antibody depletion by the agarose beads. To measure total IgG, 5 µl of sample (at a dilution equivalent to 1/500 plasma) was dried onto triplicate MaxiSorp microtitre wells (Nunc), blocked for 1 hr with 1% BSA then IgG was detected by the same secondary antibody and method used in the anti-PF4 ELISA. The anti-adenovirus antibody ELISA relies on the fact that most individuals have developed antibodies to adenovirus. It was kindly performed by Ms Yvonne Duffy at the Clinical Immunology Laboratory, Prince of Wales Hospital. Briefly, microtitre wells coated with adenovirus antigen were incubated with depleted samples and then binding of IgG was determined by incubation with an alkaline phosphatase conjugated secondary antibody. Absorbances from the total IgG and anti-adenovirus assays were standardised, relative to dilutions of each undepleted sample.

Affinity purification of HIT anti-PF4-heparin IgG A heparin-PF4-agarose affinity column was prepared by mixing 4 ml PF4-agarose (prepared as above) with 32 U heparin for 30 min then washing the agarose to remove unbound heparin. HIT plasma was heat inactivated (56°C for 30 min) and precipitate removed by centrifugation. 10 ml of this plasma was applied to the column at 110 0.2 ml/min. After washing the column with 20 ml PBS-tween, the crude antibody preparation was eluted with a linear 0.13-1.5 M NaCl gradient in 20 mM HEPES pH 7.5 at 1 ml/min. ELISA analysis of column fractions indicated that elution of anti-PF4- heparin IgG peaked at 0.6 M NaCl. This peak was then applied to a 0.5 ml Protein G sepharose column at 0.2 ml/min. After washing with 30 bed volumes of 20 mM phosphate buffer pH 7.5, 0.5 ml fractions of IgG were eluted with 0.1 M glycine pH 2.7 into tubes containing 15 µl 1.5 M Tris pH 8.8 to neutralise the pH. Protein concentration was measured using the Bio-Rad Protein Assay reagent (Bio-Rad Laboratories), which utilises a colour change in coomassie brilliant blue upon protein binding.

Purification of Normal IgG Total IgG from the pooled plasma of 4 Normal individuals was purified by affinity for protein G sepharose. A 4 ml column of heparin sepharose was washed with 20 mM PO4 buffer pH 7.0 followed by 1 M NaCl dissolved in PO4 buffer then re-equilibrated in the

PO4 buffer. 7 ml of plasma was loaded on the column at 0.5 ml/min. The column was sequentially washed with PO4 buffer, 1 M NaCl and PO4 buffer again. IgG was eluted with 0.1 M glycine pH 2.7 at 1 ml/min. 1 ml fractions were collected into tubes containing 20 µl 1M Tris pH 9. Protein concentration was measured as per the affinity purified HIT IgG.

Micro-dialysis Small volumes (250 µl) were efficiently dialysed using a micro-dialysis unit. Each unit was created from a lock-top 1.5 ml microfuge tube with its base cut off (Figure 18). Sample was sealed into the lid by 12-14 kD cut-off regenerated cellulose dialysis membrane (Spectrum Medical Industries, USA). Units were placed in dialysis buffer and stirred vigorously overnight at 4°C. Sample was retrieved by puncturing the membrane with a fine gel-loading pipette tip and drawing out the liquid.

111 Sample

Cut

(a) (b) (c)

Figure 18 - Micro-dialysis unit To create each micro-dialysis unit the upper fifth of a lock-top microfuge tube was cut off as illustrated (a). The lid of the tube provided a well that was then filled with sample to be dialysed (b). The sample was covered with a sheet of dialysis membrane, that had been soaked in water, and the lid was closed (c). This sealed in the sample but permitted dialysis through the hole formed by cutting off the base of the microfuge tube. When dialysis was completed the sample was retrieved by puncturing the membrane with a fine pipette tip and drawing out the liquid.

Preparation of F(ab’)2

F(ab’)2 was prepared by digestion of IgG with pepsin that was covalently immobilised onto agarose beads. 250 µl of affinity-purified HIT anti-PF4-heparin IgG or Normal IgG (157 µg/ml) was micro-dialysed overnight at 4°C against 240 ml of 20 mM sodium acetate buffer pH 4.7. 10 µl of immobilised pepsin (50% bead volume, Pierce) was washed with 500 µl of the acetate buffer. The dialysed IgG was mixed with 6.25 µl 10% acetic acid to lower the pH to 4.0 then incubated with the immobilised pepsin for 4 hr at 37°C with constant mixing. Pepsin was inactivated by the addition of 20 µl 1.5 M Tris pH 8.8 which raised the pH to 7.6. Pepsin was removed by centrifugation and the

F(ab’)2 was micro-dialysed overnight against PBS-tween. Protein concentration was measured using BCA assay (Pierce). SDS-PAGE of the F(ab’)2 is shown in lane 5 of Figure 31 (p.130).

Preparation of Fab Fab fragments were prepared from IgG by partial digestion of IgG with ficin that was covalently immobilised onto agarose beads. 250 µl of affinity-purified HIT anti-PF4- heparin IgG or Normal IgG (157 µg/ml) was micro-dialysed overnight at 4°C against 112 200 ml of 10 mM EDTA in 20 mM phosphate buffer pH 7.0. One tenth volume of freshly prepared 150 mM cysteine was added to the dialysed IgG as well as to the EDTA/phosphate buffer to make them 15 mM cysteine. 20 µl of immobilised ficin (50% bead volume, Pierce) was washed with the 15 mM cysteine EDTA/phosphate buffer then incubated with the IgG for 1 hr at 37°C with constant mixing. Ficin was removed by centrifugation and the digest micro-dialysed overnight against PBS-tween.

Fab fragments were separated from undigested IgG by passing the ficin digest through a miniature protein A column (Figure 19). First, the column was washed with 0.1 M Citric acid pH 3 containing 0.1% tween-20 (citric-tween). The column was equilibrated with PBS-tween then 200 µl of ficin digest was applied under gravity. The flow-through was collected and the column washed three times with 100 µl PBS. The flow-through and first wash contained the Fab fragments and were pooled. Bound protein was eluted under gravity with 3x100 µl of citric-tween into tubes containing 20 µl 1.5 M Tris pH 8.8. The first two fractions of eluate were pooled as they contained the undigested IgG and the Fc fragments.

Pipette Tip

Protein A Sepharose

Glasswool Plug

Figure 19 - Miniature protein A column A miniature protein A column was required to separate Fab from undigested IgG. This column was constructed from a fine gel-loading pipette tip that was plugged with glass wool and filled with 60 µl of protein A sepharose (Pharmacia).

113 Iodination of Proteins

125 IgG, Fab, F(ab’)2, and PF4 was iodinated with Na I using iodogen (Pierce) in appropriate facilities kindly provided by the Nuclear Medicine department at the Prince of Wales Hospital. Iodogen coated tubes were generously prepared by Mr Philip Grimsley who dried 10 µg of iodogen (in 200 µl chloroform) onto the bottom of 12 mm glass tubes. An 8 ml desalting column was prepared from Bio-Gel P-6 DG acrylamide beads (Bio-Rad) that had been briefly boiled in HBS (20 mM HEPES, 130 mM NaCl pH 7.4) and allowed to cool. An iodogen coated tube was washed three times with HBS and 200 µl of protein (10-100 µg) added for 1 min before 5 µl Na125I (4 MBq/µl, Australian Radioisotopes) was also added. The tube was incubated at room temperature for 10 min with occasional mixing. 100 µl of cytochrome C solution (20 mg/ml) was applied to the desalting column and immediately followed by the iodination reaction mixture. The column was run under gravity with HBS and 0.8 ml of flow-through collected as soon as the colour of the cytochrome C began to elute.

The incorporation of 125I was measured by mixing 5 µl of iodinated protein with 495 µl BSA (1 mg/ml) and proteins were precipitated with 500 µl of a solution containing 4% phosphotungstinic acid and 20% trichloroacetic acid at 0°C for 10 min. The proportion of precipitated radioactivity was consistently greater than 97%.

Binding studies of anti-PF4-heparin IgG Equilibrium binding of HIT IgG to microtitre wells coated with PF4 and PF4-heparin was investigated by two methods. These were the “saturation binding” technique, where the concentration of radiolabelled antibody tracer in antigen coated wells was increased until binding approached saturation, and the “competitive inhibition” method were the binding of a constant amount of radiolabelled antibody was competitively inhibited by increasing concentrations of unlabelled antibody. In both techniques, microtitre wells (MaxiSorp-BreakApart, Nunc) were coated with PF4 (20 µg/ml), PF4-heparin (5 µg/ml PF4 and 0.175 U/ml heparin) or βTG (20 µg/ml) as per the ELISA (see ELISA, p.108). These concentrations of PF4 and PF4-heparin resulted in equivalent amounts of immobilised PF4 per well because heparin promoted coating of PF4.

114 In order to measure the equilibrium binding affinity of HIT IgG, it was necessary to first determine the incubation time required for the binding of HIT IgG to PF4±heparin to reach equilibrium. Figure 20 shows that the binding of HIT IgG to microtitre wells coated with PF4±heparin reaches a plateau after about 30 min. It seemed safe, therefore, to allow 60 min for binding to reach equilibrium in the subsequent binding studies.

0.8 0.7 0.6 0.5 0.4 0.3

IgG Bound (nM) 0.2 0.1 0 0 102030405060708090 Incubation Time (min)

Figure 20 - Time-course for the binding of affinity-purified HIT IgG to PF4±heparin Microtitre wells were coated with PF4±heparin as described for the binding studies. Affinity-purified HIT 125I-IgG (20 nM) was incubated in wells with gentle mixing for 15-90 min. Wells were washed and bound radioactivity determined with a γ-counter. Binding of IgG from Patient 1 ( ) and Patient 2 ( ) to PF4-heparin is represented by closed symbols while binding to PF4 alone ( , ) is indicated by the corresponding open symbol.

Standard saturation binding studies were performed by incubating increasing concentrations of anti-PF4-heparin 125I-IgG (specific activity = 2.5 kBq/µg) in microtitre wells with gentle mixing at room temperature for 1 hr. Wells were washed three times with PBS-tween then bound IgG was measured by counting individual wells in a γ-counter. The concentration of free IgG, [Free], was calculated by subtracting the concentration of bound IgG from the total IgG concentration. Least squares non-linear

115 regression analysis was used to simultaneously find values of Kd (dissociation constant) and Bmax (number of binding sites) in Equation 1 which best fitted the observed specific binding of IgG 422. This involved using the “Solver” add-in of Microsoft Excel (version 5.0) to change the values in spreadsheet cells containing estimates of Kd and Bmax to minimise the value of a cell that calculated

− 2 ∑()Boundobserved Boundline of best fit . Any iterative calculation like this requires initial estimates of Kd and Bmax, which were obtained by inspection of the plotted data. The initial Bmax was estimated from the amount of bound IgG when all sites were saturated and Kd was estimated from the IgG concentration at half Bmax. The availability of computer programs to perform these calculations, makes this approach preferable because, unlike Scatchard analysis, it does not distort the experimental errors 423.

BFreemax• [ ] Equation 1 - Saturation binding Bound = lineof best fit Kd+[] Free

The competitive inhibition experiments involved incubating a constant concentration of 125I-labelled anti-PF4-heparin IgG (≈1 nM, specific activity 70-160 kBq/µg) and increasing concentrations of an unlabelled preparation, of the same IgG, in the microtitre wells with gentle mixing for 1 hr at room temperature. Wells were washed three times with PBS-tween and the amount of bound 125I-IgG was determined by γ-counting. I assumed that the binding affinities of 125I-IgG and unlabelled IgG were equivalent. The concentrations of free labelled IgG, [L], and free unlabelled IgG, [U], were approximated by their total concentrations. Least squares regression (described above for the saturation binding experiment) was used to fit the binding of 125I-IgG to Equation 2 and simultaneously obtain an estimate for Kd and Bmax. The initial estimates of Bmax and Kd were obtained from those calculated in the saturation binding experiment.

BLmax• [ ] Equation 2 - Competitive inhibition Bound = lineof best fit Kd++[][] U L

Equation 2 is derived from the ligand binding form of the Cheng and Prusoff 424 equation that has been used by others in algebraically equivalent forms 422,425,426.

116 Results

HIT antibodies recognise PF4-heparin and PF4 alone in ELISA In ELISA, IgG from HIT patients displayed significantly more binding to both PF4 (P<0.005) and PF4-heparin (P<<0.001) than normal individuals (Figure 21) using the two-tailed Mann-Whitney test 427. This non-parametric test is resistant to extreme values and independent of the distribution of the data. Binding of HIT IgG to PF4 alone was weaker than when heparin was also present. This binding is specific for PF4 because there was no significant difference between HIT and Normals in binding to control wells coated with either the related platelet protein βTG or with BSA. Any residual heparin in the HIT sample (if present) is unlikely to have affected the results because the plasma samples were diluted one thousand times. For example, had the plasma contained 0.5 U/ml heparin then the PF4 in the ELISA wells would have been exposed to only 5x10-5 U heparin. Given that about 70 ng PF4 bound to wells when coated with PF4 alone, achieving an optimum PF4:heparin ratio would require the addition of 2x10-3 U heparin.

117 4

3.5

3

2.5

2

1.5

1

IgG Binding (Absorbance 650nm) 0.5

0 PF4 PF4-heparin βTG BSA

Figure 21 - ELISA ELISA was used to quantitate binding of IgG antibodies in plasma from HIT patients ( ) and normal individuals ( ) to microtitre wells coated with PF4, PF4-heparin, βTG and BSA. The binding of HIT samples was significantly greater than normal samples to both PF4 (P<0.005, two-tailed Mann-Whitney test) and PF4-heparin (P<<0.001). There was no significant difference between HIT and normal individuals in binding to βTG or BSA. Bars show the mean value.

Effect of sulfoMBS activation of Covalink

The ELISA in Figure 21 is based on the original method by Amiral et al. 158 where Covalink microtitre wells were activated with sulfoMBS prior to coating with PF4- heparin. SulfoMBS was supposedly used to covalently link PF4 to the polystyrene but I question whether this was effective. SulfoMBS is a heterobifunctional cross-linker with a N-hydroxysuccinimide ester (NHS) group on one end and a maleimide group on the other. The reactions involved in covalently linking a protein to Covalink wells are outlined in Figure 22.

NHS reacts with the amine on the polystyrene, which results in the reactive maleimide group of sulfoMBS being attached to the spacer arm. When protein (eg PF4) is added to

118 the well the maleimide reacts with free sulfhydryls on the protein and covalently attaches the protein to the well. However, PF4 does not have any free sulfhydryl groups 366 so it is not clear how sulfoMBS may link it to Covalink wells. According to the product insert from Pierce, maleimide can react with primary amines but this is 1000 times slower than sulfhydryls at neutral pH. Nevertheless, maleimide will react slowly with isolated amino acids at pH 7.0 428 and it is conceivable that, in the absence of -SH groups, significant crosslinking through the lysines of PF4 may occur.

119 O

H3C O NaO3S O N

NH N O C O O N O sulfoMBS CH3

Covalink O NaO3S

N OH

O O H C 3 O N

N C O O N

CH3

HS Protein

O H C 3 O N

N C O S Protein O N

CH3

Figure 22 - Reactions involved in covalently crosslinking a protein to Covalink microtitre wells using sulfoMBS. Covalink microtitre wells consist of a polystyrene surface with a secondary amine on a 2 nm spacer arm. The N-hydroxysuccinimide ester on sulfoMBS reacts with this amine, which results in the maleimide group of sulfoMBS being covalently bound to the polystyrene. When a protein, containing a free sulfhydryl, is introduced it reacts with the maleimide and itself becomes covalently bound to the well. This reaction sequence is derived from descriptions in the Covalink Users Manual (Nunc) and the instructions for NHS-esters cross-linkers (Pierce). Note: NHS is normally said to be specific for primary amines but the Covalink Users Manual indicates that in this case NHS will react with the secondary amines of Covalink wells.

I investigated whether sulfoMBS activation of Covalink wells improved, or otherwise altered, the detection of HIT antibodies that recognised PF4 or PF4-heparin. SulfoMBS actually decreased the binding of HIT antibody to PF4-heparin (Figure 23). Whether this 120 was due to less PF4-heparin coating the wells, or if it was due to alteration of epitopes on PF4, was not investigated. Antibody binding to PF4 alone was not affected by sulfoMBS (Figure 23). These results indicated that, in my hands, there was no benefit in using sulfoMBS. Consequently, sulfoMBS was only used in the ELISA shown in Figure 21, which was performed before this revelation.

I suspect that Amiral et al. 158 originally used sulfoMBS activation because they were screening many platelet antigens for binding to HIT antibodies. PF4±heparin presumably coats the ELISA wells by passive adsorption during the overnight incubation.

4

3.5

3

2.5

2

1.5

(Absorbance 650nm) 1 Specific binding of HIT IgG 0.5

0 PF4-heparin PF4-heparin PF4 PF4 No + sulfoMBS No + sulfoMBS sulfoMBS sulfoMBS

Figure 23 - Effect of sulfoMBS activation of ELISA wells on binding of HIT antibody to PF4 and PF4-heparin Covalink microtitre wells were treated with, or without, fresh sulfoMBS (125 µg/ml) for 3 hr. Wells were then coated overnight with 20 µg/ml PF4 ± 0.67 U/ml heparin or 20 µg/ml BSA. The binding of four HIT plasma IgG was determined by ELISA. Differently shaded bars represent the binding of each HIT plasma IgG to PF4 or PF4-heparin after the background binding to BSA has been subtracted.

121 HIT IgG does not bind to native PF4 in the fluid-phase The ELISA protocol largely relies on PF4 being adsorbed onto the surface of microtitre wells, a process that may modify the conformation of PF4. In order to study the binding of IgG to native PF4 I devised a fluid-phase enzyme immunoassay (EIA) to measure antibodies recognising soluble PF4±heparin. Binding of HIT IgG to PF4-heparin was significantly (P<0.001) greater than that of normal individuals (Figure 24). In contrast, there was no significant difference in binding of HIT and normal IgG to PF4 alone in solution.

It is widely accepted that HIT antibodies do not recognise heparin itself but, hypothetically, HIT antibodies could appear to bind PF4-heparin in the fluid-phase EIA if they did recognise heparin itself and caused heparin-bound biotin-PF4 to be detected. Figure 25 shows that this is not a plausible mechanism because a mixture of heparin (0.9 U/ml) and biotin-PF4 (1 µg/ml), without any unlabelled PF4, was not recognised by HIT IgG. Only when unlabelled PF4 was added to its optimal concentration (15-20 µg/ml) did HIT IgG bind the PF4-heparin (Figure 25).

122 3.5

3

2.5

2

1.5

1

IgG Binding (Absorbance 450nm) 0.5

0 PF4 PF4-heparin

Figure 24 - Fluid-phase EIA The fluid-phase EIA was used to investigate the binding of IgG in HIT and Normal plasmas to soluble PF4±heparin. The HIT plasma IgG ( ) bound significantly more than normal plasma IgG ( ) to PF4-heparin (P<<0.001, two-tailed Mann-Whitney test). There was no significant difference between HIT and normal samples in binding to PF4 alone. Bars show the means.

123 2.5

2

1.5

1 IgG Binding (Abs 450nm)

0.5

0 0 5 10 15 20 25 30 35 40 Unlabelled PF4 Concentration (µg/ml)

Figure 25 - Effect of varying PF4 concentration on the fluid-phase EIA PF4-heparin antigen was prepared by mixing different concentrations of unlabelled PF4 with 0.88 U/ml heparin and 1 µg/ml biotinylated PF4. These antigen solutions were tested in the fluid-phase EIA with three different HIT plasmas ( , , ) that were diluted 1/100.

Depletion of HIT plasma by heparin-PF4-agarose and PF4-agarose Antibodies in HIT plasma bind to PF4 attached to microtitre wells (Figure 21) but not to native PF4 in solution (Figure 24). This suggests that PF4 may be modified by adherence to a surface, resulting in increased antibody binding. To investigate this, I used agarose beads coated with PF4 and PF4-heparin to specifically deplete HIT plasma of antibodies. The relative amount of anti-PF4-heparin antibodies remaining were determined using the fluid-phase EIA (Figure 26). As expected, in all seven patients investigated, heparin-PF4-agarose completely depleted anti-PF4-heparin antibodies. However, depletion with PF4-agarose alone resulted in three different depletion profiles. 124 In two patients PF4-agarose alone was just as effective as heparin-PF4-agarose at depleting anti-PF4-heparin antibodies (Figure 26a). Another two samples were depleted less effectively by PF4-agarose alone but eventually all the antibodies were removed (Figure 26b). The third group consisted of three samples in which PF4-agarose largely, but not completely, removed antibodies against PF4-heparin (Figure 26c). Plain agarose caused only minimal antibody depletion. Thus, antibodies that recognise PF4-heparin bind with lower avidity to PF4-agarose without heparin.

1 (a) (b) (c)

0.8

0.6

0.4

anti-PF4-heparin IgG 0.2 Relative Concentration of

0 0102030400 102030400 10203040 Volume of Agarose (µl) Volume of Agarose (µl) Volume of Agarose (µl)

Figure 26 - Depletion of anti-PF4-heparin antibodies HIT plasmas were depleted of antibodies by incubation with PF4-agarose ( ), heparin- PF4-agarose ( ) and plain agarose ( ). The concentration of antibodies remaining that bound to PF4-heparin was measured by fluid-phase EIA and is expressed relative to undepleted plasma. A total of seven HIT plasma samples were investigated. Samples that gave similar results were averaged and are plotted with SD to indicate the variability within each group. (a) In two samples PF4-agarose depleted as effectively as heparin-PF4- agarose. (b) In two samples PF4-agarose eventually depleted all the anti-PF4-heparin antibodies. (c) In three samples PF4-agarose substantially, but not completely, depleted the anti-PF4-heparin antibodies.

I was also able to elute antibodies from PF4-agarose with 2 M NaCl. These eluted antibodies recognised PF4-heparin in the fluid-phase EIA while eluate from plain agarose contained no anti-PF4-heparin antibodies (Figure 27).

125 2

1.8

1.6

1.4

1.2

1

0.8

0.6

0.4 from Agarose (Absorbance 450nm) 0.2 Concentration of anti-PF4-heparin IgG Eluted 0 PF4-agarose Plain agarose

Figure 27 - Elution of HIT anti-PF4-heparin antibody from PF4-agarose HIT plasma (50 µl) was mixed for 1 hr with 20 µl of either PF4-agarose or plain agarose that had been blocked with 1% BSA but never exposed to heparin. The agarose was then washed three times with 400 µl PBS-tween and bound antibody eluted with 100 µl 2 M NaCl in 20 mM HEPES pH 7.5. The antibody preparations were tested at a 1/10 dilution in the fluid-phase EIA for binding to PF4-heparin. The different bars represent antibody from three different HIT plasmas that bound, and were eluted from, either PF4-agarose or plain agarose.

In addition to studying the depletion of anti-PF4-heparin antibodies (Figure 26), I also investigated depletion of antibodies against PF4 alone. One depleted plasma from each of the groups recognised in Figure 26 was tested for antibodies recognising PF4 without heparin in the ELISA. A pattern identical to Figure 26 was observed for each plasma. That is, heparin-PF4-agarose was more effective than PF4-agarose at depleting antibodies that recognised solid-phase PF4 without heparin (Figure 28).

126 1 (a) (b) (c)

0.8

0.6

0.4

anti-PF4 alone IgG 0.2 Relative Concentration of

0 0 102030400 102030400 10203040 Volume of Agarose (µl) Volume of Agarose (µl) Volume of Agarose (µl)

Figure 28 - Depletion of antibodies recognising solid-phase PF4 without heparin HIT plasmas were depleted of antibodies by incubation with PF4-agarose ( ), heparin- PF4-agarose ( ) and plain agarose ( ). The concentration of antibodies remaining that bound to PF4 immobilised on microtitre wells without heparin was measured by ELISA and is expressed relative to undepleted plasma. Three HIT plasma samples, one from each of the groups recognised in Figure 26, were investigated and are identified by the same letter. (a) A sample where PF4-agarose depleted as effectively as heparin-PF4- agarose. (b) A sample where PF4-agarose eventually depleted all the antibodies that could be removed by heparin-PF4-agarose. (c) A sample where PF4-agarose substantially, but not completely, depleted the antibodies that could be removed by heparin-PF4-agarose.

The depletion of HIT antibodies was not due to non-specific depletion of IgG because there was only weak depletion of total IgG or antibodies to adenovirus (Figure 29). The total IgG assay suggests that PF4-agarose causes slight non-specific depletion (Figure 29a). In contrast, the anti-adenovirus assay suggests that heparin-PF4-agarose may cause some non-specific depletion while PF4-agarose had no effect (Figure 29b). Despite this small discrepancy between the assays, it is clear that non-specific depletion is not the cause of the substantial depletion of antibodies in Figure 26 and Figure 28, particularly at the lower volumes of agarose.

127 (a) (b) 1 1

0.8 0.8

0.6 0.6

Total IgG 0.4 0.4

0.2 anti-adenovirus IgG 0.2 Relative Concentration of Relative Concentration of

0 0 0 10203040 0 10203040 Volume of Agarose (µl) Volume of Agarose (µl)

Figure 29 - Non-specific depletion of total IgG and anti-adenovirus IgG by agarose beads. HIT plasmas were depleted of antibodies by incubation with PF4-agarose ( ), heparin- PF4-agarose ( ) and plain agarose ( ). Six depleted plasma samples were assayed for total IgG (a). Two depleted plasmas were also tested for IgG recognising adenovirus antigen (b) by Ms Yvonne Duffy (Clinical Immunology Laboratory, Prince of Wales Hospital). Mean ± standard error is plotted.

In theory, depletion of anti-PF4-heparin antibodies by PF4-agarose may have been due to the HIT plasma being contaminated with heparin that would bind to the PF4-agarose. However, protein G purified HIT IgG produced similar results to HIT plasma indicating that if there was any residual heparin in the plasma it did not affect depletion (Figure 30). This observation also leads me to reject the hypothesis that variability of depletion between individual plasmas was due to other classes of immunoglobulins competing with IgG for epitopes on the PF4-agarose.

128 1

0.8

0.6

0.4

anti-PF4-heparin IgG 0.2 Relative Concentration of

0 0 10203040 010203040 Volume of Agarose (µl) Volume of Agarose (µl)

Figure 30 - Depletion of protein G purified HIT IgG by agarose-PF4±heparin The total IgG fraction of HIT plasma was purified with protein G sepharose (as per Purification of Normal IgG, p.111) to ensure that the sample contained no residual heparin. These preparations were depleted by incubation with PF4-agarose ( ), heparin- PF4-agarose ( ) or plain agarose ( ) as described for HIT plasma. Depleted samples were assayed for antibodies that bound PF4-heparin by ELISA and binding is expressed relative to undepleted sample. The two graphs represent IgG from two different HIT patients.

Purity of affinity-purified HIT IgG, Fab and F(ab’)2 To enable investigation of the binding avidity of HIT IgG, I affinity-purified anti-PF4- heparin IgG from HIT plasma using heparin-PF4-agarose. Figure 31 demonstrates the purity of the HIT anti-PF4-heparin IgG, which ran as a single 184 kD band under non- reducing conditions (Lane 1). 184 kD is somewhat larger than 150 kD which is the accepted molecular weight for IgG but IgG from many other sources ran at the same size in this gel system. For example, normal human IgG, normal sheep IgG, and the mouse monoclonal IV.3 ran at 183 kD, 182 kD and 176 kD respectively. In this gel system, IgG and its fragments seem to run at molecular weights greater than their true size. Confirmation that the 184 kD band is in fact IgG comes from the observation that under reducing conditions it split into two bands which correspond to the heavy and light chains of IgG (Figure 31, Lane 6). The heavy and light chains are shadowed by faint bands. These are probably just artefacts of the electrophoresis but they could be minor

129 contaminants that, for some reason, are not apparent in Lane 1 which contains the non- reduced sample.

123 45 M 6

200 kD IgG 184 kD F(ab’)2 100 kD 122 kD Fc Heavy chain 57 kD 54 kD 50 kD Fab 46 kD Light chain 27 kD

10 kD

Figure 31 - SDS-PAGE of HIT anti-PF4-heparin IgG, Fab and F(ab’)2. HIT anti-PF4-heparin IgG was prepared by affinity for a heparin-PF4-agarose column and further purified on protein G sepharose. The IgG was run on a 4-15% SDS-PAGE gel (see p.86) under non-reducing (lane 1) and reducing (lane 6) conditions. In preparation for

studying the role of antibody bivalency on the binding of HIT IgG, Fab and F(ab’)2 were prepared from anti-PF4-heparin IgG and run under non-reducing conditions. Partial digestion of the IgG with ficin resulted in intact IgG, an unidentified band at ≈126 kD, Fc fragment and Fab fragment (lane 2). This partial digest was passed through a protein A column and the flow through contained Fab (lane 3). The eluate from the column

contained the intact IgG and Fc portion (lane 4). F(ab’)2 (lane 5) was prepared by complete digestion of anti-PF4-heparin IgG with pepsin. Molecular-weight markers (200 kD and 120-10 kD at 10 kD intervals) were run in lane M.

To enable investigation of antibody bivalency, Fab and F(ab’)2 fragments were obtained by enzymatic digestion of HIT anti-PF4-heparin IgG as summarised in Figure 32.

130 Ficin Fab + Fc + IgG

IgG Pepsin

F(ab’)2 + Fc fragments

Figure 32 - IgG Fragmentation Enzymatic digestion of HIT IgG with immobilised pepsin or with immobilised ficin plus

15 mM cysteine produced F(ab’)2 and Fab fragments respectively.

Fab fragments were prepared by partial digestion of anti-PF4-heparin IgG with ficin in the presence of 15 mM cysteine. This resulted in undigested IgG and Fab and Fc fragments as expected but it also produced a ≈126 kD band (Figure 31, lane 2). I would speculate that this unidentified band is IgG with just one Fab arm missing. The Fab fragments were separated from the IgG by passing the digest through a protein A column. This retained those molecules that had an Fc region and permitted Fab (Figure 31, lane 3) to flow through. The bound IgG and Fc (Figure 31, lane 4) was then eluted from the protein A. The use of a partial IgG digestion was deliberate because the intact IgG that remained from the reaction served as a control to ensure that the enzyme did not damage the antigen binding site.

F(ab’)2 fragments were prepared by complete digestion of the anti-PF4-heparin IgG with pepsin. Lane 5 of Figure 31 indicates that following digestion there was no residual IgG, only F(ab’)2 and smaller fragments that presumably correspond to Fc and perhaps

F(ab’)2 at various stages of degradation.

Importance of antibody bivalency in binding of HIT IgG to PF4-heparin Heparin could conceivably cause binding of HIT antibody to PF4 by crosslinking PF4 molecules and thus enhance the bivalent binding of IgG. The importance of antibody bivalency was investigated by comparing the binding to PF4-heparin of affinity-purified

HIT IgG, F(ab’)2 and Fab in ELISA. HIT IgG and F(ab’)2, but not the monovalent Fab,

131 bound PF4-heparin (Figure 33). The IgG and Fab were derived from the same partial digestion of HIT IgG by ficin so it is unlikely that the digestion damaged the actual antigen binding site of the Fab. Even when the Fab was tested at a ten fold higher concentration there was only weak specific binding of Fab from one of the patients (Figure 33).

4

3.5

3

2.5

2

1.5

(Absorbance 650nm) 1 IgG binding to PF4-heparin

0.5

0 HIT HIT HIT HIT Control Control Fab IgG 10x F(ab)2 Fab IgG Fab

Figure 33 - Binding of HIT IgG, Fab and F(ab’)2 to PF4-heparin ELISA was used to detect binding of affinity purified HIT anti-PF4-heparin IgG, Fab and

F(ab’)2 to microtitre wells coated with PF4-heparin. To maintain an equimolar

concentration of antigen binding sites, IgG and F(ab’)2 were assayed at 2 nM but the, monovalent, Fab was used at 4 nM. HIT Fab was additionally tested at a ten fold higher concentration (40 nM). Binding was detected with a secondary antibody that was specific for the Fab portion of human IgG and quantitated with TMB peroxidase substrate. IgG and IgG-fragments from two HIT patients are represented by solid symbols while control IgG and Fab from pooled normal plasma and from pharmaceutical IgG (Intragam, CSL, Australia) are represented by open symbols.

132 Binding studies of affinity purified anti-PF4-heparin IgG The equilibrium binding of affinity purified anti-PF4-heparin 125I-IgG to microtitre wells coated with PF4±heparin was investigated by two different methods. In the “saturation binding” method (Figure 34), I measured the binding of increasing concentrations of 125I-IgG to PF4±heparin coated microtitre wells. While the 125I-IgG did not always completely saturate the binding sites, non-linear regression still provided a reasonable estimate of Kd. Second, the “competitive inhibition” method (Figure 35) measured the decrease in 125I-IgG binding to PF4±heparin due to competition with unlabelled IgG. The Kd and Bmax for the binding of IgG to PF4 and PF4-heparin, estimated by both methods, are presented in Table 3. In summary, regardless of the method used, each patient’s IgG displayed greater functional affinity (decreased Kd) for PF4-heparin over that for PF4 alone. In contrast, there was no consistent trend in values of Bmax.

1 1

0.9 Patient 1 0.9 Patient 2

0.8 0.8

0.7 0.7

0.6 0.6

0.5 0.5

0.4 0.4

Bound IgG (nM) 0.3 0.3

0.2 0.2

0.1 0.1

0 0 0 204060800 20406080 Free IgG (nM) Free IgG (nM)

Figure 34 - Equilibrium binding isotherm of affinity purified HIT IgG to PF4±heparin Increasing concentrations of affinity purified anti-PF4-heparin 125I-IgG (specific activity = 2.5 kBq/µg) from HIT Patients 1 and 2 were incubated to equilibrium in microtitre wells coated with either PF4 ( ), PF4-heparin ( ) or βTG ( ). Binding is expressed as the concentration of IgG bound in 100 µl. Non-linear regression was used to fit the data to a single rectangular hyperbola (Equation 1, p.116). Dissociation constants for Patient 1

were KdPF4=74 nM, KdPF4-heparin=31 nM, and for Patient 2 KdPF4=20 nM, KdPF4-heparin=13 nM.

133 4% 6%

Patient 1 5% Patient 2 3% 4%

2% 3% I-IgG Bound 2% 125 1% 1%

0% 0% 0.01 0.1 1 10 100 1000 0.01 0.1 1 10 100 1000 Unlabelled IgG Competitor Unlabelled IgG Competitor Concentration (nM) Concentration (nM)

Figure 35 - Competitive inhibition of affinity purified HIT 125I-IgG binding by unlabelled IgG A constant concentration of affinity purified anti-PF4-heparin 125I-IgG (≈1 nM, specific activity = 70-160 kBq/µg) was mixed with increasing concentrations of unlabelled IgG (from the same source) and incubated to equilibrium in microtitre wells coated with either PF4 ( ) or PF4-heparin ( ). Binding is expressed as the percent of total 125I-IgG radioactivity added to the well. Non linear regression was used to fit the data to the ligand binding form of the Cheng and Prusoff 424 equation (Equation 2, p.116). The dissociation

constants for Patient 1 were KdPF4=59 nM, KdPF4-heparin=22 nM, and for Patient 2

KdPF4=33 nM, KdPF4-heparin=6.9 nM.

I note that the antibodies affinity purified from each patient may possibly be a polyclonal population of IgG, recognising different epitopes on PF4-heparin. If so, each Kd value should be interpreted as an average value representing these different populations. In addition, the process of affinity purification may have selected for higher avidity antibody populations while very low avidity (but specific) antibodies may have been washed away.

134 Table 3 - Equilibrium binding of HIT IgG Binding to solid-phase Binding to solid-phase Patient Method PF4-heparin (nM) PF4 alone (nM)

Patient 1 Saturation binding Kd = 31 Bmax =1.1 Kd = 74 Bmax =0.9

Patient 1 Competitive inhibition Kd = 22 Bmax =0.6 Kd = 59 Bmax =0.8

Patient 2 Saturation binding Kd = 13 Bmax =1.1 Kd = 20 Bmax =0.5

Patient 2 Competitive inhibition Kd = 6.9 Bmax =0.4 Kd = 33 Bmax =0.8

Dissociation constants (Kd) and number of binding sites (Bmax) for the equilibrium binding between solid-phase PF4±heparin and HIT IgG from two patients, were estimated by two methods. The “saturation binding” method measured the binding of increasing concentrations of 125I-IgG to PF4±heparin coated microtitre wells (Figure 34). The “competitive inhibition” method measured the degree to which increasing concentrations of unlabelled IgG competitively inhibited the binding of a constant concentration of 125I-IgG (Figure 35). All units are nanomolar.

Discussion

Antibodies to PF4-heparin complexes are associated with HIT 43,45,158. I confirmed the observations of others 43,45,158,159 that HIT plasmas also contain antibodies that bind to a lesser degree to surface immobilised PF4 in the absence of heparin (Figure 21). However, IgG in these plasma did not usually recognise soluble PF4 (Figure 24). The single exception to this was a patient whose IgG bound weakly to PF4 in the fluid-phase and very strongly to PF4-heparin or surface immobilised PF4. The general failure of HIT IgG to recognise soluble PF4, which is presumably in its native state, indicates that adsorption onto the ELISA microtitre wells antigenically modifies PF4 and thus facilitates antibody binding.

I am confident that the PF4 was not contaminated by heparin from the heparin- sepharose because the heparin was covalently linked to the sepharose beads which were thoroughly washed with 2M NaCl prior to adsorption of PF4. Unfortunately, demonstrating experimentally that there are not traces of heparin in the PF4 is remarkably difficult because the PF4 will interfere with any assay for heparin. Even if 135 there was a small amount of contaminating heparin then it was not sufficient on its own to promote HIT antibody binding to PF4 because HIT antibody did not bind to the PF4 in solution. I showed that HIT antibodies only bound to PF4 alone when the PF4 was immobilised on a surface (agarose beads or microtitre wells). In contrast HIT antibodies bound to PF4-heparin regardless of whether the antigen was in solution or immobilised on a surface.

Most researchers believe that HIT antibodies do not recognise heparin alone 17,95,125,148,159, however there are some reports that suggest that heparin is the target antigen 126,149. I investigated this possibility by modifying the fluid-phase EIA. The normal fluid-phase EIA involves preparing PF4-heparin by mixing heparin (≈0.9U/ml) with biotinylated PF4 (≈ 1µg/ml) and unlabelled PF4 (≈19µg/ml). This PF4-heparin complex is recognised by HIT antibodies. I modified the assay by adding different amounts of unlabelled PF4, while keeping constant the concentration of heparin and biotin-PF4. It is reasonable to assume that the biotinylated PF4 bound to the heparin and so if the HIT antibodies recognised epitopes on the heparin then this should be detectable through the attached biotin. Figure 25 illustrates that in the absence of unlabelled PF4 the HIT IgG did not bind the biotin-PF4-heparin. Binding was only detected when unlabelled PF4 was added to 15-20µg/ml (Figure 25). If the HIT IgG had bound to the heparin molecule then the observed binding would be largely independent of the PF4 concentration. Thus this data supports the most accepted view that HIT antibodies do not specifically bind to heparin alone. Instead, the extra PF4 presumably altered the PF4:heparin ratio, favouring the formation of the multimolecular PF4- heparin complex which HIT antibodies are thought to recognise 43,159,379.

HIT antibodies will bind to complexes of PF4 and various polyanionic compounds such as dextran sulfate, pentosan polysulfate and DNA 95,148,151,159. This provides some evidence against the hypothesis that heparin contributes to part of the epitope. However, such experiments do not exclude the possibility that HIT plasma contains two antibody populations, one population that recognises epitopes that incorporate heparin and another population that binds PF4 without heparin. I observed that in 4/7 HIT plasmas, agarose beads coated with PF4 alone completely depleted antibodies that recognised PF4-heparin in a subsequent assay (Figure 26a and Figure 26b). This indicates that all the anti-PF4-heparin IgG in these 4 plasmas recognise the immobilised PF4 without 136 heparin and implies that the agarose beads were able to mimic the effect of heparin by promoting the binding of antibodies to PF4. The agarose beads, or the spacer arm that binds the PF4, are unlikely to have antigenic epitopes in common with heparin. Thus it appears that these HIT antibodies predominantly bind to the PF4 component of the PF4- heparin complex and heparin does not directly form part of the epitope to which the HIT antibodies bind.

In 3/7 plasmas, the majority, but not all, of the anti-PF4-heparin IgG was depleted by PF4-agarose alone (Figure 26c). The antibodies that were not depleted may represent a population of IgG that do recognise epitopes containing part PF4 and part heparin molecules. Alternatively, they may be low affinity antibodies, which are difficult to deplete, or may specifically require heparin to produce a large conformational change in PF4 to enhance antibody affinity. Regardless, my observations are consistent with those of others, which indicate that there is antibody heterogeneity between HIT patients 48,179.

It has not been clear whether the binding of HIT antibodies to PF4-heparin and the lesser binding to solid-phase PF4 alone in ELISA is due to two distinct antibody populations in the plasma or one type of antibody that recognises both antigens but to varying degrees. If there are two types of HIT antibodies, one to solid-phase PF4 alone and one with affinity for PF4-heparin, then they should be separable by their binding to PF4-agarose. That is, antibodies to PF4 alone should be depleted more effectively by PF4-agarose than by heparin-PF4-agarose. However, this was not observed. While PF4- agarose did deplete antibodies to solid-phase PF4, depletion with heparin-PF4-agarose was even more effective (Figure 28). Furthermore, if there were two distinct anti-PF4 and anti-PF4-heparin antibody populations, then PF4-agarose should not deplete antibodies against PF4-heparin. However, not only did PF4-agarose deplete antibodies against PF4-heparin (Figure 26) but I was able to elute IgG from PF4-agarose that subsequently bound PF4-heparin in the fluid-phase EIA (Figure 27). I conclude that the anti-PF4 and anti-PF4-heparin antibodies detected by ELISA are largely the one and the same. This antibody recognises PF4 that is bound to heparin or a solid support.

The bivalent nature of HIT IgG is important for its binding to PF4-heparin. I observed that HIT IgG and F(ab’)2, but not the monovalent Fab, bound PF4-heparin (Figure 33). Even when Fab was tested at a concentration ten times higher there was little binding to

137 PF4-heparin. The enzymatic digestion did not damage the antigen binding site of the Fab fragment because the undigested IgG, from the same reaction, retained its binding ability. Others 48,151,179 have proposed that HIT IgG may bind to a cryptic/neo epitope formed by a heparin-induced conformational change in PF4. My data does not exclude this possibility but I believe an alternative model should be considered.

I propose that HIT IgG may be specific for existing low affinity epitopes on PF4 and that heparin cross-links PF4 tetramers at sufficiently high density for each arm of an IgG molecule to bind neighbouring tetramers. With IgG attached by both antigen binding arms to cross-linked PF4 tetramers the effective antibody binding avidity would be strongly enhanced compared to a monovalent interaction (Figure 36a). Under this model PF4 in solution does not bind HIT IgG because the affinity of a single antigen binding arm for a single PF4 molecule is too low (Figure 36b). The observation that other polyanionic compounds can be substituted for heparin 95,159 is explained by proposing that they also form multimolecular complexes with PF4. Similarly, PF4 that is adsorbed onto microtitre wells binds HIT antibody because each arm of an IgG can bind PF4 molecules that are immobilised close together (Figure 36c). It is noteworthy, however, that the avidity of HIT IgG for PF4-heparin is somewhat greater than that for polystyrene-adsorbed PF4 alone (Table 3). This may be due to the flexibility of heparin permitting a more favourable orientation of the PF4 that is not possible when PF4 is fixed directly to microtitre wells.

138 (a) (b) (c)

Key

Heparin

HIT IgG

PF4 tetramer

ELISA wells or agarose beads

Figure 36 - Model for the effect of IgG bivalency and antigen immobilisation on HIT antibody binding to PF4. A multimolecular PF4-heparin complex may bind HIT IgG because the heparin acts as a scaffold that cross-links PF4 at high density. The bivalent nature of IgG means that if one arm of the IgG releases PF4, the other arm holds the PF4 in close proximity and the lost bond quickly reforms (a). PF4 in solution does not bind HIT IgG because there is no stabilisation of the low affinity binding to fluid phase PF4 (b). PF4 at high density on microtitre wells or agarose beads will bind HIT IgG due to the bivalent nature of the IgG and immobilised PF4 (c). The boxes represent the favoured equilibrium state.

It has been suggested that HIT antibodies only bind PF4-heparin at the specific PF4:heparin ratio at which a multimolecular PF4-heparin complex forms 43,159,379. Figure 11 (p.94) and Figure 25 (p.124) demonstrate that HIT antibodies bind to PF4 only in a narrow range of heparin concentrations. This is compatible with a model that requires cross-linking of PF4 to facilitate bivalent binding of HIT IgG. For example, at low heparin concentrations there is insufficient PF4-heparin to detect binding. As the heparin concentration increases the multimolecular complexes form and antibody binds to the cross-linked PF4. At even higher heparin concentrations I envisage that single PF4 tetramers are wrapped in heparin and are no longer cross-linked so their functional affinity is reduced back to that of PF4 alone.

139 A potential criticism of my model of bivalent IgG binding is that PF4 is a tetramer and so epitopes would be repeated four times on the one molecule and thus IgG could easily bind bivalently to a single PF4 tetramer. In reality, PF4 is better described as a dimer of dimers (Figure 5, p.73). That is, there would be many epitopes that occur only twice on a PF4 tetramer. If these epitopes exist on opposite sides of the PF4 molecule then the flexibility of IgG may not be great enough for both Fab arms to bind simultaneously. This appears to be reasonable conjecture because Figure 37 illustrates that each Fab arm of IgG is as thick as the PF4 tetramer so bringing both arms close enough together such that each binds an epitope on opposite sides of a PF4 tetramer seems impossible. However, it should be noted that IgG is not only flexible at the hinge between Fab arms but the switch region, within each Fab, can also bend 429. It is unclear whether this would provide enough flexibility to enable both Fab arms to bind to a single PF4 molecule.

140 Fc

Hinge

Fab Switch

Figure 37 - Representation of HIT IgG binding to PF4-heparin Space filling models of IgG (yellow), PF4 tetramer (red) and heparin (48 saccharide units in blue) are drawn to scale in a hypothetical representation of the binding of HIT IgG to PF4-heparin. PF4 (1rhp.pdb) and heparin (1hpn.pdb) were obtained from the Brookhaven protein databank (www.rcsb.org). The 1hpn.pdb file contained only 12 saccharide units of heparin so RasMol (University of California Berkeley version 2.6ucb, which allows up to five molecules to be open simultaneously, http://mc2.cchem.berkeley.edu/Rasmol) was used to join four of these 12-mers by moving them on the computer screen until they gave the appearance of being linked. The IgG molecule is a composite model of human IgG1, 429 including carbohydrate, built by Padlan from F(ab’)2 fragment (2ig2.pdb) and Fc fragment (1fc2.pdb). The IgG file (igg1-eap.exe) was obtained from the RasMol web site (www.umass.edu/microbio/rasmol/padlan.htm). The IgG and PF4 were moved in RasMol so as to appear joined. The separate images of heparin and PF4-IgG were then combined in a photo manipulation program while ensuring that the relative sizes of the molecules were correct. The purpose of this Figure is simply to show the relative sizes of the HIT antibody and antigen in a plausible arrangement, therefore this illustration contains a number of inaccuracies. For example, heparin is flexible 430 and probably partially wraps around the PF4, the location of the heparin and IgG binding sites on the PF4 is purely arbitrary, flexibility within the hinge region of IgG 429 means that it can probably bind PF4 molecules that are much closer together than shown here, and PF4-heparin is not shown as a large multimolecular complex.

141 A recent report by Suh et al. 48 provides some evidence against the concept that cross- linking of PF4 is sufficient in itself to cause high-affinity bivalent binding of HIT antibody to PF4. They compared the binding of HIT antibody to PF4 immobilised on two different kinds of heparin-agarose. In the most common type of heparin-agarose, each heparin chain is covalently linked at multiple sites along its length to the agarose. HIT antibodies did not appear to recognise PF4 bound to this type of immobilised heparin. In contrast, when the heparin chains were end-linked to the agarose HIT antibody did bind to the PF4-heparin-agarose 48. This was interpreted as indicating that heparin had to be free and flexible enough to wrap around the PF4 tetramer in order to create the HIT epitope. If my hypothesis, that cross-linking PF4 is sufficient to enable HIT antibody binding, is true then both types of heparin-agarose would be expected to fulfil this role. However, my own observations demonstrate that HIT antibody recognises PF4 immobilised on non-flexible surfaces such as microtitre wells (Figure 21) or agarose beads (Figure 26). I do not have a good explanation as to why Suh et al. found immobilised heparin did not enhance HIT antibody binding to PF4 48.

My data indicate that bivalent antibody binding is important for the interaction of HIT IgG with PF4-heparin and I therefore propose that antigen immobilisation may be the mechanism by which heparin increases HIT antibody avidity for PF4. However, I do not discount the possibility that a conformational change within PF4 may also be required to create or expose HIT epitopes.

Purification of HIT IgG using end-linked-heparin sepharose coated with PF4 has recently been described 48 but antibody affinity/avidity has not been investigated. I used a simpler technique to affinity purify HIT anti-PF4-heparin IgG and for the first time report the avidity of IgG from two HIT patients using two different techniques. I observed dissociation constants of 7-30 nM for binding to PF4-heparin. This relatively high affinity is much greater than the Kd=1-10 µM reported for the interaction between soluble β2 glycoprotein I (β2GPI) and anti-β2GPI IgG from the sera of patients with anti- 406,431 phospholipid syndrome . However, the avidity of anti-β2GPI antibodies for solid- phase β2GPI is higher than for fluid-phase β2GPI. (I estimate that the antibodies have a 431 Kd≈200 nM for surface immobilised β2GPI because Tincani et al. observed that

10 µg/ml of β2GPI on polystyrene beads inhibited 50% of antibody binding to β2GPI

142 coated ELISA wells.) For comparison, very strong immunogens, such as tetanus and diphtheria toxoids, induce very high affinity IgG (Kd≈10 pM) 432,433.

The concentration of anti-PF4-heparin IgG in the plasma of Patients 1 and 2 were 500 nM and 770 nM respectively when measured by ELISA using the affinity purified IgG as a standard (data not shown). These concentrations are 10-100 fold higher than the apparent Kd of the antibodies to PF4-heparin. Even allowing for the possibility that the Kd’s I measured may be lower than the true in vivo situation, it seems likely that antigen availability and not antibody concentration would limit the binding of HIT IgG to PF4-heparin on platelets in vivo. In other words, I expect that in vivo, virtually all PF4-heparin epitopes would be occupied by antibody.

The purified HIT IgG showed increased functional affinity for PF4-heparin (Kd=7-30 nM) over surface immobilised PF4 alone (Kd=20-70 nM). However, the functional affinity for PF4 alone is still relatively high and indicates that adsorption onto the microtitre wells is almost as effective as heparin in modifying PF4 to facilitate antibody binding. While I have not quantitated the affinity of HIT antibodies for soluble PF4, the results of the fluid-phase assay indicate that it is very low. The actual mechanism by which heparin or surface immobilisation modifies PF4, to permit antibody binding, has yet to be elucidated. Binding a protein to a surface may induce a conformational change within the protein molecule that exposes or forms a new antigenic epitope 434,435. It has been suggested that heparin may induce a conformational change within the PF4 protein and expose a cryptic/neo epitope 48,151. Alternatively, immobilisation of PF4 by polystyrene or heparin may enhance the avidity of an antibody that possesses low intrinsic affinity for soluble PF4 alone. When both arms of an IgG bind to epitopes that are linked, the effective affinity of the IgG can be enhanced many hundred fold over binding by a single arm 436. Both the conformational change and antigen immobilisation mechanisms have been proposed for the anti-phospholipid antibody syndrome, where patients develop antibodies to plasma proteins that have been antigenically modified by binding to phospholipid 404,406. Further studies are required to determine whether either or both mechanisms apply to HIT.

In this chapter I report the first measurement of the functional equilibrium binding affinity of purified HIT antibodies and provide evidence that these antibodies recognise

143 epitopes on PF4 that are not directly contributed to by the heparin molecule. The avidity of HIT antibodies for these epitopes is enhanced when PF4 binds to heparin or a solid support. The exact mechanism by which avidity is increased remains to be determined.

144 Chapter 4 - Binding of purified anti-PF4-heparin antibodies to platelets and the resulting platelet activation.

Introduction

HIT plasma usually contains antibodies that aggregate platelets in the presence of heparin 14,60,96,123. In addition, antibodies against complexes of PF4 and heparin are associated with this disease 90,91,158,159,163. However, HIT plasma that aggregates platelets does not always contain anti-PF4-heparin antibodies 91,205,210. Understanding how HIT antibodies interact with platelets, and whether the anti-PF4-heparin antibodies bind platelets and cause aggregation, is important in understanding the pathophysiology of HIT. Unfortunately, conflicting models have been proposed to describe the interaction of HIT antibodies with platelets and none of the previous studies directly investigated the binding of affinity purified anti-PF4-heparin IgG to platelets. Furthermore, previous studies are too artificial and did not allow for the fact that binding of HIT IgG causes platelet activation and secretion of PF4 that may vary antibody binding during the course of the reaction.

Visentin et al. report that, using flow cytometry, IgG from some HIT patients bound to resting washed platelets and binding was greater to thrombin-activated platelets 45. However, antibody binding required that both exogenous PF4 and heparin were added at a molar ratio of approximately 10:1 respectively. Blocking the platelet Fc receptor (FcγRII) with the monoclonal antibody IV.3 abolished antibody binding. This dependence upon the Fc receptor, and that HIT IgM did not bind to platelets, was interpreted as providing evidence that only the Fc portion of HIT IgG interacts with platelets. In other words, HIT IgG forms an immune complex with circulating PF4- heparin, the complex binds to the platelet FcγRII receptor through the Fc portion of the IgG and causes platelet activation (Figure 38 - Fluid-phase antigen model).

145 Fluid-phase antigen model

Platelet

Platelet-bound antigen model

Platelet

PF4 HIT IgG Heparin FcγRII

Figure 38 - Models for the binding of HIT IgG to platelets. Fluid-phase antigen model: HIT IgG binds to complexes of PF4-heparin in solution. These immune complexes then bind and activate platelets when the Fc portion of the IgG binds to the FcγRII. This model is based on descriptions by Greinacher 44, Warkentin et al. 54, Visentin et al. 45, and Warner and Kelton 194. Platelet-bound antigen model: Complexes of PF4-heparin form on the platelet surface. The Fab portion of HIT IgG binds to this immobilised antigen. Only then does the Fc portion of the platelet-bound IgG cross-link FcγRII and trigger platelet activation. This model is based on descriptions by Amiral et al. 159, Greinacher et al. 134, Kelton et al. 148, Jackson et al. 99, and Horne and Alkins 195. Note that blocking the FcγRII with the monoclonal antibody IV.3 will prevent HIT IgG from binding platelets in the fluid-phase antigen model but not the platelet-bound antigen model. However, IV.3 will prevent platelet activation in both models.

146 The fluid-phase antigen model is disputed by Horne and Alkins who investigated the binding of iodinated total IgG from HIT patients to washed platelets 195. Antibody binding was reliably observed only with thrombin-activated platelets and was dependent upon the addition of approximately equimolar concentrations of PF4 and heparin. In contrast to Visentin et al., they observed that Fc receptors were not important for the binding of HIT antibodies. Specifically, HIT antibodies bound to platelets in the presence of IV.3. Horne and Alkins support a model in which HIT IgG binds, by its Fab portion, to PF4-heparin complexes on the platelet surface (Figure 38 - Platelet-bound antigen model).

These studies by Visentin et al. 45 and Horne and Alkins 195 are valuable but I contend that they are highly artificial and the best way to resolve the discrepancies between them is to study antibody binding to platelets in a more physiological system. Both reports investigated the binding of HIT antibodies to platelets under static conditions where the washed platelets were either fully activated or held quiescent by PGE1. There was no opportunity for secreted platelet proteins to modulate antibody binding and no information describing how binding changed during activation. Furthermore, when activated platelets were studied they were activated with an unphysiologically high concentration of thrombin rather than by HIT antibodies. The use of washed platelets, with little or no plasma, presumably necessitated the addition of exogenous PF4 to observe HIT antibody binding. While this may simplify the interpretation of results it is also a further departure from physiological conditions. In addition, the extensive washing that Visentin et al. performed, to remove primary and secondary antibodies before flow cytometric analysis, may have washed off specific antibodies. The approach used by Horne and Alkins is more desirable because platelet-bound 125I-IgG was separated from unbound by quickly centrifuging the platelets through silicone oil. However, the specific binding of HIT antibody that they report was really just a modest increase over the non-specific levels. It is difficult to see how this relatively small change could cause the extensive platelet activation that is observed in vivo or during in vitro assays that measure platelet aggregation and 14C-serotonin release.

Information as to how HIT antibodies interact with platelets may help to understand the pathogenesis of this disease so the discrepancy between the results of Visentin et al. and those of Horne and Alkins needs to be resolved. In an editorial, Aster highlights the 147 need for further studies “under conditions that mimic the in vivo situation in which resting, or partially activated, platelets circulate in a plasma milieu where they are subject to shear stress and other factors that influence their state of activation” 42. I have been able to study the binding of HIT IgG to platelets under more physiological conditions by using affinity purified anti-PF4-heparin IgG from HIT plasma. This IgG provides much greater sensitivity and is a more clearly defined antibody than the total IgG or plasma used by others 45,195.

In this chapter, I quantitate the binding of affinity purified HIT 125I-IgG to platelets during platelet aggregation. Importantly, this aggregation was induced by the HIT IgG (in conjunction with heparin) rather than artificially by thrombin as in earlier reports 45,195. Binding was studied in platelet rich plasma stirred at 37°C, so platelets were exposed to shear forces. As would be expected to happen in vivo, PF4 released from the platelets remained available to interact with heparin and the platelets. No exogenous PF4 was added. I also investigate the importance of platelet FcγRII on HIT IgG binding to platelets using this more physiological and sensitive technique. Finally, I show that activation of platelets by affinity purified HIT IgG increases the release and surface expression of platelet PF4, which explains the concurrent increase in IgG binding.

Methods

Purification of HIT IgG HIT anti-PF4-heparin IgG (HIT IgG) was affinity-purified as described in the previous chapter (see Affinity purification of HIT anti-PF4-heparin IgG, p.110). Briefly, HIT plasma was passed through a heparin-PF4-agarose column then bound antibodies were eluted with 0.13-1.5 M NaCl gradient. IgG was purified on a protein G sepharose column. Normal IgG was purified from the pooled plasma of three healthy individuals by affinity for protein G sepharose (see Purification of Normal IgG, p.111). IgG was iodinated as previously described (see Iodination of Proteins, p.114).

148 IgG binding during platelet aggregation The methods for platelet preparation and platelet aggregometry are based on those previously described 96,123. Citrated blood was acquired by collecting 9 volumes of blood into a syringe with 1 volume of 3.8% trisodium citrate. Platelet rich plasma (PRP) was prepared by centrifuging citrated blood at 170xg for 15 min. Plasma was obtained by centrifuging the remaining blood at 1600xg for 15 min. The concentration of platelets was determined with an automated blood cell counter (Sysmex NE-8000). Platelet aggregometry was performed in silicone-coated (Repel-Silane ES, Pharmacia Biotech, Sweden) glass tubes at 37°C by monitoring the increase in optical transmittance of the platelet suspension with a Chrono-Log aggregometer. Aggregation was recorded by amplifying the output of the aggregometer with a linear operational amplifier and recorded once a second on a personal computer with an 8 bit analog to digital converter (Pocket Sampler 437, Jaycar Electronics, Australia). This is described below in more detail.

The aggregation reaction involved stirring PRP (300x106 platelets/ml) with affinity- purified HIT IgG (12 µg/ml = 80 nM) and 125I-IgG tracer (≈0.07 µg/ml, ≈13 kBq/ml) in 650 µl of plasma. In some experiments the platelet FcγRII was blocked by including the mAb IV.3 (50 µg/ml, a gift from Prof. B.H. Chong). Platelet aggregation was induced by the addition of heparin (0.5 U/ml, which provides greatest sensitivity 145) or collagen (4 µg/ml, Collagenreagent Horm, Nycomed Arzneimittel, Germany). The aggregation reaction was sampled by removing, in duplicate, 80 µl of platelets with a wide-mouth siliconised pipette tip. These samples were layered onto a 800 µl cushion of 17% sucrose 438 in phosphate buffered saline (PBS) within a microfuge tube then centrifuged at 11,000xg for 2 min. The supernatant (containing free radioactivity) was carefully removed from the top down and the pellet (containing bound radioactivity) briefly rinsed with 200 µl PBS without resuspending. The bottom of the tube, containing the pellet, was cut off with dog-claw clippers and radioactivity determined with a γ-counter (Cobra II Auto-Gamma, Canberra Packard). This protocol permitted two lots of duplicate 80µl samples to be taken while maintaining sufficient platelets in the reaction tube to enable continuous monitoring of platelet aggregation. A third duplicate sample was obtained upon termination. The binding of 125I-IgG in duplicate samples was averaged and treated as a single value. The amount of IgG bound was determined by

149 multiplying the proportion of total counts that were bound by the amount of IgG added per platelet.

Recording platelet aggregation It was desirable to record the aggregation of platelets on a computer because this would make presentation of the data much easier and more attractive. Unfortunately, the aggregometer could only connect to a chart recorder. Therefore it was necessary to transform this output into a form that could be interpreted by a computer. This was achieved using an amplifier and an analogue to digital converter (ADC) as shown in Figure 39.

Platelet Analogue to Aggregometer Amplifier Digital Computer Converter

Chart Recorder

Figure 39 - Recording of platelet aggregation The output of the platelet aggregometer was amplified then fed to an analogue to digital converter (ADC) which was read by the computer using software supplied with the ADC. In addition to recording aggregation on the computer, a chart recorder was connected directly to the aggregometer to confirm that the computer trace was accurate.

The amplifier was required to increased the signal from the aggregometer to a level that could be efficiently read by the ADC. I designed and built the amplifier with valuable advice from Dr I.A. Newman (School of Mathematics and Physics, University of Tasmania, Australia). The circuit diagram of the amplifier is included in Appendix 1 (p.180). The ADC was constructed from a kit designed by G. Cattley 437. Figure 40 is a photograph of the amplifier and ADC and describes their operation in more detail.

Care was taken to confirm that the output of the aggregometer was faithfully recorded by the computer. I demonstrated that there was a direct linear relationship between the signal received the amplifier (ie. from the aggregometer) and the value recorded by the

150 computer (see Appendix 1). Furthermore, aggregation was simultaneously recorded on a standard chart recorder and this trace was visually compared with that obtained through the computer.

151 Analog to digital Input to amplifier converter from platelet aggregometer

Connection to computer

Amplifier

Figure 40 - Photograph of Amplifier and analog to digital converter The signal from the platelet aggregometer was sent to the amplifier via coaxial cable (not in photo), which terminated in two banana plugs. These plugged into the input of the amplifier which had a switch to turn it on/off and a hole through which the gain could be adjusted with a screwdriver. Internally, the offset voltage could also be set. Both the gain and offset were adjusted when the amplifier was constructed and not changed throughout the series of experiments. The output of the amplifier was carried by coaxial cable to the ADC (“Pocket Sampler”). The ADC was powered by the computer and needs no on/off switch. It has a switch to select either a 2V or 20V scale. The 2V scale was used in all experiments. A second switch selects between “sample” and “calibrate”. In “sample” mode the ADC fulfils its normal role of collecting data and transmitting it to the computer. The “calibrate” setting sends a 1.0 V signal into the ADC so that a variable resistor inside the ADC box can be adjusted so the value read by the computer is exactly 1.0 V. Flipping this switch to “calibrate” and back to “sample” was found to be a convenient way to mark an aggregometer trace because it resulted in a 1.0 V spike being recorded on the computer. This marker was easily edited out prior to graphing the aggregation trace. The exact time that reagents were added to platelets in the aggregometer was marked in just this way. The ADC communicates with the computer through a cable plugged into the parallel port of the computer. Software running on the computer read the data from the ADC once a second and saved the values to a text file. This file was easily imported into a spreadsheet/graphing program.

152 PF4 release and expression during platelet activation 650 µl of citrated PRP (300x106 platelets/ml) was stirred at 37°C in a siliconised glass aggregometer tube with (1) heparin plus HIT IgG, (2) collagen plus heparin, (3) collagen alone or (4) heparin alone. Heparin was used at 0.5 U/ml, collagen at 4 µg/ml and affinity purified anti-PF4-heparin IgG from HIT Patient 2 at 12 µg/ml. Aggregation was only observed when platelets were activated with HIT IgG, collagen plus heparin, or collagen alone. Activation was arrested after 8-9 min by adding 65 µl ETP 439 and chilling on ice (ETP is 107 mM EDTA, 12 mM theophylline, 2.8 µM PGE1). Platelets were pelleted by centrifugation (3500xg, 2 min) in the siliconised tube. The plasma supernatant was passed though a 0.2 µm filter to removed residual platelets 440 and stored at -20°C before measuring the PF4 concentration.

To measure PF4 expression, the platelet pellets were resuspended in 220 µl of a solution containing 2% BSA, 10% ETP, 50 µg/ml IV.3 and PBS. 90 µl of platelets were incubated on ice in microfuge tubes for 10 min with an equal volume of iodinated sheep anti-PF4 IgG (affinity-purified polyclonal from Cedarlane Laboratories Ltd, Canada) or sheep total IgG (purified from sheep serum by affinity for Protein G sepharose). Free 125I-IgG was removed from the platelets by direct centrifugation (9400 xg, 2 min) and two washes with PBS. The bottom of the microfuge tube, containing the pellet, was cut off with dog-claw clippers and radioactivity determined with a γ-counter. SDS polyacrylamide electrophoresis indicated that neither sheep IgG preparations contained any PF4.

PF4 Assay

A competition RIA was developed to measure the PF4 concentration in plasma derived from activated and inactivated platelets (see above). Microtitre wells that could be individually separated (MaxiSorp-BreakApart, Nunc) were coated overnight at 4°C with 100 µl of the sheep anti-PF4 IgG (1.9 µg/ml of affinity purified IgG in 0.1M Bicarbonate buffer pH 9.3). Wells were washed twice with water, air dried then blocked for 1 hr with 1% BSA in PBS-tween. Dilutions of test plasma and purified PF4 standards (0-2 µg/ml) were prepared in a solution containing 1% BSA, 0.38% trisodium citrate, 10% ETP, and PBS-tween. When the test plasma contained 0.5 U/ml heparin,

153 the same heparin concentration was maintained throughout dilution of the test and standard PF4 solutions.

110 µl of standard or unknown PF4 solution was equilibrated for 30 min with an equal volume of 125I-PF4 solution (≈0.2 µg/ml, 4 kBq/ml) containing polybrene (50 µg/ml). 100 µl of the PF4 mixture was incubated, in duplicate, for 15 min in the wells coated with anti-PF4 IgG. The wells were washed three times with PBS-tween then were broken apart and radioactivity measured in a γ-counter. The most sensitive region of the PF4 standard curve was 0.05-1 µg/ml PF4 and samples were diluted to fall within this range.

Polybrene was included in this assay because it has very high affinity for heparin and prevents heparin from blocking the binding of the sheep anti-PF4 to PF4. Consequently, this assay worked well with heparin concentrations in the range 0-1 U/ml. To counter the small residual effect of heparin, an unknown PF4 concentration was determined from an appropriate standard curve that was prepared either with or without 0.5 U/ml heparin.

Inhibition of HIT IgG binding to activated platelets by sheep anti-PF4 Activated platelets were prepared by aggregating platelet rich plasma (300x106 platelets/ml) with collagen (4 µg/ml) plus heparin (0.5 U/ml). Once the platelets were aggregated, 1/10th volume of ETP was added and platelets pelleted by centrifugation (3,500xg, 2 min). Platelets were resuspended in PBS containing 2% BSA, 6 mg/ml human IgG (Intragam, CSL, Australia), 10% ETP and 50 µg/ml IV.3 at 650x106 platelets/ml. This solution was used to block any non-specific or FcγRII-mediated binding of HIT IgG and to prevent any further platelet activation that may occur.

90 µl of platelets were incubated on ice for 30 min with 45 µl of affinity-purified sheep anti-PF4 IgG at a final concentration of 0-48µg/ml. Then HIT 125I-IgG (≈0.07 µg/ml, 4-6 kBq/ml) was added and incubated for a further 10 min on ice. Free 125I-IgG was removed from the platelets by direct centrifugation (9400xg, 2 min) and two washes with PBS. The bottom of the microfuge tube, containing the pellet, was cut off and the “bound” and “free” radioactivity determined.

154 HIT IgG binding to thrombin activated platelets The preparation of thrombin activated platelets was modified from the method of Margossian et al. 441 and is similar to that used by others in my laboratory 442. 1 µM

PGE1 was added to 20 ml citrated blood then PRP and plasma prepared as described previously (see IgG binding during platelet aggregation, p.149). Platelets were pelleted at 1300xg for 10 min and washed by resuspending in 10 ml of 5 mM PIPES buffer, pH 6.6, 1 µM PGE1, 145 mM NaCl, 4 mM KCl, 0.5 mM Na2HPO4, 1 mM MgCl2. After recentrifugation platelets were resuspended in 20 mM HEPES buffer pH 7.4, 137 mM

NaCl, 4 mM KCl, 0.5 mM Na2HPO4, 0.1 mM CaCl2 and counted in an automated blood cell counter. Washed platelets (300x106 /ml) in the HEPES buffer were incubated for 10 min ± IV.3 (50 µg/ml) then briefly mixed with 1 U/ml thrombin and allowed to stand for 5 min at room temperature. 80 µl of thrombin activated platelets were transferred to microfuge tubes and centrifuged at 9,000xg for 2 min. This created a layer of activated platelets stuck to one side of the tube and the thrombin was discarded.

The following method was used to investigate the binding of HIT IgG to fully (thrombin) activated platelets in the presence of plasma and compounds released from other platelets that had been aggregated with HIT IgG. Aggregation supernatants were produced by mixing in the aggregometer PRP, HIT IgG, 125I-IgG tracer and heparin until platelets were fully aggregated. Platelets were removed by centrifugation and discarded. More than 98% of the HIT IgG remained in the supernatant, which was mixed ± IV.3 (50 µg/ml). 80 µl of this supernatant was added, in triplicate, to tubes of thrombin activated platelets, which were then agitated gently for 15 min at 37°C on an angle to ensure that the platelet layer was covered with solution. Tubes were then centrifuged 18,000xg for 2 min and unbound radioactivity removed. The platelet layer was briefly washed with 200 µl PBS then counted in a γ−counter.

Results

HIT IgG binding during platelet aggregation

I investigated the binding of affinity purified HIT 125I-IgG to platelets at various time- points (A, B, C and D) during platelet aggregation induced by the antibody and heparin (Figure 41). Before heparin was added to the suspension of PRP and HIT IgG (time-

155 point A) the platelets remained non-aggregated and negligible binding of HIT IgG was detected. The addition of heparin triggered platelet aggregation. The beginning of aggregation (time-point B) was associated with low but detectable binding of HIT IgG. At time-point C, when platelets were approximately 50% aggregated, there was about half maximal HIT IgG binding. The greatest IgG binding was observed once platelets were fully aggregated (time-point D). The non-specific binding of Normal 125I-IgG remained low throughout, indicating minimal entrapment of IgG during aggregation. These data suggest that HIT IgG binding to platelets occurs only after platelet activation, in this case FcγRII-mediated platelet activation.

156 0.35 0.8

D 0.3 0.7

0.6 0.25

0.5 0.2 C 0.4 0.15 Heparin 0.3 B 0.1 0.2 Binding of Patient 1 IgG (fg/platelet) A Binding of Patient 2 IgG (fg/platelet) 0.05 0.1

0 0 -5 0 5 Time After Heparin (min)

Figure 41 - Binding of affinity purified anti-PF4-heparin HIT IgG to platelets during heparin-induced platelet aggregation. Platelet rich plasma (300x106 platelets/ml) was mixed with HIT IgG (12 µg/ml) and 125I-IgG tracer in a platelet aggregometer at 37°C for 12 min before heparin (0.5 U/ml) was added. The reaction was sampled 2 min before heparin (time-point A), either at the start (time- point B) or in the middle (time-point C) of aggregation, and when platelets were fully aggregated (time-point D). Samples were centrifuged through a 17% sucrose cushion to separate platelets from unbound IgG. Specific binding of Patient 1 ( ) and Patient 2 ( ) HIT IgG was measured using HIT 125I-IgG tracer while non-specific binding (open symbols) was determined with Normal 125I-IgG instead. The grey trace shows a typical aggregation profile that progresses from unaggregated to fully aggregated on an arbitrary scale. IgG bound is the mean ± SE. Specific binding of Patient 1 was derived from 7 experiments (4 sampled at time-points A, B & D and 3 sampled at time points A, C, & D) and non-specific binding from 3 experiments. Specific binding of Patient 2 was derived from 8 experiments (6 sampled at time-points A, B & D and 2 sampled at time points A, C, & D) and non-specific binding from 4 experiments.

157 Role of platelet activation The role of platelet activation was also investigated by recording the binding of trace amounts of HIT anti-PF4-heparin 125I-IgG to platelets in the absence of unlabelled HIT IgG. Platelets did not aggregate when mixed with heparin and the very low concentration of iodinated HIT IgG because there was insufficient HIT IgG to cause platelet activation (Figure 42). Aggregation only occurred when collagen was added to the platelets. The binding of the HIT 125I-IgG reflected the activation state of the platelets. That is, prior to aggregation there was negligible binding of 125I-IgG to the platelets but after collagen-induced aggregation the HIT 125I-IgG bound but Normal 125I-IgG did not (Figure 42). The results of this experiment, together with Figure 41 (p.157), indicate that platelet activation is essential for HIT IgG binding to platelets.

158 0.70 1.00

0.90 0.60 0.80

0.50 0.70

0.60 0.40 0.50 0.30 0.40 Heparin Collagen 0.20 0.30 Patient 2 IgG Bound (%) 0.20

Patient 1 and Normal IgG Bound (%) 0.10 0.10

0.00 0.00 -5 0 5 10 15 Time After Heparin (min)

Figure 42 - Binding of HIT 125I-IgG to platelets without unlabelled HIT IgG Platelet rich plasma (300x106 platelets/ml) was mixed with HIT 125I-IgG tracer (≈0.07 µg/ml) in a platelet aggregometer for 12 min before heparin (0.5 U/ml) was added. The lack of unlabelled IgG meant that the HIT IgG concentration was insufficient to cause aggregation. Therefore, aggregation was induced by addition of collagen (4 µg/ml) 12 min post heparin. The reaction was sampled 2 min before heparin, 10 min after heparin (when full aggregation would have occurred had sufficient unlabelled HIT IgG been included) and then when collagen-induced aggregation was complete. Samples were centrifuged through a 17% sucrose cushion to separate platelets from unbound IgG. Symbols represent the specific binding of 125I-IgG from Patient 1 ( ) and Patient 2 ( ) while non- specific binding ( ) was measured by the binding of Normal 125I-IgG. The grey trace shows a typical aggregation profile. IgG bound is the mean of two experiments with error bars showing the two data points from which this mean is derived.

Role of platelet FcγγγRII

The role of the FcγRII in the binding of HIT IgG to platelets was investigated by blocking the FcγRII with the monoclonal antibody IV.3. As expected, the presence of IV.3 prevented HIT IgG and heparin from inducing platelet aggregation and no binding of HIT 125I-IgG to these platelets was detected. However, after collagen was added to

159 activate the platelets, the HIT 125I-IgG did bind (Figure 43). Thus, blocking the FcγRII did not prevent binding of HIT IgG if the platelets could be activated by another pathway. In this experiment IgG binding to activated platelets in the presence of anti- FcγRII mAb (IV.3) must have occurred through the Fab region of HIT IgG, probably binding to PF4-heparin immobilised on the platelet surface. The same conclusion is reached from Figure 44 where HIT anti-PF4-heparin F(ab’)2 bound to platelets aggregated with collagen plus heparin.

160 0.18 0.60

0.16 0.50 0.14

0.12 0.40

0.10 0.30 0.08 (fg/platelet) 0.06 0.20 Heparin Collagen

0.04 0.10 Binding of Patient 2 IgG (fg/platelet) Binding of Patient 1 and Normal IgG 0.02

0.00 0.00 -5 0 5 10 15 Time After Heparin (min)

Figure 43 - Effect of blocking platelet Fc receptor on the binding of HIT IgG during platelet aggregation. Platelet rich plasma (300x106 platelets/ml) was mixed with the monoclonal antibody IV.3 (anti- FcγRII, 50 µg/ml), HIT IgG (12 µg/ml) and 125I-IgG tracer in a platelet aggregometer for 12 min before heparin (0.5 U/ml) was added. Aggregation was induced by addition of collagen (4 µg/ml) a further 12 min post heparin. The reaction was sampled 2 min before heparin, 10 min after heparin (when full aggregation would have occurred without IV.3) and then when collagen-induced aggregation was complete. Samples were centrifuged through a 17% sucrose cushion to separate platelets from unbound IgG. Specific binding of Patient 1 ( ) and Patient 2 ( ) was quantitated by the inclusion of the same HIT 125I-IgG tracer while non-specific binding ( ) was measured by the binding of Normal 125I-IgG. The grey trace shows a typical aggregation profile. IgG bound is the mean of two experiments with error bars showing the two data points from which this mean is derived.

161 0.25

0.2

0.15 Bound (fg/platelet) 2

0.1

F(ab) Heparin + Collagen

0.05

0 -5 0 5 Time after Heparin + Collagen (min)

Figure 44 - Binding of F(ab’)2 to platelets aggregated with collagen plus heparin 6 Platelet rich plasma (300x10 platelets/ml) was mixed with HIT or Normal F(ab’)2 (8 µg/ml) 125 and I-F(ab’)2 tracer in a platelet aggregometer for 12 min before heparin (0.5 U/ml) plus collagen (4 µg/ml) was added to induce aggregation. Platelets were sampled 2 min before heparin + collagen and 5 min after heparin + collagen by centrifugation through 17%

sucrose. Symbols indicate the specific binding of Patient 1 ( ) and Patient 2 ( ) F(ab’)2

and non-specific binding of Normal F(ab’)2 ( ). The grey trace shows a typical aggregation profile. Values are the mean of two experiments with error bars showing the two data points from which this mean is derived.

The action of IV.3 appears to be that it precludes HIT IgG from activating platelets. This may prevent the release of PF4 from platelet α-granules and minimise the expression of PF4-heparin on the platelet surface so there is little binding of HIT IgG. I am confident that the 50 µg/ml concentration of IV.3 was sufficient to block all FcgRII because 3 µg/ml IV.3 was sufficient to prevent a strong HIT plasma from aggregating platelets (Figure 45).

162 100% 0 µg/ml IV.3 80% 1µg/ml IV.3 60%

Heparin 40% 3µg/ml

Platelet Aggregation IV.3 20%

0% -5 0 5 10 15 20 25 30 35 40 Time After Heparin (min)

Figure 45 - Inhibition of HIT-induced platelet aggregation by IV.3 Platelet rich plasma (320 µl at 300x106 platelets/ml) was mixed with 20 µl of mAb IV.3 (resulting in final concentrations of 0, 1 or 3 µg/ml) and 150 µl of a HIT plasma that was known to produce a strong aggregation response. Heparin (0.5U/ml) was added and platelet aggregation recorded.

Figure 43 and Figure 44 demonstrate that HIT IgG can bind to platelets through the Fab region but they do not indicate what proportion of the binding is by Fab and what by Fc. In order to quantitate the contribution of Fc-FcγRII interaction to the binding of HIT IgG. I investigated binding to fully activated platelets that were pre-blocked either with, or without, IV.3. Platelets were activated with thrombin, in the presence or absence of IV.3. These activated platelets were incubated (± IV.3) with supernatant derived from platelets that were aggregated by HIT IgG and heparin. These supernatants provided a source of PF4, HIT IgG, 125I-IgG tracer and heparin in plasma at concentrations equivalent to my earlier experiments. Figure 46 shows that blocking FcγRII with IV.3 had little effect on the specific binding of HIT IgG to fully activated platelets.

163 1

0.9

0.8

0.7

0.6

0.5

0.4

0.3 IgG Bound (fg/platelet) 0.2

0.1

0 Patient 1 Patient 2 Patient 1 Patient 2 Specific Specific Non- Non- specific specific

Figure 46 - Effect of Fc receptor blockade on the binding of HIT IgG to thrombin activated platelets. Thrombin-activated washed platelets (24x106 platelets pretreated ± IV.3) were incubated ± IV.3 (50 µg/ml) for 15 min with 80 µl of plasma supernatant that was derived from platelet aggregation induced by heparin plus HIT IgG. These supernatants contained HIT IgG (12 µg/ml), 125I-IgG tracer, heparin (0.5 U/ml) and platelet products released during aggregation. IgG binding in the absence (open bars) and presence (dark bars) of IV.3 was determined after briefly rinsing the platelets with PBS. Specific binding of HIT IgG was quantitated by the inclusion of the same HIT 125I-IgG tracer while non-specific binding was measured by the binding of Normal 125I-IgG. Bars show the mean of two experiments with error bars showing the two data points from which this mean is derived.

Specificity of affinity purified HIT IgG To confirm that the HIT IgG was indeed recognising PF4, I blocked the binding of HIT IgG to activated platelets by preincubating the platelets with affinity purified sheep anti- PF4 IgG. The binding of HIT 125I-IgG was increasingly inhibited with higher concentrations of anti-PF4 (Figure 47). At 48 µg/ml anti-PF4 the binding of HIT IgG from Patients 1 and 2 was reduced to one quarter and one eighth, respectively, of the binding without anti-PF4. 164 0.6% 5%

0.5% 4%

0.4% 3%

0.3%

2% 0.2%

Binding of HIT IgG from Patient 1 1% 0.1% Binding of HIT IgG from Patient 2

0.0% 0% 0 1020304050 anti-PF4 IgG concentration (µg/ml)

Figure 47 - Inhibition of HIT IgG binding to platelets by anti-PF4. Platelets, which had been activated by collagen plus heparin, were resuspended in PBS containing 2% BSA, 6 mg/ml human IgG, 10% ETP and 50 µg/ml IV.3. 60x106 platelets were blocked for 30 min with various concentrations of affinity-purified sheep anti-PF4 IgG before HIT 125I-IgG was added for 10 min. All incubations were performed at 0°C to minimise further platelet activation. Free 125I-IgG was removed by centrifugation and two washes with PBS. The binding of HIT IgG from Patient 1 ( ) and Patient 2 ( ) is expressed as a percent of the total radioactivity added. The background binding of Normal 125I-IgG was only 0.05%.

Release of PF4 from platelets To show that PF4 is released during platelet activation, I quantitated the PF4 released from platelets during incubation of PRP with various agonists (Figure 48). The plasma from unstimulated platelets, anticoagulated with citrate, contained 0.19 µg/ml PF4. When platelets were aggregated by the addition of either HIT IgG plus heparin, collagen alone, or collagen plus heparin, the plasma contained about 5 µg/ml PF4. Treatment of platelets with heparin alone released a lesser amount of PF4 and the resulting plasma

165 contained 0.82 µg/ml PF4. The released PF4 may be absorbed onto the platelet surface, resulting in elevated surface expression of the protein.

7

6

5

4

3

2

Plasma PF4 Concentration (µg/ml) 1

0 Unactivated HIT IgG & Collagen & Collagen Heparin alone Heparin Heparin alone

Figure 48 - Release of PF4 following platelet activation. 650 µl of citrated platelet rich plasma (300x106 platelets/ml) was either not activated or activated with heparin and HIT IgG, collagen and heparin together, collagen alone, or heparin alone by stirring in an aggregometer. Activation was arrested by adding ETP and chilling on ice. Platelets were removed by centrifugation (for use in Figure 49) and the plasma PF4 concentration measured by competitive RIA. Bars show mean ± SE of three experiments.

Increased expression of PF4 on the platelet surface following activation The relative surface expression of PF4 on activated and non-activated platelets was measured by the binding of sheep anti-PF4 125I-IgG (Figure 49). Unstimulated platelets displayed little specific binding of anti-PF4 antibodies. Aggregation of platelets with either HIT IgG plus heparin, collagen alone, or collagen plus heparin resulted in a marked increase in PF4 expression. Platelets treated with heparin alone had only a modest increase in surface PF4 compared to unstimulated platelets.

166 0.9%

0.8%

0.7%

0.6%

0.5% I-IgG (% of total CPM) 125 0.4%

0.3%

0.2%

0.1% Binding of anti-PF4

0.0% Unactivated HIT IgG & Collagen & Collagen Heparin Heparin Heparin alone alone

Figure 49 - Expression of PF4 on the platelet surface following activation. Citrated PRP was either not activated or activated with heparin and HIT IgG, collagen and heparin together, collagen alone or with heparin alone. Activation was arrested by adding ETP and chilling on ice. Plasma was removed by centrifugation (for use in Figure 48). Relative surface expression of PF4 was determined by the binding of sheep anti-PF4 125I-IgG (dark bars). The open bars represent the non-specific binding of non-immune sheep 125I-IgG. This graph illustrates the percent of 125I-IgG bound to platelets as a proportion of the total radioactivity added and shows the mean of two experiments with error bars showing the two data points from which this mean is derived.

These data support the notion that activated platelets express PF4 that binds heparin and HIT IgG. The bound IgG could activate other platelets through FcγRII and initiate a chain reaction that leads to a rapid increase in platelet aggregation and HIT IgG binding.

Role of heparin Figure 50 demonstrates that heparin is required to facilitating the binding of HIT IgG to aggregated platelets. Platelets aggregated with collagen alone did not bind HIT IgG.

167 Binding only occurred when heparin was included with the collagen. This supports the concept that PF4 must form a complex with heparin before it is recognised by HIT IgG.

0.4 0.9

0.35 0.8

0.7 0.3

0.6 0.25 0.5 0.2 0.4

(fg/platelet) 0.15 Collagen 0.3 ± Heparin 0.1 0.2 Binding of Patient 2 IgG (fg/platelet) Binding of Patient 1 and Normal IgG 0.05 0.1

0 0 -5 0 5 10 Time After Collagen (min)

Figure 50 - Heparin dependence in the binding of HIT IgG to collagen aggregated platelets. Platelet rich plasma (300x106 platelets/ml) was mixed with HIT or Normal IgG (12 µg/ml) and 125I-IgG tracer in a platelet aggregometer for 12 min before collagen (4 µg/ml) ± heparin (0.5 U/ml) was added. Platelets were sampled 2 min before collagen, 4 min after collagen (when platelets were consistently fully aggregated) and 10 min after collagen by centrifugation through 17% sucrose. Solid symbols indicate the specific binding of Patient 1 ( ) and Patient 2 ( ) IgG and non-specific binding of Normal IgG ( ) in the presence of heparin. The corresponding open symbols represent binding without heparin. The grey trace shows a typical aggregation profile. Values are the mean of two experiments with error bars showing the two data points from which this mean is derived.

I note that in Figure 50 the amount of IgG bound to platelets was double that observed when FcγRII were blocked by IV.3 in Figure 43. This probably reflects differences in the degree of platelet activation between the two figures and not a component of Fc

168 binding. Specifically, in Figure 50 platelets were activated by both collagen and HIT IgG so probably expressed more PF4-heparin antigen than in Figure 43 where platelets were activated by collagen alone because activation by HIT IgG was prevented with IV.3. Support for this view comes from Figure 49 where platelets activated by HIT IgG plus heparin expressed more PF4 than those activated by collagen plus heparin. Furthermore, blocking FcγRII had little effect on binding of HIT IgG to fully activated platelets (Figure 46).

Discussion

HIT is associated with antibodies directed against complexes of PF4-heparin in 75- 100% of cases, 90,91,159,163. In this chapter I demonstrate that HIT IgG, affinity purified on heparin-PF4-agarose, will induce heparin-dependent platelet aggregation (Figure 41). This finding extends that of Greinacher et al. who showed that HIT antibodies, isolated by affinity for endothelial cells, could bind PF4-heparin in ELISA and activate platelets 43. My results are important because I isolated antibodies using purified PF4 immobilised on agarose. In contrast, endothelial cells are covered with many different antigenic determinants, including a number in common with platelets 167-173, and Greinacher et al. may have co-purified a separate platelet-activating factor along with an anti-PF4-heparin antibody. My data provide the strongest evidence that the anti-PF4- heparin antibody is the heparin-dependent platelet aggregating factor known to be present in the plasma of patients with HIT and implies that it may cause in vivo platelet activation and consequently thromboembolism.

To date, there has been no data describing the dynamic interaction of HIT IgG with platelets. I provide fundamental data on the binding of affinity-purified HIT anti-PF4- heparin IgG to platelets during platelet aggregation. I used concentrations of platelets 112, heparin 288-290,292 and specific anti-PF4-heparin IgG (see Chapter 3 Discussion, p.143) that are achievable in vivo. Importantly, no exogenous PF4 was added and PF4 released from the platelets was available to modulate further antibody binding to the platelets. My findings are particularly significant because the platelet aggregation was induced by the HIT IgG itself, rather than by thrombin. The use of affinity purified antibody provided greater sensitivity than previously available.

169 Figure 41 indicates that before the addition of heparin there is little or no binding of HIT IgG to platelets. I only detected HIT IgG bound to platelets once heparin was added to the platelets and aggregation begun. The HIT IgG binding then increased sharply until platelets were fully aggregated. There is clearly a close correlation between the degree of aggregation and the amount of HIT IgG bound. This confirms that platelet aggregation by HIT IgG is a dynamic process. Activation promotes antibody binding that, in turn, causes more platelet activation, ultimately resulting in maximum antibody binding and full platelet aggregation. I have also modified the binding experiment by adding heparin to a mixture of PRP and HIT 125I-IgG tracer (≈0.07 µg/ml), omitting the higher concentration of unlabelled HIT IgG. As expected, the HIT antibody concentration was too low to induce platelet aggregation. I did not detect any specific binding of the HIT 125I-IgG to the platelets (Figure 42). Only when the platelets were subsequently aggregated by collagen did I observe the HIT 125I-IgG bind. I can only speculate that the level of binding of HIT IgG to the platelets before aggregation was very low.

PF4 is strongly implicated in forming the platelet antigen that HIT IgG recognises because the IgG that I used was purified by affinity for PF4-heparin. Confirming this, I observed that binding of HIT IgG to activated platelets was inhibited by preincubating the platelets with a sheep anti-PF4 IgG (Figure 47). Furthermore, activation of platelets by HIT IgG plus heparin, or by collagen, triggered a substantial increase in the expression of PF4 on the platelet membrane (Figure 49). Importantly, incubating PRP with heparin alone was sufficient to modestly elevate the surface expression of PF4. Thus, on heparinised platelets there is likely to be a small amount of PF4-heparin to which undetectable amounts of HIT IgG bind.

Many reports demonstrate that HIT antibodies optimally bind to PF4-heparin in a narrow range of PF4:heparin ratios. In my hands, this ratio corresponds to at least 20µg PF4 per unit of heparin, regardless of whether the PF4-heparin is immobilised on ELISA wells or free in solution. While, earlier reports contend that HIT antibodies only bind to platelets in a narrow range of PF4:heparin concentration ratios 45,195,443. I now demonstrate that the ratio of PF4:heparin is perhaps not as important in a more physiological and sensitive system. I added 0.5 U/ml heparin to platelet rich plasma but the PF4 was derived solely from that already present in the plasma or the platelets. The 170 plasma PF4 concentration increased from ≈0.19 µg/ml in citrated platelet rich plasma to ≈5.6 µg/ml when platelets were fully aggregated by HIT IgG (Figure 48). Consequently, heparin was always in excess but the increase in PF4 concentration would have moved the PF4:heparin ratio towards the optimum. It is likely that this contributed to the increased binding of HIT IgG during platelet aggregation.

The 0.19 µg/ml PF4, that I detected in the citrated platelet rich plasma, is much higher than the 2-20 ng/ml that has been reported as normal blood concentrations (see PF4 in the Plasma, p.65). This is probably because blood drawn for PF4 determination should be collected with special anticoagulants and precautions to prevent any platelet activation. In contrast, preparation of platelet rich plasma can cause some PF4 release because citrate is not as effective at preventing platelet activation 319. For example, Engstad et al. report that citrated platelet rich plasma contained 0.44µg/ml PF4 after incubation at 37°C for 1 hr 444. The PF4 concentration that I observed after platelet activation by collagen is similar to the 4.6µg/ml reported by others 310.

Previously, I (Figure 21, p.118) and others 43,45,159 have observed that HIT IgG will bind weakly to PF4 alone immobilised onto microtitre wells in the absence of heparin. Therefore, I investigated the importance of heparin in binding of HIT IgG to platelet surface PF4. I observed that binding of anti-PF4-heparin IgG to collagen aggregated platelets only occurs in the presence of heparin (Figure 50). This is despite the fact that platelets aggregated by collagen alone express significant amounts of PF4 on their surface (Figure 49). I conclude that PF4 on the platelet surface may not be modified in the same way as PF4 adsorbed to microtitre wells. Instead heparin is required to facilitate HIT antibody binding, perhaps by inducing a conformational change within PF4.

Aster 42 indicates that understanding the orientation of IgG on the platelet surface is important because if the Fab region of IgG can bind platelets then anti-PF4-heparin antibodies of IgA or IgM classes may also bind. While these antibody classes can not activate the FcγRII, they may opsonise platelets for destruction in vivo. This may explain why HIT can occur in patients with only IgA or IgM anti-PF4-heparin antibodies 165, provided platelets in vivo have been partially activated by a mechanism unrelated to HIT. Some researchers favour a model where HIT IgG forms an immune complex with

171 PF4-heparin in solution and the IgG in the immune complex then binds to the platelet FcγRII 44,45,54. Others envisage that HIT IgG binds to PF4-heparin that is already on the platelet membrane and then activates platelets through the FcγRII on the same or adjacent platelet 99,134,148,159,195. Blocking the platelet Fc receptor with the monoclonal antibody IV.3 is known to inhibit HIT antibody induced platelet activation and aggregation 138,139,151 but this alone does not help distinguish between the two models. I set-out to resolve this controversy by measuring the binding of HIT IgG to platelets using my more sensitive and physiological technique.

Figure 43 indicates that, as expected, IV.3 prevented HIT IgG from aggregating platelets in the presence of heparin. These non-aggregated platelets did not bind HIT 125I-IgG. Platelet aggregation was then induced by the addition of collagen, which acts through a mechanism independent of FcγRII. Importantly, once aggregated, the platelets bound

HIT IgG despite the presence of IV.3. Similarly, F(ab’)2 fragments of the HIT IgG also bound to platelets that were aggregated by collagen and heparin (Figure 44). This implies that the binding of HIT IgG to platelets occurs when the F(ab’)2 portion of the IgG recognises PF4-heparin that is already on the platelet surface. Consequently, HIT IgG binding is dependent upon platelet activation, which is needed to increase PF4 expression on the platelet surface, and not on the availability of FcγRII. The effect of IV.3 is simply to prevent HIT IgG from activating platelets through the Fc receptor.

In order to quantitate the respective contributions of F(ab’)2 and Fc binding I investigated the binding of HIT IgG to fully activated platelets ± IV.3. The rationale for this was that fully activated platelets could not be further activated by HIT IgG, which would otherwise promote further antibody binding. Thus, the supernatant from platelets aggregated by HIT 125I-IgG plus heparin was incubated with thrombin activated platelets ± IV.3. If there was any substantial binding of PF4-heparin-IgG immune-complexes to platelet FcγRII then IV.3 would reduce the binding to the platelets. Instead I observed little difference in HIT IgG binding to platelets with or without IV.3, which supports my conclusion that it is largely the F(ab’)2 portion of HIT IgG that binds to activated platelets. Interestingly, it has been accepted for some time that the antibodies associated with quinine/quinidine induced thrombocytopenia bind to antigen on the platelet surface 392,393.

172 I provide definitive evidence that affinity-purified anti-PF4-heparin IgG from HIT patients can cause heparin-dependent platelet aggregation. For the first time, I also report the dynamic binding of HIT IgG to platelets while the IgG (plus heparin) induces platelet aggregation under conditions that are as close as possible to those found in vivo. IgG binding was dependent upon (1) the availability of PF4 on the platelet surface, which increases upon platelet activation and (2) upon the addition of heparin. I was not able to detect HIT IgG binding to non-aggregated platelets incubated with heparin but I demonstrated that such platelets do express low levels of PF4 that may bind tiny amounts of IgG. It may be that in vivo the weak platelet-activating effect of heparin (see Effect of heparin on platelets, p.63) results in sufficient PF4-heparin on the platelet surface to initiate binding of HIT IgG. Blocking the platelet FcγRII did not prevent HIT IgG from binding when the platelets were activated with collagen. This indicates that the Fab region of HIT IgG will bind to PF4-heparin on the platelet membrane in a plasma milieu. My data support a mechanism of platelet activation by HIT IgG that is summarised in Figure 51. Initially, upon the addition of heparin, tiny amounts of PF4- heparin complexes form on the platelet surface. The PF4 may be derived from that released from the endothelium by heparin or may come from platelets that have been weakly activated by a concomitant infection, vascular damage or other triggers of coagulation, which produces thrombin. The release of sufficient PF4 may in fact be a rare event and could explain why only a few of the patients with anti-PF4-heparin antibodies actually develop thrombocytopenia. I propose that once PF4-heparin has formed on the platelet membrane the Fab portion of HIT IgG binds to this antigen. The Fc region of the bound HIT IgG then cross-links FcγRII on adjacent platelets, which triggers platelet activation and degranulation. The PF4 that is released binds more heparin and forms more antigen on platelets. Thus positive feedback accelerates platelet activation until all the platelets are fully aggregated (Figure 51).

173 Platelet

1

3 4 2

PF4 HIT IgG Heparin FcγRII

Figure 51 - Dynamic model of platelet activation by HIT IgG (1) Complexes of PF4-heparin form on the platelet surface. (2) HIT IgG binds to the platelet bound PF4-heparin through the Fab region. (3) The Fc portion of the bound IgG cross-links FcγRII on adjacent platelets and produces platelet activation. (4) PF4, released from the activated platelets, forms more complexes with heparin on platelets and promotes antibody binding. In this model, blocking FcγRII with IV.3 would still permit the Fab part of IgG to bind platelets but IV.3 would inhibit platelet activation.

174 Chapter 5 - Overview

Immune heparin-induced thrombocytopenia (HIT) has received considerable interest over the years, not least because of its paradoxical association with thromboembolism. Warkentin 183 has described it as “the prototypic IgG-mediated platelet activation disorder”. The development of thrombocytopenia in a patient who is receiving heparin poses a number of dilemmas for the treating physician. In such a case the possibility of HIT must be considered but thrombocytopenia can be caused by many factors other than heparin so laboratory tests can be useful in establishing a diagnosis. Functional tests (eg. 14C-serotonin release assay and platelet aggregometry) measure the degree to which test plasma activates normal platelets in the presence of heparin. Immunoassays use ELISA to measure the binding of HIT antibody to complexes of PF4 and heparin and potentially other antigens. Immunoassays don’t require viable platelets, are generally quicker to perform and require less technical expertise than functional assays.

If a diagnosis of HIT is likely then heparin should be ceased. Failure to do so runs the risk of worsening thrombosis but this raises the issue of what to replace heparin with because, presumably, the patient still needs anti-thrombotic cover. Some potential anticoagulants are warfarin, ancrod, hirudin, LMWH, danaparoid. However, these are not without their disadvantages. Warfarin may, in the short term, promote thrombosis by inhibiting the (anticoagulant) protein C pathway. Ancrod is not a particularly effective anticoagulant and is not available for use in Australia. LMWH often cross-reacts with HIT plasma in functional assays and it is generally recommended that if there is cross- reaction then LMWH should not be used in HIT.

There is little data concerning the degree to which HIT antibodies cross-react with LMWH or danaparoid in a immunoassay. One of the difficulties in investigating such cross-reactivity is that ELISA wells coated with PF4 complexed to either heparin, LMWH or danaparoid will bind different amounts of PF4 (Figure 14, p.97). This makes it difficult to tell if altered antibody binding is caused by the anticoagulant altering antibody affinity or if it is just a result of decreased antigen availability. I overcame this problem by developing a fluid-phase enzyme-immunoassay in which the amount of PF4 available for antibody binding was constant regardless of the anticoagulant. This assay

175 was marginally more sensitive than a commercially available anti-PF4-heparin ELISA in detecting HIT antibodies. Using my fluid-phase assay I observed that about 88% of HIT plasma that bound PF4-heparin also bound PF4-LMWH (Figure 15, p.98). In contrast, the binding of the plasmas to PF4-danaparoid was considerably weaker and only half the plasmas were deemed to cross-react with danaparoid (Figure 15). However, this is considerably greater than the 0-20% cross-reactivity with danaparoid that is observed in functional assays. Some of the HIT patients had received danaparoid following heparin withdrawal, before this study was performed. By reviewing the clinical course of these patients it became apparent that the immunoassay was a poor predictor of clinical outcome because the thrombocytopenia resolved during the course of danaparoid in many of the patients who where positive for antibodies to PF4-danaparoid (Figure 17, p.101). This data should help avoid the unnecessary withholding of danaparoid from HIT patients who show cross-reactivity only in an immunoassay. Danaparoid cross- reactivity in a functional assay is a different matter. Two patients who were treated with danaparoid were positive for antibodies that cross-reacted with danaparoid in both a functional assay and the fluid-phase immunoassay. One of these recovered while receiving danaparoid but the other died. Obviously it is difficult to draw conclusions from just two patients but my data indicate that caution should be used if danaparoid is to be given to HIT patients who cross-react in a functional assay.

The fluid-phase assay also contributes to the understanding of the mechanism of HIT. It provides the first explicit demonstration that HIT IgG will bind to soluble complexes of PF4-heparin. In all other immunoassays the PF4-heparin has been immobilised on ELISA wells. This demonstrates the plausibility of the hypothesis that, in HIT, platelets are activated when soluble complexes of IgG-PF4-heparin bind to platelets via the FcγRII. This is a valuable contribution even though I have subsequently demonstrated that it is more likely that the IgG actually binds PF4-heparin on the platelet surface.

One advantage of the fluid-phase assay is that it has lower background compared to the ELISA or, more accurately, the fluid-phase assay shows less variation among the absorbances from the Normal plasmas. This means that one can be more confident that moderate absorbances from test samples should be reported as a positive result. The reason for this better performance stems from the design of the fluid-phase assay where HIT antibodies are reacted with biotin-PF4-heparin in solution then all IgG is 176 precipitated by the addition of protein G sepharose and the bound biotinylated PF4 is detected. In contrast, the ELISA depends upon a very sensitive secondary antibody to detect the small amount of HIT antibody that has bound to PF4-heparin on the microtitre well. It takes very few antibodies binding non-specifically to the ELISA wells to substantially increase the background.

HIT antibodies bind to PF4 alone in ELISA (Figure 21, p.118). This raises the issue of whether HIT antibodies recognise native PF4 or if adsorption of PF4 to ELISA wells promotes antibody binding. I used the fluid-phase assay to resolve this and demonstrated that HIT IgG does not bind soluble/native PF4 without heparin. If HIT antibodies are not directed against native PF4 then why do the antibodies bind to PF4-heparin? I demonstrated that agarose beads coated with PF4 alone, largely or completely, depleted HIT plasma of IgG that recognised PF4-heparin in a subsequent assay. This demonstrates that the covalent immobilisation of PF4 to the uncharged agarose was able to substitute for heparin, which implies that heparin does not form part of the epitope(s) that most of the HIT antibodies recognise. So, if the heparin molecule does not contribute to the HIT epitope then perhaps the antibodies that bind PF4-heparin are the same IgG molecules that bind PF4 alone adsorbed on ELISA wells. This appears to be the case because agarose beads coated with PF4-heparin were more effective than those coated with PF4 alone at depleting HIT plasma of antibody that recognised PF4 in a subsequent ELISA (Figure 28, p.127).

One way that heparin or attachment to either microtitre wells or agarose beads may modify PF4 to facilitate HIT antibody binding is to induce a conformational change in PF4 that exposes or creates a neoepitope. An alternative hypothesis, that I provide some evidence for, is that immobilisation of PF4 at sufficiently high density enables the bivalent binding of HIT IgG to adjoining PF4 tetramers. The injection of heparin could allow low affinity antibodies to PF4, that would normally cause no problem, to bind PF4-heparin complexes with relatively high avidity and eventually produce thrombocytopenia. I observed that HIT IgG and F(ab’)2 but not the monovalent Fab fragments of HIT IgG bound to PF4-heparin. I contend that the bivalent nature of IgG and the multivalent nature of PF4-heparin is required to increase the functional affinity of the antibody-antigen interaction but I have not determined if this is the main

177 mechanism in HIT or if a conformational change in PF4 is also required for antibody binding.

A key step in understanding the interaction between antibody and antigen is obtaining a measure of the binding avidity. I have been able to provide the first estimate of the binding avidity of HIT IgG. This first required a technique to affinity purify the anti- PF4-heparin IgG from HIT plasma. This was achieved using an affinity column consisting of heparin-PF4-agarose followed by further purification on protein G sepharose. The avidity of anti-PF4-heparin IgG from two HIT patients was measured using two different techniques so as to provide a more accurate estimate. I observed that these HIT IgG had dissociation constants of 7-30 nM for PF4-heparin and 20-70 nM for PF4 alone adsorbed to microtitre wells. These avidities are higher than had perhaps been assumed, given the relatively low avidity that antiphospholipid syndrome antibodies have for β2GPI.

Having investigated the interaction of HIT antibody with PF4-heparin at the molecular level, I set out to determine if, and how, the anti-PF4-heparin IgG bound to platelets and whether this antibody could in fact cause platelet aggregation and thus potentially be pathogenic. I demonstrated that 12 µg/ml (80 nM) affinity-purified HIT anti-PF4- heparin IgG was sufficient to cause complete platelet aggregation when 0.5 U/ml heparin was added. This unequivocally indicates for the first time that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to cause platelet aggregation. Other plasma constituents may be required but they are available from normal plasma. This data strongly implicates the anti-PF4-heparin antibodies in the pathology of HIT but it must be recognised that, to date, there is no direct evidence that the antibodies which cause platelet activation in vitro do so in vivo.

HIT IgG presumably binds to platelets in order to trigger platelet activation but there has been disagreement as to the orientation of the IgG on the platelet surface. Some researchers propose that the Fab portion of HIT IgG binds to PF4-heparin immobilised on the platelet surface, while others favour a model where the Fc portion of IgG-PF4- heparin immune complexes bind to the Fcγ receptors of the platelet. Previous papers have provided evidence to support each model but are somewhat unconvincing, containing minimal hard data or poor sensitivity. I investigated the binding of affinity-

178 purified HIT anti-PF4-heparin IgG to platelets, which provided far greater sensitivity than previously available. The binding was measured in a plasma milieu at 37 °C with platelets subject to shear forces, thus the conditions were also more physiological than previous reports.

I provide the first data that detail the binding of HIT IgG to platelets during the course of platelet aggregation induced by that IgG. The binding of anti-PF4-heparin IgG seemed to reflect the degree of platelet aggregation induced by the IgG. That is, prior to the addition of heparin there was no binding or aggregation, after heparin was added and aggregation just begun there was low but detectable binding, which increased as the aggregation progressed. It seems likely that neither IgG binding nor platelet aggregation precedes the other, in fact they probably promote each other. Platelet aggregation by HIT IgG or collagen also triggers a large increase in the release and surface expression of PF4 concurrent with the elevated binding of HIT IgG.

I addressed the issue of the orientation of IgG on the platelet surface by specifically blocking the platelet Fc receptor with the mAb IV.3. Blocking this receptor prevents HIT IgG from activating platelets but I observed that it did not prevent HIT IgG from binding to the platelets. Similarly, HIT F(ab’)2, which lacks Fc, also binds to activated platelets. This indicates that HIT IgG can bind to PF4-heparin that is already on the platelet surface and antibody binding is not dependent upon Fc/FcγRII interactions. The FcγRII does, however, play a critical role in the activation of platelets by HIT IgG that is bound to neighbouring cells. One implication of HIT IgG being able to recognise antigen on the platelet surface is that this provides a mechanism by which IgA and IgM HIT antibodies could bind and opsonise platelets.

The improved understanding of the interaction between HIT antibody and platelets permits the refinement of the models that are used to explain the activation of platelets in HIT. I contend that the binding of HIT IgG to PF4-heparin on the platelet and the subsequent activation of platelets via the FcγRII releases more PF4 which adheres to more platelets and promotes antibody binding and activation. Thus platelet activation by HIT IgG is a dynamic process with strong positive feedback.

179 Appendix 1 - Technical details of amplifier and ADC

The equipment used to record platelet aggregation is illustrated schematically in Figure 39 (p.150). The purpose and operation of the amplifier and ADC that were used to record platelet aggregation were also described earlier (Recording platelet aggregation, p.150). This appendix describes more technical aspects of the design of the amplifier

Linearity of amplifier The output of the platelet aggregometer was typically 0 V when platelets were unaggregated and 0.08 V when fully aggregated. In some instances, unaggregated platelets can appear to disaggregate further, resulting in an output as low as -0.01 V. Therefore, the amplifier was designed to convert voltages in the range of -0.01 V to +0.1 V into an output of 0-2 V. Figure 52 demonstrates the validity of the amplifier- ADC-computer components because it shows a direct linear relationship between the signal received by the amplifier and the value recorded on the computer.

180 2

1.8

1.6

1.4

1.2

y = 17.989x + 0.1953 1

0.8 Output of Amplifier (V) 0.6

0.4

0.2

0 -0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 Input to Amplifier (V)

Figure 52 Calibration curve for amplifier and analog to digital converter Voltages from -0.01 V to +0.1 V, measured using a digital voltmeter, were fed into the amplifier. The output of the amplifier was recorded on a computer via the analog to digital converter. Clearly, there is a direct linear relationship between the input to the amplifier (typically the signal from the aggregometer) and the value recorded by the computer. The slope of the line indicates that the gain of the amplifier is about 18 and the y-intercept reflects the 0.2 V offset.

Circuit diagram The amplifier was built around the LM 741 Op Amp and the circuit diagram is shown in Figure 53. This circuit is designed to take an input signal from the platelet aggregometer and amplify it about 18 times to result in a signal of up to 2V that can be read using an analog to digital converter.

181 The potential divider, R8 and R9, provide a +4.5V (+Vcc), 0 V (ground), and -4.5V (- Vcc) power supply from a single 9V battery 445. The LM 741 OpAmp is set up as a non- inverting amplifier. The gain is determined by the values of R1, R2 and V1 according to the formula:

()RV11++ R 2 Gain = ()RV11+

Thus V1 is used to fine-tune the gain to about 18. C1 filters out any high frequency noise. The risk of interference was further minimised by enclosing the whole circuit in an aluminium box that was connected to the 0 V / ground. This in turn was connected to the earthed chassis of the aggregometer via the outer sheath of the coaxial cable that carried the input signal. The two silicon diodes across the input of the LM 741 provide overload protection by limiting the input voltage to around 0.6 V 445. The potential divider of R6, R7 and V2 provide an adjustable offset voltage. This means that when the input signal is 0 V the actual input voltage to the LM 741 can be set to be slightly higher so the output of the amplifier is around 0.2 V. This method of determining the offset is based on a circuit recommended to give a stable offset to an inverting amplifier 445. In theory, it is not ideal to use with the non-inverting amplifier shown here. Specifically, it is unsuitable when the source of an input signal has a high output resistance because the amplifier circuit will influence the input signal. Fortunately, I have observed that the signal from the platelet aggregometer is not affected by the amplifier (data not shown) so this circuit is entirely suitable for this application.

182 +Vcc R7 51kΩ

V2 Ω 2k R5 11kΩ R6 51kΩ -Vcc

Input + R3 LM 741 Output 11kΩ - R2 + C1 180kΩ 1µF

R1 +Vcc 4.7kΩ

R8 + C2 V1 2.2kΩ 220µF 10kΩ

9V

R9 + C3 2.2kΩ 220µF

-Vcc

Figure 53 - Schematic Diagram of Amplifier Circuit

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