Defining the role of protease-activated receptors in thrombosis
Creators: name: Lee, Hannah
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Introduction: Arterial thrombosis is the leading cause of morbidity and mortality in industrialised countries. Platelets are the key cellular component of arterial thrombi and are therefore the main target of antithrombotic therapies. Current anti-platelet agents, most commonly aspirin and/or clopidogrel, exhibit low efficacy in the prevention of arterial thrombosis, and novel anti-platelet agents are therefore sought. Thrombin potently activates platelets via protease-activated receptors (PARs) and antagonists of the primary thrombin receptor on platelets, PAR1, are leading candidates for novel anti-platelet therapies. However, recent results of the initial Phase 3 clinical trials of the first-in-class PAR1 antagonist, vorapaxar, showed a lack of clinical benefit when the PAR1 inhibitor was added to existing anti-platelet therapies, largely due to an increase in bleeding which outweighed any antithrombotic effect. These findings suggest that there remains much to learn about the roles of PARs in thrombosis and how best to utilise PAR antagonists in the setting of arterial thrombosis. Aim: The studies of thesis used genetic mouse models and pharmacological intervention to define the role of PARs in thrombosis. Key Findings: First, the role of PAR-dependent platelet activation in thrombus formation was examined in the pathological setting of elevated blood shear rates. Two models of in vivo thrombosis and an ex vivo whole blood flow approach were used in PAR4-/- mice, whose platelets do not respond to thrombin, to show that the contribution of PAR-mediated platelet activation to thrombosis is reduced when blood shear rates are elevated, due to a decrease in platelet incorporation into growing thrombi. Second, treatment of PAR4-/- mice with either of the existing antiplatelet agents, aspirin or clopidogrel, provided marked protection against this high shear thrombosis. However, only treatment with aspirin provided this antithrombotic effect without a significant impact on bleeding, as assessed by two independent in vivo assays of haemostasis. Third, the roles in thrombosis of the two thrombin receptors on human platelets, PAR1 and PAR4, were examined. To do this, existing PAR1 antagonists were characterised and a PAR4 antagonist was developed. The experimental PAR1 antagonist, SCH79797, but not the clinically trialled PAR1 antagonist, E5555, induced PAR-independent effects on platelet morphology and function that impact its value as an experimental tool. An anti-PAR4 function-blocking antibody was developed and found to provide highly specific inhibition of thrombin-induced PAR4 activation on human platelets, as assessed by platelet aggregation. E5555 and the anti-PAR4 antibody were then used to examine the specific functions of PAR1 and PAR4, respectively, on human platelets. It was found that PAR4 selectively promotes thrombin-induced PS externalisation on human platelets, and that this effect may contribute to platelet procoagulant activity in the setting of thrombus formation. Summary: The studies of this thesis examined pathological conditions and anti-platelet drug interactions in order to identify the conditions under which PAR inhibition may be of most clinical benefit. Furthermore, using novel pharmacological tools, it was demonstrated that PAR4 performs a function distinct from PAR1 in human platelets. Together, these studies provide insights into how PAR-mediated platelet activation might be best manipulated for the prevention of arterial thrombosis.