CYTOSKELETON AND PLATELET CLEARANCE

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Platelets, harvested for transfusion, have a relatively short “shelf life” of 5 days. With increasing storage, platelets develop structural and functional changes called “platelet storage lesions”. These changes result not only in reduced hemostatic response to agonists but also in an accelerated clearance from the circulation in vivo. Several mechanisms have been proposed including clustering of the platelet glycoprotein Ib complex on the surface of platelets, alterations in glycans leading to recognition by hepatic Ashwell-Morell receptor and shedding of platelet glycoprotein Ibα. Exposure of phosphatidylserine (PS) on stored platelet surface is a well known tag for clearance from the circulation. In resting platelets, PS is located in the inner leaflet of the membrane bilayer and there is transbilayer movement from the inner to the outer leaflet during storage and during platelet activation. Both a senescence (or apoptosis)- induced and an activation-dependent pathways of PS exposure have been described. Exposure of PS in senescent platelets occurs through the mitochondrial Bcl-2 family of proteins and (in contrast to activation-induced PS exposure) it does not require elevation of cytosolic Ca+ and it involves cytochrome c release and subsequent activation of caspase 3. PS exposure occurs simultaneously with the generation of microparticles and stored platelet preparation contain PS-rich microparticles. The molecular mechanisms that maintain the phospholipid asymmetry in resting platelets, as in most mammalian cells, are not known. We propose a novel mechanism for the loss of phospholipid asymmetry during storage. We posit that the cortical actin network confines PS to the inner leaflet and hypothesize that during storage the reorganization of cytoskeleton releases normal constraints on plasma membrane bilayer leading to the loss of asymmetry and transbilayer movement of anionic phospholipids. The cytoskeletal proteins, tightly bound to anionic phospholipids such as actin, are also exteriorized to the cytoplasmic surface possibly with a putative scramblase-dependent mechanism. The overall objective of the grant is to delineate the mechanisms that maintain phospholipid asymmetry in fresh platelets and its loss during storage. Specific aims of this proposal are: The specific aims of the proposal are (1) to determine the role of the cortical actin network in maintaining the phospholipid asymmetry, (2) to define the mechanism of regulation of cofilin-1-induced PS exposure in stored platelets and (3) to determine the effect of cofilin-1 inhibition on platelet survival.