Project Summary/Abstract Platelets are arguably the most important cellular participant in hemostasis and thrombosis. Many therapeutic strategies target platelet molecules to inhibit their functions in thrombosis. In particular, two membrane adhesion receptors, glycoprotein (GP) Ib-IX and GPIIb-IIIa (integrin IIb3), cooperatively mediate platelet adhesion and thrombus formation under high shear flow, and thus are ideal targets for inhibiting thrombosis in stenotic arteries as well as in microvasculature. Also, all anti-platelet drugs currently being used to treat thrombosis clinically have adverse hemorrhagic side effects, because the platelet plug formation required to stop bleeding is also inhibited. Under the support of this grant in its past period, we and others showed that both GPIbα and IIb3 are mechanoreceptors capable of receiving mechanical cues. We found that GPIb mechano-signaling leads to integrin inside-out signaling, which activates IIb3 from the inactive state having a bent conformation and a closed headpiece (BC) with a low affinity and short bond lifetime for ligand to an intermediate state having an extended conformation but a closed headpiece (EC) with an intermediate affinity and intermediate bond lifetime for ligand, which is distinct from the fully active state of IIb3 often seen in platelets stimulated by soluble agonists that has an extended conformation and an open headpiece (EO) with a high affinity and long bond lifetime for ligand. Significantly, the intermediate state of IIb3 mediates the formation of “biomechanical thrombi”, consisting of transient and unstable platelet aggregates resembling the dynamic outer layer of an in vivo thrombus formed under high shear blood flow, which is distinct from platelet aggregates underneath the outer layer that are further stimulated by released soluble agonists and where IIb3 are further activated to the fully active EO state. Further activation from the intermediate state to the fully active state can also be induced by IIb3 outside-in mechano- signaling, which also induces further activation of platelets. These data highlight the role of force-induced signaling – mechano-signaling – of, by, and between GPIb-IX and IIb3, and their unique importance in thrombus formation. Our overarching hypothesis is that a balanced regulation of mechano-signals of GPIbα and IIb3, and their synergy with biochemical signals induced by soluble platelet agonists, holds the promise to control thrombosis without unsetting hemostasis. The specific aims are to elucidate 1) the roles of 14-3-3ζ and filamin A on GPIbα ligand-binding, unfolding and mechano-signaling, 2) the mechanism of GPIb-IX mechanosensing- induced intracellular signaling pathway leading to integrin activation, and 3) the roles of G13, talin1 and Rap1 on GPIIb-IIIa binding, conformation and mechano-signaling. These studies will characterize the ligand-binding and signaling of of two major platelet mechanoreceptors, G...