This project focuses on investigations of platelet signaling in response to shear stress. Preliminary data indicate that intact platelets subjected to pathological shear stress respond in two unique and functionally significant ways. These responses are 1) the vWF-independent phosphorylation of actin binding protein (ABP)- 280 and 2) the vWF- dependent release of a 14-3-3zeta adaptor protein from the cytoplasmic domain of GpIbalpha. The experiments proposed will examine if vWF- mediated platelet adhesion and aggregation are triggered by ABP-280 phosphorylation that develops as a direct effect of shearing the cell surface, and will establish the mechanism by which ABP-280 phosphorylation that develops as a direct effect of shearing the cell surface, and will establish the mechanism by which ABO-280 phosphorylation that develops as a direct effect of shearing the cell surface, and will establish the mechanism by which ABP-280 phosphorylation modulates both inside-out and outside-in signaling.
The specific aims of this proposal are as follows. 1) Test the hypothesis that shear stress-induced phosphorylation of actin binding protein 280 modulates its association with the platelet GpIb-IX-V complex. We will determine if ABP-280 phosphorylation affects the affinity and/or stoichiometry of GpIbalpha binding, and identify the kinases that are directing ABP-280 phosphorylation. 2) Test the hypothesis that ABP-280 binding to the cytoplasmic domain of GpIbalpha modulates both inside- out signaling (vWF binding) and outside-in signaling (GpIIb-IIIa activation). We will determine how these responses are influenced by domains within the cytoplasmic tail of GpIbalpha, by the association of GpIbalpha with a 14-3-3zeta adaptor protein, and by dynamic molecular associations between phosphoinositide 3-kinase, 14-3-3zeta and GpIb-IX- V. 3) Test the hypothesis that shear stress-induced vWF binding causes the oligomerization of Gp-IX-V resulting in Syk activation that modulates GpIIb-IIIa. We will determine if shear-induced vWF binding to GpIb-IX-V causes a GpIbalpha-based signal complex to organize that causes Syk activation leading to PI-3-kinase activation affecting GpIIb- IIIa function. Results of these studies should allow us to identify trigger mechanisms for shear-induced platelet adhesion and aggregation, and begin to establish the sequence of platelet biochemical responses leading to pathological arterial thrombosis.
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