This is one of three collaborative ROls submitted in response to RFA HL-99-015 on Arterial Thrombosis. The applicants proposal's overall goal is to define the signaling mechanisms by which platelets are activated, thereby providing a framework for therapeutic development and risk factor identification. The goal of this individual project is to define the role of thrombin signaling in hemostasis and thrombosis. Thrombin activates platelets via proteaseactivated G protein-coupled receptors (PARs). The applicants shall use mice deficient in these receptors to ask: 1) Is thrombin signaling in platelets necessary for normal hemostasis? PAR3 and PAR4 are the thrombin receptors known to participate in mouse platelet activation. It appears that mPAR3 does not mediate transmembrane signaling but instead acts as a cofactor for mPAR4 cleavage and activation by low concentrations o f thrombin. Thus the applicants expect PAR4 to be required for thrombin signaling in mouse platelets. The applicants will use PAR4-deficient mice to test the importance of thrombin signaling in hemostasis. 2) Does attenuation or ablation of thrombin signaling in platelets inhibit thrombosis? Human platelets use PAR1 and PAR4 for thrombin signaling, and it is unknown whether inhibition of PAR1 and/or PAR4 would be useful for preventing or treating thrombosis. PAR3- deficient mouse platelets are analogous to PARl-inhibited human platelets (both rely on PAR4 for thrombin signaling). PAR4-deficient mouse platelets will likely prove analogous to human platelets in which all thrombin signaling has been blocked. The applicants will use PAR3- and PAR4-deficient mice to determine whether partial or complete inhibition of thrombin signaling in platelets protects against arterial and microvascular thrombosis. 3) Is endothelial cell activation by thrombin or other proteases important in hemostasis and thrombosis? The applicants hypothesize that activation of endothelial PAR1 and PAR2 promotes thrombosis and inflammation by promoting platelet and leukocyte rolling and adhesion. The applicants will determine whether mice deficient in PAR1 and/or PAR2 are protected in models of inflammation and microvascular and arterial thrombosis. 4) Do gain-of-function mutations in platelet G protein-coupled receptors (GPCRs) promote thrombosis? The discovery of prothrombotic mutations in genes that regulate cellular behaviors has lagged that in genes encoding the plasma proteins. Several human diseases are mediated by gain-of-function mutations in GPCRs. The applicants will use mice bearing gain-of-function mutations in PAR4 to determine if such GPCR mutations, alone or in combination with mutations in other genes, might be a basis for thrombophilia.
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