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Role of Phosphoinositide 3-Kinase in Platelet Responses To ROLE OF PHOSPHOINOSITIDE 3-KINASE IN PLATELET RESPONSES TO PLATELET-ACTIVATING FACTOR by RONALD WILLIAM LAUENER B.Sc, Simon Fraser University, 1985 M.Sc, The University of British Columbia, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 1997 © Ronald William Lauener, 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 '( ABSTRACT Platelets are necessary for hemostasis but are also involved in a broad range of pathophysiologic processes such as atherosclerosis and thrombosis. Platelet activating factor (PAF), an inflammatory mediator, is an important physiological regulator of platelet function. The major objective of this work was to determine the role of phosphoinositide 3-kinase (PI 3-kinase) in platelet responses to platelet activating factor. We show, for the first time in platelets, that PAF activates PI 3-kinase over a rapid time course that correlates closely with the aggregation response. The potent PI 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-l-benzopyran-4-one (LY294002) were used to probe the dependence of PAF-induced aggregation and dense granule release on PI 3-kinase. Both compounds markedly inhibited PAF-induced aggregation; however, only at low activation states giving reversible aggregation (primary phase) did this correlate with PI 3-kinase inhibition. Secretion, measured as release of 3H- hydroxytryptamine, was inhibited to a maximum of 30 % and only at low concentrations of PAF. We suggest that PI 3-kinase activation is important for reversible (primary) aggregation of platelets in response to PAF, perhaps by contributing to the 'inside-out' activation of platelet glycoprotein Ilbllla, the fibrinogen receptor. PI 3-kinase only plays a minor role in PAF induced dense granule release at low activation states. Both responses are dramatically less dependent on PI 3-kinase activity when high concentrations of PAF are used, suggesting greater contribution from other pathways. Tyrosine kinases appear to be important regulators of PI 3-kinase at high PAF levels as stimulation results in a greater than 10 fold increase in PI 3-kinase activity associated with tyrosine-phosphorylated proteins. The p85 regulatory subunit of PI 3-kinase is not tyrosine-phosphorylated. Rather, the enzyme associates rapidly with a major tyrosine- phosphorylated 115 kDa protein, a potential regulator of PI 3-kinase activation in platelets. This protein was not immunoreactive with several antibodies to proteins in this Ill molecular weight range known to be tyrosine phosphorylated and to associate with PI 3- kinase on cell activation. iv CONTENTS Page ABSTRACT ii CONTENTS iv LIST OF FIGURES ix LIST OF ABBREVIATIONS xi ACKNOWLEDGMENTS xii INTRODUCTION 1 A. Platelets 1 A.1 Platelet Physiology 1 All Thrombopoiesis-The Origin of Platelets 1 A. 1.2 Platelet Ultrastructure/Anatomy 3 A. 1.3 The Platelet in Primary Hemostasis 5 A. 1.4 Congenital Disorders of Hemostasis 8 A. 1.5 Adjunct Roles for Platelets in Other Diseases 9 A.2 Mechanisms of Stimulus-Response Coupling (Signal Transduction) 10 in Platelets A.2.1 Phospholipase C 11 2+ A.2.1.1 IP3/Ca in Platelet Function 12 A.2.1.2 Diacylglycerol/Protein kinase C in Platelet Function 13 A.2.2 Phospholipase D 14 A.2.3 Phospholipase A2 15 A.2.4 Platelet Inhibition via Activation of Adenylate Cyclase 16 V B. Platelet-activating Factor (PAF) 17 B. 1 Physiology and Metabolism of Platelet-activating factor 17 B.2 The Platelet-activating factor Receptor and Signal Transduction 19 B. 3 Physiological Responses of Platelets to Platelet-activating factor 22 C. Phosphoinositide 3-kinase 22 C. l Conventional Polyphosphoinositide Metabolism 23 C.2 Discovery of a New Phosphoinositide kinase (type I PI 3-kinase) 24 C.3 3-Phosphoinositides are Formed In vivo in Oncogene-transformed 25 and Growth-factor stimulated cells C.4 Structural and Functional Characterization of Phosphoinositide 3- 26 kinases C.4.1 The p85 Regulatory Subunit 27 C.4.2 The pi 10 Catalytic Subunit 29 C.5 Regulation of Phosphoinositide 3-kinase 30 C.5.1 Translocation 31 C. 5.2 Allosteric Regulation of PI 3 -kinase 31 C.5.3 Phosphorylation 32 C.5.4 Regulation of PI 3-kinase by G-proteins 33 C.5.4.1 Small G-proteins 33 C.5.4.2 Heterotrimeric G-proteins 34 C.6 Function of Phosphoinositide 3-kinase 34 C.6.1. Correlation of Cellular Responses with PI 3-kinase activity 35 C.6.2. Cloning of PI 3-kinase Provides New Functional Insights 36 C.6.3. Inhibitors of Phosphoinositide 3-kinase 38 vi C. 6.4. Cellular Functions of Phosphoinositide 3-kinase 39 C.6.4.1. Mitogenesis 39 C.6.4.2. Apoptosis 40 C. 6.4.3. Intracellular Vesicular/Protein Trafficking 40 C.6.4.4. Regulation of the Cytoskeleton 41 C.7. Molecular Targets of Phosphoinositide 3-kinase 41 C. 8. Metabolism of 3-phosphoinositides 42 D. Platelets and Phosphoinositide 3-kinase 43 D. 1. Activation of PI 3-kinase in Agonist-stimulated Platelets 43 D.2. Regulation of Phosphoinositide 3-kinase in Platelets 44 D.3. Function of Phosphoinositide 3-kinase in Platelets 48 E. Current Problem 51 OBJECTIVES 52 METHODS 53 A. Platelet Isolation 53 A.1 Rabbit Platelets 53 A.2. Human Platelets 53 B. Platelet Aggregation 54 C. Platelet Secretion (dense-granule release) 54 D. Measurement of PI 3-kinase Activation in Platelets 55 D. 1. Detection and Quantitation of Intracellular Phosphatidylinositol 55 (3,4,5) trisphosphate D. 1.1. Labelling of platelets with 32P-orthophosphate 55 Vll D. 1.2. Extraction of Total Cellular Lipids 55 D.1.3. TLC Analysis of Phosphatidylinositol (3,4,5) trisphosphate 56 D. 1.4. Validation of TLC Method for Measurement of 56 Phosphatidylinositol (3,4,5) trisphosphate D.2. Detection and Quantitation of Intracellular 57 Phosphatidylinositol (3,4) bisphosphate D.3. In vitro Determination of Phosphatidylinositol 3-kinase activity 58 D.3.1 Preparation of Platelet Lysates and Immunoprecipitation 58 D.3.2 Kinase Reaction 58 D.3.3 Thin-layer Chromatography 59 E. Immunoblotting of Platelet Lysates and anti-PI 3-kinase 59 Immunoprecipitates RESULTS 61 A. Activation of PI 3-kinase in PAF-stimulated platelets 61 A. 1 Analysis of 3-phosphoinositide Formation in PAF-stimulated 61 platelets A. 2 PI 3-kinase Activity Associated with Tyrosine-phosphorylated 66 proteins B. Role of PI 3-kinase in PAF-stimulated Platelets 69 B. 1. Correlation of PI 3-kinase Activation with Platelet Functional 69 Responses. B.2. Effect of Selective Inhibition of PI 3-kinase on Platelet Functional 73 Responses B.2.1. Validation of Wortmannin and LY294002 as Inhibitors of 73 PI 3-kinase in Platelets B.2.2. Correlation of PI 3-kinase Inhibition with Inhibition of Platelet 87 Functional Responses viii B.2.2.1 Aggregation 87 B.2.2.2 Secretion 90 C. Co-immunoprecipitation of PI 3-kinase with Tyrosine-phosphorylated 94 Proteins in PAF-stimulated Platelets DISCUSSION 104 A. Activation of PI 3-kinase in PAF-stimulated Platelets 104 A. 1. Direct Measurement of 3-phosphoinositides 104 A. 2. PI 3-kinase Activity Associated with Tyrosine-phosphorylated 106 Proteins B. Effect of PI 3-kinase Inhibitors on Platelet Responses to PAF 109 B. 1. Validation of Wortmannin and LY294002 as Inhibitors of Platelet 110 PI 3-kinase B.2. Aggregation 112 B.3. Secretion 115 C. Co-immunoprecipitation of PI 3-kinase with Tyrosine-phosphorylated 116 Proteins in Activated Platelets D. Mechanisms leading to Activation of Platelet PI 3-kinase following 120 Ligation of the PAF Receptor E. A Model for the Involvement of PI 3-kinase in Platelet Activation 122 Leading to Reversible Aggregation F. Recommendations for Future Work 123 CONCLUSIONS 126 REFERENCES 129 ix LIST OF FIGURES Page Figure 1. PIP3 is elevated in PAF-stimulated platelets. 62 Figure 2. PAF induces a concentration-dependent increase in PIP3 in platelets. 64 Figure 3. Identification of PIP3 in PAF-stimulated platelets. 67 Figure 4. PI 3,4P2 formation is induced in PAF-stimulated platelets. 68 Figure 5. PI 3-kinase activity associated with tyrosine-phosphorylated 70 proteins is increased in PAF-stimulated platelets Figure 6. Aggregation and activation of PI 3-kinase are tightly linked 72 events in PAF-stimulated platelets. Figure 7. Attainment of irreversible aggregation is correlated with a 74 large increase in PI 3-kinase activity associated with tyrosine- phosphorylated proteins. Figure 8. Wortmannin and LY294002 are potent inhibitors of rabbit 78 platelet PI 3-kinase in vitro. Figure 9. Wortmannin inhibition of PI 3-kinase is retained after detergent- 79 lysis of platelets, immunoprecipitation and numerous washings. Figure 10. Wortmannin rapidly permeates rabbit platelets. 81 Figure 11. Concentration-dependence for wortmannin inhibition of 83 PI 3-kinase activity in anti-p85 immunoprecipitates without removal of wortmannin from the medium before platelet lysis. Figure 12. Wortmannin and LY294002 potently inhibit PAF-induced 84 formation of PIP3 in platelets. Figure 13. Wortmannin is unstable in nutrient cell-culture medium at 86 physiological pH. Figure 14. Inhibition of aggregation by wortmannin depends on the 88 activation state of the platelets Figure 15.
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