Determining the Platelet Signaling Pathway(s) Critical in Venous Thrombosis Pathogenesis" it might change in the process of editing

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Project summary Venous thrombosis (VT) affects close to a million Americans annually. One of the main triggers of VT is flow restriction, which leads to vascular inflammation and initiation of thrombus formation. Venous thrombi contain platelets and changes in platelet count correlate with VT outcome in humans and mice. However, little is known about the molecular mechanisms by which these cells contribute to venous thrombogenesis. The goal of my project is to systematically investigate on how major platelet signaling pathways facilitate platelet/leukocyte adhesion, thrombus growth, and thrombus consolidation following vascular stenosis in mice. My main hypothesis is that the platelet signaling pathways required during venous thrombogenesis are similar to those required for securing vascular integrity at sites of inflammation. Specifically, my study aims to delineate if and how platelet G-protein-coupled receptors (GPCRs), immunoreceptor tyrosine-based activation motif (ITAM) receptors, and the Rap1-talin1-integrin signaling axis contribute to VT development in mice. Consistent with my main hypothesis, I expect ITAM signaling and talin1 to be more important for venous thrombogenesis than GPCRs and Rap1. The following transgenic mouse lines with documented defects in platelet integrin signaling will be used: CLEC2 (Clec2fl/flpf4-Cre+), Rap1 (Rap1afl/flRap1bfl/flpf4-Cre+) and Talin1 (Tln1fl/flpf4-Cre+). Pharmacological inhibition of the collagen receptor, GPVI, the ITAM signaling molecule, Bruton’s tyrosine kinase (Btk), and 1 and 3 integrins will also be used. In vivo, VT will be induced by partially ligating the inferior vena cava (IVC stenosis), the gold standard model for VT studies in mice. Mice will be sacrificed after 48 hrs of flow restriction and thrombi harvested and weighed. To investigate the role of platelets in the initiation of VT, I will use intravital imaging of the IVC 2 to 3 hours after ligation and quantify platelet and leukocyte adhesion to the IVC wall. Whole blood clot contraction and susceptibility to lysis studies will provide important information on the role of specific platelet signaling pathways during thrombus consolidation and resolution. Successful completion of the proposed studies will elucidate platelet signaling pathways that contribute to the initiation, propagation, and stability of venous thrombi. This information may lead to the identification of new platelet-based therapeutic targets for the prevention/treatment of VT.