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The Role of Platelet Thrombin Receptors PAR1 and PAR4 in Health and Disease

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The Role of Platelet Thrombin Receptors PAR1 and PAR4 in Health and Disease Linköping University Medical Dissertations No 1261 The role of platelet thrombin receptors PAR1 and PAR4 in health and disease Martina Nylander Division of Clinical Chemistry Department of Clinical and Experimental Medicine Linköping University, Sweden Linköping 2011 © Martina Nylander, 2011 Published papers are reprinted with the permission from the copyright holder. The role of platelet thrombin receptors PAR1 and PAR4 in health and disease Cover: A drawing made by the author, illustrating PAR1 & PAR4 cell signaling. Printed in Sweden by LiU-tryck, Linköping, Sweden, 2011 ISBN: 978-91-7393-067-3 ISSN: 0345-0082 ”Life is a mystery” -Julien Offray de La Mettrie To all my dear friends standing steady on earth or flying in the sky ABSTRACT Blood cells are continuously flowing in our systems maintaining haemostasis in the arteries and veins. If a vessel is damaged, the smallest cell fragments in the blood (platelets) are directed to cover the wound and plug the leakage to prevent blood loss. Most of the time platelets stop the blood leak without any difficulties. During other, pathological, circumstances, platelets continue to form a thrombus, preventing the blood flow and may cause myocardial infarction or stroke. Thrombin is the most potent platelet agonist and is a product created in the coagulation cascade. This thesis is focused on the interactions between the two platelet thrombin receptors; protease activated receptors 1 (PAR1) and PAR4 in vitro. We have investigated potential differences between these receptors in several situations associated with cardiovascular disease. First we studied interactions between PAR1 and PAR4 and the oral pathogen Porphyromonas gingivalis (which secretes enzymes, gingipains, with properties similar to thrombin). Here we showed that P. gingivalis is signaling mainly, but not exclusively, via PAR4. Our second study showed that the cross-talk between the stress hormone epinephrine and thrombin occur exclusively through PAR4 if the key-substance ATP is present and cyclooxygenase-1 inhibited by aspirin. The third study investigated platelet secretion, with focus on the protein plasminogen activator inhibitor 1(PAI-1), an inhibitor of the fibrinolytic process responsible for dissolving a formed clot. Here we showed that PAI-1 secretion and synthesis was more sensitive to stimulation through PAR1 than PAR4. Finally this thesis describes differences between PAR1 and PAR4 in cell-signaling pathways regulating the stability of a platelet aggregate, where PAR4 seems to be of importance to create stable platelet aggregates and that this stability is dependent on ADP activation via P2Y12 and cell signaling via PI3-kinase. Until now, PAR1 has been considered to be the most important thrombin receptor, due to its high affinity for thrombin. However, there must be a reason why platelets express two different thrombin receptors. This thesis highlights several situations where PAR4 plays a complementary and important role in platelet signaling and haemostasis. In conclusion, this thesis suggests that PAR4 plays a major role in calcium signaling and the induction of sustained aggregation, while PAR1 shows a more prominent role in platelet 5 secretion and synthesis. This thesis also reveals new interactions between platelet thrombin receptors and the ADP-, ATP- and epinephrine receptors. The results described in this thesis contribute to an increased knowledge of the platelet thrombin receptors and their interplay in situations such as infection, stress, fibrinolysis, and platelet aggregation. 6 POPULÄRVETENSKAPLIG SAMMANFATTNING I människokroppen flyter blodet genom kärlen för att upprätthålla homeostasen och andra jämvikter. Ibland händer det att en skada uppstår i kärlväggen, och det första som händer är att blodplättarna (trombocyterna) samlas vid skadan och fäster samt sprider ut sig och bildar en plugg för att stoppa blödningen. När skadan väl är tilltäppt så kommer läkningsprocessen att börja återställa alla kärlväggsceller och blodflödet igen. Ibland händer det dock att processen inte balanseras på rätt sätt, och följden kan då bli att trombocytpluggen blir större och till slut bildar en trombos, en blodpropp, som i sin tur kan orsaka hjärtinfarkt eller slaganfall.. Trombin är ett enzym som bildas när koagulationskaskaden aktiveras, och dess funktion är att aktivera trombocyterna och aktivera koagulationen ytterligare för stabilisering av den pågående temporära trombocytpluggen. Trombocyten i sig har två specifika receptorer som trombin binder in till, PAR1 och PAR4, och det är skillnaden mellan dessa receptorer som undersökts i avhandlingen. Avhandlingen beskriver trombocyten och dess trombinreceptorer PAR1 och PAR4 och visar på ett antal situationer där dessa spelar olika roller i trombocyten. Trombocyter tillsammans med bakterien Porphyromonas gingivalis samt stresshormonet adrenalin visar sig ge upphov till en trombocytaktivering främst via PAR4, och till en mindre del via PAR1. I trombocyter som behandlats med aspirin visar sig adrenalin också kunna ge upphov till trombocytaggregation i närvaro av mycket låga doser av trombin, denna gång via PAR4 och inte alls via PAR1. Avhandlingen tar även upp skillnader mellan PAR1 och PAR4 för frisättningen av ett viktigt protein som förhindrar nedbrytningen av blodkoagel i kärlet, PAI-1. Här visar vi att PAI-1- frisättning är känsligare för aktivering via PAR1 än PAR4. Slutligen visar denna avhandling att det finns tydliga skillnader mellan trombinreceptorerna vad gäller trombocytaggregatens stabilitet där PAR4 verkar spela den största rollen för att stabilisera och bibehålla ett bildat aggregat. Om man betraktar det fylogenetiskt och patofysiologiskt så borde det finnas en orsak till varför trombocyten har två trombinreceptorer. Tidigare har det föreslagits att PAR1 skulle vara den enda trombinreceptor som är av betydelse inom hemostas- och trombosområdet. I 7 denna avhandling visar vi att den andra trombinreceptorn, PAR4, också kan vara av betydelse för trombocytsignalering, trombocytaktivering och hemostas. Avhandlingens slutsats är att trombinreceptorn PAR4 kan spela en större roll för trombocytaktivering och proppbildning än vad man tidigare trott. Avhandlingen beskriver även nya skillnader mellan PAR1 och PAR4 och deras interaktioner med ADP-, ATP- och adrenalin-receptorerna. Resultaten från avhandlingen bidrar till mer förståelse för trombocyten och dess trombinreceptorer vid situationer såsom infektion, stress, fibrinolys och trombocytaggregation. 8 TABLE OF CONTENTS ABSTRACT ………………………………………………………………………………. 5 POPULÄRVETENSKAPLIG SAMMANFATTNING ………………………………… 7 ABBREVISTIONS ……………………………………………………………………….. 10 LIST OF PAPERS ………………………………………………………………………... 13 INTRODUCTION ……………………………………………………………………….. 15 Haemostasis …………………………………………………………………………... 15 Platelets ……………………………………………………………………………….. 17 Protease-activated receptors …………………………………………………………… 18 Purinergic receptors……………………………………………………………………. 25 Adrenergic receptors…………………………………………………………………... 26 Haemostasis & Thrombosis ………………………………………………………….. 26 AIMS OF THE THESIS …………………………………………………………………. 29 METHODOLOGY……………………………………………………………………….. 31 Materials ……………………………………………………………………………… 31 Isolation of human platelets………………………………………………………….. 32 Measurement of platelet aggregation ……………………………………………….. 32 Culture and preparation of Porphyromonas gingivalis …………………………….. 32 Measurement of cytosolic calcium …………………………………………………... 33 Measurement of dense granule secretion …………………………………………… 33 Western Blot ………………………………………………………………………….. 33 Fluorescence Microscopy ……………………………………………………………. 35 Enzyme-linked immunosorbent assay ……………………………………………… 35 mRNA assay …………………………………………………………………………... 35 Experimental design …………………………………………………………………. 36 RESULTS AND DISCUSSION ………………………………………………………….. 39 PAPER I ......................................................................................................................... 39 PAPER II ....................................................................................................................... 40 PAPER III ...................................................................................................................... 41 PAPER IV …………………………………………………………………………….. 42 GENERAL DISCUSSION ………………………………………………………………. 43 PRINCIPAL FINDINGS ………………………………………………………………… 51 ACKNOWLEDGEMENTS ……………………………………………………………... 53 REFERENCES……………………………………………………………………………. 55 9 ABBREVIATIONS AC adenylyl cyclase Ab antibody ACD Acid citrate dextrose ADP adenosine 5’-diphosphate ASA acetyl salicylic acid ATP adenosine 5’-triphosphate bFGF basic fibroblast growth factor cAMP cyclic adenosine monophosphate COX cyclooxygenase DAG 1,2 diacylglycerol DVT deep vein thrombosis ELISA Enzyme-linked Immunosorbent Assay GAPDH glyceraldehyde 3-phosphate dehydrogenase GP glycoprotein GPCR g-protein coupled receptor GP glycoprotein HEPES N-[2-hydroxyethyl]-piperazine-N’-[2-ethanene-sulfonic acid] IP3 inositol 1,4,5-triphosphate Kgp lysine-specific protease KRG Krebs-Ringer glucose solution mRNA messenger Ribonucleic acid NCCE non-capacitative Ca2+-entry NO nitric oxide OMV outer membrane vesicles PAI-1 plasminogen activator inhibitor 1 PAR1 protease-activated receptor 1 PAR1-AP PAR1-activating peptide (amino acid sequence: SFLLRN) PAR4 protease-activated receptor 4 PAR4-AP PAR4-activating peptide (amino acid sequence: AYPGKF) PDGF platelet-derived growth factor PGI2 prostacyclin = prostaglandin I2 PI3-K phosphatidyl inositol 3 kinase PIP2 phosphatidylinositol 4,5-biphosphate 10 PKC protein kinase
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