The control of multimer size

John Eshantha Pimanda

A thesis submitted in fulfillment of the requirements for the degree of

Doctor of Philosophy

Faculty of Medicine, University of New South Wales

December 2003 Acknowledgements

A courageous six year old girl with congenital TTP participated in our treatment trial; I thank her and her parents for their trust. Dr. Julian Paxton, Mr. Andrew Schultz, Ramsay Health and the staff at St. John of God pathology and the children's ward at Mildura Base Hospital made the study possible. Over twenty five patients with a past diagnosis of TTP participated in a study to identify mutations in the -1 ; I thank them and their physicians for their time and effort.

I thank Professor Philip Hogg for supervising my work. The power of his ideas and generosity of spirit have taught me much. I have enjoyed the three years spent in his laboratory. Professor Colin Chesterman has guided my development as a haematologist and a scientist. I have benefited from the clarity of his reasoning and value his mentorship.

Professor Beng Chong encouraged me to pursue a career in research at an early stage in my clinical training; Drs. Robert Lindeman and Michael Buckley gave of their time and expertise. Ms. Sue Evans, Ms. Janet Argyl, Mr. Robert Casten and Ms. Bernadette O'Reilly in the haematology department at the Prince of Wales Hospital were ever generous with their time and expertise. The staff of the bank accommodated my most unreasonable requests. Mr. Peter Taylor and the staff in the molecular genetics department provided valuable technical advice and practical help. Professor Michael Berndt, Monash University and Dr. Emanuel Favaloro, ICPMR Westmead were gracious with time, materials and ideas.

My colleagues at the Centre for Vascular Research were a joy to work with. Dr Xing-mai Jiang and Ms. Akiko Maekawa introduced me to the fundamentals of molecular biology and protein expression. Drs. Angelina Lay, Mark Raftery and Troels Wind furthered my understanding in protein chemistry. Ms. Lisa Sun and Ms. Lakmini Weerakoon helped with material preparation and Mr. Geoff Kershaw (at the Royal Prince Alfred Hospital) and Mr. Tim Ganderton with running VWF multimer gels. Dr. Susan Maastricht and Ms. Christine Sutter facilitated the animal work. The National Health and Medical Research Council of Australia provided financial support for which I am most grateful.

My parents in law, Rita and Hector Welgampola, with their presence have been a major asset to the completion of this thesis. My parents, Frank and Sriyani Pimanda continue to contribute selflessly to my happiness as they always have. I dedicate this thesis to my wife, Miriam

ii Publications arising from this thesis

Refereed journal articles published

Pimanda JE, Maekawa A, Wind T, Paxton J, Chesterman CN, Hogg PJ. Congenital

Thrombotic Thrombocytopenic Purpura in association with a mutation in the second

CUB domain of ADAMTS13. Blood 2003 (prepublished online September 25)

Pimanda JE, Annis DS, Raftery M, Mosher DF, Chesterman CN, Hogg PJ. The von

Willebrand factor-reducing activity of thrombospondin-1 is located in the calcium binding/C-terminal sequence and requires a free thiol at position 974. Blood. 2002 Oct

15; 100(8):2832-8.

Pimanda JE, Chesterman CN, Hogg PJ. A Perspective on the Measurement of

ADAMTS13 in Thrombotic Thrombocytopaenic Purpura. European Journal of

Haematology. 2003 Apr; 70 (4):257-62.

Pimanda J, Hogg P. Control ofvon Willebrand factor multimer size and implications for disease. Blood Rev. 2002 Sep; 16 (3):185-92

Pimanda JE, Lowe HC, Hogg PJ, Chesterman CN, Kachigian LM. Novel and emerging therapies in cardiology and haematology. Current Drug Targets­

Cardiovascular & Haematological Disorders, 2003; 3 (2):101-123

iii Refereed journal articles submitted

Pimanda JE, Ganderton T, Lawler J, Kershaw G, Maekawa A, Chesterman CN, Hogg

PJ. Role ofthrombospondin 1 in control ofvon Willebrand Factor multimer size in mice.

Referenced abstracts

Pimanda JE, Xie LJ, Chesterman CN, Hogg PJ. Control ofvon Willebrand Factor

Multimer Size. Blood 2001 Nov 16, 98(11): 160a.

Pimanda JE, Annis DS, Raftery M, Mosher DF, Chesterman CN and Hogg PJ. The von Willebrand Factor Reducing Activity ofThrombospondin-1 Centres around a Free

Thiol at Position 974. Blood2002 Nov 16, 100(11): 980a.

Pimanda JE, Kershaw G, Lawler J, Chesterman CN, Hogg PJ. The control of von

Willebrand Factor multimer size by Thrombospondin-1. Journal of Thrombosis and

Haemostasis 2003 (in press).

Presented abstracts

Xie LJ, Pimanda JE, Chesterman CN, Hogg PJ. The control ofvon Willebrand factor multimer size by thrombospondin-1. Haematology Society of Australia and New

Zealand/ Australasian Society of Blood Transfusion/ Australasian Society of

Thrombosis and Haemostasis Joint Annual meeting.(HSANZ/ ASBT / ASTH) Brisbane.

September 2001.

iv Pimanda JE, Annis DS, Raftery M, Mosher DF, Chesterman CN and Hogg PJ.

Localization of the von Willebrand factor reducing activity of thrombospondin-1. The

Australian Society of Medical Research Scientific Meeting. Sydney. June 2002.

Pimanda JE, Annis DS, Raftery M, Mosher DF, Chesterman CN and Hogg PJ. The von Willebrand Factor Reducing Activity ofThrombospondin-1 is located in the

Calcium-binding/C-globular Domain and Requires a Free Thiol at Position 974.

HSANZ/ASBT/ASTH Joint Annual Meeting. Adelaide. September 2002.

Pimanda JE, Kershaw G, Lawler J, Chesterman CN, Hogg PJ. The control of vWF multimer size by thrombospondin-1. A study of Plasma and VWF multimer size in thrombospondin-1 null mice. The Royal College of Pathologists of Australasia­

Pathology Update 2003. Sydney. March 2003.

Pimanda JE, Ganderton T, Kershaw G, Lawler J, Chesterman CN, Hogg PJ. Role of thrombospondin-1 in the control of VWF multimer size in mice. The Australian

Vascular Biology Society and Australian Atherosclerosis Society-annual scientific meeting. Ballarat, Victoria, September 2003.

Pimanda JE, Ganderton,T, Kershaw G, Lawler J, Chesterman CN, Hogg PJ. Role of thrombospondin-1 in the control ofVWF multimer size in mice. HSANZ/ASBT/ASTH­ joint annual scientific meeting, Christchurch, New Zealand, October 2003

V Table of contents page

List of abbreviations 1 Abstract 4 Preface 6

Chapter 1: Literature review 8 Introduction 9

1.1 von Willebrand factor 10 1.1.1 Synthesis 10 1.1.2 Storage and secretion 12

1.2 Control of plasma VWF multimer size-by proteolytic cleavage 13 1.2.1 ADAMTS13-a novel VWF cleaving 15 1.2.1.1 Tracing the ADAMTS13 gene 15 1.2.1.2 Phylogeny of ADAMTS13 17 1.2.1.3 The ADAMTS13 proenzyme 17 1.2.1.4 The domain structure of ADAMTS13 and relevance to function 19 1.2.1.5 The Tyr1605 and Met1606 cleavage site resides within the A2 domain of VWF 21 1.2.1.6 Glycosylation of ADAMTS13 23 1.2.1.7 Measurement of ADAMTS13 activity in plasma 24

1.3 Control of plasma VWF multimer size -by a VWF reductase 29 1.3.1 The thrombospondin gene family 35 1.3.1.1 Evolution of the thrombospondin gene family 37 1.3.1.2 The structure ofTSP-1 and relevance to function 38 1.3.1.3 The structure and function relationship between TSP-1 and VWF 44

vi 1.3.1.4 TSP-1 as an inhibitor 45 1. 3 .1. 5 The phenotype of the TSP-1 null mouse 46

1.4 Control of platelet VWF multimer size 47

1.5 VWF multimer size and disease 50 1.5.1 Deficiency of high molecular weight VWF multimers and 50 1.5.2 Persistence of high molecular weight VWF multimers and thrombosis 52 1.5.2.1 The thrombotic microangiopathies 52 1.5.2.2 VWF: atherosclerosis and arterial thrombosis 56 1.5.2.3 Pre-eclampsia, eclampsia and the HELLP syndrome 58

Chapter 2: The VWF reducing activity ofthrombospondin-1 60 2.1 Summary 61 2.2 Introduction 62 2.3 Materials and methods 64 2.4 Results 69 2.5 Discussion 81

Chapter 3: Role ofthrombospondin-1 in the control of VWF multimer size in mice 85 3.1 Summary 86 3.2 Introduction 87 3.3 Materials and methods 90 3.4 Results 97 3.5 Discussion 112

vii Chapter 4: Role ofthrombospondin-1 in TTP 117 5.1 Summary 118 5.2 Introduction 119 5.3 Patients and methods 120 5.4 Results and discussion 125

Chapter 5: TTP in association with a mutation in the second CUB domain of ADAMTS13 135 4.1 Summary 136 4.2 Introduction 136 4.3 Materials and methods 138 4.4 Results 141 4.5 Discussion 146

Chapter 6: Summary and future directions 150

References 157

viii List of abbreviations and acronyms used

AEBSF 4-(2-aminoethyl) benzensulfonyl fluoride

ADAM ~ gisintegrin-like ~d metalloprotease

ADAMTS ~ .-like ~nd metalloprotease with thrombo.s_pondin type 1

motif

BSA bovine serum albumin

CBA collagen binding activity

CK cysteine knot

COMP cartilage oligomeric matrix protein

COS-7 cell line (monkey, African green, Kidney) fibroblast like

CP123 procollagen and properdin (type 1) repeats 1-3

CPA cone and plate (let) analyzer

CUB £Omplement components Cl r/Cls, sea yrchin epidermal growth

factor and .b_one morphogenetic protein

DDAVP 1-desamino-8-D-arginine vasopressin

DTNB 5, 5 '-dithiobis (2-nitrobenzoic acid)

E3CaG third type 2, type 3 and C-terminal globular domain

EDTA ethylenediaminetetraacetic acid

EGF epidermal growth factor

ELISA enzyme linked immunosorbant assay

ER endoplasmic reticulum

FFP fresh frozen plasma

1 GSH reduced glutathione

HBS HEPES buffered saline

HELLP haemolysis, elevated liver function and low proteins

HEPES (N-[2-Hydroxyethyl] piperazine-N' -[2-ethanesulfonic acid])

HIV human immunodeficiency virus

HMWM high molecular weight multimers

HUS haemolytic uremic syndrome

HUVEC human umbilical vein endothelial cell

IAP integrin associated protein

IRMA immuno radiometric assay

LDLR low density lipoprotein receptor

MALDI-rTOFMS matrix-assisted laser desorption/ionization reflectron time of flight

mass spectrometry

MMP matrix

MPB 3-(N-maleimidylpropionyl) biocytin

NEM N-ethylmaleimide

NOCl N-terminal globular domain and procollagen domain ofTSP-1

NTCB 2-nitro-5-thiocyanobenzoic acid

P3E123 third properdin (type 1), type 2 repeats 1-3

PDI protein disulfide

PFA-100 platelet function analyzer -100

PGK phosphoglycerate kinase

2 PIH pregnancy induced hypertension

PMSF phenylmethanesulphonyl fluoride

PPACK D-Phe-Pro-Arg-chloromethyl ketone

PVDF polyvinylidene difluoride

RGD Argenine-Glutamic acid-Aspartic acid

RPMI Roswell Park Memorial Institute

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TGA therapeutic goods administration

TGFp tumor growth factor-P

TMA thrombotic microangiopathy

TRAP thrombospondin related protein

TSP thrombospondin

TSR thrombospondin type 1 repeat

TTP thrombotic thrombocytopenic purpura

ULVWF ultra large von Willebrand factor

UNC-SC UNC-SC protein from Caenorhabditis elegans uPA urokinase-like plasminogen activator

VWF von Willebrand factor

VWFCP von Willebrand factor cleaving protease

WPB Weibel-Palade body

3 Abstract

Plasma von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of to sites of vascular injury, however only the very large VWF multimers are effective in promoting platelet adhesion at arterial shear. VWF also plays an important role in platelet-platelet interaction and promotes growth.

The multimeric size of VWF can be controlled by proteolysis at the Tyr1605-Met1606 peptide bond by ADAMTS13 or cleavage of the disulfide bonds that hold VWF multimers together by thrombospondin-1 (TSP-1). We have demonstrated that the reductase activity of TSP-1 centers on a free thiol at Cys 974 in the calcium binding/ C­ terminal globular domain.

To clarify the role of TSP-1 in the control of VWF multimer size in-vivo, we studied the pattern of VWF multimers in TSP-1 null mice. The average multimer size of plasma

VWF in TSP-1 null mice was significantly smaller than in wild type mice and suggests that TSP-1 regulates the activity of ADAMTSl 3 in-vivo. Platelet VWF multimer size was reduced upon lysis or activation of human and wild type murine platelets but not

TSP-1 null murine platelets. This difference was reflected in a significantly faster rate of shear-induced aggregation of the TSP-1 null platelets.

Patients with TTP, a disorder characterized by the persistence ofULVWF multimers in plasma and the thrombotic occlusion of arterioles were screened for mutations in the

TSP-1 gene and a deficiency in plasma TSP-1 concentration. No significant mutations

4 or plasma deficiency ofTSP-1 were identified. Furthermore the infusion of a TSP-1 rich preparation of plasma, prepared from platelet concentrates, failed to reduce the plasma

VWF multimer size in a patient with congenital TTP.

A severe deficiency of ADAMTS 13 is associated with TTP but its structure-function relationship to VWF is incompletely understood and the functional role of the

ADAMTS13 CUB domains is debated. We identified and studied a novel mutation in the second CUB domain in a child with congenital TTP. Secretion of the mutant protein was severely impaired but retained VWF cleaving protease activity comparable to that of the wild-type protein.

5 Preface

Plasma von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury, however only the very large VWF multimers are effective in promoting platelet adhesion at arterial shear. VWF also plays an important role in platelet-platelet interaction and promotes thrombus growth.

A deficiency of the largest forms ofVWF is associated with type 2A which manifests as a bleeding diathesis. To the contrary, persistence of the ultra large forms ofVWF in plasma is associated with a thrombotic disorder, thrombotic thrombocytopenic purpura (TTP). Therefore maintaining VWF multimers within an optimal size range is of fundamental physiological importance. Two control mechanisms appear to operate in regulating VWF multimer size in physiology. The proteolytic cleavage ofVWF by a VWF cleaving protease and the dismantling ofVWF multimers by inter chain disulfide bond reduction mediated by a VWF reductase.

The VWF cleaving protease has now been identified as AD AMTS 13 and a severe deficiency of this enzyme is associated with both congenital and acquired forms ofTTP.

The structure and function relationship between ADAMTS 13 and substrate VWF is under investigation in a number of laboratories. The development of a simple assay for measuring enzyme activity in plasma and the production of a recombinant enzyme or enzyme fragment to treat TTP are of potential clinical utility. The physiological variation of plasma ADAMTS 13 concentration and the variable reduction of enzyme

6 activity reported in some case series of TTP are suggestive of other factors that modulate the enzyme or trigger the syndrome.

Thrombospondin-1 (TSP-1) has been identified as a VWF reductase. The structure and function relationship between TSP-1 and VWF is not known. The biological significance ofTSP-1 deficiency in relation to the control ofVWF multimer size is also not known. The primary objectives of this thesis were to identify regions in TSP-1 that are critical for its reductase activity and to explore the consequences of its deficiency in

TSP-1 null mice. It is recognized that VWF is stored in two compartments in the body; endothelial cells and platelets. As both compartments contribute to normal haemostasis but ADAMTS13 is absent from platelets, we were particularly interested in the control of platelet VWF by platelet TSP-1.

The role ofTSP-1 in modulating the phenotype ofTTP is also not known. We studied a population of TTP patients for mutations in the TSP-1 gene and aberrations in the concentration of plasma TSP-1. We focused on a patient with congenital relapsing TTP to first, identify a disease causing mutation in the ADAMTS13 gene and study the functional properties of the mutant protein and second, to investigate TSP-1 as an alternate therapy.

The individual studies that address these questions are presented as they were submitted for publication. A degree of repetition is therefore unavoidable and the methods relating to each study are detailed in the relevant chapters.

7 Chapter 1

Literature review

8 INTRODUCTION

Plasma von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury, however only the very large VWF multimers are effective in promoting platelet adhesion at arterial shear. VWF, either as an intrinsic component of the subendothelial matrix (Stel et al., 1985; Wagner, 1990) or deposited from plasma onto subendothelial collagen (Savage et al., 1998) or other substrates (de

Groot et al., 1988) binds to the platelet membrane glycoprotein Iba. receptor and tethers platelets to the vessel wall. Binding of platelet and or plasma VWF and fibrinogen to the platelet integrin a.rlbP3, recruits additional platelets and enables progression of the platelet thrombus. GP Iba. bound VWF appears to act in synergy with a.rlbP3 bound fibrinogen in sustaining platelet accrual (Ruggeri et al., 1999). Homotypic self­ association of VWF immobilized to the subendothelial matrix with soluble and platelet bound VWF may also play a role in arresting platelets in flowing blood (Savage et al.,

2002). VWF is not required to initiate platelet adhesion to collagen fibrils at wall shear rates that are typically generated in the venous circulation (Houdijk et al., 1985; Savage et al., 1998) but is essential at flow rates that are typically generated in the arterioles of the normal circulation (Tangelder et al., 1988) or in diseased larger arteries with narrowed lumina (Back et al., 1977). When VWF binds to collagen fibrils at high shear the multimers unfurl exposing binding sites within the individual VWF subunits for their respective ligands in the subendothelial matrix and platelet surface (Siedlecki et al.,

1996). The larger the VWF multimer, the higher the number of exposed binding sites

9 and greater the potential for VWF-ligand interaction and formation of a competent platelet thrombus (Doucet-de Bruine et al., 1978; Meyer et al., 1980; Ruggeri and

Zimmerman, 1980). Large VWF multimers bind to activated platelets and collagen with up to -100-fold higher affinity than monomeric fragments (Furlan, 1996). The unusually large VWF multimers secreted by endothelial cells have been shown to be more effective than the largest plasma forms in inducing platelet aggregation under high fluid shear (Moake et al., 1986).

VWF also functions as a carrier for blood clotting factor VIII and not only prolongs its survival in the circulation but also concentrates this factor at sites of vascular injury.

Both small and large multimers may be equally competent at carrying factor VIII in plasma (Hoyer, 1981 ).

1.1 VON WILLEBRAND FACTOR

1.1.1 Synthesis

VWF production is restricted to endothelial cells and megakaryocytes. The VWF gene is located on the short arm of 12 and the primary translation product includes a 22 amino acid signal peptide and a large 741 amino acid propeptide sequence.

Within the endoplasmic reticulum (E.R. ), individual pro-VWF molecules are linked in a tail-to-tail configuration by disulfide bonding at the C-terminal ends to form pro-VWF dimers (Wagner et al., 1987) (figure 1). The tail-to-tail linked pro-VWF dimers are then

10 transported to the Golgi apparatus where they are modified by glycosylation and

sulfation and are multimerized in a head-to-head configuration by the formation of

additional disulfide-bonds at the N-tenninal ends. The propeptides may function as a

disulfide isomerase to promote the mu.ltimerization of the pro-VWF dimers in the acidic

environment of the trans Golgi network (Mayadas and Wagner, 1989). After

multirnerization the propeptides are removed by proteolysis and are released along with

mature VWF from the endothelial cells. Recent observations from platelet adhesion on

stimulated endothelial cells suggest that UL VWF strings could be several millimeters

long. The length of these UL VWF multimers is much greater than previous estimates by rotary shadowing electron microscopy for plasma derived UL VWF multimers which

were considered large at ~2µM (Moake, 2002a).

Heparin N ~ - t Ls s s:.___ - - ls-s . ~l~:s:s~s-s ~ l N \/ C l l NFactor VIII Collagen GPllb/llla GPlbN/IX Sulfatides

Figure I. Schematic diagram of the domain structure of VWF and its ligands

The individual 250 kDa monomers are linked in a tail-tail orientation by C-terminal disulfide linkage to form VWF dimers, which are in tum linked in a head-head orientation by N-terminal linkage to form VWF multimers.

11 1.1.2 Storage and secretion

VWF produced by endothelial cells is either secreted constitutively or stored in Weibel­

Palade bodies and released in response to secretagogues such as thrombin, fibrin, histamine and complement factors 5b-9 (figure 2). The constitutively secreted VWF is composed of dimers and small multimers in contrast to that released from the storage compartments, which is only of high molecular weight (Sporn et al., 1986; Sporn et al.,

1987). Weibel Palade bodies release their VWF in a basolateral direction, towards the extra-cellular matrix, consistent with the finding that the VWF multimer size in the subendothelium is higher than in plasma (Sporn et al., 1987). Biomechanical disruption of the endothelial monolayer also disrupts the polarity ofWeibel-Palade body release with the high multimer VWF appearing both at the luminal side, where it promotes haemostasis, as well as the basolateral side where it helps attach the injured endothelium to the vessel wall (Wagner, 1990).

12 A B

C

Figure 2. Weibel Palade bodies in an endothelial cell

A . Immunofluorescence micrograph ofa cultured endothelial cell stained for VWF. Electron micrograph ofa WPB in (B) longitudinal section and (C) cross section

1.2 CONTROL OF PLASMA VWF MUL TIMER SIZE - BY PROTEOLYTIC

CLEAVAGE

Unless physicaUy disrupted or stimulated by secretagogues such as thrombin and fibrin, the largest VWF multimers synthesized by the endothelial cells are directed towards the basement membrane and away from blood (Wagner, 1990). The infusion of 1-deamino-

8-O-arginine vasopressin (ODA VP) is followed not only by an increase in the overall concentration of VWF in plasma but also by an increase in the proportion of large and

13 ultra large multimers that are rapidly cleared because of proteolytic degradation (Batlle et al., 1987). A small but consistent proportion of VWF in normal plasma is composed of 189-, 176-, 140-kD proteolytic fragments of the 250kD mature VWF subunit

(Zimmerman et al., 1986). Following the infusion ofDDAVP and transient increase in

VWF, there is a gradual, time dependent decrease in the concentration of the intact VWF subunits with a corresponding increase in the concentration of the proteolytic fragments

(Batlle et al., 1987). Type 2A von Willebrand disease is characterized by a selective loss of the ultra large VWF multimers. Some patients within this category have increased plasma concentrations of the 176- and 140-kD proteolytic fragments suggestive of increased susceptibility to enzymatic cleavage (Zimmerman et al., 1986).

Analysis of the 176- and 140-kD fragments by epitope mapping using generated against synthetic peptides and by amino acid sequencing of the 176-kD fragment indicated that there was cleavage of the VWF subunit at Tyr1605 and Met1606

(Dent et al., 1990). Despite the ability of a number of leukocyte- and platelet- derived to cleave VWF in-vitro, it was not until the independent observations by

Furlan and Tsai in 1996, that a protease was described that was able to cleave VWF at this specific site and generate these proteolytic fragments (Furlan, 1996; Tsai, 1996).

The proteolytic cleavage patterns for VWF of a number of proteases have been described. These include plasmin (Hamilton et al., 1985), trypsin (Houdijk et al., 1986), a.-chymotrypsin (Chopek et al., 1986), (Chopek et al., 1986), porcine pancreatic elastase (Chopek et al., 1986), human leukocyte elastase (Berkowitz et al.,

1988), calpains from porcine erythrocytes and kidney (Berkowitz et al., 1988) and

14 Staphylococcus aureus V8 protease (Girma et al., 1986). Although these can degrade VWF in-vitro with a resultant loss oflarge multimers they fail to generate the fragments produced in vivo and are of doubtful physiological significance.

1.2.1 ADAMTS13- a novel VWF cleaving protease

1.2.1.1 Tracing the ADAMTS13 gene

The specific VWF cleaving protease (VWFCP) has now been purified and identified as a new member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motif) family (Fujikawa et al., 2001). ADAMTS13 cleaves

VWF at Tyr1605 -Met1606 to generate the 176 KDa and 140 KDa fragments (figures 3 and

5). VWF is resistant to cleavage by this enzyme unless first denatured by urea or guanidine in-vitro or by arterial shear in-vivo. The purification of the protein and the partial identification of the amino acid sequence ofVWFCP, helped trace the VWFCP gene to . The domain structure ofVWFCP and its listing as ADAMTS13 was based on the composition of the transcriptional unit on chromosome 9 (Zheng et al.,

2001 ). An independent approach, using polymorphic microsatellite markers in families with VWFCP deficiency, was used to trace the gene to chromosome 9q34 (Levy et al.,

2001 ). ADAMTS 13 is produced primarily in the liver reportedly by hepatic stellate cells (Lee et al., 2002). The 4.7-kb mRNA transcript of this protein has been found consistently in the liver and is absent from the brain, kidney, spleen, testes, ovary and heart (Levy et al., 2001; Zheng et al., 2001). A 2.4-kb truncated form of the VWFCP mRNA, possibly a result of alternative splicing of the initial RNA transcript, is found in

15 the placenta and skeletal muscle (Levy et al. , 2001 ; Zheng et al. , 2001) and is of unknown functional significance.

A B ~: :m 140

Figure 3. ADAMTS13 mediated proteolysis ofVWF

A. I% agarose gel of non-reduced VWF incubated with various dilutions of plasma under conditions that promote ADAMTS 13 activity. There is a concentration dependant breakup of the high multimer forms. B. 8% SDS-PAGE under reducing conditions. With increasing concentrations of ADAMTS 13 activity the parent 250kDa monomer is cleaved to yield the 176 and 140 kDa fragments.

16 1.2.1.2 Phylogeny of ADAMTS13

ADAMTS13 is the most divergent member of the 19 proteins that make up the

ADAMTS family which belongs to the metzincin superfamily of metalloproteases

(Stocker et al., 1995). These proteins share common structural domains including, a hydrophobic signal sequence, a propeptide, a metalloprotease domain, a disintegrin-like region, a thrombospondin-1 repeat, a cysteine-rich domain and a spacer domain. In common with other members of the family, ADAMTS13 has a number of additional thrombospondin-1 repeats after the spacer domain but its two CUB domains at the carboxy-terminus are unique (Zheng et al., 2001) (figure 4). Candidate ADAMTS13 cDNA sequences have been identified in birds, fish (Fugu rubripes) and mammals

(Homo sapiens, Mus musculus, Rattus norvegicus) underscoring its biological significance (Majerus et al., 2003). Members of the related ADAM family of membrane associated proteases share similar propeptide, metalloprotease and disintegrin domains but lack thrombospondin-1 repeats and have different C-terminus domains (Stone et al.,

1999). The matrix metalloproteases (MMPs) share the propeptide and metalloprotease domains but differ in other respects (Visse and Nagase, 2003 ).

1.2.1.3 The ADAMTS13 pro-enzyme

The ADAMTS13 propeptide is uncommonly short consisting of 41 amino acid residues in contrast to the -200 residues in other members of the ADAMTS family and is cleaved at a conserved furin site prior to release (Majerus et al., 2003). The proteolytic activity of metalloproteases can be controlled by the propeptide by a "cysteine-switch"

17 mechanism whereby a conserved Cys residue coordinates the Zn2+ ion

(Loechel et al. , 1999). In addition to conferring latency to the pro-enzyme, propeptides also facilitate protein folding and secretion (Cao et al. , 2000; Loechel et al. , 1999). The

ADAMTS 13 propeptide appears dispensable in relation to both the above. The single

Cys residue in the human ADAMTS 13 propeptide is not conserved in mice and does not confer latency on proADAMTS13 and deletion of the propeptide does not impair protein secretion (Majerus et al., 2003).

SP~ MP

Figure 4. Domain structure of the ADAMTS13 protein. The 1427-aa precursor protein contains a signal peptide (SP), a propeptide (Pro), a metalloprotease domain (MP), a disintegrin-like domain (Dis), a type 1 TSP motif(T), a cysteine-rich (Cys) and spacer domain, seven additional type I TSP motifs and two CUB (1

ADAMTS13 I

Figure 5. ADAMTSl3 cleaves VWF at a specific site in the A2 domain.

Tyr842-Met843 of the mature protein corresponds to residues Tyr1605-Met1 606 when the amino acid numbering is from the initiating methionine residue.

18 1.2.1.4 The domain structure of ADAMTS13 and relevance to function

ADAMTS13 activity can be inhibited by chelation of either Zn2+ or Ca2+. The metalloprotease domain includes the characteristic HE**H**G**HD sequence which includes the three His residues that coordinate the Zn2+ ion, the conserved Met249 in a proposed Met turn, and residues Glu83, Asp173 , Cys281 and Asp284 which are predicted to coordinate a Ca2+ ion (Zheng et al., 2001). A number of ADAMTS13 mutations lie within the metalloprotease domain and would be predicted to be functionally inactive.

The mutants within this domain that have been expressed (TI 961 and R268P) have impaired secretion (Kokame et al., 2002; Motto et al., 2002) and the VWFCP activity of the fraction that is secreted has not been reported.

The spacer domain after the Cysteine-rich domain is functionally essential (Soejima et al., 2003; Zheng et al., 2003) as recombinant ADAMTS13 truncated N-terminal to this domain loses VWFCP activity. VWF is resistant to ADAMTS13 mediated hydrolysis unless exposed to fluid shear stress (Tsai et al., 1994). Most functional assays of

ADAMTSl 3 activity use urea or guanidine denaturation ofVWF to facilitate proteolysis

(Furlan et al., 1996; Tsai, 1996). Mutants, truncated C-terminal to the spacer domain retain full enzyme activity when tested using assays that chemically denature VWF

(Soejima et al., 2003; Zheng et al., 2003). A polymorphism in the Cys rich domain

(P475S) is efficiently secreted and retains low but significant VWFCP activity (Kokame et al., 2002). Unless this polymorphism changes protein stability it is likely that subtle changes in structure within the Cys rich domain also affect the interaction between

19 ADAMTSI 3 and VWF. This polymorphism has an allelic frequency of 5.1 % in a

Japanese population sourced from the division of Hypertension and Nephrology at the

National Cardiovascular Center in Suita, Japan (Kokame et al., 2002). Although the association between impaired processing ofULVWF and TTP has been established its association with other vascular disorders is still obscure and is discussed later in this chapter. This population with low ADAMTS13 activity and impaired VWF processing would be of particular interest with regard to further investigation of this putative association.

The implication that thrombospondin-1 repeats 2-8 and the CUB domains are not required for VWFCP activity in-vivo, is a subject for debate. The TSP-1 repeats (TSR) in platelet purified TSP-1, engage a number of cellular (CD36, glycosoaminoglycans) and extra cellular binding molecules (collagen, fibronectin, fibrinogen, TGF-13) (Adams and Tucker, 2000). The C-terminal TSRs of ADAMTSl and 12 are also reported to bind the extracellular matrix. Targeting ADAMTS13 to sites of vascular injury would facilitate the regulation of VWF multimer size and the subsequent growth of a platelet thrombus. Although the binding of ADAMTS13 to the extracellular matrix would seem intuitive and consistent with other described ADAMTS proteases, recombinant

AD AMTS 13 has been shown not to bind the surface of COS-7 cells or the extracellular matrix of COS-I or RFL-6 rat lung fibroblast cells (Zheng et al., 2003). Nevertheless

ADAMTS13 binding or lack thereof to vascular endothelial cells or a subendothelial matrix bed is yet to be clarified. It has also been suggested that ADAMTS13 has a yet

20 uncharacterized protein chaperone that helps target the enzyme to the vascular bed

(Zheng et al., 2003).

The role of the C-terminal CUB domains in light of the normal in-vitro VWFCP activity of the truncated mutants, is uncertain. The analogy that the CUB domains of procollagen C-proteinase bind to procollagen was used to predict that the CUB domains of ADAMTS13 conferred substrate specificity (Zheng et al., 2001). This is pertinent in view of many splice variants of ADAMTS13 that have been cloned that encode for a truncated protein after the metalloprotease domain, the spacer domain , the second TSR, or the first CUB domain (Levy et al., 2001; Soejima et al., 2001; Zheng et al., 2001) and are of uncertain physiological relevance. A description that certain mouse strains encode AD AMTS 13 truncated after the seventh TSR and therefore lack the CUB domains without an obvious abnormality suggests that these domains are not essential for substrate recognition or VWFCP activity (Banno et al., 2003). The validity of this premise is based on the assumption that the ADAMTS13 null state is lethal in mice but this has yet to be reported. Synthetic peptides based on TSR and CUB domain sequences on the other hand have been reported to inhibit the ability of ADAMTS13 to cleave ULVWF strings released from histamine stimulated cultured endothelial cells in a parallel plate flow chamber(Bemardo et al., 2003).

1.2.1.5 The Tyr1605 and Met1606 cleavage site resides within the A2 domain ofVWF.

ADAMTS13 coated fluorescent beads have been reported to interact directly with both immobilized VWF and ULVWF strings at venous and arterial shear (figures 6 and 7).

21 Soluble ADAMTS13 is reported to bind preferentially with the A3 domain ofVWF

(Dong et al. , 2003). The current model for ADAMTSl 3 mediated proteolysis ofVWF predicts that the tensile stress imposed on endothelial cell bound VWF by the shear forces of flowing blood exposes cryptic binding sites within the A3 domain for circulating ADAMTSl 3. The VWF bound enzyme then cleaves the substrate at the adjacent A2 domain. The loss of tensile strength within the cleaved fragment then precludes it from further proteolysis. It is predicted that the binding of multiple

ADAMTS13 molecules to A3 domains within the one ULVWF string at random intervals would account for the variable size of cleaved fragments in plasma (Dong et al. , 2003). Given its essential role, it is possible that the spacer domain of ADAMTS 13 docks with the A3 domain ofVWF and positions the metalloprotease domain to cleave

VWF at the A2 domain. Using overlapping fragments of recombinant VWF, a minimal region (1660-1668) around the A2 domain (1498-1665) has been reported to be essential for cleavage at Tyr1605 and Met1 606 (Kokame et al. , 2003a).

Figure 6. Videomicrographs of strings of ULVWF with adherent fluorescent labeled platelets in a parallel plate flow chamber. A. Stimulated endothelial cells release strings of UL VWF that f01m a template for the adherence of platelets in fl owing a blood. B. The strings ofULVWF were measured to be in excess of a millimeter in length and appear to stretch and relax under flow. From Dong et al Blood 2002; I 00 ( 12):4033-9

22 Fluorescence microsco y

direction of flow

Figure 7. ADAMTS13 coated fluorescent labeled beads adhere to ULVWF strings under flow. The shear force of flowing blood stretches strings ofULVWF to expose cryptic sites on the A3 domains of multimeric VWF for ADAMTS 13 binding. The upper panel demonstrates clusters of ADAMTS 13 coated fluorescent labeled beads bound to UL VWF. The arrowheads in the lower panel point to clusters of platelets which are adherent to UL VWF between ADAMTS 13 coated beads (mrnws). From Dong et al J Biol Chem. 2003; 278(32) 29633-9

1.2.1.6 Glycosylation of ADAMTS13

Based on its amino acid composition, ADAMTS13 has a predicted size of-145 kDa but has an observed size of- 190 kDa. This is at least in part due to protein glycosylation.

The protein has 10 potential N-glycosylation sites of which at least 6 are modified with

23 complex-type oligosaccharides, which are likely to be sialylated. This is predicted to account for its long (2-3 day) plasma half life. There is also the potential for C­ mannosylation and O-fucosylation of Ser/Thr residues on the TSRs {detailed in

(Majerus et al., 2003)}. The rate limiting step to ADAMTS13 secretion is its exit from the endoplasmic reticulum as the N-oligosaccharides of the protein in cell lysates is endo-H sensitive in contrast to the secreted protein (Majerus et al., 2003). The intra cellular pro enzyme has been reported to retain VWFCP activity as the multimer size of

VWF, secreted from HeLa cells co transfected with ADAMTS 13 and VWF, was significantly smaller than VWF secreted from cells transfected with VWF alone

(Majerus et al., 2003). This is noteworthy given the earlier observation that

ADAMTS13 rich cell lysates were unable to cleave plasma purified VWF possibly due to the presence of an inhibitory substance in the lysate (Plaimauer et al., 2002).

1.2.1.6 Measurement of ADAMTS13 activity in plasma

The two original methods developed by Furlan et al (Furlan et al., 1996)and Tsai (Tsai,

1996) respectively, are functional assays that are based on the principle that plasma

ADAMTS 13 when activated by BaCh or CaCh, cleaves denatured VWF. The loss of high molecular weight VWF multimers, as a result of proteolytic cleavage, is then assessed by 1% agarose gel electrophoresis. This method reported by Furlan et al

(Furlan et al., 1996) uses a calibration curve generated by dilutions of pooled normal plasma to express VWF cleaving protease activity as a percentage of normal.

24 The method developed by Tsai (Tsai, 1996) applies a similar principle but measures

ADAMTS13 activity by the generation of a proteolytically cleaved VWF fragment rather than the loss of the high molecular weight multimers. Proteolytic cleavage of the

VWF subunit between Tyr1605 and Met1606 generates N and C-terminal disulfide linked dimers (200kD and 350kD respectively) that can be separated by SOS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) under non-reducing conditions. The fragments are detected using 1251 labeled anti-VWF antibodies and resolved by autoradiography. Measurement of the relative intensity of the 350kD C-terminal dimer between a reaction mix incubated with and without EDT A (inactive and active respectively) is used to quantify ADAMTS13 activity. The measurement of the end product of proteolytic cleavage rather than the mere loss of the large VWF multimers is a potential advantage. In-house threshold sensitivities of <3-5% activity for Furlan's method and

The requirements for purified, protease free VWF (used as a substrate for plasma

ADAMTS13) and the ability to consistently perform gel electrophoresis of high quality place these assays beyond the resources of most routine diagnostic laboratories.

Variations of the assay use collagen binding affinity (Gerritsen et al., 1999) or ristocetin co-factor activity (Bohm et al., 2002) as a surrogate measure of cleaved VWF multimer size. The 2-site immunoradiometric assay (IRMA) measures the products ofVWF hydrolysis by using a combination of monoclonal antibodies. MoAb 453 which is directed against the C-terminal part ofVWF is used to capture VWF and the presence

25 (uncleaved VWF) or absence (cleaved VWF) of the N-terminal fragment is probed using a mix of 1251-anti-N-terminal MoAbs (Gerritsen et al., 1999; Obert et al., 1999).

A criticism of these assays is the unphysiological conditions used to denature substrate

VWF and activate ADAMTS13. Attempts to incorporate shear stress as a more physiological alternative have led to the description of a couple of assays that are still under development. The aggregation of normal whole blood platelets in a cone and plate(let) analyzer (CPA) at high shear (1800sec-1) is increased in the presence of TTP plasma in contrast to normal plasma and forms the basis of one of these tests (Shenkman et al., 2003) and the other uses a parallel-plate flow chamber and video-microscopy to quantify the cleavage of VWF multimers as they are secreted from histamine stimulated endothelial cells (Amar et al., 2002). Independent verification of these methods using well characterized patient samples, with known ADAMTS 13 activity is necessary prior to the wider application of either of these tests. Furthermore the second of these assays will also need to be modified, if it is to be of use in a routine diagnostic laboratory.

The identification of a VWF fragment D1596-R1668 (VWF73) as a minimal region recognized as a specific substrate for ADAMTS13 is an exciting development. It is reported that this fragment can be used as a substrate to detect plasma ADAMTS13 activity after 20 minute incubation without need for either urea or guanidine -HCl

(Kokame et al., 2003b). Purified VWF-A2 is also susceptible to ADAMTS13 mediated hydrolysis and has been used with N and C terminal tags as a substrate to measure

enzyme activity in an ELISA format (Cruz et al., 2003a; Cruz et al., 2003b).

26 The presence of an inhibitor to ADAMTS 13 can be determined by a mixing study, which involves the measurement of protease activity in a mixture of normal plasma incubated with patient plasma. Inactivation of protease activity in 1ml of normal plasma by 1 ml of patient plasma is defined as 1U of protease inhibitor. The presence of an inhibitor can be further clarified when necessary by affinity chromatography of IgG from patient plasma and testing the eluate for ADAMTS13 inhibition. Anti­

ADAMTS13 immunoglobulins that do not cause functional inhibition but precipitate clearance of the enzyme from the circulation have been detected using an ELISA based method (Scheiflinger et al., 2003). These antibodies would not be detected by a mixing test and could account for the variable frequency of inhibitors (48-80%) that have been reported in acquired TTP (Furlan et al., 1998a; Tsai and Lian, 1998; Veyradier et al.,

2001).

A quality control exercise using 30 plasma samples with ADAMTS13 activity ranging from <3% to >100% found good concordance for detecting a severe deficiency (<5%) between 5 laboratories using immunoblotting, IRMA, collagen binding and ristocetin binding methods. One false negative and two false positives were recorded by two laboratories using a collagen binding assay. The results were less concordant for

ADAMTS13 in the normal, mild and moderately reduced groups. There was also good agreement for detecting strong inhibitors. As the diagnosis of thrombotic thrombocytopenic purpura is strongly supported with a severe deficiency (<5%) of

ADAMTS13 and the detection of inhibitory antibodies are confirmatory, the test is of

clinical utility.

27 With the identification of the ADAMTS13 gene and the expression of recombinant

ADAMTS13 there is the potential to generate anti-human ADAMTS13 antibodies. The introduction of an ELISA (enzyme linked immunosorbant assay) based method that can detect ADAMTS13 (as the antigen) in plasma with greater precision and supersede the cumbersome assays in use today has been delayed due to unexpected technical difficulties. The stable expression of the protein in several mammalian cell lines has been associated with low level expression due in part to the apparent toxicity of

ADAMTS13 to the cells used to express the protein (Majerus et al., 2003). Generating polyclonal antibodies has also proved difficult although there are now at least two anti­ human ADAMTS13 mAbs described (Motto et al., 2003; Plaimauer et al., 2002). It is salient to note though that the mutations in ADAMTS13 in familial recurrent TTP were identified based on a reduction of the functional activity of the enzyme and not the antigen per se (Levy et al., 2001 ). The presence of a dysfunctional protein with no protease activity that is detected by ELISA could pose a problem. This is underscored by a recent report by Kokame et al (Kokame et al., 2002) of 4 mutations in the

ADAMTS13 gene in 2 Japanese families with congenital TTP. The mutant proteins were expressed in mammalian cells and it was noted that 2 of these mutants (R268P and

C508Y) were not secreted from cells but that the other 2 (Q449stop and P475S) were secreted normally but demonstrated minimal activity. Furthermore, genotype analysis of

364 Japanese subjects revealed that approximately 10% were heterozygous for P475S.

It is likely therefore that although an ELISA based assay that quantifies ADAMTS13

28 with greater precision will be of value in acquired TTP which constitute the majority of patients, a role for a functional assay will remain.

1.3 CONTROL OF VON WILLEBRAND FACTOR MULTIMER SIZE - BY

DISULFIDE-BOND REDUCTION

Disulfide bonds stabilize the native conformation of proteins and maintain protein integrity by limiting access to oxidants and proteolytic enzymes. Although the disulfide bonds in mature proteins were once thought to be permanent, it now appears that disulfide cleavage has significant consequences for protein function (Hogg PJ., 2003).

One of the first examples of disulfide cleavage described in a secreted protein was in thrombospondin-1. The thiol-disulfide , protein disulfide isomerase

(PDI), facilitates disulfide interchange in TSP-1 (Hotchkiss et al., 1996). Different disulfide-bonded forms of TSP-1 exist in vivo and have different adhesive properties

(Sun et al., 1992; Hogg et al., 1997). Disulfide bonds in plasmin, a serine proteinase that mediates cell migration and thrombolysis, are also manipulated in the extracellular space. Angiostatin, an inhibitor of angiogenesis, is an N-terminal fragment of plasmin that is generated following the cleavage of two disulfide bonds in the fifth kringle domain of plasmin (Hogg et al 1997). This disulfide-bond cleavage is facilitated by phosphoglycerate kinase (PGK), an enzyme of the glycolytic pathway that is secreted in a regulated manner by tumour cells (Lay et al. 2000).

29 The conformation and function of cell surface receptors are also regulated by disulfide bond cleavage. The disulfide bond in the D2 domain of CD4+ T cells is redox active and can be cleaved by thioredoxin, which is secreted by these cells (Matthias et al.,

2002). The entry of the human immunodeficiency virus type 1 (HN-1) appears to be regulated by the redox state of the D2 domain. Most integrins contain an on/off switch that regulates ligand binding. Enzymatic cleavage of disulfide bonds and generation of free thiols in integrins a1IbJ33 and a2J31 are required for these receptors to engage their ligands (Lahav et al., 2002; Lahav et al., 2003). Protein disulfide isomerase (POi) has been reported to catalyze the adhesion of a 2J3 1to a specific peptide from type 1 collagen

(Lahav et al., 2003).

Dithiol-disulfide exchange is one of three possible mechanisms that have been identified or suggested as mechanisms for disulfide-bond cleavage (figure 8b) (Hogg PJ., 2003).

Alkaline hydrolysis and acid-base assisted hydrolysis are two other mechanisms that could operate in vivo. It has been proposed that PGK facilitates disulfide bond cleavage in plasmin by triggering alkaline hydrolysis (Lay et al., 2002). Acid-base assisted hydrolysis is as yet only a theoretical concept (Brandt et al., 1999).

30 (a) D11h1ol - d1suH1de redox exchange (b) Alkaline hydrolysis

R2 OH s- s f.;J~Olt"" -~\--• R2 -~ w R, R,-s-

(c) Acid-base assisted hydrolysis

Figure 8b. Possible mechanism of disulfide-bond cleavage. (a) Dithiol-disulfide redox exchange: a thiol group of the oxido-reductive dithiol attacks the substrate disulfide bond. (b) Alkaline hydrolysis: a hydroxide ion attacks the disulfide bond. (c) Acid-base assisted hydrolysis: the carboxylate anion

Ri-Co2• attacks one of the sulfur atoms of the disulfide bond with the assistance of the nearby

ammonium cation R3-NH/. From Hogg PJ Trends in Biochemical Sciences 2003; 28(4): 210-214

VWF multimer size can be controlled by the reduction of the disulfide-bonds holding the subunits together. Human umbilical vein endothelial cells (HUVECs) grown in both sernm free and serwn containing media, release UL VWF when exposed to either steady venous-like or pulsatile a1terial-like wall shear stresses. Plasma cryosupernatant was reported to prevent the accumulation of ULVWF , released from the luminal surface of

HUVECS. A range of protease inhibitors could not inhibit this activity in cryosupernatant and it was hypothesized that a disulfide bond reductase in plasma cryosupematant regulated VWF multimer size (Frangos et al., 1989). This VWF depolymerizing activity was subsequently reported to be ablated by the adclition of thiol­ blocking agents (Phillips et al. , 1993).

31 The conditioned media of cultured human umbilical vein, human dermal microvascular and bovine aortic endothelial cells were also shown to contain an activity that reduced the average multimer size of plasma or purified VWF (Xie et al., 2000). This reducing activity was ablated by pre-treatment with heat or thiol blocking agents, but not by a range of serine, cysteine, aspartic or metallo-proteinase inhibitors. Significantly, the reduction ofVWF multimer size was accompanied by the generation of new thiols and there was no evidence for additional proteolytic processing ofVWF. This observation led us to suggest that endothelial cells, which produce VWF, also produce an enzyme that reduces the disulfide-bonds linking VWF subunits, thereby reducing the average

VWF multimer size (Xie et al., 2000). In support of this hypothesis, incubating VWF with known protein reductants, protein disulfide isomerase or thioredoxin resulted in the reduction of the average multimer size ofVWF along with the generation ofnewthiols.

The reducing activity secreted by endothelial cells was associated with a protein with an anionic pi that bound to S-Sepharose (a strong cation exchange matrix), heparin­

Sepharose and activated thiol Sepharose (a matrix that binds proteins containing accessible thiol groups), but not to Q-Sepharose (a strong anion exchange matrix) (Xie et al., 2001 ). 30L of conditioned medium from cultured human microvascular endothelial cells (HMEC-1) was concentrated to 350mL using a 10-kD cutoff membrane. The medium was applied to a column ofheparin-Sepharose, washed with three bed volumes of HEPES buffer and the bound proteins were eluted with a NaCl

gradient. The fractions containing VWF reductase activity were concentrated and gel

filtered using a column ofSephacryl S-300 HR. The samples were resolved on 4-15%

32 SDS-PAGE under reduced and non reduced conditions. A protein, with a molecular mass of -500 kDa which reduced to -170 kDa after reduction and alkylation, was noted.

This subunit structure was very similar to the trimeric glycoprotein, thrombospondin-1.

This association was strengthened by the demonstration that the protein in the conditioned medium was recognized by an anti-TSP-1 monoclonal , and immuno-precipitation of TSP-1 from the conditioned medium accounted for all the

VWF reductase activity in the medium (Xie et al., 2001). Furthermore, the incubation of purified TSP-1 with recombinant VWF resulted in the generation of new thiols in VWF and diminution of VWF multimer size. The first step in reduction of a disulfide bond is nucleophilic attack on the substrate disulfide bond by a reductant thiol which results in formation of a disulfide-linked complex between the substrate and the reductant.

Release of the reductant from the complex requires nucleophilic attack on the mixed

(reductant-substrate) disulfide linkage by another thiol, usually of the reductant. The reductant can be trapped in disulfide linkage with the substrate if the thiol responsible for separating the complex is inhibited (figure 8). Up to eightfold more TSP-1 bound to

VWF in the presence of NEM, a thiol blocking agent.

TSP-1 regulates the function ofVWF by reducing its multimer size. It is also conceivable that cleavage of disulfide bonds within the VWF monomer regulates its affinity for collagen and platelet integrins.

33 TSP-1

C974 ? r(vwF+

1 ~~n:~~:'i~nal 1~change

Ca1· I SH

> > < <

Figure 8a Model for TSP-1 mediated reduction of VWF. A free sulfhydryl residue on TSP-1 launches a nucleophilic attack on the VWF interchain disulfide bond. A mixed interchain disulfide is formed as an intermediate complex from which the reductant (TSP-1) is released by reduction initiated by another free thiol in TSP-1 .

34 1.3.1 The thrombospondin gene family

The are a family of matrix glycoproteins that assist in cell-cell and cell-matrix communication by modulating the action of various cytokines at cell surfaces. Thrombospondins, by sequestering calcium ions at the cell surface, acting synergistically with growth factors and by associating with various cell surface components may direct the assembly of macromolecular complexes that facilitate the transduction of signals which, in tum, modulate cell adhesion, movement, proliferation and differentiation (reviewed in(Adams et al., 1995)). The TSP family consists of five members in vertebrates, TSP-1-4 and TSP5 (also known as cartilage oligomeric matrix protein-COMP). Thrombospondins -1 and -2 are produced by endothelial cells and play key roles in vascular biology (Iruela-Arispe et al., 1993). TSP-1 is also important in lung homeostasis as evident by the inflammatory infiltrates and epithelial hyperplasia in the lungs of TSP-1 null mice (Lawler et al., 1998). TSP-2 is produced by developing myoblasts and is strongly expressed in vascular smooth muscle cells and in linings of the coelomic cavities. TSP-3 is present in abundance in the central nervous system and possibly plays a role in neurite outgrowth and neuron migration. TSP-4 and COMP are concerned primarily with the development of bone and cartilage. The study of the temporal and spatial distribution of the different thrombospondins during embryonic development requires monoclonal antibodies that can discriminate between the different thrombospondin types for use in immunohistochemistry or alternatively, the tissue extraction ofRNA at different stages of embryonic development. The early studies into the tissue distribution of thrombospondin were performed in an era when the existence

35 of the various types was not known and are hampered by the use of cross-reacting polyclonal antibodies (Adams et al. , 1995).

TSP-1 , but not TSP-2, is also produced by megakaryocytes and is concentrated in platelet a- granules (figure 9). Platelet a- granules have peripherally located electron lucent tubular structures akin to those found within Weibel-Palade bodies, which serve as storage sites for platelet VWF (Cramer et al., 1985). Thrombospondin- 1 is located in a zone of intermediate electron density, away from VWF (Suzuki et al., 1990) (figure

10), but they are released simultaneously following platelet activation and degranulation.

This is of particular significance with regards to the potential role of platelet TSP-1 in regulating platelet VWF multimer size.

~.. :.::-~::<·-':'· (>'>:1:-:-~::- i -;:~ ~ ;5~)::;;:s::::;,:,::;;)(' ~>-~~:;-:':)

I' ''.1,·::1_ ,:•;,::::::·:~1x,:~ ~-:?<=~<*~.. : I : I,,,: l <>-~-:\~~-i

Figure 9. Electron micrographs of resting and activated platelets and details of their ultrastructure. Activation of pl atelets results in a reorientati on of pl atel et granules to fac ilitate release of their contents to the platelet sw·face. From George JN Lancet 2000; 355 (921 4 ): 1531-9

36 Figure 10. Ultra structure of platelet alpha granules. Immunogold particles label VWF only in the electron lucent zone of alpha granules. The mrnws in the inset highlight tubular structw·es reminiscent of those found in Weibel-Palade bodies in endothelial cells. TSP-1 was detected in the zone of intermediate electron density marked by the double anows in the main figure. The single anow denotes the nucleoid zone and the three arrows the electron lucent zone which contains VWF. From Suzuki et al Histoche111ist1v 1990: 34(4): 337-344

1.3.1.1 Evolution of the thrombospondin gene family

Thrombospondin-1 and -2 have a similar molecular architecture and are designated subgroup A thrombospondins. They are two of 41 TSR containing proteins in the that are part of a superfamily that includes the complement factors, C8 and C9; the malaria protein, TRAP and the axon guidance proteins, F-spondin and UNC-

5C (Adams and Tucker, 2000). TSP-1 and-2 are trimeric and each subunit is composed of multiple domains: NH2- and COOH-terminal globular domains, a procollagen-like

37 domain and three types of repeated sequence motifs, designated type 1 (TSR), 2

(Epidermal growth factor like) and 3 (Calcium binding) repeats (Lawler and Hynes,

1986) (figure 11 ). TSP-3, -4 and COMP are pentameric and similar to each other in lacking a TSR and are designated subgroup B thrombospondins. COMP differs from the other subgroup B thrombospondins in lacking a NH2 globular domain (Oldberg et al. ,

1992).

A phylogenetic tree constructed for the thrombospondins indicate that the present day have evolved from a primordial gene by a series of exon shuffling, duplication and deletion over approximately 900 million years (Lawler et al. , 1993 ). In addition to the five thrombospondins identified in vertebrates a single thrombospondin has been identified in Drosophilia melanogaster (Adams et al., 2000; Adolph, 2001) and TSRs in

Caenorhabditis elegans.

1.3.1.2 The structure of thrombospondin-1 and relevance to function

TSP-1 subunit

Figure 11. The domain structure of a TSP-1 subunit The subgroup A th rombospondins, TSP- l and TSP-2 are t1imeri c and share a simil ar molecul ar architectu re comprising a unique heparin-binding domain at the N-terminus fo llowed by a connecting region th at links three subunits v ia inter-chain disulfide bonds, a procoll agen-like module, three properdin-l ike or type l m odules, three epidem1al growth fac tor-l ike or type 2 modules, seven type 3 repeats ( l 2 un ique calcium binding loops) an d a unique C -tem1inal sequence

38 The NH2-terminal globular domain has a strong affinity for heparin and related glycosoaminoglycans and a peptide from this region (amino acid residues 17-35) causes a loss of focal adhesion plaques that are formed by endothelial cells and fibroblasts

(Murphy-Ullrich et al., 1993). This is important to its role in tissue remodeling as signified by the impaired wound healing evident in TSP-1 null mice (Lawler et al.,

1998). Soluble TSP-1, applied to fibroblasts in culture, is rapidly endocytosed and reappears in a degraded form in the culture medium and matrix (McKeown-Longo et al.,

1984). The internalization ofTSP-1 is thought to involve the heparin binding motifs interacting with cell surface receptors as it is inhibited by heparin (McKeown-Longo et al., 1984).

The NH2-terminal domain is followed by a region that includes Cys 242 and Cys 245, the residues involved in the formation of interchain disulfide bonds that link the individual subunits to form trimeric TSP-1 (Sottile et al., 1991 ).

The procollagen domain is reported to be involved with the assembly of the TSP-1 triple helix (Sottile et al., 1991 ). A peptide from this region has also been reported to be antiangiogenic possibly by impairing synthesis of collagen (Tolsma et al., 1993).

Each TSP-1 monomer contains three type 1 repeats (TSRs) which mediate cell attachment, protein binding, glycosaminoglycan binding, inhibition of angiogenesis, activation ofTGFf3 and the inhibition of matrix . The crystal structure of the TSRs reveals a novel, antiparallel, three-stranded fold that consists of alternating stacked layers of tryptophan (W) and arginine (R) residues from respective

39 strands, capped by disulfide bonds on each end (Tan et al., 2002). Peptides derived from the second and third TSR which include the CSVTCG sequence were reported to be antiangiogenic by binding to CD36 on endothelial cells (Dawson et al., 1997). The sequences flanking this stretch, W**W**W (a putative GAG ) and

GVITRIR (highly conserved arginine residues) are also reported to inhibit angiogenesis

(Dawson et al., 1999; Iruela-Arispe et al., 1999). The positively charged, exposed tryptophans and arginine residues form the recognition face ofTSR and are likely to mediate the interaction with the negatively charged face of CD36 (Tan et al., 2002). The

Cys residues of the CSVTCG sequence are involved with two separate disulfide linkages and the Thr residue is O-glycosylated. The reported activities of the synthetic peptide therefore is unlikely to correlate with the three dimensional structure of TSR (Tan et al.,

2002). The TGFJ3 activating motif involves the highly basic tripeptide, RFK (Schultz­

Cherry et al., 1995) which is situated in the linker region between TSRl and 2.

Unfortunately this region could not be modeled due to conformational changes that are predicted to have occurred due to the expression of high concentrations ofTSRl-3 (Tan et al., 2002).

The TSP-1 monomer has three type 2 (EGF like) repeats. The spacing of the six cysteine residues that characterize the EGF-like domain is conserved only in the last type 2 repeat (Adams et al., 1995). The second type 2 repeat contains a conserved sequence for J3-hydroxylation and is predicted to bind a calcium ion (Handford et al.,

40 1991 ). The binding of calcium to this site may induce a conformational change that can be detected using a monoclonal antibody A6.1 (Adams et al., 1995).

The calcium binding type 3 repeats are rich in aspartic acid residues which are positioned in a characteristic pattern that enables the negatively charged hydroxyl groups of the aspartate residues to interact with the calcium ion. TSP-1 has seven type 3 repeats and 12 potential calcium binding sites in each monomer and each molecule has been reported to bind 35 +/- 3 calcium ions. Calcium binding is corporative and induces a conformational change. The binding affinity ofTSP-1 to calcium is -120µM at 22°C which is considerably lower than calmodulin (Lawler and Simons, 1983 ). TSP-1 operates in an extracellular environment in which the ambient calcium concentration is

105 higher than the intracellular environment in which calmodulin functions. One of the functions of thrombospondins is to sequester calcium in the extracellular matrix and at the cell surface and possibly regulate membrane proteins by the transfer of calcium ions.

Both human and murine thrombospondin-1 have an RGD integrin binding motif in the last type 3 repeat, the activity of which can be modulated by calcium and by thiol­ disulfide isomerization (Lawler and Simons, 1983; Sun et al., 1992). TSP-1 is a tight binding competitive inhibitor of cathepsin G and neutrophil elastase. The reactive centers reside within the type 3 calcium binding repeats and the potency of inhibition varies markedly with the concentration of calcium and is also modulated by disulfide isomerization (Hogg et al., 1994; Hotchkiss et al., 1996).

41 Disulfide exchange in TSP-1 is complex and incompletely understood. Each molecule ofTSP-1 contains 3 free thiols, presumably one on each monomer (Speziale and

Detwiler, 1990). TSP-2 differs from the other thrombospondins in not containing unpaired cysteine residues (Bornstein et al., 1991). The TSP-1 thiol is remarkably fluid and can reside on any one of 12 different cysteines in the type 3 repeats and the C­ terminal globular domain (Speziale and Detwiler, 1990). Nevertheless TSP-1 purified in buffers containing 0.1 mM Ca2+ is a homogeneous TSP-1 population in that the free thiol is at Cys974 in the C-terminal globular domain (Speziale and Detwiler, 1990).

Protein disulfide isomerase, an enzyme present on the platelet surface has been shown to catalyze TSP-1 disulfide exchange and can modulate its function (Hotchkiss et al.,

1996). The C-terminal globular domain and the type 3 repeats appear to function as a single unit. Residues RFYVVMWK (also named 4Nl-1) and IRVVM from the C­ terminal domain have been identified as cell binding motifs (Kosfeld and Frazier, 1993).

The 4Nl-1 and 4N1K (RFYVVMWKK) peptides bind a widely expressed protein identified as integrin-associated protein (IAP) or CD4 7 and has been reported to activate integrin a.1lbP3 and to synergize a.2P1 mediated platelet activation (Chung et al., 1997;

Chung et al., 1999). These peptides have more recently been reported to induce platelet activation and subsequent a.1lbP3 activation by an FcR y-chain associated signaling pathway that is independent ofCD47 (Tulasne et al., 2001). The authors also report that the peptide induces platelet agglutination that is independent of GPl ba. and a.1lbf33. These studies have been performed using synthetic peptides and should be interpreted with caution as the conformation of the C-terminal globular region is sensitive to Ca2+ and

42 disulfide isomerization, which may affect the availability of this sequence for receptor interaction.

43 l.3.1.3 The structure and function relationship between TSP-1 and VWF

As detailed in the previous section, thrombospondin-1 is a trimeric protein and each of its subunits contains an unpaired cysteine. It is proposed that these free thiols law1ch a nucleophilic attack on the VWF intersubunit disulfide-bonds, resulting in their reduction

( figure 12). The progressive reduction of intersubunit disulfide-bonds will lead to the dismantling of the VWF multimer (Xie et al., 2001) (figure 13). The interaction ofTSP-

1 with VWF is proposed to occur between the type 1 repeats of TSP-1 and the A3 domain of VWF. The basis of this proposal is the sequence similarity between the TSP-

1 binding motif on CD36 (GPYTYR VRFLA) and the A3 domain of VWF

(DALGF A YR YL T) and the inhibition of TSP-1 mediated VWF reduction in the presence of synthetic A3 and TSR peptides (Xie et al. , 200 l ). The location of the free thiol that mediates VWF reduction is not known but is suspected to exist on Cys 974 at

0 .1 mM Ca2+ (Xie et al ., 2001 ). The binding interaction is further explored in this thesis using insect cell expressed TSP-1 fragments rather than synthetic peptides. The location of the free thiol and VWF reductase activity at different concentrations of Ca2+ is also addressed (Pimanda et al. , 2002). Figure 12. The target interchain disulfides It is uncertain whether the N- or C­ terminal interchain disulfide linkage or both are susceptible for TSP-1 ~p~n N mediated reduction. The intra-chain I', .~ - - ls-s~ S-5~· .s~s.s1-:- - - disulfides could also be potential S.S A2 C '- ' 1 l ! N '- I N / Factor VII targets that affect VWF ligand Collagen GPIIMRa GPtb/Vnx suratides binding without altering multimer size.

44 vWF/',a. T;P-1 .._, TSP-1= \

Lumen

Basement Membrane

Figure 13. A schematic diagram of TSP-1 mediated reduction of plasma VWF. It is predicted that there is a proportionate reduction in the avidity with which TSP-1 binds the VWF multimer as it diminishes in size. This would imply that the largest multimers would make better substrates for TSP-1 mediated reduction. The reduction is theoretically reversible but may not apply in the dynamic milieu in-vivo. Pimanda et al Blood Rev. 2002 Sep 16(3) 185-192

1.3.1.4 TSP-1 as an

TSP-1 has been shown to bind and inhibit the activities of the neutrophil enzymes, neutrophil elastase (Hogg et al. , 1993b) and cathepsin G (Hogg et al., 1993a), and the fibrinolytic enzymes, plasm.in and urokinase (Hogg et al., 1992; Mosher et al., 1992a).

Neutrophil elastase and cathepsin G are released by activated neutrophils and have been implicated with tissue destruction at sites of inflammation (Malech and Gatlin, 1987).

Cathepsin G is a strong platelet agon.ist acting via PAR-4 (protease activated receptor-4)

45 on the platelet surface resulting in platelet activation and degranulation (Sambrano et al.,

2000). TSP-1 inhibits cathepsin G mediated platelet aggregation (Hogg et al., 1993a).

There are reports for and against the inhibition of plasmin and uPA by TSP-1 (reviewed in (Hogg, 1994)) although there is agreement that TSP-1 inhibits uPA-mediated fibrinolysis (Hosokawa et al., 1993; Mosher et al., 1992b). TSP-1 has also been reported to inhibit the activity of matrix metalloproteinases by preventing the activation of

MMP2 and MMP9 zymogens (Bein and Simons, 2000).

The release ofTSP-1 from activated platelets (Baenziger et al., 1971), fibroblasts (Jaffe et al., 1983), endothelial cells (Mosher et al., 1982), smooth muscle cells (Majack et al.,

1985) and activated neutrophils (Kreis et al., 1989) to the milieu of damaged tissue, and the ability to regulate the activity of the above enzymes in the extracellular matrix is pertinent to the role ofTSP-1 in wound healing.

1.3.1.5 The phenotype of the TSP-1 null mouse (Lawler et al., 1998)

The homozygous matings produce significantly fewer cumulative litters per breeding pair (3.4 +/-1.7) as well as offspring per litter (4.8+/- 1.9) than the wild-type matings

(7.2+/- 2.5 and 6.2 +/- 2.2 respectively). TSP-1 deficient mice display a mild and variable lordotic curvature of the spine from birth and abnormalities in pulmonary homeostasis from four weeks of age. The changes in the lung are characterized by an organizing pneumonia, haemosiderosis, epithelial hyperplasia, increased collagen and elastin deposition. No major histological changes were noted in the brain, heart, aorta, liver, kidney, spleen, stomach and intestines. The TSP-1 null mice have an elevated

46 leukocyte count, particularly of the monocyte and eosinophil lineages, normal haemoglobin levels and platelet counts. Measurement of platelet function was limited to the assessment of bleeding times which were comparable and the response of washed platelets to thrombin which was equivalent. The TSP-1 null mice are also reported to show impaired wound healing with delayed organization and prolonged neovascularization of skin wounds (Agah et al., 2002; Polverini et al., 1995)

1.4 CONTROL OF PLATELET VWF MULTIMER SIZE

Platelet VWF constitutes 8-15% of total circulating VWF (Howard et al., 1974) and does not exchange with the plasma pool (Sultan et al., 1978). Whereas endothelial cell/plasma VWF serves as the initial bridge between collagen and GPl ba. in tethering platelets to the subendothelium at sites of vascular damage, the release and surface expression of platelet VWF is important for platelet-platelet interaction and the subsequent growth of a thrombus (Ruggeri, 1993 ). Platelet VWF plays a key role in platelet aggregation by mediating the tethering and translocation of platelets on platelets with a.nbJ33 mediating stable cell arrest (Kulkarni et al., 2000) (figure 14). Fibrinogen and fibronectin, two other adhesive proteins released by activated platelets contribute to the growth of a stable thrombus (reviewed in (Ruggeri, 2000)).

47 f lntegrin a1Jl3 (inadive) V lntegrin a1J33(adive) W GP lbNAX ,'vWf Fibrinogen

~..... ,.... •.·· Adhesion

Figure 14. A schematic representation of platelet -vessel and platelet-platelet interaction in the formation of a platelet thrombus.

Following the formation of the initial platelet layer at the vessel interface, platelet VWF forms a secondary template upon which platelets tether. translocate and form stable adhesions in the process of forming a platelet aggregate.

The VWF multimers released from platelets are not uniform in size but include fonns which are larger than those normally present in plasma (Fernandez et al. , 1982). The reason for the variation in size of platelet VWF multimers has not been established.

Platelet lysates do not contain ADAMTS 13 (Furlan et al. , 1996) but do contain proteases such as calpain and plasmin which can degrade VWF but without yielding the characteristic VWF fragments present in plasma (Berkowitz et al., l 988; Moore et al.,

1990). Furthermore as TSP-1 null platelets also contain VWF multimers of varying size

( detailed in chapter 3 of this thesis) it is unlikely to be due to reductase activity. The

48 Quebec platelet disorder is an autosomal dominant platelet disorder characterized by alpha granule protein degradation and a bleeding phenotype due to the excessive production of a urokinase-type plasminogen activator (uP A) (Kahr et al., 2001 ). The increased degradation of VWF multimers that is evident in this syndrome could be an extreme of normal. Alternatively the multimers could be synthesized and packaged in varymg sizes.

It is estimated that 60-70% of platelet VWF reassociates with the platelet surface following release with the concentration VWF in the interstices of a platelet aggregate being approximately 50 times higher than plasma (Fernandez et al., 1982). Furthermore the larger multimers bind preferentially to the platelet surface in a time dependant manner when platelets are activated with thrombin in the presence of calcium

(Fernandez et al., 1982).

As plasma and platelet VWF interact in determining the degree of thrombus growth in­ vivo, quantifying the contributions from each compartment is complex. Chimeric vWD pigs with normal platelet VWF but low plasma VWF have very prolonged bleeding times and pigs with the reverse phenotype have mildly prolonged bleeding times

(Nichols et al., 1995). This is consistent with observations in humans with low plasma

VWF and normal platelet VWF who have either normal or mildly prolonged bleeding times (Mannucci et al., 1985). As endothelial cell/ plasma VWF is required to initiate platelet adhesion it is suggested that at least 10-20% plasma VWF activity is required for

49 platelet VWF to compensate for the plasma deficiency and sustain normal thrombus growth (Mannucci, 1995).

Although the preferential association of the largest multimers on the surface of activated platelets would be consistent with providing a greater number of adhesive contacts for platelet-platelet interaction and thrombus growth, it is unclear whether a degree of processing to yield an optimal sized VWF multimer occurs in vivo. ADAMTS13 is not present in lysates of washed platelets (Furlan et al., 1996) and due to the absence of shear within the interstices of a growing thrombus, is unlikely to play a role in determining platelet VWF multimer size upon release. I have proposed in this thesis that

TSP-1 provides a degree of modulation of platelet VWF multimer size. It is feasible that proteolytic enzymes present in platelets may do the same in vivo.

1.5 VWF MULTIMER SIZE AND DISEASE

1.5.1 Deficiency of high molecular weight VWF multimers and bleeding von Willebrand Disease (vWD) is associated with a quantitative or qualitative defect in

VWF with an associated bleeding diathesis. The disorder is currently categorized as 3 major types: type 1 (partial) and type 3 (complete) are quantitative defects ofVWF whereas type 2 is a qualitative defect ofVWF, usually present in normal quantities

(Fressinaud et al., 2002; Sadler, 1994). vWD type 2A comprises all variants lacking

HMWM in plasma and combines defects in dimerization (subtype IID)(Schneppenheim

50 et al., 1996), multimerization (subtype IIC)(Holmberg et al., 1998), intracellular transport and secretion (subtype IIA; group 1 )(Ginsburg and Sadler, 1993), enhanced proteolysis by ADAMTS13 (subtype IIA; group 2)(Ginsburg and Sadler, 1993), impaired dimerization with reduced proteolysis (subtype IIE)(Schneppenheim et al.,

2001) and a number of other less well defined subtypes(Sadler, 1994). vWD type 2B is characterized by enhanced ristocetin-induced platelet aggregation (RIP A) often with reduced plasma HMWM. Type 2M which is characterized by a specific defect in platelet/ VWF interaction and type 2N in FVIII/ VWF interaction have a normal range of multimers (Sadler, 1994 ). Type 2M "Vicenza" is an unusual subtype characterized by low plasma VWF antigen and supra normal multimers with apparently normal proteolysis (Mannucci et al., 1988).

Approximately 70 mutations in the VWF gene contributing to the type 2A vWD phenotype have been described (http://www.shefac.uk/VWF/mutations/). These include point mutations at Cys2771 and Cys2773, which along with Cys2811; contribute to the

C-terminal disulfide links between VWF (Katsumi et al., 2000). There is also a cluster of mutations around Tyr1605-Met1606 which presumably increase the sensitivity of

VWF to its cleaving protease (Sadler et al., 2000).

Acquired Type 2A vWD has been described in patients with valvular heart disease, with normalization of the VWF multimer pattern and cessation of bleeding (e.g. from gastro intestinal ) following valve replacement (Anderson et al., 1996; Sadler,

2003; Vincentelli et al., 2003; Warkentin et al., 1992). The pressure gradient across the

51 narrowed aortic valve creates a shear force that is thought to stretch VWF and increase its susceptibility to ADAMTS 13 mediated proteolysis.

1.5.2 Persistence of ultra large VWF multimers and thrombosis

1.5.2.1 The Thrombotic Microangiopathies (TMAs)

Figure 15. A blood film of a patient with TTP The red cell fragments result from the perfusion of blood through partially occluded arterioles and capillaries. There is also a paucity of platelets which are consumed in the diffuse thrombosis.

Thrombotic Thrombocytopaenic Purpura (TTP) and the Haemolytic uraemic syndrome

(HUS) are thrombotic microangiopathies in which a proportion of individuals ( ~ 1/2-1 /3) have a higher than normal average VWF multimer size (figure 15). It is suspected that a triggering event that results in endothelial injury together with the release and persistence of ultra large VWF multimers contribute to diffuse arteriolar platelet clumping (Moake, 2002). The presence of ultra-large von Willebrand Factor multimers in the plasmas of patients with chronic relapsing TTP during remission that disappear during an attack led to the implication of these multimers in the pathogenesis of the

52 platelet rich, fibrin poor thrombi that occlude arterioles and are the hallmark of this disorder (Moake et al., 1982) (figure 16). During the acute thrombotic event the larger and more functionally active VWF multimers are sequestered in the platelet thrombus and are cleared from plasma resulting in at times a lower than average VWF multimer size.

Ptn A

Figure 16. The VWF multimers in the plasma of a patient with congenital TTP During remission, the multimers in plasma are larger than in normal plasma and approximate the size in endothelial cells. There is a loss of the largest multimers during a relapse as they are sequestered in the thrombus. The absent plasma factor in congenital TTP has now been identified as ADAMTSl3. From Moake et al N Engl J Med 1982; 307 (23): 1432-5

A proportion of patients with TTP/ HUS have either a congenital or acquired deficiency of ADAMTSl 3. (Furlan et al., 1998a; Tsai and Lian, 1998) (figure 17). A number of mutations in the ADAMTSl 3 gene, associated with absent or very low VWFCP levels, have been described in the familial form of the disease (Levy et al., 2001 ). The sensitivity, specificity and positive predictive value of ADAMTS 13 deficiency in the acquired TMAs are currently being evaluated. The complexity of the task is compounded by the ongoing debate as to the distinction, if any, between TTP and non-

53 diarrhoeal HUS (Furlan et al., 1998b; George, 2000) and a variability of ADAMTS 13 levels in TTP associated with different clinical categories (Vesely et al., 2003; Veyradier et al., 2001 ). The lack of an easily performed test and inter assay variability add to the complexity (Studt et al., 2003). With the purification of the protein and the identification of the gene, a simpler and more reproducible test should replace the rather cumbersome tests that are currently in use. Considering the TMAs as a whole, less than half the patients have <10% VWFCP activity (Veyradier et al., 2001) and there is considerable physiological variation with age, pregnancy and in pathological states such as cirrhosis, chronic renal insufficiency, and systemic inflammation (Mannucci et al.,

2001). Less than 5% of normal ADAMTS13 activity may be specific for TTP (Bianchi et al., 2002) but the diagnostic sensitivity can be as low as 13% (Vesely et al., 2003).

TTP in association with certain clinical syndromes such as stem cell transplantation is not associated with a severe reduction in plasma ADAMTS 13 (George et al., 2002). It is possible that the degree of endothelial damage is sufficient to overwhelm the capacity to cleave VWF, assuming the aetiology of the diffuse thrombosis in this instance also involves VWF. A variable proportion of patients with acquired TTP (48-80%) have inhibitory antibodies that neutralize ADAMTS13 (Furlan et al., 1998a; Tsai and Lian,

1998; Veyradier et al., 2001).

The clinical response to fresh frozen plasma and cryosupernatant is due, at least in part, to the replacement of deficient VWFCP, which is estimated to have a circulating plasma half-life of2-3 days. In sporadic TTP/HUS, the presence of an enzyme inhibitor and rapid clearance of the transfused protease and/or ongoing endothelial damage results in

54 the need for prolonged therapy. Observations in a child with chronic relapsing TTP receiving prophylactic FFP, have demonstrated that as little as 5% of protease activity may be sufficient to degrade the ultra large VWF multimers (Barbot et al., 2001 ). The factors contributing to the significant proportion of TMAs with normal VWFCP activity still need to be evaluated. The role of TSP-1 deficiency in the aetiology of TMA was investigated as part of this thesis.

Normal Subject Patient with Thrombotic Th rombocy tope11ic Purpura

Adh, J~r.,n 3nd ..'l< 1t1r1 ,tJ.lt1~1n <,f pl1t,1l1 1l!rl --- c1~.1V·•:l 11n11su:,Uv 1,:uo• n•udcu,wrs ,~r,.,,,,n --~­ Will~I , 13nfl fa, 1, ,r Uncl,•.Jv11d un11~ 1.·,11y ~ lnr,~ -mul!im•·r> 1of ,,.,.) ---- , W1llelJ1;m11 laclor

AOAMTS13 AOAMTS 13

Enrloth Ii.ii EndotMial II cen __...... ,.,-. } Socruu n of mul11 mer~ fr t:' m Wurbol-P.)IJJ,, hot!~

A B

Figure l 7. A schematic representation of events in TTP The absence or severe reduction of ADAMTS 13 in TTP permits the release of UL VWF multimers into the circulation without prior processing. The UL VWF multimers precipitate the formation of occlusive thrombi. Other co-factors such as Factor V lei den and factor H deficiency could play a role. TTP associated with certain clinical syndromes such as stem cell transplantation is not associated with a reduction in ADAMTS I 3. From Moake. JL. N r:nfd J Med 2002: 347 (8) 589-600

55 1.5.2.2 VWF: atherosclerosis and arterial thrombosis

The contribution ofVWF to atherogenesis is unclear. It has been postulated that VWF, by promoting platelet adherence to the subendothelial matrix, facilitates the action of cytokines in promoting smooth muscle cell migration and proliferation (Ruggeri, 1997).

Foamy macrophages, a feature of the atherosclerotic plaque, are derived from circulating monocytes translocating to the subendothelium. It is possible that VWF participates in the recruitment of monocytes, either directly or indirectly via platelets (Diacovo et al.,

1996; Theilmeier et al., 1999). The VWF propolypeptide, which is released along with mature VWF from endothelial cells, is a ligand for a.4~1, an integrin used by monocytes to adhere to endothelial cells. The propolypeptide, therefore may also participate in the recruitment of monocytes (Methia et al., 2001 ).

Early reports of resistance to atherosclerosis in vWD pigs (Fuster and Bowie, 1978;

Fuster et al., 1982) have been tempered by studies demonstrating no difference in neo­ intimal proliferation in arteries subjected to sheer stress (Nichols et al., 1998). The background genetic heterogeneity of the vWD pigs has complicated the interpretation of these studies (Nichols et al., 1992) .. Mice can be made susceptible to atherosclerosis by breeding low density lipoprotein receptor (LDLR)-deficient mice and feeding them a diet rich in fat and cholesterol. Mice lacking both the VWF gene and the LDLR gene

(LDLR ·1·: VWF ·1·) had 40% less atheroma at arterial bifurcation points than their counterparts with the VWF gene (LDLR ·1·: VWF +/+) when fed a diet rich in fat and

56 cholesterol (Methia et al., 2001 ). This study supports the view that VWF plays a significant role in atherogenesis at points of turbulent blood flow.

In relation to the role ofVWF in predicting future coronary events, conflicting results have emerged from two large clinical trials. The Northwick Park Heart Study concluded that an elevated baseline VWF antigen (Ag) level was not predictive of future ischaemic heart disease (Meade et al., 1994) but the Caerphilly Heart Study (Rumley et al., 1999) demonstrated an increased odds ratio of 1.84 (p=0.02). High mean VWF Ag levels at the time of a myocardial infarction have been shown to predict recurrence (Jansson et al., 1998). Although the role ofVWF in plaque generation and instability is uncertain, it is of critical importance in the development of an occlusive thrombus following plaque rupture (Ruggeri, 2000; Sporn et al., 1987). Stroke and myocardial infarction have been described in vWD but at a lower than expected frequency. A risk analysis based on vWD subtypes has not been performed. The role ofVWF multimer size as opposed to total Ag in these events is also not known. Subtle variation in the average VWF multimer size may be a risk factor in cardiovascular disease but proving the effect of a single variable in a complex lesion is difficult and prone to error. With the advent of a sensitive ELISA assay for the measurement of VWFCP levels it may be possible to investigate a link between impaired processing of ultra large VWF multimers and arterial disease. It is notable that in a survey of allelic variants in a large set of vascular biology genes in families with premature coronary artery disease, a missense variant in thrombospondin-1 (N700S) was associated with an adjusted odds ratio for coronary artery disease of 11.90 (P=0.041) in homozygous individuals, who also had the lowest

57 level ofthrombospondin-1 by plasma assay (P=0.0019). The N700S polymorphism causes a local change that sensitizes the calcium binding repeats to removal of Ca2+ and thermal denaturation (Hannah et al., 2003). Whether this association reflects a perturbation of control of VWF multimer size is not known.

1.5.2.3 Pre-eclampsia, eclampsia and the HELLP syndrome

Microangiopathic haemolysis, severe thrombocytopaenia, renal impairment and neurological dysfunction are common to TTP-HUS, pre-eclampsia/eclampsia and the

HELLP (haemolysis, elevated liver function tests, and low platelets) syndrome. Despite the common clinical syndrome they probably have distinct aetiologies as suggested by the markedly different strategies used in therapy. Clinical suspicion of TTP-HUS requires urgent intervention with plasma exchange while pre-eclampsia and HELLP syndrome typically resolve spontaneously following delivery. There is a fall in

ADAMTS13 levels during the second and third trimesters of normal pregnancies

(Mannucci et al., 2001) with 12-31 % of women with TTP-HUS presenting during or immediately after pregnancy (McMinn and George, 2001). Although ADAMTS13 levels can be less than 5% in pregnancy associated TTP-HUS, the diagnostic sensitivity is only -20% and is a poor predictor ofresponse to plasma exchange as patients with levels >25% achieved a similar therapeutic outcome (Vesely et al., 2003). Variations in

ADAMTS13 in pre eclampsia and HELLP syndrome are not known at present. The

VWF Ag level is elevated in parallel with the degree of severity of pre eclampsia probably as a result of the marked endothelial cell activation. A full range ofVWF

58 multimers is present (Bergmann et al., 1991; Brenner et al., 1989) but unlike in TTP­

HUS the ULVWF fraction is not specifically increased (Thorp et al., 1990). Whether the increase in the total concentration of ultra large VWF multimers contributes to the progression of the disease is not known. A case of severe PIH in a patient with type 2A vWD has been described (Jones et al., 1999) but whether the overall incidence of PIH is reduced in vWD is also not known.

59 Chapter 2

The von Willebrand Factor reducing activity of

thrombospondin-1

60 2.1 SUMMARY

Plasma von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury, however only the very large VWF multimers are effective in promoting platelet adhesion in flowing blood. The multimeric size of VWF can be controlled by the glycoprotein, thrombospondin-1 (TSP-1), which facilitates reduction of the disulfide bonds that hold VWF multimers together. The thrombospondin family of extracellular glycoproteins consist of five members in vertebrates, TSP-1-4 and TSPS/COMP. TSP-1 and TSP-2 are structurally similar trimeric proteins composed of disulfide-linked 150-kDa monomers. Recombinant pieces of TSP-1 and TSP-2 incorporating combinations of domains that span the entire subunit were produced in insect cells and examined for VWF reductase activity. VWF reductase activity was present in the Ca2+-binding repeats and C-terminal sequence of

TSP-1, but not ofTSP-2. Alkylation ofCys974 in the C-terminal TSP-1 construct, which is a Ser in TSP-2, ablated VWF reductase activity. These results imply that the reductase function ofTSP-1 centers around Cys974 in the C-terminal sequence.

61 2.2 INTRODUCTION

Platelet adhesion to von Willebrand factor (VWF) in the subendothelium of a damaged blood vessel is the initial step in formation of a haemostatic plug at high shear rates

(reviewed in (Sadler, 1998)). VWF also acts in synergy with fibrinogen in the formation of inter-platelet adhesive links to form a stable thrombus at arterial shear rates (Ruggeri et al., 1999; Savage et al., 2002). As a carrier for pro-coagulant factor VIIl, VWF prolongs its survival in the circulation by protecting it from inactivation by activated protein C and factor Xa. VWF is synthesized by vascular endothelial cells and megakaryocytes and circulates in blood as a series of multimers containing a variable number of-500 kDa homodimers (Counts et al., 1978). The largest VWF multimers have a molecular mass of -20,000 kDa, comparable in length to the diameter of a medium platelet (2 µM), and are released from endothelial cells following stimulation.

The assembly ofVWF multimers follows a stepwise process. Pro-VWF dimers are assembled in the endoplasmic reticulum via disulfide bridges between Cys residues located in the Cys-knot like domains at the C-terminal ends of the pro-VWF subunits.

Inter-subunit disulfide bonds involve one or three of the Cys residues at positions 2008,

2010 and 2048 (Katsumi et al., 2000). These tail-to-tail linked pro-VWF dimers are subsequently multimerized within the Golgi apparatus by head-to-head linkage by disulfide bonds near the N-terminal domains (Wagner et al., 1987). Inter-dimeric disulfide bonds involve Cys379 and one or more of the Cys residues at positions 459,462,

62 and 464 (Dong et al., 1994). After multimerization, the VWF propeptides are removed by proteolysis (Wagner et al., 1987).

Only large multimeric forms of VWF are haemostatically active (Furlan, 1996). The unusually large VWF multimers secreted by endothelial cells have been shown to be more effective than the largest plasma forms in inducing platelet aggregation under conditions of high fluid shear (Moake et al., 1986). This functional importance of multimer size relates to the affinity of VWF for its ligands. Large VWF multimers bind with a -100 fold greater affinity to both collagen and platelets than monomeric VWF

(Furlan, 1996). Some thrombotic disorders are characterized by altered VWF multimer size. Thrombotic thrombocytopenic purpura (TTP) is often associated with unusually large VWF multimers in the blood, which are thought to precipitate intravascular platelet clumping (Moake, 1997; Moake et al., 1982). Conversely, lower than average multimer size characterizes the bleeding diathesis of type IIA von Willebrand disease.

Modulation of VWF multimer size is, therefore, critical to the control of its haemostatic activity.

We recently reported that the homotrimeric glycoprotein, thrombospondin-1 (TSP-1)

(for review see (Lawler, 2000)), reduces the average multimer size of plasma or purified

VWF both in vitro and in vivo (Xie et al., 2000; Xie et al., 2001). Incubation ofTSP-1 with VWF results in formation ofthiol-dependent complexes of TSP-1 and VWF, generation ofnewthiols in VWF and reduction in the average multimer size ofVWF.

Moreover, the ratio of the concentrations ofTSP-1 and VWF in plasma reflect the

63 average multimer size ofVWF. The higher the plasma TSP-1 :VWF molar ratio the smaller the average VWF multimer size. These results indicate that TSP-1 regulates the multimeric size and therefore haemostatic activity ofVWF. We show herein that the

VWF reductase activity of TSP-1 resides in the Ca2+ -binding and C-terminal sequences and requires a free thiol at Cys974.

2.3 MATERIALS AND METHODS

Proteins and reagents

2-nitro-5-thiocyanobenzoic acid (NTCB), 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB),

N-ethylmaleimide (NEM), reduced glutathione (GSH) and EDTA were from Sigma, St.

Louis, MO. 3-(N-maleimidylpropionyl) biocytin (MPB) was from Molecular Probes,

Eugene, OR. Purified human plasma VWF and recombinant human VWF was used in this study. Plasma VWF was a gift from Dr. Michael Berndt, while recombinant VWF produced in baby hampster kidney cells and purified by immuno-affinity chromatography was a gift from Dr. Eric Huizinga. VWF concentration was measured by BCA protein assay (Pierce, Rockford, IL ). TSP-1 was purified from human platelet concentrates as described previously (Murphy-Ullrich and Mosher, 1985) with some modifications (Hogg et al., 1997). Buffers containing 0.1 mM CaCh were used throughout the chromatographic purification of TSP-1. TSP-1 concentration was

64 calculated using an absorption coefficient for a 1% solution at 280 nm of 10. 9. All other reagents were of analytical grade.

TSP-1 and TSP-2 fragments

Modular pieces of TSP-1 and TSP-2 were expressed using a baculovirus expression system and purified by nickel chelate chromatography as described (Misenheimer et al.,

2001; Misenheimer et al., 2000; Mosher et al., 2002). Briefly, DNA encoding for TSP-1 proteins NoC-1 (residues 1-356), CP123-1 (294-529), P3E123-1 (473-673), E3CaG-1

(630-1152) and delNo-1 (294-1152) and TSP-2 proteins delNo-2 (290-1154) and

E3 CaG-2 ( 632-1154) were cloned into the baculovirus transfer vector pAcGP67. coco.

The Baculogold transfection module (BD Biosciences Pharmingen, San Diego, CA) was co-transfected with a pAcGP67.coco clone into SF9 cells. Individual viral clones were isolated, and high titre viral stocks(> 108 pfu.rnL-1) were obtained. The TSP-derived proteins were expressed by infection of High Five cells in SF900II serum free media at a

MOI (multiplicity of infection) of -5 followed by growth at 27°C for 65 to 72 hours.

The His-tagged proteins were purified from the conditioned media by nickel chelate chromatography.

Assays for VWF Multimer Size

VWF (8 nM) was incubated with TSP-1 or TSP-1/TSP-2 fragments (0.8, 8 or 80 nM) for

1 hat 37°C. All dilutions were made with 50 rnM HEPES, 0.125 M NaCl, pH 7.4 buffer

(HEPES-buffered saline) containing 0.1 rnM CaClz. Aliquots (30 µL) of the reactions

65 were diluted 20-fold in 20 mM imidazole, 5 mM citric acid, 0.12 M NaCl, pH 7.3 buffer containing 5% bovine serum albumin and assayed for collagen binding affinity and

VWF antigen as described by Favaloro et al. (Favaloro et al., 1991) and Xie et al. (Xie et al., 2000). Reactions were assayed in triplicate for collagen binding affinity and VWF antigen and the ratio of the two measurements was reported. The overall error was calculated by adding the relative errors (1 SD) of each measurement. The data groups were compared using a one-way ANOVA and a Tukey post-hoe test was applied to compute significance between groups.

Citrated normal plasma was incubated with an equal volume of purified platelet TSP-1,

E3CaG-1 or E3CaG-2 in HEPES-buffered saline containing 50 mM CaCh and 10 µM

D-Phe-Pro-Arg-chloromethylketone (Calbiochem-Novabiochem, Bad Soden, Germany) for 1 hat 37°C. The final concentrations ofTSP-1, E3CaG-1 and E3CaG-2 were 40 nM,

400 nM and 400 nM, respectively. Aliquots of the reactions (10 µL) were resolved on

1% agarose gel electrophoresis (Ruggeri and Zimmerman, 1980), transferred to polyvinylidene difluoride (PVDF) membrane (DuPont NEN, Boston, MA), blotted with

2 µg.mL- 1 of peroxidase-conjugated anti-VWF polyclonal antibodies (DAKO,

Carpinteria, CA) and visualized using chemiluminescence (DuPont NEN, Boston, MA).

Assay for Formation of new Thiols in VWF

The biotin-linked maleimide, MPB, was used to measure reduction ofVWF disulfide bond(s) by TSP-1 or TSP-1/fSP-2 fragments. The protocol was essentially as described

66 by Xie et al. (Xie et al., 2000). Briefly, aliquots (250 µL) of the incubation mixtures used to measure VWF multimer size were labeled with MPB (100 µM) for 10 min at

37°C and the unreacted MPB was quenched with GSH (200 µM) for 10 min at 37°C.

The MPB-labeled VWF was incubated in microtiter plate wells coated with anti-human

VWF polyclonal antibodies (DAKO, Carpinteria, CA), and the biotin label was detected using StreptABComplex/HRP (DAKO, Carpinteria, CA). The reactions were assayed in triplicate and the mean and 1 SD is reported.

Quantitation of thiols in E3CaG-1

The number ofthiols in E3CaG-l were measured using DTNB. E3CaG-l (-10 µM) was incubated with DTNB (-1 mM) in 0.1 MHEPES, 0.3 MNaCl, 10 mM EDTA, pH 7.0 buffer for 10 min at room temperature and the TNB was measured from the absorbance at 412 nm using a Molecular Devices Thermomax Plus (Palo Alto, CA) microplate reader. The extinction coefficient for the TNB dianion at pH 7.0 is 14,150 M-1cm-1 at

412 nm (Riddles et al., 1983 ).

Alkylation of E3CaG-1 with maleimides

E3CaG-l (0.5 mg_mL- 1) was incubated with 10 mM NEM for 20 h in HEPES-buffered saline containing 0.1 mM CaCh, and the excess NEM was removed by dialysis against the same HEPES buffer. The unreacted and alkylated E3CaG-l (10 µg_mL- 1) was labeled with MPB (100 µM) for 30 min at room temperature in HEPES-buffered saline containing 10 mM EDTA. Samples of the labeled proteins (0.2 µg) were resolved on 8-

67 16% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, transferred to PVDF membrane, blotted with a 1 :2000 dilution of streptavidin-peroxidase (DAKO, Carpinteria, CA) and visualized using chemiluminescence.

Mapping the free thiol in E3CaG-1

NTCB specifically S-cyanylates Cys thiols and the peptide bond on the N-terminal side of the cyanylated Cys is then cleaved under mildly alkaline conditions (Jacobson et al.,

1973; Wu et al., 1996), E3CaG-1 (0.2 mg.mL-1) in 0.1 M Tris, pH 8.0 buffer containing either 0.1 mM or 2 mM Ca2+ was incubated with NTCB (20 mM) for 60 minutes at 37°C to cyanylate the Cys thiols. The pH of the reaction was adjusted to 9.0 using 3.0 M Tris base and incubated at 37°C for a further 60 minutes. The resulting fragments were reduced with dithiothreitol, alkylated with iodoacetamide and resolved by 8-16% SDS­

PAGE. The Coomassie-stained fragments were cut from the gel and analyzed by mass spectrometry.

Peptide mass fingerprinting

The SDS-PAGE gel pieces were completely destained in 1 :1 acetonitrile and 25 mM

NHJICQ3, (4 x 200 µl, 30 min) then acetonitrile (100 µl, l0min) and dried in a vacuum

centrifuge. The gel pieces were rehydrated in 20 µL of 10 µM NHJICO3 containing

-10 µg.mL- 1 trypsin and incubated at 37°C overnight. Aliquots (0.5 µl) of each sample were added to a matrix (1 µL of 10 mg.mL-1 2,5-dihydroxybenzoic acid) and spotted on

68 to a 100 well sample plate and analyzed by matrix-assisted laser desorption/ionization reflectron time of flight mass spectrometry (MALDI-rTOFMS) as described (Raftery et al., 2001). The molecular mass profiles of the trypsin-digested fragments were then compared with the theoretical tryptic digestion ofE3CaG-1.

N and C terminal VWF dimers as substrates for TSP-1 reductase activity

The glycosylated N terminal VWF Al domain dimers, incorporating residues 445-733 were produced in Drosophila melanogaster cells and were a gift from Dr. Zaverio

Ruggeri and the C terminal CK dimers incorporating residues 1957-2050 were produced in High FiveTM cells and were a gift from Dr J Evan Sadler (Katsumi et al., 2000). The proteins were incubated with TSP-1 for lh at 37°C and the products resolved by 4-20%

SDS-P AGE and stained with either coomassie blue or a silver stain.

2.4RESULTS

TSP-1 and TSP-2 fragments

The domain structures ofTSP-1 and TSP-2 and the recombinant pieces used in our experiments are shown in Fig. 1A. Overlapping TSP-1 constructs span the entire subunit. The TSP-2 constructs focused on the region that was active in the TSP-1 constructs. The SDS-PAGE profile of the reduced pieces are shown in Fig. lB.

Without reduction, all migrated similarly except for NoC-1, which was trimeric.

69 TSP-1 subuntt B A

1 861 ~~ 160· 1162 294 6211 m 80- • ~ 1 :...... 1El!C!0-1 f...... : , ..... ~ ...... 70- 11.62 294 ~-----i~t------50- 40- 30- TSP-2 subuntt 20-

m 1~ ...... j El!C!0-2 f...... :

11.6 4 290 >... ···································· .....-.·.········· ··idelND-21 =···•·

s Figure 1. TSP-1 and TSP-2 fragment molecular architectw-e , TSP-l and TSP-2 , share a similar A The subgroup A thrombospondins by a connecting region that domain at the N-terminus fo llowed comprising a unique heparin-binding e module, three properdin-like or disulfide bonds, a procollagen-lik links three subunits via inter-chain , seven type 3 repeats ( 12 unique growth factor-like or type 2 modules type I modules, three epide1mal contains the sequence. The NoC-1 fragment loops) and a unique C-tem1inal calcium binding at mature proteins. TSP-1 begins . The residue numbers are for the c01mecting region and is trime1ic 1 and TSP-2 fragments. Samples . B SOS-PAGE profile of the TSP- Asn 1, while TSP-2 begins at Glyl 7 and 8) fragments were reso lved TSP-1 (lanes 2-6) and TSP-2 (lanes ( 4 µg ) of TSP-1 (lane I) and the Blue. The positions of conditions and stained with Coomassie on 6-18% SOS-PAGE under reducing M, markers are shown at left.

70 The VWF reducing activity ofTSP-1 was contained in the E3CaG-1 fragment

Increasing concentrations of TSP-1 and TSP-1 constructs were incubated with VWF for

1 hr and VWF reductase activity was identified by the concurrent reduction of VWF multimer size and generation of new thiols in VWF. The collagen binding affinity to

VWF antigen ratio (CBA/VWF Ag) was used as a surrogate measure of the average

VWF multimer size. Reduction of disulfide bonds in VWF was measured from incorporation of the biotin-linked maleimide, MPB.

VWF reductase activity was limited to two TSP-1 constructs, delNo-1 and E3CaG-1, which both contain the third type 2 repeat, the 7 type 3 domains and the C-terminal sequence (Fig. 2). CP123-1 and P3E123-1 were devoid ofVWF reductase activity over the same concentration range used for intact TSP-1, delNo-1 or E3CaG-1.

The NoC-1 fragment was unusual in that it reduced the CBA/VWFAg ratio but did not result in the formation of new thiols in VWF (Fig. 2). This result was confirmed over a

5-log concentration range ofNoC-1 (Fig. 3A). Calcium ions were not required for the

effect of the NoC-1 fragment on the CBA/VWFAg ratio, which is in contrast to the

requirement for calcium ions for the VWF reductase activity of TSP-1 (Fig. 3B) (Xie et

al., 2000; Xie et al., 2001). In addition, incubation ofVWF with NoCl did not change

VWF multimer size measured by agarose gel electrophoresis (not shown). These results

suggest a mechanism for the effect of NoC-1 other than the reduction ofVWF multimer

size. The simplest explanation is that NoC-1 competed with VWF for binding to

71 collagen, although this would imply that the affinity of NoC-1 for collagen is significantly higher than the affinity of intact TSP-1 for collagen.

0.8

------U) -0 ~ 0.6 * C: * * * & * * ~ ;f 0.4 :::, 3 -0 .1 n ~ 0 ~ rt> IDu 0.2 a ------[ r- rr - 2- 0.0 r r r I 11 -.0 - CO CO 0 CO CO 0 CO CO 0 CO CO 0 rCO CO 0 CO CO 0 :z 0 co 0 co 0 co 0 co 0 co 0 co L____J L____J L____J L____J L____J L____J TSP-1 delNo-1 NoC-1 CP123-1 P3E123-1 E3CaG-1 nM

Figure 2. The VWF reducing activity ofTSP-1 is contained in the E3CaG-l fragment VWF (8 nM) was incubated with 0.8, 8 or 80 nM TSP- l , delNo-l , NoC-l, CP123-l , P3 E123-l or E3CaG- l. A liquots of the reactions were anal yzed for the average VWF multimer size and for the generation of new thiols in VWF. The average VWF multimer size was estimated from the ratio of the collagen binding activity and VWF antigen level (open bars). Generation of new free thiols in VWF was assessed from incorporati on of the biotin-linked maleimide, MPB (closed bars). The dotted lines represent no change in VWF multimer size (top line) or thiols in VWF (bottom line). The asterisks indicate significant difference to control (p < 0 05)

72 A ,0, 1! B . . .- ...... - ...- · ... ~ V) O.! o., 0 a- ~ .. ::J r,"' (') 1 • .. ~O.G 1 ~. 10 l ·' t ~ u. Cl. 1r l f g 3: ~ I "'::J Ju l (D 3 u t ' 'ii: ,0.0 5 0 ~ V) 0.2 (D U 0.2 13. ()' ~ V) .- <>: z f- ~ 0.0 ,.00 c;_ Q (I> a 00 w 00 0 «> «> «>. 6 0 w «>. «> 0 0 «> 0 z 0 0 0 «> 0 '+ ~ : .-"" .-"" l~ ..,.J c;_ l) 6CliG-1, nM NoC-1, nM 0 "'f- z + +

Figure 3. The etl'ect ofNoC-1 on the collagen binding of VWF is independent of calcium ions. A VWF (8 nM) was incubated with 0.8 to 80 nM E3CaG-l or 0.008 to 800 nM NoC-1 . Aliquots of the reactions were analyzed for the average VWF multimer size (open bars) and for the generation of new thiols in VWF (closed bars). The dotted lines represent no change in VWF multi.mer size (top line) or thiols in VWF (bottom line). The asterisks indicate significant difference to control (p < 0.05). The small increase in MPB labeling (closed bars) at high concentrations of NoC-1 was not significantly different to control (p > 0.05) B VWF (8nM) was incubated with TSP-1 or the NoC-1 fragment (80 nM) in the absence or presence of EDTA ( I O mM). Aliquots of the reactions were analyzed for the collagen binding affinity and VWF antigen level. The aste1isks indicate significant difference to contrnl (p < 0.05).

73 TSP-2 fragments did not contain VWF reducing activity

Increasing concentrations of recombinant TSP-1 or TSP-2 constructs were incubated with VWF for 1 hr and VWF reductase activity was identified by the concurrent reduction ofVWF multimer size and generation of new thiols in VWF. Neither the delNo-2 nor E3CaG-2 fragments expressed VWF reductase activity (Fig. 4A).

The reduction of the average VWF multimer size by E3CaG-1 but not E3CaG-2 was confirmed by resolving aliquots of the reaction mixtures on 1% agarose gel electrophoresis and Western blotting for VWF (Fig. 4B).

A B 08 •

z ~: 0:, gl ,: 0) ~ 1 ·2 3 4 TIP-1 delNo -2

Figure -t TSP-2 fragments did not contain VWF reducing activity A VWF (8 nM ) was incubated with 0. 8, 8 or 80 nM TSP-1 , de!No-2 or E3CaG-2 . Aliquots of the reacti ons were analyzed fo r the average VWF multirner size ( open bars) and for the generation of new th iols in VWF (closed bars). The dotted lines represent no change in VWF multimer size (top line) or thiols in VWF (bottom lin e) The asteri sks indicate significant difference to control (p < 0 05) B Citra ted nom1 al plasma was incubated with 40 nM TSP-1 (lane 2), 400 nM E3CaG-I (lane 3) or 400 nM E3CaG-2 (lane 4) and ali quots of the reacti ons were resolved on I % agarose gel electrop horesis. The VWF was Western blotted usin g pero:xidase conjugated anti-VWF polyclonal antibodies.

74 Alkylation of the Cys thiol in E3CaG-1 ablated the VWF reducing activity

Each TSP-1 subunit contains a single free thiol at Cys974 when TSP-1 is purified in buffers containing 0.1 mM Ca2+ (Speziale and Detwiler, 1990; Sun et al., 1992). The homologous residue in TSP-2 is Ser, and the Cys in E3CaG-2 are all in disulfides

(Misenheimer et al., 2001). The number ofthiols in E3CaG-l was quantitated using

DTNB and found to be 0.94 mol thiol per mol ofE3CaG-l. E3CaG-l, therefore, contains a single free thiol, like the intact TSP-1 subunit.

The thiol in E3CaG-l in buffer containing 0.1 mM Ca2+ was alkylated with NEM, and the fragment was tested for VWF reductase activity. Extent of alkylation of E3CaG-l was assessed by labeling with the biotin-linked maleimide, MPB, and detecting incorporation of the label by blotting with streptavidin-peroxidase. MPB labeled

E3CaG-l but not the alkylated protein (Fig. 5A), which indicated that the majority of the thiols in the E3CaG-l preparation were blocked by NEM. TSP-1 or unreacted or alkylated E3CaG-l was incubated with VWF for 1 hr, and VWF reductase activity was identified by the concurrent reduction ofVWF multimer size and generation of new thiols in VWF. Alkylation ofE3CaG-l ablated VWF reductase activity (Fig. 5B).

75 B A -··· ------f -·· 1~------1; 1 -.0.6 , 1; ,I 190 120 - ·. ! ~ 85- }{ ~ ! 60 _::,,..., ,,,-,"'i' +-E JCaG-1 Q. ,; 0 04 -l i!; 50- 40 - -i! ,:i: ' ... 20- r ; cc 0,2 1 1 2

U 0.0 _..,j w.. ca1--. ···_·., _.·-·- ·..1··· ·..- · --..,. _ _.....,:l..,_..____....>..J'--"'fl i ::.o Nil 80 8 80 80 nM L..,,,,,.,..,., TSP.-1 EJCaG-1 NEM­ EJCaG-1

the VWF reducing activity Figure 5. Alkylation of the Cys thiol in E3CaG-l ablated - l ) was incubated with MPB, resolved on A E3CaG- l or NEM-alk ylated E3CaG- I (NEM-E3CaG detect the biotin label. The positions ofM, SOS-PAGE and blotted with streptavidin-peroxidase to ed with 80 nM TSP-1 , 8 or 80 nM E3CaG-l markers are shown at left . B VWF (8 nM) was incubat analyzed for the average VWF multimer or 80 nM NEM-E3CaG-I Aliquots of the reactions were VWF (closed bars). The dotted lines size (open bars) and fo r the generation of new thiols in thiols in VWF (bottom line). The asterisks represent no change in VWF multimer size (top line) or indicate sign ificant difference to control (p < 0.05)

76 The Cys thiol in E3CaG-1 was at position 974

Specific chemical cleavage and mass spectrometry was used to establish the position of the free thiol in the E3CaG-1 fragment (Jacobson et al., 1973; Wu et al., 1996). NTCB specifically S-cyanylates unpaired Cys residues at pH 8. Cleavage of the peptide bond

N-terminal to the cyanylated Cys is achieved by transfer/migration of the Cys residue's nitrogen to the cyano group at pH 9, forming a 2-iminothiazolidine-4-carboxylyl (ITC) peptide. This cleavage will occur at every cyanylated Cys, and therefore the number of

ITC peptides corresponds to the number of unpaired Cys in the protein.

E3CaG-1 in pH 8.0 buffer containing either 0.1 mM or 2 mM Ca2+ was reacted with

NTCB and the peptide bond N-terminal of the cyanylated Cys was then cleaved at pH 9.

The protein was reduced and alkylated and resolved by SDS-PAGE. The E3CaG-1 fragment migrated at - 75 kDa, and NTCB cleavage in buffer containing either 0.1 mM or 2mM Ca2+ yielded peptides of -60 and -20 kDa (Fig. 6 inset). The expected molecular mass of the E3CaG-1 fragment was 58849 Da and NTCB cleavage at Cys 974 should yield an N-terminal fragment of 38278 Da and an ITC peptide of20726 Da.

There is agreement between the expected and observed mass of the ITC peptide. The discrepancy in size on SDS-PAGE of the parent molecule and the N terminal peptide is probably due to the high aspartate content (17%) of these fragments.

The three fragments were cut from the gel, digested with trypsin and the resulting peptides analysed by MALDI-rTOFMS (Fig. 6). Residues Asp914-Arg962 mapped to the -60 kDa fragment. This is consistent with NTCB cleavage ofE3CaG-1 at Cys 974.

77 NTCB cleavage at Cys892 or Cys928, for example, would have resulted in part or all of these amino acids being located within the ITC fragment. Furthermore, NTCB cleavage at Cys892 or Cys928 would have resulted in N-terminal/ITC peptides of29011/29838

Da and 32939/25910 Da respectively, which is contrary to the peptide masses resolved by SDS-PAGE. These results indicate that the free thiol in E3CaG-1 was at Cys974, which is the same position of the free thiol in intact TSP-1 (Sun et al., 1992).

78 A 0 • ., 0.'1 2 2 ea~·. mM .. NTCG 160- -

-11 •o-DO- - - -·

ii::.= •u 1•- , :z "' 4 • B ... R-'d- 161>'".L 1 --57 "137,4 , 1 79~ .. ,:ar:to ::Q:>¥. ::C V'l4-i>4'1 1104. 8 -;z,.950 1 :)7::)..0 1t01-9G:f - ..... 1816. 3 '1001·• '1010 -.-, 1017• 1023 - l&.~-..:z 'loao.-'IO'T3 ... 92,4.,0 107...... 1080 7~. 7"7.t. o ,_.,ou, - B~nd ' -- C 1- 1074 I M,. 1092 2 ~-G7 --~ ~ - ,~7~., en C: 3~.3 e1 ..-1 s - -~o f '°"'·· c:: •::SY::\ .O o~-,~::i: ...,..,, ~ - -_____,_ ...... - .....---...-.,.__..._.,...... ,~...,,,---.-..--B-+--n-n_d _ , •_·..,. .-r ..· --f r ,.,...... ~ _.,.,.'I"'' i,. _ ::, 100,.,ou, - .,.,,,,., {'tNlt"' ·,c:n?-1023 'll!!i-.2 I 0:2A-1 030 - "If'_,.., IOOD. O 1037-1040 ,~s, :2. ,~;07'-' 1"4M07 lliiil'f(I.:, - 82,,4 .8 107-1080 T72 ,G IOOG-10V1

- _I j Bond Iii r-·... l ~ ~- I -.- m/.z - - -

Figure 6. The Cys thiol in E3CaG-1 is at position 974. (A) E3CaG-I in pH 8.0 buffer containing either 0.1 mM (lanes 2 and 3) or 2 mM Ca2+ (lanes 4 and 5) was untreated (lanes 2 and 4) or reacted with NTCB (lanes 3 and 5). The peptide bond N-terminal of the cyanylated Cys was then cleaved at pH 9. The protein was reduced and alkylated and resolved by SDS-PAGE (inset). The M, markers are shown in lane l. The three Coomassie-stained fragments were cut from the gel, digested with trypsin and the resulting peptides analysed by MALDI-rTOFMS. The top (B), middle (C) and bottom (D) profiles are of Bands i, ii and iii , respectively, from lane 5. Band i is undigested E3CaG-1, while bands ii and iii are the result of a single cleavage of band i. The M,'s of the major peptides were matched to the E3CaG-l amino acid sequence. The amino acid residues of selected peptides are shown in the tables at the right of the profiles. From Pimanda et al Blood 2002; 100(8) 2832-8

79 The VWF Al domain-'45-733 and CK domain 1957-2050 dimers are not reduced by TSPl

TSP-1 reduces the N and/ or C terminal inter-dimeric disulfide bonds in purified VWF as evidenced by a reduction in VWF multimer size by gel electrophoresis and collagen binding. Whether the Nor C tenninal inter-dimeric disulfide linkages are preferentially targeted for reduction is not known (Fig. 7). Neither the VWF A 1 domain dimers nor the CK domain dimers were susceptible to TSP-1 mediated reduction (Fig. 8). This could reflect a requirement for TSP-1 to dock with specific domains within the VWF molecule not present in the constructs tested and/or a requirement for shear induced strain of the linking disulfides for the efficient reduction of VWF by TSP-1 .

vWF-CP ~-- TSP-1 --~

r- CK dimer i7 A1 dimer

,--c-1 I N I s-s-r&-&-111111111~:~~~1~ ~m,6),.,.S-S1 -,A\- C '----.. N V ) ) Factor VIII Sulfatides Collagen GPllb/llla GPlbN/IX

Figure 7. The VWF Al domain445-733 and CK domain1957-2050 dimers are not reduced by TSP-1 A schematic diagram of the domain structure and orientation ofmultimeric VWF. ADAMTS 13

cleaves VWF at Tyr842-Met843. The N and/ or C terminal interdimeric disulfide bonds are potential targets for TSP-1 reductase activity.

80 Figure 8. The VWF Al domain445.733 and

CK domain1957. 2050 dimers are not reduced by TSPl ·- - ·,.. , ;iai. .... - · • -450- The VWF A [ 44'·733 N tem1inal dimer -go- (- 90kDa; lanes 3, 4) and the VWF CK1951· 2050 C terminal dimer (-35kDa; lanes 5 and 6) -35- were incubated with (lanes 4 and 6) or without (lanes 3 and 5) TSP-l . The proteins were resolved under non-reducing conditions on 4-20% SDS-PAGE and silver stained. TSP-l (- 450kDA; lanes 2, 4 and 6) barely 1 2 3 4 5 6 enters the gel. Lane I was loaded with a molecular weight marker. There is no detectable reduction by TSP-1 of either the A I or CK dimer to its monomeric fonn.

2.5 DISCUSSION

The TSP's are a family of extracellular glycoproteins that participate in cell-cell and cell-matrix communication. They approximate and regulate cytokines at cell surfaces and play a role in the growth and differentiation of tissues. The TSP family consists of five members in vertebrates, TSP-1-4 (Bornstein et al., 1991; LaBell et al., 1992; Lawler et al., 1993; Lawler and Hynes, 1986; Vos et al., 1992)and TSPS (also known as cartilage oligomeric matrix protein) (Old berg et al., 1992). A single member, dTSP, has also been identified in Drosoph;/a (Adams et al. , 2000; Adolph, 2001 ). Based on their molecular architecture, the TSP gene family can be divided into 2 groups. TSP-1 and

TSP-2 (subgroup A) are structurally similar trimeric proteins, composed of identical disulfide-linked 150-kDa monomers (Fig. 1). The members of subgroup B, TSP-3, TSP-

4 and TSP-5/COMP, are pentameric and differ from subgroup A in that they lack the

81 procollagen and properdin modules and contain an extra EGF-like module (reviewed in

(Adams, 2001). The aspartate rich, Ca2+-binding repeats and C-terminal sequence are common to all TSP's and have been extraordinarily well conserved. Using baculovirus­ expressed recombinant overlapping constructs of TSP-1 that span the entire subunit and parallel TSP-2 constructs, we have shown that VWF reductase activity resides in the

Ca2+-binding repeats and C-terminal sequence of TSP-1 (E3CaG-1 ), but not in the parallel sequence of TSP-2 (E3CaG-2). Alkylation of the free thiol at Cys974 in the C­ terminal TSP-1 fragment ablated VWF reductase activity.

Each subunit of TSP-1 contains a free thiol (Speziale and Detwiler, 1990). TSP-2, in contrast, does not contain unpaired Cys (Bornstein et al., 1991; LaBell et al., 1992). The

TSP-1 thiol is remarkably fluid and can reside on any one of 12 different Cys in the

Ca2+-binding repeats and C-terminal sequence ofTSP-1 upon release from platelets and chelation of Ca2+ with EDT A (Speziale and Detwiler, 1990). In contrast, TSP-1 purified in buffers containing 0.1 mM Ca2+ is a homogeneous TSP-1 population in that the free thiol is at Cys974 (Sun et al., 1992). A model of the Cys in the Ca2+-binding repeats and

C-terminal sequence ofTSP-1 is shown in Fig. 9.

82 C-terminal sequence

Ca 2•-binding repeats

Figure 9. Model of the Caz+-binding loops and C-terminal sequence ofTSP-1 and positions of reactive Cys. Small circles represent the relative positions of the 19 Cys in the Ca2+-binding loops and C-terminal sequence. Open circles, shaded circles and solid circles represent Cys not involved in disulfide exchange, moderately involved in disulfide exchange and heavily involved in disulfide exchange, respectively, in Ca2+-depleted TSP-1 (Speziale and Detwiler, 1990). The free thiol is at Cys974 in TSP-1 purified in bufferscontaining0.l mM Ca2+ (Sun et al., 1992) and in E3CaG-l. The dotted lines represent the likely disulfide bonding based on the disulfide connectivity ofE3CaG-2 (Misenheimer et al., 2001) Cys974 may exchange with Cys928 and/or Cys892 (arrows).

We have proposed that nucleophilic attack by a TSP-1 thiol on a VWF inter-subunit disulfide bond results in reduction of the disulfide bond with formation of an intermediate disulfide-linked complex between TSP-1 and VWF. Attack by a second

TSP-1 thiol results in release ofVWF and formation of an intramolecular disulfide bond in TSP-1. We suggest that Cys974 is the TSP-1 thiol that mediates these events. This is supported by the demonstration that the free thiol in the E3CaG-1 fragment resides predominantly, if not exclusively, at Cys 974 and that alkylation of this thiol ablates

83 VWF reductase activity. It remains to be determined whether Cys974 operates in isolation or that exchange ofCys974 with Cys928 and/or Cys892 (Fig. 7), or other TSP-

1 Cys, is important for VWF reductase activity.

Misenheimer et al. (Misenheimer et al., 2001) have determined the disulfide connectivity ofE3CaG-2. The disulfide pairing of the 18 Cys in the Ca2+-binding repeats and C-terminal sequence is sequential, that is a 1-2, 3-4, 5-6, etc. pattern. The corresponding disulfide connectivity in the Ca2+-binding repeats and C-terminal sequence ofTSP-1 is shown in Fig. 7. It will be informative if the unpaired Cys974 in

TSP-1 results in different disulfide connectivity.

TSP3 (Vos et al., 1992), TSP4 (Lawler et al., 1993), TSP5/COMP (Oldberg et al., 1992) and dTSP (Adams et al., 2000; Adolph, 2001) each contain an unpaired Cys, although it is not at a position equivalent to Cys974 in TSP-1 but is very close to the C-terminus. It may be that these other TSP family members also have VWF reductase activity, although their tissue distribution (cartilage, bone, ligaments, lung and brain) does not support this function in vivo (Iruela-Arispe et al., 1993; Tooney et al., 1998; Tucker et al., 1995). In contrast, TSP-1 is readily demonstrable in and around blood vessels, which is where VWF acts and the regulation of its multimer size has functional relevance.

84 Chapter 3

Role of thrombospondin-1 in control of von Willebrand

factor multimer size in mice

85 3.1 SUMMARY

Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF can be controlled by proteolysis at the

Tyr842-Met843 peptide bond by ADAMTS13 or cleavage of the disulfide bonds that hold

VWF multimers together by thrombospondin-1 (TSP-1 ). The average multimer size of plasma VWF in TSP-1 null mice was significantly smaller than in wild type mice. In addition, the multimer size ofVWF released from endothelium in vivo was reduced more rapidly in TSP-1 null mice than in wild type mice. TSP-1, like ADAMTS13, bound to the VWF A3 domain. TSP-1 in the wild type mice, therefore, may compete with ADAMTS13 for interaction with the A3 domain and slow the rate ofVWF proteolysis. TSP-1, unlike ADAMTS13, is stored in platelet a-granules and is released upon platelet activation. Accordingly, platelet VWF multimer size was reduced upon lysis or activation of human and wild type murine platelets but not TSP-1 null murine platelets. This difference was reflected as an increase in collagen and VWF-mediated aggregation of the TSP-1 null platelets under both static and shear conditions. These findings indicate that TSP-1 influences plasma and platelet VWF multimeric size differently and may be more relevant for control of the platelet VWF pool.

86 3.2 INTRODUCTION von Willebrand Factor (VWF) serves a critical role in hemostasis by facilitating the initial tethering of platelets to the subendothelium at high shear (reviewed in (Sadler,

1998)). VWF is synthesized by megakaryocytes and endothelial cells and circulates in blood as a series of multimers made up of a variable number of disulfide linked 500-kDa homodimers (Counts et al., 1978). Only the larger multimers ofVWF are effective in hemostasis as a selective deficiency of these forms is associated with a bleeding diathesis, type IIA von Willebrand disease (Furlan, 1996). The intracellular assembly of

VWF multimers is a stepwise process. First, individual pro-VWF subunits are linked in a tail to tail orientation by C-terminal disulfide bonds to form pro-VWF dimers, which are then linked in a head to head orientation by N-terminal disulfide bonds to form VWF multimers. The largest VWF multimers have a molecular mass in excess of20,000 kDa

(reviewed in (Wagner, 1990)).

The VWF secreted constitutively from endothelial cells is composed of dimers and small multimers in contrast to the ultra large multimers that are released from the storage compartment (Sporn et al., 1986). The Weibel-Palade bodies in endothelial cells and the a.-granules of platelets release ultra large VWF in response to vascular injury and platelet activation. The largest VWF multimers in plasma are smaller than those stored within endothelial cells and platelets (Moake et al., 1982). Two mechanisms operate in regulating VWF multimer size. Shear dependent hydrolysis of VWF multimers by the

VWF cleaving metalloproteinase, ADAMTS13 (Furlan et al., 1996; Tsai, 1996;

87 Fujikawa et al., 2001) (~ gisintegrin-like ~nd metalloprotease with thrombo~pondin type

1 motif) and cleavage of the linking disulfides by the plasma and platelet glycoprotein, thrombospondin-1 (Xie et al., 2001 ).

A severe deficiency of ADAMTS13 is associated with congenital and acquired forms of thrombotic thrombocytopenic purpura (Levy et al., 2001; Furlan et al., 1998; Tsai and

Lian, 1998), a disorder characterized by a schistocytic hemolytic anemia, a consumptive and variable degrees of renal and neurological impairment. The persistence of unprocessed ultra large VWF multimers in the circulation is thought to precipitate platelet clumping in arterioles and capillaries resulting in tissue ischaemia

(Moake et al., 1982). The full length ADAMTS13 transcript is expressed predominantly in the liver (Levy et al., 2001; Zheng et al., 2001) and appears to cleave ultra large VWF multimers as they are secreted from endothelial cells and undergo conformational change when exposed to high shear (Dong et al., 2002a).

The thrombospondins are a family of extracellular glycoproteins that function in cell-cell and cell-matrix communication and modulate cellular phenotype (Lawler, 2000). We reported that the supernatant of cultured endothelial cells possessed VWF reductase activity and identified TSP-1 as a VWF reductase (Xie et al., 2000; Xie et al., 2001).

More recently we showed that the VWF reducing activity of TSP-1 centers on a free thiol at Cys974 in the Ca2+-binding C-terminal sequence of the protein (Pimanda et al.,

2002). The role of TSP-1 in regulating VWF multimer size in vivo and its relationship to ADAMTS13 activity was unknown.

88 To clarify these issues, we have characterized the plasma and platelet VWF multimer pattern in TSP-1 _,_ and TSP-1 +t+ C57Bl/6J mice. Incubation of VWF with TSP-1 in vitro results in smaller VWF multimers. Surprisingly, TSP-1 contributes to the persistence of larger VWF multimers in the circulation possibly by negatively regulating

ADAMTS 13 activity. On the other hand, platelet VWF multimer size was reduced upon lysis or activation of wild type platelets but not TSP-1 null platelets. This difference has functional relevance as the TSP-1 null platelets exhibited an increase in collagen and

VWF-mediated aggregation under static and shear conditions. We discuss the implications of these findings with regards to the initiation and development of an arterial thrombus.

89 3.3 MATERIALS AND METHODS

Proteins and Reagents. Leupeptin, D-Phe-Pro-Arg-chloromethyl ketone (PPACK) and

4-(2-aminoethyl) benzensulfonyl fluoride (AEBSF) were from Calbiochem (La Jolla,

CA) and bovine thrombin, N-ethylmaleimide (NEM), ethylenediaminetetraacetic acid

(EDT A), 1, 10-phenanthroline, phenylmethanesulphonyl fluoride (PMSF), Triton X-100 and Tween-20 were from Sigma (St Louis, MO). Aprotinin was from Bayer AG

(Leverkusen, Germany) and 1-desamino-8-D-arginine vasopressin (desmopressin) was from Ferring AB (Limhamn, Sweden). TSP-1 was purified from human platelet concentrates (Lawler et al., 1998); the recombinant TSP-1 fragments, CPI 23-1 (residues

294-529) and E3CaG-1 (630-1152), were expressed in insect cells and were a gift from

Dr. Deane Mosher (Kundu et al., 1995). The residue numbers are for the mature protein.

The recombinant VWF A3 domain was expressed in E. coli and purified from bacterial inclusion bodies and was a gift from Dr. Miguel Cruz (Cruz et al., 1995). The anti-TSP-

1 monoclonal antibody, mAb 133, was a gift from Dr. Joanne Murphy-Ulrich and

HB8432 was produced from a murine hybridoma cell line obtained from American Type

Culture Collection, Bethesda, MD. All other reagents were of analytical grade.

Animals. Wild type (TSP-1 +/+) and TSP-1 null (TSP-1-1-) C57BL/6J mice were used in this study. TSP-1 _1_ mice (Lawler et al., 1998) were from Dr. Jack Lawler's laboratory

(Boston, Mass) and raised in the biological resource centre at the University of New

South Wales (Sydney, NSW). Animals aged between 6 weeks and 6 months and of both

90 sexes were used. The experimental procedures were approved by the Animal Care and

Ethics Committee of the University of New South Wales.

Preparation of plasma. The mice were anaesthetized with isoflurane (Abbott

Laboratories, IL) and blood collected by cardiac puncture using a I mL syringe and a

26-guage needle and mixed with 3.8% trisodium citrate anticoagulant in a 6:1 vol/vol ratio. For the desmopressin time course study, free flowing blood from the saphenous vein was collected via heparin-coated capillary tubes and mixed with citrate anticoagulant as above. To evaluate human plasma and platelet VWF, blood was collected by venepuncture using an 18-guage needle and a 20 mL syringe and mixed with citrate anticoagulant.

Platelet rich plasma was obtained by centrifuging blood at 150 g for 20 min. The platelet rich plasma was then spun at 500 g for a further 20 min to obtain a platelet pellet. The supernatant was re-centrifuged at 2000 g for a further 15 min to prepare platelet poor plasma. A platelet count was performed to confirm the absence of platelets and aliquots of platelet poor plasma were frozen at -80°C till further use. For the experiments using pooled mouse plasma, equal volumes of blood were pooled prior to centrifugation.

Preparation of washed platelets. The platelets obtained from platelet rich plasma were washed thrice with 5 mM Pipes, pH 6.8 buffer containing 0.145 M NaCl, 4 mM KCl, 0.5 mM NaiHPO4, 1 mM MgC}z and 5 mM dextrose. The platelet pellet after the final wash was resuspended in 20 mM Hepes, pH 7.4 buffer containing 0.137 M NaCl, 4 mM KCl,

91 0.5 mM NaiHPO4, 0.1 mM CaChand 5 mM dextrose (platelet Hepes) and the platelet count adjusted to 2 x 109 per mL.

Preparation of a platelet lysate. Suspensions of washed platelets were centrifuged at

7000 g for 2 min and the platelet pellet resuspended in 20 mM Hepes, pH 7.4 buffer containing 0.14 M NaCl, 1% Triton X-100, 0.05% Tween 20, 10 µM leupeptin, 2 mM

PMSF, 1 mM AEBSF, 1 mM phenanthroline and 10 µM aprotinin (lysis buffer), and incubated at 3 7°C for 3 0 min. 5 mM EDT A and 10 mM NEM were added to the lysis buffer either before or 5 min into the incubation period. The suspensions oflysed platelets were then centrifuged at 7000 g for 2 min and aliquots of the supernatant stored at -80°C till further use.

Thrombin activation of platelets and preparation of platelet releasate. A suspension of washed platelets was pre-incubated at 37°C for 30 min. Aggregation was performed at

37°C by adding 1 U.mL"1 thrombin to a continuously stirred suspension of platelets.

The platelets were stirred for a total of 10 min with the addition of 5 µM PPACK, 10 µM leupeptin, 1 mM AEBSF, 1 mM phenanthroline, 2 mM PMSF and 10 µM aprotinin at 2 min and 5 mM EDT A and 10 mM NEM at 5 min. The suspension was centrifuged at

7000 g for 2 min and aliquots of the supernatant stored at -80°C till further use. The aggregated platelet pellet was then lysed with lysis buffer containing 5 mM EDT A and

10 mM NEM and incubated at 37°C for 30 min. The lysed platelets were then centrifuged at 7000 g for 2 min and aliquots of the supernatant stored at -80°C.

92 The measurement of VWF multimer size. Two measurements of multimer size were performed; agarose gel electrophoresis and collagen binding. The samples were assayed using an in-house method at the Royal Prince Alfred Hospital (Sydney, Australia) modified from Ruggeri et al (Ruggeri and Zimmerman, 1980). Briefly, aliquots of murine and human plasma, platelet lysate and releasate were diluted 10-fold in a 200 mM Tris, pH 6.8 buffer containing 10% glycerol, 1 % SDS, 2 mM EDTA and 0.01 % bromophenol blue and warmed to 56°C for 20 min. 20 µL of the mix was loaded onto a

1% agarose gel (Seakem™ HGT(P) agarose, Cambrex, Santa Rosa CA) in Tris-glycine­

SDS electrophoresis buffer, pH 8.3 mounted on a Protean™ II xi cell system (Bio-Rad,

Hercules, CA) and electrophoresed at 4°C at 125 V for 5 h. The gels were fixed and the non-specific binding sites blocked with skim milk, followed by an overnight incubation with 1251-labeled anti-human VWF polyclonal antibodies (Dako, Carpinteria, CA). The gels were then washed in distilled water, dried on gelbond film (BMA, Rockland, ME) and exposed to Kodak BIOMAX MS film. The autoradiographs were developed at 48 h.

Densitometry was performed using a Fluor-S Multilmager and Quantity One software from Bio-Rad, Hercules, CA.

Aliquots of murine and human plasma, platelet lysate and platelet releasate were diluted

10-fold in 20 mM imidazole, pH 7.3 buffer containing 0.12 M NaCl, 5 mM citric acid

(ELISA buffer) and 5% bovine serum albumin (BSA) and the VWF collagen binding affinity and VWF antigen level were each assayed in triplicate as described by Favaloro et al. ( Favaloro et al., 1991 ). Murine VWF was detected using a 1000-fold dilution of peroxidase-conjugated anti-human VWF polyclonal antibodies (Dako, Carpinteria, CA)

93 in ELISA buffer containing 0.1 % BSA The human samples were diluted 100-fold in

ELISA buffer containing 5% BSA The optical densities (O.D.) of the ELISA plate were monitored in the plate reader and the reactions stopped at 0.2-0.3 O.D. rather than at a fixed time point for improved accuracy of the individual measurements. Although this results in a variation of the CBANWF: Ag ratio of plasma and platelet VWF between experiments, this was considered less important than the accuracy of each measurement for a given experiment.

Binding ofTSP-1 to VWF A3 domain. VWF A3 domain (1 µM solution in 0.1 M

NaHCO3, pH 7.3) was adsorbed to Nunc PolySorp 96-well plates overnight at 4°C in a humid environment. Wells were washed thrice with ELISA buffer/0.1 % BSA, nonspecific binding sites blocked by incubating wells with 200 µl of ELISA buffer/5%

BSA for 90 min at 37°C, and then washed twice with ELISA buffer/0.1 % BSA

Dilutions of TSP-1 or the E3CaG-1 fragment were prepared in 50 mM Hepes buffer, pH

7.4 containing 0.14 M NaCl, 1 mM CaClz, 1 mg.mL·1 PEG 6000 and 1% BSA

Reactions were in 100 µl for 60 min at room temperature with orbital shaking. Wells were washed four times with ELISA buffer/0.1 % BSA and incubated with 100 µl of 5

µg.mL· 1 mAb 133 for 60 min at room temperature with orbital shaking. Wells were washed three times with ELISA buffer/0.1 % BSA and incubated with 100 µl of 1: 1000 dilution of rabbit anti-mouse peroxidase-conjugated antibodies for 30 min at room temperature with orbital shaking. Wells were washed three times with ELISA buffer/0.1 % BSA and the peroxidase detected as previously described ( Favaloro et al.,

1991 ). mAb 133 recognizes an epitope within E3CaG-l. To detect binding ofTSP-1 to

94 VWF A3 in the presence of CP123-l or E3CaG-l, HB8432 was substituted for mAbl33.

HB8432 recognizes an epitope within the epidermal growth factor-like domains of TSP-

1 (Prater et al., 1991) and does not react with either CP123-l or E3CaG-l (data not shown).

Static platelet aggregometry. Pooled platelet rich plasma was prepared as described and the platelet count adjusted to 300 x 109 per L using platelet poor plasma. TSP-1 +I+ and

TSP-l -1- mice platelet rich plasmas were incubated at 3 7°C for 10 min and the platelet aggregation response to SKF HormlM collagen (Nycomed, Ismaning, Munchen) and

ADP (Chrono-Log Corporation, Havertown, PA) measured over 10 min in a platelet aggregometer (Chrono-Log Corporation). The role ofVWF in the formation of a platelet aggregate was investigated by pre-incubating TSP-l-1- mice platelet rich plasma with rabbit anti-human VWF polyclonal antibodies (DAKO, Carpinteria, CA) or control normal rabbit immunoglobulin at 37°C for 30 min prior to the addition of collagen.

Shear-induced platelet aggregometry. 1 mL of whole blood was collected by cardiac puncture from 6 12-week old female TSP-1 +I+ and TSP-1-1- mice and added to 140 µL citrate-phosphate-dextrose anticoagulant. Shear-induced platelet aggregation was performed using a PFA-1001M test system (Kundu et al., 1995) (Dade Behring, Illinois) and a cartridge coated with collagen (fibrillar type 1 equine tendon) and ADP.

Administration of desmopressin. Desmopressin was diluted in sterile saline and infused over 30 min via the tail vein using a 10 mL syringe, "Flowline" SpringfusorlM syringe driver and Springfusor flow control tubing (Pacific Medical Supplies, VIC, Australia)

95 and a 30-guage ½ inch needle. For the time course experiment, the concentration of desmopressin was adjusted to deliver 3 µg per kg at a fixed volume of250 µLover 30 min to 6 TSP-1 +/+ or TSP-1 _,_ mice. Blood was sampled 1 h before desmopressin infusion and at 1 and 6 h post infusion. The first two collections were by saphenous vein bleeds and the third by cardiac puncture. In separate experiments, desmopressin was infused over 30 min to 3 TSP-1 +I+ or TSP-1 _,_ mice and blood collected by cardiac puncture at 1 h in one study and 6 h in another.

Statistics. Comparative data are presented as means± SO. Statistical significance was calculated with a Student's t test for all analyses.

96 3.4RESULTS

The average multimer size of VWF in plasma and platelet samples was estimated using two different measures. Samples were resolved on 1% agarose gel electrophoresis and the VWF detected using 1251-labeled anti-human VWF polyclonal antibodies. The densities of the resulting multimer patterns were quantified and expressed as band intensity as a function of size. To compensate for small variations in protein loading and to better compare average VWF multimer size in a given experiment, the optical densities of the individual lanes were normalized so that the total density for each lane was the same. The collagen binding of the VWF (CBA) was also measured and expressed relative to the total VWF concentration (VWF Ag) in the sample. The

CBANWFAg ratio correlates with the average molecular weight of the intermediate and high VWF multimer forms for a given concentration of VWF (Favaloro et al., 1995).

The overall error for the CBANWF Ag ratio was calculated by adding the relative errors

(1 SD) for the individual CBA and VWF Ag measures.

TSP-1 influences the average multimer size of only the very large VWF multimers (Xie et al., 2000; Xie et al., 2001 ), which are difficult to resolve by gel electrophoresis or other means. Proteolysis of VWF by ADAMTS 13, on the other hand, is readily apparent using this technique. The difficulty in resolving the very large VWF multimers can lead to uncertainly in the interpretation of the electrophoresis experiments.

Importantly, however, the relative average VWF multimer size measured by gel

97 electrophoresis in our study always correlated with the CBNVWFAg ratio in a given experiment.

Plasma VWF multimer size was smaller in TSP-1 null than in wild type mice.

To evaluate the contribution ofTSP-1 to the control of plasma VWF multimer size, we measured VWF multimer size in the plasmas ofTSP-1 +;+ and TSP-1 _1_ mice. Based on the in vitro evidence that TSP-1 reduces VWF multimer size (Xie et al., 2000), we anticipated that in its absence the multimers might be larger in the TSP-1 _1_ mice. To the contrary, in both individual and pooled murine plasma, VWF multimer size was significantly smaller in TSP-1-1- mice plasma.

The ultra large VWF multimers in the plasmas of individual mice were larger in TSP-

1+t+ mice than in TSP-1-1- mice (Fig. IA). This difference was confirmed by densitometry (Fig. IB). The CBA to VWFAg ratios of the TSP-1 +t+ cohort plasmas was also significantly higher than for the TSP-l-1- cohort (Fig. 1C).

To allow for individual variations in multimer size between mice within a cohort, VWF multimer size was also measured in pooled plasma. Blood was collected by cardiac puncture from 12 (6 male and 6 female) TSP-1 +I+ or TSP-l-1- mice and an equal volume of blood from the two groups was pooled. This was repeated using 3 other groups of mice. The VWF multimer size in the pooled plasmas of the TSP-1-1- mice was significantly smaller than in the TSP-1 +t+ mice in each group (Figs. ID and E).

98 A C 1.0

C) 0.8 .,... ~ LI. ••••• i 0.6 cc 0.4 u 0.2

0.0 rsp.1+/+ TSP.1·1·

D E 1 2 3 4 5 6 7 8 -rsp.1+1+ = rsp.1-l· B 0.8 .. .: 1.2 .. .. C - lanes 1-4 >I- .. _:!: ::, >I- .. II lanes 5-8 ~ 0.6 .. .t i - ! o.e . ~ .. ~ 0.4 .. Cl J ,! :,,~ cc i 0 .'1 u 0.2 ~ 0.0 0.0 High M' -+ LO'N M A B C D 1 2

Figure I. Plasma VWF multimer size is smaller in TSP-1 null than in wild type mice.

A. VWF multimer size in the plasmas ofTSP-1 +I+ (lanes 1-4) and TSP-1 -1- (lanes 5-8) mice measured by agarose gel electrophoresis. B. Densitometry traces of the YWF multimer patterns shown in A. C.

VWF multimer size in the plasmas of 11 TSP-1 +i. and TSP-1 -1- mice measured by collagen binding.

D. YWF multimer size of pooled plasma from 4 groups (A-D) ofTSP-1 +t+ and TSP-1 _,_ mice measured by collagen binding. Each group comp1ises 12 (6 male and 6 female) mice. E. VWF multimer size of the group A plasma pools measured by agarose gel electrophoresis. ***, p<0.00 1; ****, p<0.000 1

99 The multimer size of endothelium-derived VWF was reduced more rapidly in TSP-1 null than in wild type mice.

Desmopressin is a synthetic analog of arginine vasopressin. The infusion of desmopressin to mice (Sweeney et al., 1990) and humans (Richardson and Robinson

1985) results in a rapid increase in plasma VWF, which persists for 6 h or more.

Desmopressin is thought to act via vasopressin receptors on endothelial cells to stimulate the release ofVWF from endogenous stores. The increase in plasma VWF is accompanied by an increase in the concentration of ultra large VWF multimers in the circulation (Battle et al., 1987).

The intravenous infusion of desmopressin to TSP-1 +t+ and TSP-1 _1_ mice resulted in an increase in plasma VWF levels which persisted at 6 h. The increase in plasma VWF concentration in TSP-1 +t+ mice (Fig. 2A), however, was accompanied by a greater relative increase in the ultra large VWF multimers than was observed in TSP-1--1- mice

(Fig. 2B). The average multimer size of plasma VWF was larger in TSP-1 +t+ (Fig. 2C) than TSP-1-t- (Fig. 2D) mice at both 1 and 6 h post desmopressin treatment. This result indicated that ultra large VWF multimers released from stimulated endothelial cells in vivo are more efficiently processed in the absence of plasma TSP-1.

100 A C TSP-1+/+ 1 h post desmopressin ~ 1.2 - JSP-1'~ 2.D -~C C - TSP-1" :::, :::, ~ ? 1.5 .; D.I "'C .,C Q Q" 1Jl l .~ 0.4 " 6.5 "., a:" a: 6.1 , =-.• 6.1 -1 1 6h High t4 ~ Low 14 2 3 4 5 6 High 111- ~ Low '4 B D TSP-1 -1- 6 hpost desmopressin

~ -~ 2.0 -TSP-f" 'i: 2.5 C - TSP-1" :::, :::, ? 2.1 ?1.5.. "'C .,C ., 1.5 Q o 1.0 1.0 -~i -~ 'i !0.5 a: 1.5 a:" ... !!_ ""' 0.0 ~!: 1.0 -1 1 6h HighM-~Lowt.1- 1 2 3 4 5 6 High M- ~ Low f4

Figure 2. The multimer size ofendothelium-derived VWF is reduced more rapidly in TSP-1 null than in wild type mice. The variation in plasma VWF multimer size over time in response to desmopressin administration to a TSP-1 ° ' (part A) or TSP-1 - (part B) mouse was assessed by collecting blood at - 1, I and 6 h. Blood was also collected from 6 TSP- I si+ (lanes 1-3) or TSP-1 1· (lanes 4-6) mice I h (part C) or 6 h (part D) post desmopressin infusion. VWF multimer size was measured by agarose gel electrophoresis and densitometry. The results shown in part A was representative of6 experiments.

I 01 TSP-1 bound to the A3 domain ofVWF.

Interaction ofTSP-1 and TSP-1 fragments with the A3 domain ofVWF was measured in an ELISA format. The binding of TSP-1 and E3CaG-1 (which contains the

Ca2+-binding repeats and C-terminal sequence and harbors the VWF reductase activity) to immobilised A3 was specific, as it was competed for by soluble A3 (Figs 3A-D).

Apparent dissociation constants for binding ofTSP-1 or E3CaG-1 to soluble A3 of-3 and -6 µM, respectively, was estimated from the competition experiments. As expected, soluble E3CaG-1, but not the CP123-1 fragment (which contains the procollagen-like module and three properdin-like or type 1 modules), competed with TSP-1 for binding to immobilised A3 (Fig. 3E and 3F). These results indicate that TSP-1, like

ADAMTS13 [Dong et al., 2003], interacts with the A3 domain ofVWF.

102 A TSP-1 subunit ~~~ 294 529 ~52 ~ ---~

B D F

E 1.0 E 1.e C C : 0_12 ~ 0.04 pM TS P-1

lo 0.08 u "C r~ 0::.4 // j::// "' "'5 0.04 ! 0.2 ! 1.2 ] <( <( <( 0.0 1.0 , 0.00 0.0 0.1 1.2 1 2 0.0 0.5 1.0 1.5 2.0 TS P-1, p:M EJ CaG -1,/lM E3CaG -1, ,,rn

C E G

E o.5 0.04pM TS P.1 ~ I .J 0.46 11M EJCaG -1 E 0.J C C ~ 0.4 r, "'...0 ~1.2\ .; 0.2 ; O.J \ u "C ~ 0.2 Jl.1 ~ ,e" 0.1 j 0,1 ' <( i <( 0.0 "-----. ,.o 5 10 15 20 5 10 15 1/Wf A1 , pi/I VWFAl, ,11M

Figure 3. TSP-1 binds to the A3 domain ofVWF. Interaction of TSP- 1 and TSP-1 fra gments with the A3 domain o f VWF was measured in microtitre plate well s using anti-TSP-1 monoclonal antibodies. TSP-1 (part B) and E3CaG-I (part D) bound to immobilised VWF A3 . Binding o f both proteins was specific as it was competed for by soluble A3 . The concentration of soluble A3 th at reduced binding of TSP-1 (0.04 µM, prni C) or E3CaG-I (0 4 6 µM , part E) to immobilised A3 by halfwas-3 and -6 µM , respecti vely. Soluble E 3CaG-l also competed with TSP- 1 (0 04 µM) for binding to immobilised A3 (p rn·t F) E3CaG-l (0 8 µM) but not the CP 123 -1 fragm ent (2 µM) competed fo r binding o f TSP-1 to immobili sed A3 (prn·t G) The data points and brn·s represent th e mern1 and range of duplicate dete1min ations.

103 Platelet VWF multimer size was reduced upon lysis of wild type but not TSP-1 null platelets.

To study the role ofTSP-1 in regulating platelet VWF multimer size, we prepared platelet lysates from TSP-1 +t+ and TSP·1 _,_ mice. The platelet lysates were prepared in the presence of a range of protease inhibitors, including phenanthroline that inactivates

ADAMTS 13 [Tsai, 1996] and either with or without the TSP-1 inhibitors, EDTA and

NEM. The VWF reductase activity of TSP-1 is inactivated by EDTA, which depletes the C-terminal Ca2+-binding repeats of this ion, and by NEM, which alkylates the critical thiol at Cys974. The average VWF multimer size was partially reduced in TSP-1 +t+ but not in TSP-1 _,_ platelets when lysed without the TSP-1 inhibitors (Fig. 4). Lysis ofTSP-

1_,_ platelets in the presence of 10 µg.mL- 1 human platelet TSP-1 resulted in reduction of

VWF multimer size (Fig. 4D). This result indicated that addition of human TSP-1 corrects for the absence of endogenous murine TSP-1 with respect to control of VWF multimer size.

104 A B TSP-1+/+ - platelet lysate + EDTA - platelet lysate · EDTA 1 .8 - plasma

f:::, ~ ! 1 .2 4) c::::, 4) .a: 0.6 l, ~ 0 .0 High M -+ Low M

C TSP-1-I- -platelet lysat,. + EDTA - platelet lysate - EDTA 1 .8 plasma ~ C :::,

~ 1 .2 !a, C a, 1 2 3 4 5 6 ::, 0 .6 ~ i 0 .0 D High M -+ Low M - TSP-1+/+ =TSP- 1-f- ...... - ~-lt.f ......

- ltA*-..-- ~--- I 1.0 : ..:. : - ' ' ~ 0.8 ' u.. ;o ~ 0.6 .. cc< 0.4 u 0.2

0.0 #~ -q

platelet lysate

Figure 4. Platelet VWF multimer size is reduced upon lysis of wild type by not TSP-1 null platelets.

A. Washed TSP-1 H+ (lanes 2 and 3) and TSP-1 -1 (lanes 5 and 6) mice platelets were lysed with a range ofproteinase inhibitors with addition of the TSP-1 inhibitors EDTA and NEM at O (lanes 2 and 5) or 5 (lanes 3 and 6) min, and VWF multimer size measured by agarose gel electrophoresis. TSP- 1' ' (lane I) and TSP-1 (lane 4) mice plasma is shown for comparison. Band C. Densitometry traces of the VWF multimer patterns shown in A D. VWF multimer size of the samples shown in A measured by collagen binding. The lysis ofTSP-1 +i+ platelets resulted in significant reduction in VWF multimer size, which was not evident in TSP-1 platelets. **, p<0.01 ; ***, p<0.00 I

105 TSP-1 null platelets exhibited an increase in collagen and VWF-mediated aggregation under both static and shear conditions.

The aggregation of platelet rich plasma pooled from TSP-1 +;+ or TSP-1 _1_ mice in response to increasing concentrations of collagen and ADP was measured by optical density in a platelet aggregometer (Fig. 6). The TSP-1 -1-, but not TSP-1 +t+ platelets, aggregated in response to 5 µg.1nL- 1 collagen and ADP, although the ADP-mediated aggregation was less pronounced than with collagen. The aggregation in response to collagen was inhibited by anti-VWF antibodies; confirming a central role for VWF in the process.

A B ~------~TSP-1+/+ TSP-1+1+ 100 I I-- --~ 100 Cl) Cl) .s C di 80 ~ 80 ~ ! CJ) (J) 'if. 60 'if. 6

40 4 8 10 .-----,-TSP-1-I------~ TSP-1-I- Time, min , I -- - 0.5 rng .rnL·' control pAb l"'c- ~----1 anti-vWF pAb, mg.ml·' -0.1 - 0 .5

0 4 8 10 Time, min collagen, µg.rn L·1 ADP, µM - 1 - 1 - 2.5 -2.5 - 5 - 5

Figm·e 6. TSP-1 null platelets aggregated in r·esponse to a lower concentration of collagen. The aggregation of platelet rich plasma pooled from 6 female TSP-1 +t+ or TSP-1 _1_ mice in response to increasing concentrations of collagen and ADP is shown in part A. The TSP-1 1 , but not TSP-1 "T platelets, aggregated in response to 5 µg.mL·' collagen and ADP. The aggregation in response to collagen was inhibited by anti-VWF antibodies (part B).

107 The PFA-1 oo™ analyzer (fig. 7 A) measures whole blood flow through an agonist­ coated capillary under high shear (-6,000 s- 1). Collagen and ADP-coated capillaries were used in this study (fig. 7B and 7C). The machine has been validated as a sensitive measure of VWF multimer size and as a screening tool in the diagnosis of VWD

[Fressinaud et al., 1998; Favaloro et al., 1999]. The closure time corresponds to the occlusion of capillary flow through the cartridge due to platelet aggregation. The closure time corresponds to the occlusion of flow through the capillary due to platelet aggregation and is prolonged in the absence of ultra large VWF multimers as in type 2A

VWD. VWF binding to glycoprotein lb and/or anhl3m appears to be the major determinant of closure time [Watala et al., 2003]

A closure time was not recorded for the 6 female TSP-1 +!+ mice due to either the maximum test time of 3 00 sec or the maximum syringe travel (> 250 sec in this sample) being reached. The failure of the wild type murine platelets to occlude the capillary was most likely a consequence of their small.size. The machine is calibrated for human platelets, which are two to three times bigger than murine platelets [Schmitt et al., 2001].

The closure time values for the TSP-1-1- mice ranged from 160-200 sec (mean± SD;

174.8 ± 16.8 sec). Applying 250 sec as the maximum closure time for the control group, p < 0.001 (fig 7D).

108 PFA-100

C

0 ~ ~ c· 300 0 .; Ol ~ 200 Ol Ol ~

~ 100 .,.~ a.

Figure 7 D

109 The average multimer size of the VWF in human platelets is smaller than in murine platelets.

Variation in the pattern and processing ofVWF multimer size in human and murine platelets was evaluated by lysing platelets in the absence or presence of the TSP-1 inhibitors, EDT A and NEM. There was a partial reduction in multimer size of both human and murine platelet VWF upon lysis in the absence of the TSP-1 inhibitors (Fig.

6). This is consistent with the results shown in Fig. 4. Notably, the VWF multimers in human platelets are significantly smaller than in C57BL/6J murine platelets.

110 A B - plateletlys..te+ EDTA Human - platelet lysa!e - EDTA 3 -plasma

High ~ -+ low 1v1-

C - platelet lysa!e + EDTA Murine - platelet lysate - EDTA -plasma

3 - 2 1 2 3 4 5 6

High ru\- -+ Low IVI- D •Human DMurine ,----... --, •--r---~ ' .. 2 .0 ' ""t a, 1.6 ...~ ,.:. ! 1.2 ~ 0 .8 ... =u 0 .4

0 .0 'I,-/ ~~ ~ .,.~ -~~ platelet lysate

Figm·e 7. The average multimer size of the VWF in human platelets was smaller than in murine platelets. A. Washed human (lanes 2 and 3) and wild type murine (lanes 5 and 6) platelets were lysed with a range of proteinase inhibitors with addition of the TSP-1 inhibitors EDT A and NEM at 0 (lanes 2 and 5) or S (lanes 3 and 6) min, and VWF multimer size measured by agarose gel electrophoresis. The 0 min time point is referred to as 'platelet lysate + EDT A' , while the 5 min time point is referred to as ' platelet lysate - EDT A'. Human (lane 1) and murine (lane 4) plasma is shown for comparison. B and C. Densitometry traces of the VWF multimer patterns shown in A. D. VWF multimer size of the samples shown in A measured by collagen binding The lysis of both human and murine platelets resulted in significant reduction in VWF multimer size. **, p<0.01; ***, p<0.001 .

111 3.5 DISCUSSION

Plasma VWF is largely of endothelial cell origin [Jaffe et al., 1974]. The smaller size and presence of proteolytic fragments in plasma support the premise that VWF stored in endothelial cells are modified after release [Tsai et al., 1989; Zimmerman et al., 1986].

TSP-1 reduces VWF multimer size; therefore we anticipated that unless ADAMTS13 could adequately compensate for its absence, plasma VWF multimers in TSP-1 null mice would be larger than in wild type mice. To the contrary, we found that the multimer size was significantly smaller in TSP-1 null mice. In addition, endothelium­ derived VWF was more efficiently processed in the plasma of TSP-1 null mice than in wild type mice. These findings indicate that TSP-1 possibly inhibits the activity of

ADAMTS13 in vivo. A preliminary report of the in vitro inhibition of ADAMTS13 by

TSP-1 supports this premise [Dong et al., 2002b].

Mechanisms by which TSP-1 inhibits ADAMTS13 are speculative and include interference with ADAMTS13 anchorage to the endothelial cell surface [Dong et al.,

2002a], binding to and inhibition of ADAMTS13 activity and/or the inhibition of its interaction with VWF. Binding of ADAMTS13 to the A3 domain ofVWF exposed to the shear force of flowing blood [Dong et al., 2003] has been proposed to precede cleavage of the Tyr842-Met843 peptide bond in the adjacent A2 domain. Our finding that

TSP-1 also binds the A3 domain ofVWF suggests a mechanism by which TSP-1 may compete with ADAMTS13 for interaction with VWF. The C-terminal TSP-1 repeats are not required for activity in static assays [Zheng et al., 2003; Soejima et al., 2003] but

112 may be necessary for anchorage of ADAMTS13 to endothelial cells and VWF proteolysis in vivo. The regulation of enzyme activity by TSP-1 is not without precedent. TSP-1 has been shown to bind and inhibit the activities of the neutrophil enzymes, neutrophil elastase [Hogg et al., 1993a], cathepsin G [Hogg et al., 1993b] and the fibrinolytic enzymes, plasmin and urokinase [Hogg et al., 1992; Mosher et al., 1992].

TSP-1 has also been reported to inhibit the activity of matrix metalloproteinases by preventing the activation ofMMP2 and MMP9 zymogens [Bein and Simons, 2000].

We reported earlier that intra-peritoneal administration ofTSP-1 to Balb c mice (to achieve a plasma concentration of -1 µg.mL- 1) results in a lower average plasma VWF multimer size [Xie et al., 2001]. It would appear, therefore, that although supra­ physiological concentrations ofTSP-1 result in reduction ofVWF multimer size, physiological plasma concentrations (-0.05 µg.mL- 1 in humans) are inhibitory.

Platelet a-granules are described as consisting of nucleoid and electron-lucent zones

[Wencel-Drake et al., 1985]. Based on electron density, the electron-lucent matrix can be further subdivided into intermediate and light zones. Platelet VWF is localized within tubular structures in the light zone located at one pole of the a-granule. TSP-1 along with fibrinogen and fibronectin, on the other hand, appears to be dispersed within the intermediate zone [Suzuki et al., 1990]. This compartmentalization ofVWF has been reported in both human and murine platelets [Schmitt et al., 2001]. The concentration of VWF and TSP-1 within separate but closely related compartments raises the possibility that these glycoproteins mix during platelet degranulation.

113 Considering that platelet lysates do not contain ADAMTS13 activity [Furlan et al.,

1996], we investigated a role for TSP-1 in regulating platelet VWF multimer size. To account for possible contamination of our platelet preparations with plasma

ADAMTS 13, we included the metalloproteinase inhibitor, phenanthroline [Tsai, 1996] in our cocktail of protease inhibitors. Phenanthroline does not inhibit the VWF reductase activity ofTSP-1 (data not shown). When human and wild type murine platelets were lysed or activated with thrombin there was a reduction in VWF multimer size, which was not observed with TSP-1 null platelets. These results indicated that the multimer size of platelet a-granule VWF is altered by a TSP-1 dependant process during platelet lysis and activation.

Endothelial cell derived (plasma) VWF is required for the initial tethering of platelets to the subendothelium at high shear by its association with collagen and the platelet glycoprotein lb-V-IX complex. Platelet VWF, on the other hand, plays an important role in platelet aggregation by mediating the tethering and translocation of platelets on platelets [Kulkarni et al., 2000]. The individual and exclusive contributions of the different VWF compartments are controversial. Chimeric pigs with normal platelet

VWF but low plasma VWF have markedly prolonged bleeding times, while pigs with the reverse phenotype have mildly prolonged bleeding times [Nichols et al., 1995]. This is consistent with observations in humans with normal plasma VWF and low platelet

VWF who have either normal or mildly prolonged bleeding times [Mannucci et al.,

1985]. Platelet VWF can compensate for deficiency in plasma VWF to some extent. It has been estimated that I 0-20% plasma VWF activity is required for platelet VWF to

114 compensate for the plasma deficiency and sustain normal thrombus growth [Mannucci,

1995).

The deficiency in processing of VWF multimer size following activation of TSP-1 null platelets was reflected as more pronounced aggregation in response to collagen and/or

ADP under both static and shear conditions. The TSP-1 null platelets aggregated in response to lower concentrations of collagen or ADP than wild type platelets, which was inhibited by anti-VWF antibodies. The PFA-100 is highly specific for aberrations in platelet function under high shear and abnormalities in VWF-GPiba. and VWF-a.uhl33 interaction [Harrison et al., 1999). We recorded a shorter closure time for TSP-1 null whole blood using the ADP/collagen cartridge. It is possible that the PFA-100 analyzer detects changes in platelet VWF in preference to plasma VWF, which would help explain the faster rate of aggregation in association with the larger VWF multimers released from TSP-1 null platelets despite the presence of smaller VWF multimers in

TSP-1 null plasma. The normal tail bleeding times reported in TSP-1 null mice despite the lower average plasma VWF multimer size could represent a degree of compensation by platelet VWF. We cannot exclude TSP-1 mediated effects on the activation status of a.1Ihl33 which could impact on the rate of aggregation of the null platelets.

Notably, the average multimer size of human platelet VWF is significantly smaller than the murine protein, although both are processed by endogenous TSP-1. This result suggests that the human and murine proteins are processed to different degrees, which should be kept in mind when using mice to answer questions about control of human

115 VWF multimer size. It is possible that the degree of processing ofVWF also varies between mice strains.

The presence of ultra large VWF at sites of vascular injury is important for the initial tethering of platelets to the subendothelial matrix. Inhibiting ADAMTS13 activity at these sites would be desirable in facilitating these initial events. TSP-1 is very adhesive and binds to collagen [Galvin et al., 1987], laminin [Lawler et al., 1986], fibronectin, fibrin and proteoglycans [Lahav et al., 1984], and is well suited to interacting with other proteins that work in the extracellular matrix. As platelet activation and aggregation follow, there is a progressive release of platelet VWF. The exponential increase in TSP-

1 that accompanies platelet activation could help regulate the rate of thrombus growth by controlling platelet VWF multimer size in the developing platelet thrombus. The relatively subtle differences in control of VWF multimer size observed herein are consistent with the lack of an obvious haemostatic disorder in the TSP-1 null mice. It remains to be determined, however, whether these changes attain greater significance in determining the rate of thrombus growth in damaged or diseased arteries.

116 Chapter 4

Role of thrombospondin-1 in thrombotic

thrombocytopenic purpura

117 5.1 SUMMARY

The role of TSP-1 in thrombotic thrombocytopenic purpura is unknown. A severe deficiency of ADAMTS13 (<5%) is specific for the diagnosis ofTTP but the overall diagnostic sensitivity can be as low as 13%. The diagnostic sensitivity also varies with the underlying clinical syndrome that is associated with TTP and is highest in the idiopathic category and lowest in stem cell transplantation and bloody diarrhoea associated TTP. An increased frequency of other variables such as Factor V Leiden and

Factor H deficiency has been reported in association with TTP-HUS. The persistence of ultra large VWF multimers in the circulation has been implicated in the pathophysiology ofTTP-HUS. As TSP-1 is an independent modulator ofVWF multimer size, I measured the plasma concentration ofTSP-1 and screened selected exons of the TSP-1 gene for mutations in patients with TTP. Furthermore to test the capacity ofTSP-1 to modulate plasma VWF multimer size, I also infused a customized TSP-1 rich plasma preparation to a patient with severe ADAMTS13 deficiency and congenital TTP during a period of clinical remission when her plasma ULVWF multimers were characteristically larger than normal. The plasma concentrations of TSP-1 were elevated during the acute phase ofTTP consistent with diffuse platelet activation. The plasma

VWF multimer size was inversely correlated with TSP-1 concentration. Several polymorphisms were identified in the TSP-1 gene but were of equal frequency in TTP and the control population. The infusion of TSP-1 rich plasma did not result in the predicted increase ofTSP-1 in the patient's plasma, limiting further analysis with regard to variation in VWF multimer size.

118 5.2 INTRODUCTION

Thrombotic thrombocytopaenic purpura (TTP) and Haemolytic uraemic syndrome

(HUS) are thrombotic microangiopathies characterized by a schistocytic haemolytic anemia and consumptive thrombocytopaenia with varying degrees of neurological and renal impairment. Although most presentations are sporadic and triggered by an assortment of factors, including infections, drugs, vaccines, pregnancy, malignancies, connective tissue disorders and possibly bone marrow transplantation (George et al.,

2002), an autosomal recessive relapsing TTP-HUS syndrome, also referred to as the

Schulman-Upshaw syndrome, has been recognized since 1960 (Schulman et al., 1960;

Upshaw, 1978). Impaired processing and persistence of ultra large von Willebrand factor multimers in plasma due to a lack of a VWF cleaving protease or reductase was postulated as the cause of this syndrome in 1982 (Moake et al., 1982 ).

Familial relapsing TTP-HUS has now been determined to result from a deficiency of the

VWF cleaving proteinase, ADAMTS 13 (Levy et al., 2001 ). The commoner sporadic form of the disease occurs in association with an acquired deficiency of the enzyme often in association with circulating inhibitors (Furlan et al., 1998b; Tsai and Lian,

1998). Although a severe deficiency of ADAMTS13 (<5% activity) is reported to be specific for TTP, the diagnostic sensitivity at this threshold can be as low as 13%

(Vesely et al., 2003; Veyradier et al., 2001). Difficulties associated with the reliable measurement of enzyme activity and the physiological variation of AD AMTS 13 levels may account for some of this variability. Furthermore TTP in association with

119 haemopoeitic stem cell transplantation, drugs and a bloody diarrhoea prodrome are usually not associated with severe ADAMTS13 deficiency (Vesely et al., 2003). The role of the VWF reductase, thrombospondin-1 in modulating the phenotype ofTTP­

HUS is not known. It should be borne in mind that the clinical syndrome of acquired

TTP-HUS is likely to be a manifestation of an imbalance between a variety of factors that are released from the damaged endothelium and the capacity to modulate them and not limited to the ineffective modulation of VWF multimer size alone.

To investigate the role ofTSP-1 in TTP we adopted the following approach

1. Measurement of plasma TSP-1 concentration and VWF multimer size in patients with congenital/acquired TTP and comparison with healthy normal volunteers.

2. Mutation screen of selected exons of the TSP-1 and VWF genes in patients with a clinical diagnosis ofTTP.

3. Monitoring changes in plasma TSP-1 and VWF multimer size following the infusion of a customized TSP-1 enriched plasma preparation to a patient with congenital

TTP during remission

5.3 PATIENTS AND METHODS

Patients with congenital TTP

Patient A- an 8 year old Caucasian girl diagnosed at 10 months of age. Her episodes are usually triggered by an intercurrent respiratory tract infection. She is monitored with

120 a monthly full blood count and administered 10-40 mL.kg"1 of fresh frozen plasma when her platelet count falls below 30 x 109 per L. She demonstrates a predictable response to plasma, 24-48 h post infusion, with an elevation in her platelet count and normalization of her blood film. On average, she receives plasma infusions every 2-3 months with the longest documented remission lasting approximately 6 months. She has inherited critical mutations in the ADAMTS13 gene from each of her asymptomatic parents

(587C/f and 4143-4144insA) and has circulating enzyme levels of< 10%. These mutations and their consequences are discussed in greater detail in chapter 5 of this thesis.

Patient B- a six year old girl born to first cousins of Y emenite background, developed thrombocytopenia and microangiopathic hemolysis soon after birth. She is treated with plasma infusion (1 0mL/kg) every 2 weeks to maintain a normal platelet count and hemoglobin level. She is homozygous for a 1 783-1784 delTT mutation in the

ADAMTS13 gene and has circulating enzyme levels of< 10% (Savasan et al., 2003).

Patients with acquired TTP

French TTP Registry- The registry is coordinated by Dr. Paul Coppo at the Laboratoire

Hematopoiese et Cellules souches, Institut Gustave Roussy, Villejuif, France. The validity and consistency of the initial diagnosis of TTP was reviewed by an independent committee. Twenty one consecutive patients with idiopathic TTP from January 1 to

December 31, 2001 were evaluated. Frozen plasma samples were transported to

Australia on dry ice and were received in good order.

121 Australian DNA bank ofTTP patients- Consultant haematologists at tertiary referral hospitals in Australia were asked to provide a sample of blood from patients with a present or past diagnosis of TTP for the purpose of DNA extraction. The study was approved by the ethics committees of The Prince of Wales Hospital and The University of New South Wales. DNA was extracted from 25 TTP patients using a standard in­ house protocol in the department of molecular genetics at the Prince of Wales Hospital.

Control samples were selected from a batch of DNA extracted to screen for the fragile X syndrome.

Preparation of platelet poor plasma

Venepuncture was performed without a tourniquet using an 18-guage needle. The blood was collected into 3.8 % trisodium citrate anticoagulant and 0.4U apyrase in a 9:1 vol/vol ratio. Topical 1% lignocaine was used at the discretion of the phlebotomist. The samples were placed on ice and centrifuged within 20 minutes of collection at 4000g for

10 minutes at 4°C. Two thirds of the supernatant was aspirated and re-centrifuged as above. The platelet poor plasma supernatant was aliquoted and frozen at -80°C.

Assay for TSP-1

The mAb 133 monoclonal antibody (l00µL ofl0µg/mL in 0.lMNaHCO3, pH 8.3 buffer) was adsorbed to Nunc PolySorp 96-well plates overnight at 4°C in a humid environment. Wells were washed once with 20mM Hepes, 0.14M NaCl, pH 7.4 buffer

(HBS) containing 0.05% Tween 20 (HBS/fween), nonspecific binding sites blocked by

122 adding 200µL of2% BSA in HBS and incubating for 90 min at 37°C, and then washed twice with HBS/Tween. Normal plasma was depleted ofTSP-1 by immunoaffinity chromatography on HB8432-Sepharose CL-4B matrix and then spiked with known amounts of purified platelet TSP-1. The spiked plasmas and normal or patient plasmas were diluted in HBS/Tween and 100µL aliquots added to antibody coated wells and incubated for 1h at room temperature with orbital shaking. Plasmas were assayed in triplicate. Wells were washed thrice with HBS/Tween and l00µL of a 1 :500 dilution of rabbit anti human TSP-1 polyclonal antibody added and incubated for 1h at room temperature with orbital shaking. Wells were washed three times with HBS/Tween, and

100µL of 1 : 1000 dilution of a HRP conjugated swine anti rabbit IgG antibody in

HBS/Tween was added and incubated for 30 min at room temperature with orbital shaking. Wells were washed three times with HBS/fween and the peroxidase detected using Sure Blue™ TMB microwell peroxidase substrate. Control wells contained TSP-1 depleted plasma.

Screening for mutations in TSP-1 and VWF genes

Exons 7, 8, 9, 16 and 17 of the TSP-1 gene and exon 31 of the VWF gene were selected for mutation analysis based on our understanding of TSP-1/ VWF interaction and TSP-1 reductase activity (Pimanda et al., 2002; Xie et al., 2001 ). Genomic DNA extracted from patients with a history of TTP was used in the study. The selected exons were

PCR amplified with flanking intron primer sets using Platinum® Pfx DNA polymerase

(Invitrogen, Carlsbad, CA) (Table lA). The single strand conformation polymorphism

123 (SSCP) technique was used to distinguish variation in the nucleotide sequence based on the conformation of the amplified DNA fragments. The GeneGel Excel 12.5/24 Kit and the GenePhor Electrophoresis Unit (Pharmacia Biotech-Pfizer, New York, NY) were used to this end in accordance with the manufacturer's guidelines. Briefly, the PCR products (1 µl) were diluted in 1OrnM Tris, lrnM EDTA buffer (2µ1) and denatured 1: 1 in denaturing solution (3µ1 of99% formamide and 1% xylene cyanol mix) at 95°C for 5 minutes. The samples were placed directly on ice to prevent reannealing of the single stranded product and electrophoresed on GeneGel Excel 12.5/24 at 8°C using lOmA for

5 hours. The gels were fixed in 10% Ethanol and the DNA fragments resolved using a silver stain. Patient and control PCR products with distinct patterns by SSCP were sequenced in both directions using a BigDye Terminator Kit (Applied Biosystems,

Foster City, CA) at the sequencing facility at the University of New South Wales.

Preparation and infusion of TSP-1 enriched plasma

The storage of platelets at room temperature and subsequent centrifugation results in a supernatant enriched in TSP-1 with concentrations similar to that of serum (Dawes et al.,

1983). Appropriately screened, day 5 platelet concentrates from the Australian Red

Cross Blood Service were centrifuged and the supernatant transferred under a closed seal, first into an intermediate transfer bag (Baxter, Deerfield, IL) and then following a second centrifugation step into a cryobag (Baxter, Deerfield, IL) for storage (Fig 2).

The platelet concentrates were centrifuged at 2000g for 5 minutes at 25°C. A Terumo sterile docking system was used to connect the tubing of the bags under a closed seal.

124 An aliquot of the supernatant in the intermediate bag was used to measure the TSP-1 concentration. The patient was admitted to the children's ward at Mildura Base Hospital and blood collected at 18h, 14 h and 20 minutes prior to the commencement of the infusion. The TSP-1 rich plasma supernatant (- 3mg of TSP-1 in IOmL) was infused over 15 minutes under close medical and nursing supervision and blood collected at prescribed intervals for the measurement of plasma TSP-1, VWF multimer size and a

Full Blood Count. Biochemical parameters including renal and liver function tests were measured on a daily basis.

5.4 RESULTS AND DISCUSSION

Plasma TSP-1 concentration is not reduced in patients with congenital or acquired

TTP

Blood was collected from fifty healthy subjects to establish a normal range. The TSP-1 concentrations in this sample population ranged from 39.4-357.2ng/mL with a mean concentration of 195.5ng/mL and a standard deviation of78.6ng/mL. The plasma TSP-1 concentrations during remission and relapse in patient A with congenital TTP were

264.9ng/mL and 850.Sng/mL respectively. The TSP-1 concentration in patient B with congenital TTP prior to her regular scheduled plasma infusion was l00ng/mL. The

ADAMTS13 activity was less than 10% in both patients.

125 In plasma samples from the French TTP registry collected prior to the commencement of plasma exchange, the TSP-1 concentrations ranged from 133.7-2128.9ng/mL with a mean concentration of 805ng/mL and a standard deviation of 626.5ng/mL. There was an inverse correlation between plasma TSP-1 concentration and VWF multimer size in this sample group (Fig 1; r = -0.5443; p < 0.01). The increased concentrations of plasma

TSP-1 probably reflect the heightened degree of platelet activation in TTP. The inverse correlation between plasma TSP-1 concentration and VWF multimer size is probably a reflection of the degree ofVWF/platelet rich thrombus formation and clearance of the

ULVWF multimers from the circulation. The independent breakup of the ULVWF multimers by the highest concentrations of plasma TSP-1 in association with reduced

ADAMTS13 levels cannot be excluded. The ADAMTS13 levels were measured in

France and ranged from <5% to 70% (normal 45-125%). There was no correlation between the level of ADAMTS13 and plasma TSP-1.

126 1.50

1.25 • • • • 1.00 t •

~> 0.75

0I

0.50 • • 0.25

0.001 ....------0 SOO 1000 1500 2000 2Stl0

TSP-1, ng.mL'1

Figure 1. Inverse correlation between TSP-1 concentration and VWF multimer size in acquired idiopathic thrombotic thrombocytopenic purpura. TSP-1 concentrations were measured in platelet poor plasma samples obtained from a TIP registry in France (n=2 l ). The pre treatment TSP-1 levels were significantly increased compared with healthy normal subjects. The reduction in VWF multimer size in association with increasing concentrations of TSP-1 probably reflects the degree of platelet activation and thrombus growth with clearance of the UL VWF multimers from the circulation.

127 Lack of association between TTP and mutations in selected regions of the TSP-1 and VWF genes

We postulated that the VWF reductase activity of TSP-1 centered on a free thiol at residues 892, 928 or 974 in the Ca binding C terminal region of the protein. We also predicted based on sequence similarity between the A3 domain ofVWF and CD36 (an established cell surface receptor for thrombospondin type 1 repeats) that there is an initial binding interaction between VWF A3 and the type 1 repeats of TSP-1 (Pimanda et al., 2002; Xie et al., 2001 ). This view was supported by the inhibition ofVWF reductase activity using synthetic peptides fashioned on the amino acid sequence of the predicted binding sites.

Exons 7, 8 and 9 of the TSP-1 gene incorporate the type 1 properdin like domains and

Exons 16 and 17, the cysteine residues at 892, 928 and 974. Exon 31 of the VWF gene incorporates the sequence motif on the A3 domain (1°08D ALGF AVRYL 1018) that bears a similarity to the TSR 1 binding sequence ofCD36 (89GPYTYRVRFLA99). These exons of patients A and B with congenital TTP were sequenced and proved normal. Following the discovery of the ADAMTS13 gene both these patients were shown subsequently to have loss of function mutations in this gene (Savasan et al., 2003).

Given the large cohort of patients with acquired TTP (n=25) a screening strategy (SSCP­ see diagram below) was employed that limited DNA sequencing to those with suggested differences from control DNA The only mutation that resulted in a change in the protein was G 1567 A on exon 9 of TSP-1 and was present at an equal frequency in both

128 the patient and control populations and has subsequentl y been identified as a polymorphjsm in the Gen Bank SNP database (Table I B).

Single Strand Conformation Polymorphism (SSCP) and Heteroduplex Formation Allele a Allele b a b a' b'

PCR Amplification b---- and ] xn denaturation a' ------b' ------

? Allele a=b / "" ? Allele a-:/:. b due to ~ GIA mutation

SSCP < or= 2 < or= 4 Patterns When DNA re-anneals the single strands form l

-- G - - --A -- Homoduplex ------C ------T ------

and/or G A __I \__ __I\__ Heteroduplex -----\ /------\ 1----- T C

129 A Exon (size, bp) Forward Primer Reverse Primer TSP-1 Exon 7 (225) CTGATGAACCTCTGTTTCCC CGTCCCTGAGAGGCTAAGATG TS P-1 Exon 8 (234) CATTTGTGACCATCAACTCTGTAC ACCCTTGCAGCGGACCTCACAC TSP-1 Exon 9 (229) GTTGCCTGTGGTTCATCTTC TTTCATCCTGGGCTGCGTGG TSP-1 Exon16 (285) ACCCTCCATTTACATCTCTC TAGAAAGTGGCTCCCATGAC TSP-1 Exon 17 (294) TTAGAATTTTGCTGAACTCTTGC CTCTTAGGATCTAAGAACTC vWF Exon 31 (200) CAGTCAGTACTGACTTGGC CCACTGAATCCACAGAGACG

B

Position Variation Protein Exon SNP

1290 A/G Lys/Lys 7 Gen Bank

1329 G/A Pro/Pro 8 N/D

1410 TIC Asn/Asn 8 GenBank

1563 err Asn/Asn 9 N/D

1567 G/A Thr/Ala 9 Gen Bank

2868 err Asp/Asp 17 Gen Bank

Table 1. (A) List of primers used for the amplification of TSP-1 and VWF exons. (B) List of mutations identified. The G 1329A and C 1563T missense mutations are novel but do not result in a change in the amino acid sequence.

130 The infusion ofTSP-1 enriched plasma does not affect plasma VWF multimer size

To test the hypothesis that TSP-1 regulates plasma VWF multimer size in-vivo, we sought and received parental consent and institutional ethics clearance to infuse a customized preparation of TSP-1 enriched plasma to a six year old child with relapsing

TTP. Permission to administer this preparation was granted by the Therapeutic Goods

Administration (TGA), Canberra, Australia. The largest VWF multimers are the best substrate for VWF reductase activity (Pimanda and Hogg, 2002). Therefore a period of disease remission during which the patient characteristically demonstrates high circulating VWF multimers was chosen as the most appropriate time to initiate therapy.

The infusion was generally well tolerated except for a complaint of transient malaise 10 minutes into the infusion which resolved with cessation of the infusion at 15 minutes.

The elevation in TSP-1 concentration was only- 10% above the baseline (Fig 3A). The infusion of a similar preparation was reported to have resulted in a rapid redistribution phase followed by a slower clearance with an estimated half life of9h for TSP-l(Dawes et al., 1983). Based on the delivered dose ofTSP-1 (-3mg) and the patients calculated plasma volume (-lL), a ten fold increase should have been observed had the infused

TSP-1 been retained solely in the plasma compartment. TSP-1 is an adhesive protein with pronounced binding to cell surface receptors on activated endothelium, platelets and exposed matrices (Asch et al., 1993; Galvin et al., 1987). It is uncertain whether transfused platelet release components accelerated the clearance of TSP-1. Plasma

VWF is largely sourced from endothelial cells (Wagner, 1990). The stimulation of

131 endothelial cells results in the release of ULVWF. The changes measured in plasma

VWF multimer size following the infusion were negligible (Fig 3B). However, if the lack of a substantial increase in plasma TSP-1 could be attributed to endothelial cell activation by the platelet releasate, it is possible that the anticipated increase in circulating ULVWF multimers was also abrogated by the infusion. Although the infusion of purified or recombinant hTSP-1 rather than a platelet releasate would have been preferable this would be difficult to justify given our more recent observation that

TSP-1 potentially inhibits ADAMTS13 activity.

132 A

C B

\

~--- .., .. ,.,onn~~ If • ,-

for infusion Figure 2. Preparation ofTSP-1 enriched plasma and a were a donor unit of platelets, a transfer pack (A) The three components used in the preparation under a docking system that enables the joining of tubes cryo bag for storage. (B) A Terumo sterile bag to the unit to the transfer bag and then the transfer closed seal was used to first join the platelet was to pellet the platelets and only the supernatant cryo storage bag. The platelet unit was centrifuged was tubes. The transfer bag (containing the supernatant) transferred to the transfer bag via the joined tranferred to the storage bag. These double in turn centrifuged before its supernatant was stored to minimize the number of platelets in the final centrifugation and transfer steps were adopted product (C).

133 A -+-TSP-1 - o- CBAtvWF:Ag

30 ,2.,0

.5

I , , I a,---,,---.--u. O .20 .1s -10 .5 o o 10 20 2s 1s 12s ns hr-s from PCP infusi

- o- CBAJvWF'. Ag

40 ~ -0 B

.5 - 0 0 .... 3----o------·-·--o ~·o-~------o -·¾. o,, ~ ..... ~ C: < !;:) 0 32S ~ V ~ ·- 7':l ]l l» s 300 {D a:,:<:: .5 275 L 250+------.----t I 1 1 I ..,.____ ...,._,_.-o .o ·20 - 15 -10 - 5 0 0 10 20 25 75 125 175 h rs from PCP ir\fus.ion

Fig.3. Variation in plasma TSP-1, VWF multimer size and platelet count following the infusion ofTSP-1 enriched plasma. A I OmL aliquot of PCP containing approximately 3mg of TSP-1 was infused over 15 minutes to a 6 year old girl with a history of congenital thrombotic thrombocytopenic purpura during remission. The changes in plasma TSP-1, VWF multimer size and platelet count were negligible.

134 Chapter 5

Congenital thrombotic thrombocytopenic purpura in association with a mutation in the second CUB domain of

ADAMTS13

135 4.lSUMMARY

Severe deficiency of the von Willebrand Factor (VWF) cleaving proteinase,

ADAMTSI 3, is associated with the development of thrombotic thrombocytopenic purpura. Several mutations spread across the ADAMTS 13 gene have been identified in association with a deficiency ofVWF cleaving proteinase activity in patients with congenital TTP. The spread of these dysfunctional mutations and the domain structure of ADAMTS13 are suggestive of a complex interaction between the enzyme and its substrate. We have studied a patient with congenital TTP who is a compound heterozygote for the Thrl 96Ile mutation in the metalloproteinase domain and a frameshift mutation (4143-4144insA) in the second CUB domain that results in loss of the last 49 amino acids of the protein. The VWF cleaving proteinase activity of the truncated enzyme was comparable to that of the wild-type enzyme but its secretion from transfected COS-7 cells was -14% of the wild-type.

4.2 INTRODUCTION

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy characterized by a schistocytic hemolytic anemia and consumptive thrombocytopenia with varying degrees of neurological and renal impairment. The presence of ultra-large von Willebrand Factor multimers in the plasmas of patients with chronic relapsing TTP during remission that disappear during an attack led to the implication of these multimers in the pathogenesis of the platelet rich, fibrin poor thrombi that occlude

136 arterioles and are the hallmark of this disorder (Moake et al., 1982). The VWF cleaving metalloproteinase, ADAMTS13, hydrolyses the ultra-large VWF multimers as they emerge from endothelial cells and undergo conformational strain due to shear stress in arterioles and capillaries (Amar et al., 2002). A severe deficiency ofVWF cleaving proteinase activity is associated with the pathogenesis of congenital TTP (the Schulman­

Upshaw syndrome) (Furlan et al., 1998a; Levy et al., 2001).

A number of mutations, spread throughout the ADAMTS13 gene, have been reported in association with congenital TTP (Levy et al., 2001 ;Kokame et al., 2002;Antoine et al.,

2003;Schneppenheim et al., 2003;Assink et al., 2003;Savasan et al., 2003) (see Table 1).

The CUB domains of the C-terminus of ADAMTS13 are of uncertain physiological relevance. The findings that the CUB domains are not required for VWF cleaving proteinase activity measured under static conditions (Zheng et al., 2003; Soejima et al.,

2003) and that certain strains of mice have a variant form ofmurine ADAMTS13 that lacks the CUB domains (together with the seventh and eighth TSP-1 repeats), supports the premise that these domains are dispensable in vivo (Banno et al., 2003). However, a report that peptides from the CUB domains inhibit VWF cleaving proteinase activity under flow, but not static, conditions suggests a functional role for these domains

(Bernardo et al., 2003).

We studied a patient with congenital TTP who is a compound heterozygote for the

Thrl 96Ile mutation in the metalloproteinase domain and the insertion of a nucleotide

(A) at 4143-4144. This insertion results in a frameshift in the second CUB domain and

137 loss of the last 49 amino acids of the protein. The CUB mutation had little effect on the specific activity of the enzyme but its secretion from COS-7 cells was impaired.

4.3 MATERIALS AND METHODS

Case history

We investigated an 8 year-old Caucasian girl with congenital TTP. She was diagnosed at 18 months of age at which stage she received her first infusion of plasma. She is monitored with a monthly full blood count and administered 10-40 mL.kg-1 of fresh frozen plasma when her platelet count falls below 30 x 109 per L. She demonstrates a predictable response to plasma, 24-48 hrs post infusion, with an elevation in her platelet count and normalization of her blood film. On average, she receives plasma infusions every 2-3 months with the longest documented remission lasting approximately 6 months.

Assay of plasma VWF cleaving proteinase activity.

An assay based on the collagen binding activity of digested VWF (Gerritsen et al., 1999) was used to measure the VWF cleaving proteinase activity in the plasma of the patient

(during remission) and her family. Briefly, citrated platelet poor plasma samples were diluted 1/10, 1/20 and 1/40 in 5mM Tris, pH 8.0 with 1.5 M urea. Aliquots of 100µL were preincubated with l0µL, 93 mM BaCb for 30 min at 37°C to degrade endogenous

VWF. Substrate VWF (50 µL of 50 µg.mL- 1) was then added and incubated for 2h at

37°C. The reactions were stopped by adding l0µL, 0.825 M Na2SO4. The mixtures

138 were centrifuged and the supematants diluted 100-fold in 20mM imidazole, 5 mM citric acid, 0.12 M NaCl, pH 7.3 buffer containing 5% bovine serum albumin and assayed for collagen binding affinity. This assay is a surrogate measure ofVWF multimer size

(Favaloro et al., 1991) and has been detailed in a previous chapter. Serial dilutions of pooled normal human plasma were used to generate a calibration curve. The VWF cleaving proteinase activity of 10 control subjects ranged from 70-130%.

ADAMTS13 sequence analysis.

DNA was extracted from peripheral blood leukocytes and all the exons and intron-exon boundaries of the ADAMTS13 gene were amplified by PCR as previously described

(Kokame et al., 2002). The sequencing reactions were performed at the University of

New South Wales sequencing facility and the study was conducted with parental consent and institutional ethics approval.

Transient expression of the ADAMTS13 mutant.

The cDNA construct of the open reading frame of ADAMTS13 was a gift from Dr Evan

Sadler (Zheng et al., 2001) and the 4143insA mutant was created using PCR based mutagenesis. The constructs were subcloned into the mammalian expression vector pCXN and transiently expressed in COS-7 cells. The DNA sequence of all inserts was confirmed by sequencing. The cells were grown in RPMI (GIBCO/Invitrogen,

Carlsbad, Ca) with 10% fetal calf serum and transfected at-80% confluence using

FuGENE6 (Roche Diagnostics, Basel, Switzerland). Serum-free medium was applied to confluent cells at 24 h and the conditioned medium collected at 48 h was clarified by

139 centrifugation and concentrated 50-fold using Amicon Microcon YM-30 (Millipore,

Bedford, MA). The cells (1 x 106 per mL) were harvested, counted and lysed with 0.5%

Triton X-100 in 20 mM Tris, pH 7.5 buffer containing 0.15 M NaCl, 5 mM EDTA and 1 mM Pefabloc. The samples were resolved on 8-16% SDS-PAGE under reducing conditions and visualized by Western blot using an anti-ADAMTS13 murine monoclonal antibody (242/H2) provided by Dr Friedrich Scheiflinger (Plaimauer et al.,

2002).

Assay of ADAMTS13 activity.

VWF cleaving proteinase activity was determined as previously described (Furlan et al.,

1996; Plaimauer et al., 2002) with minor modifications. Test samples were diluted in 10 mM Tris, pH 7.4 buffer containing 0.15 M NaCl and incubated with 10 mMBaC}zfor 5 min at 37°C. Aliquots (100 µL) of each dilution were mixed with VWF (50 µL of50

µg.mL" 1) and the mixtures dialyzed for 24 hs using circular dialysis membranes floating on 50 mL of 5 mM Tris, pH 8.0 buffer containing 1.5 M urea at 37°C. The reactions were quenched by adding 5 µL of 0.5 M EDT A. Proteolysis ofVWF was measured by resolving reactions on 8% SDS-PAGE under reducing conditions and blotting the VWF and fragments with peroxidase-conjugated anti-VWF polyclonal antibodies (Dako,

Carpinteria, CA) (Tsai HM., 1996). Densitometry was performed using a Fluor-S

Multilmager and Quantity One software from Bio-Rad, Hercules, CA on a digitalized image of the gel. The optical densities of the 250 kDa (parent VWF) and 176 kDa

(hydrolyzed VWF) bands in each lane were measured and normalized to account for variations in protein loading. The generation of the 176 kDa VWF fragment by

140 ADAMTS13 mediated proteolysis was used as a measure ofVWFCP activity. This was expressed as a ratio of optical density units {176kDa (post hydrolysis)-l 76kDa (pre hydrolysis)/ 250kDa (post hydrolysis)+ 176 kDa (post hydrolysis)} for each reaction.

Dilutions of purified rADAMTS13 of known concentration were used for assay calibration. The calibration curve was fitted to a sigmoid curve using the GraphPad

Prism software package. The ratios of optical density units for representative dilutions of the conditioned media (1/20 for the wild type and 1/10 for the mutant) were used to read the VWFCP activity off the calibration curve. The results are expressed as µg.mL- 1 ofVWFCP activity after accounting for the 50 fold concentration of the conditioned media.

The reactions were also resolved on 1% agarose to confirm the loss of the high molecular weight multimers of VWF following hydrolysis. The gels were blotted with

1251-labeled anti-human VWF polyclonal antibodies and the multimers visualized by autoradiography using an in-house method at the Royal Prince Alfred Hospital (Sydney,

Australia) modified from Ruggeri et al (Ruggeri et al., 1980) and detailed in a previous chapter.

4.4RESULTS

ADAMTS13 mutations in TTP patient

Two silent mutations (420T/C and 1716NG) and three additional mutations (587C/T,

1342G/C and 4143-4144insA) were identified in this patient (Fig. 1). The nucleotides

141 are numbered from the A of the initiation Met codon. Both silent mutations are reported in GeneBankTM as SNPs. The 587Cff (Thr196Ile) missense mutation has been previously identified in patients with familial TTP and the recombinant mutant protein is reported to have less than 25% VWF cleaving proteinase activity of the wild-type (Levy et al., 2001;Motto et al., 2002). The 1342G/C (Gln448Glu) missense mutation on the other hand is not associated with reduced VWF cleaving proteinase activity and is a SNP with a reported heterozygosity of 31.2-42.5% (Kokame et al., 2002; Antoine et al.,

2003). The insertion of a nucleotide (A) at 4143-4144 results in a frameshift in the second CUB domain after Ser1381 followed by a predicted 5 amino acid extension

(Arg-Glu-Gln-Pro-Gly) and premature termination of the protein with the loss of 49 amino acids.

142 A I-1 I-2 --~------;,/ a~ ~. ....__}:;7' 70% 50% 11-l- []l"-2

<10% 60%

B

587CIT 4143-4144insA T1961 S1381+REQPG 1 l SP Pro! lffl

Fig. 1. VWF cleaving proteinase activity and ADAMTS13 mutations in a patient with congenital TTP. A. The pedigree with th e ADAMTS 13 activity levels of the father (I-1 ), th e mother ( l -2), the patient (Il-1 ) and her brother (II-2). The values are expressed as a% of VWF cleaving proteinase activity in nonnal pooled human p lasma. B. The 1427 amino acid precursor protein of ADAMTS 13 contains a signal peptide (SP), a propeptide (Pro), a metalloprotease domain (MP), a disintegrin-like domain (Dis), a type I TSP module (T), a cysteine-rich (Cys) and spacer domain, seven additional type I TSP modules and two CUB ~omplement components C 1r /C Is, sea !lrchin epidennal growth factor and B_one morphogenetic protein) domains. The mutation at Thr I 96Ile on exon 6 is positioned within the metalloprotease domain and the mutation after Ser 138 1o n exon 29 truncates the second CUB domain of the protein.

143 Exon6 Exon29

G T C A N CC G C T G A G T C A N AAAA C

Patient

G T C A CCC ~ G C T 1 G A G T C A G A G A G C

Wild type

The secretion and VWFCP activity of the 4143insA mutation

The CUB mutation impaired secretion of the enzyme from transfected COS-7 cells but had Little effect on VWF cleaving proteinase activity. The rate of secretion (Fig. 2A) and the specific activity (Fig. 2B) of the mutant was - 14% and - 85%, respectively, of the wild-type enzyme. Whether this in vitro activity translates into full functional activity in vivo is unknown.

144 Secreted A 180 · -+- ADAMTS 13

180 · 2 3 4 5

B wild-type mutant rAOAMTS13 .,,.,, .,_I> .,_I> p. g .ml ·1 ,:_. ..:_f ,/,rJ> <)I ,:_'r ,:_'? ,:_ , ,:_'fr ,:_'f ~ I)• ,l ,;,f' .. ",

1% agarose

wild-type mutant rAOAMTS13

~ ./ ,l " ~ ,:,-"~ p. g .ml ·1 ,;-~ W,i' -~~= !~: :. =N•)'ff 140 2 3 .,4 5 6 7 8 -· 8%SDS-PAGE

Fig. 2. Expression and activity of the ADAMTSl3 mutant. A. COS-7 cells were transfected with plasmids encoding wild-type or mutant ADAMTS I 3. The ADAMTS 13 in the conditioned media (upper panel) and cell lysates (lower panel) were analyzed by Western blotting. Controls were cells transfected with empty vector (lane I), or wild-type (lane 3) or mutant (lane 5) cDNA inserted in the reverse orientation. The band density of the secreted mutant (lane 4) was - 14 % of the wild-t ype protein (lane 2) B. ADAMTS 13 activity in the conditioned medium of the transfected COS-7 cells was assayed using purified VWF. Reactions were resolved on I% agarose under non-reducing conditions and visualized by autoradiography (upper panel) and SDS­ PAGE under reducing conditions and the cleaved VWF visualized by Western blotting (lower panel). Purified rADAMTS I 3 (lanes 6-8) was used to calibrate the assay. Wild-type conditioned medium (lanes 2 and 3) had -0.36 ~tg .mL·1 VWF cleaving proteinase activ it y, while mutant conditioned medium (lanes 4 and 5) had -0.04 µg .mL· 1 activity. Based on a secreti on efficiency of 14% (part A), the VWF cleav ing proteinase activity of the mutant was - 85% of the wild-type protein.

145 4.5 DISCUSSION

The reported VWF cleaving proteinase activities in patients with hereditary TTP and

ADAMTS13 mutations are all less than 10%. The plasma VWF cleaving proteinase activity in our patient was below the detection threshold of 10% and has been noted to be <2-5% in other compound heterozygotes with this mutation [Schneppenheim et al.,

2003). Asymptomatic heterozygotes, including the mother of our patient have VWF cleaving proteinase levels ranging from 21-55% (Schneppenheim et al., 2003). The

CUB mutation impaired secretion of the enzyme from transfected COS-7 cells but had little effect on VWF cleaving proteinase activity. The rate of secretion (Fig. 2A) and the specific activity (Fig. 2B) of the mutant was -14% and-85%, respectively, of the wild­ type enzyme. It is possible that the plasma level of this mutant is further compromised after secretion due to increased clearance. Whether this in vitro activity translates into full functional activity in vivo is unknown. Complete absence of enzyme activity is thought to be lethal in utero (Levy et al. 2001 ). A recent report that two brothers with congenital TTP are homozygous for this mutation ( Ass ink et al., 2003) supports the notion that the mutant retains at least some of its biological activity in vivo.

146 Fig 3. A three-dimensional representation of the structure of acidic seminal fluid protein illustrating the CUB domain architecture. S-strands are shown as ribbons (blue) and the connecting loops as Ca traces (grey). The expected position of the free cysteine in the first CUB domain of ADAMTSI 3 is indicated with an arrow. The side chains of disulfides (SS I and SS2) are shown in yellow. Note that SS2 can exist in the reduced dithiol form in the acidic seminal fluid protein, and is absent from the second CUB domain in ADAMTS 13 . The two central S-strands that are deleted in the first or second CUB domain of ADAMTS 13 as a result of the 3769-3770insT or 4143-4 I 44insA mutation, respectively, are shown in red. C, C-terrninus.

Jn addition to the 4143-41441nsA mutation in the second CUB domain, another frameshift mutation 3769-3770lnsT that leads to truncation at Leul257 in the first CUB domain and a predicted extension of 34 amino acids has been described in association with congenital TTP (Levy et al., 200 I). The structural consequences of the truncations in the CUB domains were evaluated by searching the Superfamily 1.61 database of all known three-dimensional protein structures (Gough et al., 2001 ) (http://supfam.org)

147 using either residues 1131-1298 or residues 1287-1427 as templates. These stretches correspond to the first and the second CUB domain and their flanking regions, respectively. This analysis suggested that residues 1192-1286 and 1299-1407 have structural similarity to the CUB domains of the spermadhesins, porcine major seminal plasma glycoprotein Psp-1 and bovine acidic seminal fluid protein, respectively. The

CUB domain is a sandwich of two five-stranded ~-sheets with one or two disulfides

(Romero et al., 1997). The mutations 3769-3770insT and 4143-4144insA remove the central ~-strands from both sheets in the affected CUB domain thereby destroying its architecture.

The first CUB domain of ADAMTS13 contains five cysteines of which one (Cys1275) is predicted to be unpaired and surface exposed in the loop connecting the two central ~­ strands in this domain (Fig.3 . Furthermore, the second disulfide in the acidic seminal fluid protein CUB domain can exist in the cleaved dithiol form (Romero et al., 1997).

The first ADAMTS13 CUB domain, therefore, may contain one to three free thiols.

This is noteworthy, considering that the Cys974 thiol ofTSP-1 can reduce VWF multimer size by facilitating reduction of inter-subunit disulfide bonds (Pimanda et al.,

2002). It is possible, therefore, that the first CUB domain has a similar activity. CUB domains in general are involved in protein-protein and protein-carbohydrate interactions

(Bemocco et al., 2003; Thielens et al., 2001 ;Arlaud et al., 2001), but the specific ligands for the CUB domains of ADAMTS13 are not known. The sequences of the CUB domains of ADAMTS13 did not align sufficiently with those of other human proteins to predict the identity of these ligands.

148 Exon Nucleotide Amino acid change Reference 2 130 err Q44Stop Antoine et al 3 286 e/G H96D Levy et al 3 304 err R102e Levy et al 6 587 err T196I Levy et al 7 695 T/A L232Q Schneppenheim et al 7 703 Gle D235H Assink et al 7 718-724 del Del G+e Assink et al 7 788 e/G S263e Schneppenheim et al 7 803 Gle R268P Kokame et al 8 932 GIA e311Y Assink et al 9 1058 err P353L Schneppenheim et al Assink et al 10 1169 GIA W390Stop Schneppenheim et al 10 1193 GIA R398H Levy et al 12 1345 err Q449Stop Kokame et al 12 1370 err P457L Assinket al 13 1582 AJG R528G Levy et al 13 1584 +5G Splice Levy et al 13 1523 GIA e508Y Kokame et al 17 2014 err R692e Levy et al 18 2195 err A732V Antoine et al 19 2279delG Frameshift stop AA776 Assink et al 19 2376-2401 del Frameshift Levy et al 20 2549-2550delAT Frameshift stop Schneppenheim et al 21 2728e/f R910stop Schneppenheim et al Assink et al 22 2851T/G e951G Levy et al 24 3070T/G e1024G Levy et al 24 3100A/f R1034stop Schneppenheim et al 26 3638 GIA e1213y Levy et al 27 3769-3770insT Frameshift Levy et al 28 4006 err R1336W Antoine et al 29 4143insA Frameshift stop AA 1386 Schneppenheim~~ Assink et al Pimanda et al

Table 1

149 Chapter 6

Summary and future directions

150 The first objective of this thesis was to solve the structure-function relationship between

TSP-1 and VWF. To this end we have demonstrated that the reductase activity ofTSP-1 centers on a free thiol at Cys 974. The conformation of the C-terminal globular domain and the position of the free thiol in TSP-1 have been reported to vary with the ambient

Ca2+ concentration. We found that the free thiol was at Cys 974 at both 0. lmM and 2 mM Ca2+ and was required for the reductase activity ofTSP-1 (Pimanda et al., 2002).

Contrary to the preliminary evidence that the CSVTCG motif in the TSR engaged a complementary sequence on the A3 domain ofVWF, using insect cell derived overlapping fragments of TSP-1 we have narrowed the binding interaction to the

E3CaG-l (incorporating the last type 2 repeat, the type 3 calcium binding repeats and the C-terminal globular domain) fragment (see chapter 3, Pirnanda et al., submitted).

This is consistent with the emerging view, supported by data from the recently solved crystal structure of the TSR, that the CSVTCG motif is unlikely to form part of its recognition face. The E3CaG-1 fragment engages the A3 domain ofVWF but this binding may not be exclusive as it can also engage M3-VWF. The minimal region required for VWF binding and reductase activity has not been determined and will

require testing further truncations of the E3CaG-l fragment for VWF reductase activity.

These studies are in progress.

A second objective of this thesis was to clarify the importance of TSP-1 in the

physiological control ofVWF. As detailed in chapter 3 we have demonstrated that in

mice, TSP-1 inhibits rather than facilitates the breakup of plasma VWF in the presence

of other operative mechanisms (including but possibly not restricted to ADAMTS13) in-

151 v1vo. We propose that this inhibition occurs in part due to TSP-1 competing with

ADAMTS13 for a common binding site in the A3 domain of VWF. It is also feasible that TSP-1 inhibits ADAMTS13 by a direct binding interaction as it does with neutrophil elastase and cathepsin G, and is worthy of further investigation. Cultured endothelial cells can be stimulated to release VWF in a parallel plate flow chamber and the adherence of fluorescent labeled platelets to ULVWF strings under shear can be recorded using video-microscopy. This test system is used by Dr. Jing-fei Dong at the

University of Texas in Houston, Texas, to study ADAMTS 13 mediated cleavage of

ULVWF under near physiological conditions. We are studying, with Dr Dong, the role ofTSP-1 inhibition of ADAMTS13 mediated cleavage ofVWF strings in his test system. In three separate experiments, TSP-1 null plasma was less efficient (60- 70% of wild type plasma) in cleaving VWF strings under flow. This contrasted with marginally increased VWFCP activity in TSP-1 null plasma using two different static assays. It is possible that the bi-functional role ofTSP-1, ofVWF reductase activity and

ADAMTS13 inhibition become important at different concentrations ofTSP-1. This possibility will be tested by adding exogenous human TSP-1 to TSP-1 null murine plasma at increasing concentrations and studying the cleavage of VWF strings. These results will be further clarified using recombinant ADAMTS13, platelet derived full length TSP-1 and recombinant fragments of TSP-1 in this test system.

We have also found that TSP-1 is a regulator of platelet VWF multimer size (chapter 3;

Pimanda et al., submitted). The absence of ADAMTS13 in platelets and the absence of

shear in the interstices of a developing thrombus provide both a need for an alternate

152 control mechanism and an arena for TSP-1 to function. Our results show enhanced aggregation of TSP-1 null platelets in response to collagen under both static and shear conditions. We propose that this is in part due to the larger VWF multimers in TSP-1 null platelets but also recognize a possible role for the control of platelet integrins by

TSP-1. TSP-1 null mice do not have a bleeding or thrombotic phenotype and are reported to have normal bleeding times and display a normal platelet aggregation response to thrombin. Our observations will form the basis of more detailed investigation into the properties ofTSP-1 null platelets in the laboratory of Dr Shaun

Jackson at Monash University, Melbourne. Preliminary results show that TSP-1 null platelets spread faster on glass than do wild-type platelets and the rate of platelet spreading on collagen and fibrinogen will also be measured. The role of platelet VWF in TSP-1 null platelets will be further investigated by studying platelet-platelet interaction at different shear rates under video microscopy. The performance of TSP-1 null platelets in the vicinity of an injured blood vessel is also not known and will be studied in a murine model of vessel injury.

Factors that regulate ADATMS13 enzyme activity are of interest as a majority of patients with TTP do not have a severe deficiency despite responding to plasma exchange. Our findings indicate that normal or even increased concentrations of plasma

TSP-1 cannot compensate for the absence of ADAMTS13 activity (chapter 4). The treatment ofTTP with supra-physiological doses of TSP-1 is unlikely to be of benefit, particularly in light of the potential inhibition of ADAMTS13 by TSP-1 and the results reported in Chapter 4 of this thesis. It is envisaged that recombinant ADAMTS13 will

153 be used in the future treatment ofTTP. Improved and easily performed assays of

ADAMTS13 activity and ADAMTS13 inhibitors that guide the rational use of recombinant products are needed.

The structure -function relationship between ADAMTS13 and VWF and factors that control this interaction are under scrutiny in a number of laboratories. The C-terminal

TSRs and CUB domains of ADAMTS13 are of uncertain importance as truncated mutants retain normal VWFCP activity using non-flow based assays of enzyme function. Peptides derived from the TSR and CUB domains of ADAMTS13 have been used to inhibit the activity of ADAMTS13 under flow and could possibly be developed as ADAMTS13 inhibitors to promote thrombosis in bleeding diatheses. We studied a patient with congenital TTP associated with a mutation in the second CUB domain which results in the truncation of 49 amino acid residues from the C-terminus of the protein. The secretion of the mutant was severely impaired but the expressed protein retained normal in-vitro function.

VWF multimer size is currently measured using 1% agarose gel electrophoresis or estimated using the collagen binding assay. TSP-1 reduces only the ULVWF multimers, and measuring variations in size of these -20,000 kDa multimers is difficult and the changes are often subtle. Nevertheless these subtle changes on gel electrophoresis translate to functional differences as indicated by the more robust changes measured using the collagen binding assay. Improved methods of rapidly and reliably measuring

VWF multimer size are needed.

154 VWF is not an established target for anti-thrombotic drug development and modulating the concentration and/ or multimer size of a plasma protein to this end would be an interesting challenge. The spectrum of pathologies associated with either a deficiency of

VWF Ag or abnormalities in multimer size and the proven efficacy of anti-a.rlbJ33 therapy would support VWF as a worthy target for drug development. As the activity of

ADAMTS13 appears to be modulated by the substrate itself, this enzyme has promise as a potential antithrombotic with a favourable therapeutic index. It has to its credit a long circulating half life (2-3 days) although the wide normal range of this enzyme in plasma would suggest that it is already present in excess. VWF is the only known substrate for

ADAMTS13 but others may exist and could be an issue in drug development. Our work suggests that TSP-1 is more important in the control of platelet VWF multimer size.

Unless platelet or megakaryocyte VWF multimer size can be controlled in-situ, our current understanding of thrombus growth would suggest that exogenous therapy would be of little practical value. As platelets are already rich in TSP-1 a further elevation of its content is unlikely to be of benefit. The inhibition of ADAMTS13 by TSP-1 or its fragments on the other hand have potential as pro-thrombotic agents for use in bleeding diatheses. The current practice ofDDAVP infusion to stimulate endothelial VWF release in the treatment of bleeding diatheses could be complemented with agents that inhibit ADAMTS13. This would preserve the effective fraction ofVWF multimers that are released in response to DDAVP for primary haemostasis.

I have detailed, in the course of this thesis, the role of two proteins that control VWF multimer size. This story is not complete but in evolution. The next phase would be to

155 identify factors that by regulating the interaction of these proteins at the blood­ endothelial interface influence the final outcome.

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