The role of protease activated receptors on platelets

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PROJECT SUMMARY/ABSTRACT Venous thrombosis (VT) and its major complication, pulmonary embolism (PE), are often grouped together and called venous thromboembolism (VTE). VTE is a major health problem that affect nearly 600,000 people each year. The historical drivers of VTE are blood stasis, endothelial dysfunction, and hypercoagulation (Virchow’s triad). It is now recognized that platelets and neutrophils have a critical initiating role. The molecular mechanisms are only being uncovered. Given that hypercoagulation is a risk factor and thrombin activated protease activated receptor 4 (PAR4) promotes procoagulant platelets, phosphatidyl serine (PS) exposure and subsequent thrombin generation, we propose that PAR4 is an important contributor to VTE. The long-term goals of this research program are to uncover the mechanisms of PAR4 activation at the molecular level and test these mechanism in vivo to inform disease processes and potential drug development. The scientific premise of this proposal is based our preliminary data showing that extracellular loop 3 (ECL3) of PAR4 coordinates with the ligand binding site (LBS) during PAR4 activation. Further, mutations in either ECL3 or the LBS disrupt PAR4 signaling to the same degree. This points to an essential role for ECL3 in PAR4 activation. The overall objective of this proposal is to 1) to determine how PAR4 contributes to the initiation and propagation of venous thrombosis using mouse models, 2) conduct proof-of-concept studies using PAR4 antagonist to treat VT, 3) to translate our recent structural insights on the PAR4 activation mechanism to PAR4 function in vivo. We will do this by taking advantage a PAR4 variant in human platelets and a new mouse model. Our overall hypothesis is that the sustained signaling from PAR4 on platelets is a driver of VTE and reduced PAR4 signaling from hypo-reactive variants or pharmacological inhibitors will lead to protection from VTE. Our innovative approach will take advantage of a new mouse model that recreates a polymorphism in ECL3 and will allow us to determine the mechanism of how this polymorphism contributes to platelet function and thrombosis. The completion of the proposed studies will accomplish two major advances. 1) we will be the first to specifically examine the role of PAR4 in venous thrombosis. 2) we will continue to push our basic understanding of PAR activation mechanisms by testing the observations from our structural studies in vivo to determine how these mechanisms operate in physiological and pathophysiological contexts.