Platelet-Neutrophil Interaction
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PLATELET-NEUTROPHIL INTERACTION: NEUTROPHILS MODULATE PLATELET FUNCTION Richard William FAINT 1992 A thesis submitted to the University of London for the degree of Ph.D The Department of Haematology University College and Middlesex School of Medicine University College London 98 Chenies Mews London WC1E 6HX ProQuest Number: U049250 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U049250 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 2 ABSTRACT Platelets are vital for the arrest of bleeding following tissue injury. Neutrophils play an important role in host defence against bacterial infection. In the milieu of the acute inflammatory response both cellular and non-cellular elements may interact to modify behaviour. Evidence suggests that leukocytes may play an active role in the modulation of platelet function. This interaction may be abnormal in certain pathological states. This study examined the effect of purified neutrophils upon both washed platelets and platelet-rich plasma, as well as in whole blood in vitro. Neutrophils were found to alter platelet behaviour by several mechanisms. These included transcellular metabolism of eicosanoids. Neutrophils utilized platelet-derived arachidonate to release increased amounts of leukotrienes. Other arachidonate metabolites resulted from platelet-neutrophil interaction and these differed quantitatively and qualitatively from those arising from either cell-type alone. Another mechanism was the release of a nitric oxide-like factor by neutrophils. Nitric oxide inhibits platelet adhesion and aggregation via guanylate cyclase stimulation. Platelet ATP secretion and TXB2 release were also impaired. The effect of authentic nitric oxide in whole blood was investigated. Platelet aggregation in whole blood, in the presence of L-arginine (the substrate for nitric oxide synthesis), was found to be inhibited by a nitric oxide-like factor. Neutrophils, under different conditions, were potent inducers of platelet calcium flux, aggregation and secretion. This activity was mediated by a neutrophil-derived protease, most likely to be cathepsin G. These different mechanisms of platelet-neutrophil interaction were investigated in patients with Multiple System Organ Failure Syndrome, where clinical observations have implicated both platelet and neutrophil involvement. Some patients exhibited platelet-neutrophil interaction abnormalities. The interaction of platelets with neutrophils may help to explain some of the pathophysiological events associated with different clinical states. 4 TABLE OF CONTENTS SECTION PAGE ABSTRACT 2 LIST OF TABLES 9 LIST OF FIGURES 10 ACKNOWLEDGEMENTS 13 ABBREVIATIONS 14 CHAPTER 1 GENERAL INTRODUCTION 1.1 Introduction 15 1.2 Platelet responses 19 1.2.1 Adhesion 19 1.2.2 Shape change 20 1.2.3 Aggregation 21 1.2.4 Secretion 21 1.2.5 Dense granule secretion 22 1.2.6 Alpha granule secretion 23 1.2.7 Lysosome secretion 23 1.2.8 Arachidonate liberation 24 1.3 Physiologically important platelet agonists 25 1.4 Neutrophil responses 27 1.4.1 Adherence and migration 28 5 1.4.2 Chemotactic receptor activation 29 1.4.3 Proteolytic enzymes 31 1.4.4 The NADPH oxidase system and oxygen metabolites 33 1.4.5 Bioactive lipids 35 1.5 Intracellular regulatory processes 36 1.5.1 G-proteins 38 1.5.2 Inositol phosphates and cell signalling 40 1.5.3 Diacylglycerol and protein kinase C 41 1.5.4 Inhibition by cyclic nucleotides 42 1.6 Cell-cell interactions 43 1.6.1 Eicosanoid metabolism 44 1.6.2 Classification of eicosanoid mediated cell interactions 46 1.6.3 PAF-acether formation 48 1.6.4 PF4- and PDGF-induced neutrophil chemotaxis 49 1.6.5 Oxygen metabolites and proteolytic enzymes 50 1.6.6 Nitric oxide metabolism 52 1.7 Pathological implications of platelet-PMN interactions 54 1.7.1 Platelet-neutrophil interactions in septicaemic shock 55 1.8 Aims of thesis 58 CHAPTER 2 METHODS 2.1 Blood samples, collection and handling 59 2.2 Full blood, differential, platelet and leukocyte counts 60 6 2.3 Preparation of platelet-rich plasma (PRP) 60 2.4 Preparation of washed platelets 60 2.4.1 Gel filtration 60 2.4.2 Centrifugation with inhibitors 61 2.5 Preparation of washed, purified neutrophils 61 2.6 Methods for studying platelet function 63 2.6.1 Turbidometric platelet aggregation 63 2.6.2 Whole blood impedance aggregation 64 2.6.3 Measurement of adenine nucleotides in ethanol extracts 65 2.6.4 Measurement of ATP secretion by PRP and washed platelets 67 2.7 Measurement of intracellular calcium flux 68 2.8 Cyclic GMP measurement by radioimmunoassay 70 2.9 Preparation of authentic nitric oxide solution 73 2.10 Thromboxane B2 radioimmunoassay 74 2.11 6-Keto-Prostaglandin Flot radioimmunoassay 75 2.12 Leukotriene B4 radioimmunoassay 77 2.13 High performance liquid chromatography (HPLC) of eicosanoids 79 2.13.1 Preparation of [1-14C] sodium arachidonate 79 2.13.2 Labelling of platelets with [1-14C] sodium arachidonate 79 2.13.3 Labelling of neutrophils with [1-14C] sodium arachidonate 80 2.13.4 Incubation and activation of samples 80 2.13.5 Extraction of arachidonate metabolites 81 2.13.6 HPLC of eicosanoids 82 7 2.13.7 Quantitation of eicosanoids by ultra violet absorbance 84 2.14 Statistical analysis 85 CHAPTER 3 Platelet aggregation is inhibited by a nitric oxide-like factor released from human neutrophilsin vitro 3.1 Introduction 87 3.2 Methods 89 3.3 Results 91 3.4 Discussion 107 CHAPTER 4 The inhibition of whole blood aggregation in vitro by an endogeneous nitric oxide-like factor 4.1 Introduction 116 4.2 Methods 117 4.3 Results 118 4.4 Discussion 121 CHAPTER 5 Neutrophils stimulate platelet intracellular calcium flux and aggregationin vitro 5.1 Introduction 128 5.2 Methods 129 5.3 Results 131 8 5.4 Discussion 144 CHAPTER 6 Interaction between platelets and neutrophils for eicosanoid production 6.1 Introduction 148 6.2 Methods 149 6.3 Results 151 6.4 Discussion 171 CHAPTER 7 A preliminary investigation of platelet-neutrophil interactions in Multiple System Organ Failure (MSOF) 7.1 Introduction 175 7.2 Methods 177 7.3 Results 180 7.4 Discussion 191 CHAPTER 8 Summary and Conclusions 8.1 Summary 194 8.2 Conclusions 196 CHAPTER 9 References 198 9 LIST OF TABLES 1.1 Major factors secreted by platelets 1.2 Major platelet activating agents 1.3 Neutrophil granules and their major contents 2.1 Ultraviolet absorption characteristics of some arachidonic acid metabolites 7.1 Clinical diagnosis of patients studied 7.2 Platelet aggregation to thrombinin the presence of neutrophils 7.3 Platelet aggregation to thrombinin the presence of SOD and neutrophils 7.4 Platelet aggregation to thrombin in the presence of L-arginine and neutrophils 7.5 Platelet aggregation to thrombin in the presence of N^nitro-L-arginine and neutrophils 7.6 Platelet aggregation to thrombin in the presence of haemoglobin and neutrophils 7.7 95% Confidence intervals for the effect of NO release 7.8 95% Confidence intervals for the stimulation of platelet aggregation by neutrophils 7.9 Platelet aggregation in the presence of neutrophils stimulated with FMLP 7.10 Platelet aggregation in the presence of neutrophils stimulated with LTB4 7.11 Platelet aggregation in the presence of neutrophils stimulated with PAF 7.12 Platelet aggregation induced by thrombin in the presence of neutrophils 10 LIST OF FIGURES 1.1 Signal transduction during platelet activation 1.2 The arachidonate cascade 2.1 Calibration curve for determining intracellular [Ca2+] 3.1 The effect of neutrophils upon platelets stimulated with different agonists 3.2 The effect of neutrophil concentration on platelet aggregation 3.3 Thrombin concentration and platelet aggregation in the presence of neutrophils 3.4 The effect of different NO concentrations on thrombin-induced platelet aggregation 3.5 Characterisation of a neutrophil-derived inhibitory factor on platelet aggregation 3.6 The effect of neutrophils on thrombin-induced platelet ATP secretion 3.7 The effect of neutrophils on thrombin-induced platelet TXB2 release 3.8 The effect of unstimulated neutrophils on platelet cyclic GMP 3.9 The effect of neutrophils stimulated with FMLP on platelet cyclic GMP 3.10 The effect of neutrophils stimulated with LTB4 on platelet aggregation 3.11 The failure of N°-nitro-L-arginine to completely restore platelet aggregation 3.12 The characterisation of another neutrophil-derived inhibitor of platelet function 3.13 Inhibition by neutrophils of platelet aggregation is prevented by adenosine deaminase 3.14 A dose response curve for collagen-induced aggregation in the presence of adenosine 3.15 The activity of different supernatants on collagen-induced platelet aggregation 4.1 The effect of nitroprusside and authentic nitric oxide on aggregation in blood 4.2 The effect of red blood cells on inhibition of aggregation by nitric oxide 4.3 The effect of L-arginine and phosphodiesterase inhibitors upon aggregation in blood 4.4 A dose response curve for the inhibition by L-arginine of aggregation in blood 4.5 The effect of N^nitro-L-arginine on aggregation in