Rho GTPase inhibitors for refrigerated platelet storage

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ABSTRACT The use of platelet transfusions has increased dramatically since 1980s, but a safe, long-term platelet storage method remains missing. Current practice has platelets stored at 20 to 24¿C after preparation, which has a limited lifetime up to 5 days primarily due to concerns about bacterial contamination. Refrigerated storage reduces platelet life-span because it causes glycoprotein-Ib (GPIb) receptors to cluster on specific microdomains of the platelet membrane. Recognition of specific glycated/syalylated residues on clustered glycoproteins by macrophage b2 integrins and hepatocyte Ashwell-Morell receptors results in platelet phagocytosis by the host and removal from circulation. Thus, prevention of glycoprotein clustering represents a useful target for chemical intervention. Platelet glycoproteins are intimately associated with intracellular cytoskeleton. Their clustering depends on the formation of lipid raft in the platelet membrane which in turn depends on the dynamics of the highly regulated processes of actomyosin assembly/disassembly. Rho family GTPases, including RhoA, Rac1 and Cdc42, are a class of GTP-binding enzymes that are central regulators of F-actin polymerization/depolymarization, and have been shown to control lipid raft formation and composition. Therefore, changes in Rho GTPase activities may influence platelet membrane lipid raft assembly and glycoprotein composition. Based on preliminary and published data, we hypothesize that reversible targeting of Rho family GTPases by small molecule inhibitors can prevent cytoskeleton-dependent refrigeration storage lesions in platelets and result in increased platelet survival. In this project, we will analyze the cytoskeletal functions and in vivo viability of murine and human platelets after refrigeration in the presence or absence of various combinations of specific inhibitors for Cdc42, Rac1 and RhoA GTPases. Combination of cell biology, drug discovery and platelet analysis expertise as demonstrated by multiple co-publications of the co-PIs resulting in an innovative, outside-the-box method for intervention in platelet storage technology. Chemical drug targeting of Rho GTPase activities through interference of protein-protein interaction is a revolutionary approach allowing for reversible inhibition of cytoskeletal rearrangements for which our group has pioneered and own the intellectual property licensed to P2D Inc.