Platelets are vital for hemostasis. The platelet component of hemostasis is impaired by common medications (aspirin), genetic disorders (von Willebrand's Disease), and platelet deficiency (cancer, leukemia, chemotherapy, immune thrombocytopenia). Platelets are a component of pathological thrombosis (myocardial ischemia and infarction, stroke, peripheral limb ischemia, reperfusion injury), raising possibilities for strategic impairment of platelet function in the prevention of these diseases if basic understanding of platelet structure and function is improved. The goal of this proposal is to utilize newly available biochemical and morphological techniques to explore the anatomy and regulation of platelet activation with emphasis on the role of actin in this process. To understand this morphological transformation we will define: (1) the detailed ultrastructure of the resting platelet actin skeleton by combining rapid-freezing and freeze-drying with immunogold to locate important membrane proteins; (2) anatomical alterations that occur in this resting skeleton with the stimulation of platelets with agonist, the most prominent of which is actin filament assembly; (3) the role of cytosolic calcium in regulating actin assembly; and (4) locate the site of GPIb/IX binding on actin-binding protein (ABP) and investigate the regulation of this interaction. Recent advances, some of which we have contributed, permit us to explore these events at the morphological and biochemical level. We propose a comprehensive interdisciplinary study on to define the resting platelet cytoskeleton and understand the mechanism of platelet actin assembly during activation of the cells.
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