Project 2: Summary Platelet αIIbβ3 has been considered the prototypic integrin whose quintessential feature is its nearly instantaneous conversion from an inactive bent conformation on circulating platelets to an extended ligand binding conformation following vascular trauma. This global reorganization is initiated by platelet agonist-stimulated biochemical reactions that disrupt an intramolecular clasp composed of portions of the αIIb and β3 cytosolic, transmembrane, and extracellular stalk domains. Recent work suggests that αIIbβ3 is not representative of all integrins and that various integrins differ in the stringency of their regulation. Unlike αIIbβ3, some integrins may be constitutively active. Project 2 addresses topics related to the protein-protein interactions that maintain integrins in their basal states, intra-molecular interactions in Specific Aim 1 and inter- molecular interactions in Specific Aim 2. In Specific Aim 1, intramolecular constraints located in the interface between the αIIb and β3 extracellular stalks will be identified using a novel structural bioinformatics method to predict interacting interfacial “hot spots”. The relative importance of the predicted hot spots will then be determined by expressing hot spot mutants in CHO cells and in iPSC-derived human megakaryocytes produced in collaboration with Project 4. This experimental approach will then be used to compare αIIbβ3 to the integrins αvβ3, α2β1, and αvβ8 and in collaboration with Project 1, to characterize the interaction between the PH and BEACH domains of Nbeal2 in studies designed to understand the pathogenesis of α granule defect in the gray platelet syndrome. A second set of integrin constraints located in the transmembrane domain interface will be studied based on preliminary data indicating that β3 uses different motifs to interact with αIIb and αv. Novel computational methods will then be used to derive two- dimensional kinetic parameters for these interactions in collaboration with Project 3. Lastly, we will use high-resolution cryo-electron microscopy to correlate our computational and experimental results with the global conformation of full-length integrins. Specific Aim 2 will the identify and quantitively evaluate the protein-protein interactions responsible for αIIbβ3-mediated fibrin clot contraction. The studies are based on the observation that agonist stimulation causes platelet calpain activation and the degradation of platelet cytosolic proteins, in particular the proteins talin and vinculin that link αIIbβ3 to the actin cytoskeleton. Proposed studies will test the hypothesis that talin cleavage by calpain enables vinculin binding, thereby generating sufficient traction force to contract αIIbβ3-bound fibrin clots. This hypothesis will also be tested in vivo using mouse thrombosis models and calpain-deficient mice in collaboration with Project 3.