This application is a new submission extending prior research funded by HL102482, initially funded in 2009 to identify the genetic basis for inter-individual variation in platelet function that contributes to ischemic occlusion of arteries. We generated a human reference platelet transcriptome, developed a public, user-friendly interactive web tool to query platelet function and RNA-protein associations, and discovered platelet aggregation through the protease activated receptor-4 (PAR4) thrombin receptor was greater in blacks than whites. We showed this difference was due to a PAR4 Ala120Thr substitution with racially divergent allele frequencies (Thr120: .63 blacks; .19 whites). The Thr120 variant had increased sensitivity to thrombin and demonstrated ex vivo thrombus formation under arterial, but not venous, shear stress. Genotyping >12,000 patients demonstrated the Thr120 allele was associated with an increased risk of ischemic stroke and less bleeding. More recent work has led to new data relevant to basic and clinical aspects of platelet PAR biology, and the overall goals of the current application are to study (1) PAR4 interactions with other GPCRs and proteases and (2) the effect of human PAR4 (hPAR4) and the Ala120Thr variant in an in vivo model of brain ischemia/reperfusion (I/R) injury. PAR4 has slower signaling kinetics than PAR1, and our new data shows that PAR4 co-immunoprecipitates with platelet PGI2 receptor (IP). Compared to PAR4 Ala120, Thr120 is relatively resistant to desensitization in the presence of prostacyclin (PGI2), and generates less cAMP after activation in human platelets and primary megakaryocytes (MKs). We hypothesize IP cross-talks with PAR4, and Aim 1 will assess the role of IP/Gas in PAR signaling and desensitization in genetically altered human MKs and platelets from our novel humanized PAR4 mouse lines. The neutrophil serine protease, cathepsin G (CatG), is a potent platelet agonist that activates platelet PAR4, but not PAR1, and we show CatG induces more platelet activation of PAR4 Thr120 than Ala120. For the first time, we identified CatG enzymatic cleavage sites in PAR4, one of which generates a novel tethered ligand, SRALLLGWVPTR, which induces human platelet aIIbb3 activation, granule release and aggregation. Aim 2 will study CatG-platelet PAR4 interactions. Platelet PAR4, not PAR1, is critical for leukocyte recruitment, rolling and adhesion, and our preliminary data show that murine brain I/R injury in our humanized PAR4 mouse line is hPAR4- and neutrophil-dependent. The role of hPAR4 in stroke and brain hemorrhage after I/R injury has been poorly studied, and the goal of Aim 3 is to utilize our hPAR4 mouse lines to determine how hPAR4 mediates platelet and neutrophil activities in a murine stroke model, and to assess the pharmacogenetic effect of the Ala120Thr variant on hPAR4-mediated infarct, hemorrhage and platelet signaling pathways. Successful completion of the proposed studies is expected to enhance the understanding of the molecular basis of inter-individual variation in human platelet biology and PAR4-expressing tissues, and provide groundwork for individualized anti-platelet therapies in disorders with racial predilections.

Narrative The goal of this research is to understand better the molecular genetic mechanisms that contribute to blood platelet reactivity and stroke outcomes, and to develop better targets for drug development to treat ischemic vascular disease based on an individual’s physiology.