Platelet transfusion in supportive care of cancer patients has seen a significant increase since 1980s, but a safe, long-term platelet storage method remains missing. Current FDA-mandated practice allows platelets to be stored at 20 to 24C after preparation with a limited lifetime up to 5 days due to concerns about bacterial contamination. Refrigerated storage drastically reduces platelet life-span because it causes glycoprotein-Ib (GPIb) receptors to cluster on specific microdomains of the platelet membrane. Recognition of specific de-glycated/de-syalylated residues on clustered glycoproteins by von Willebrand factor, macrophage ?2 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 way for chemical intervention. Platelet glycoproteins are intimately associated with intracellular cytoskeleton and their glycosylation depends on the location and activity of specific glycosyl-transferases. 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 cytoskeletal rearrangements. RhoA is the founding member of the Rho GTPase family that are central regulators of cytoskeletal dynamics, and has been shown to control lipid raft formation and composition. Therefore, changes in RhoA activity may influence platelet membrane lipid raft assembly and glycoprotein composition. Our preliminary studies by genetic and pharmacological means show that cold receptor upregulates RhoA activity, which in turn induces platelet activation. We have discovered a lead RhoA inhibitor, Rhosin/G04, that is specific to RhoA activation and is able to mimic RhoA knockout to prevent clathrin-independent internalization of lipid raft enriched in glycosyl transferase and cold-induced GPIb clustering. We hypothesize that RhoA inhibition can prevent platelet cold-induced GPIb clustering and consequent clearance by host upon transfusion, and that development of a reversible RhoA inhibitor can be translated to a new regimen of platelet cold storage. In this proposal, we will determine the mechanism of action by the lead RhoA inhibitor, Rhosin/G04, and demonstrate the therapeutic benefits of Rhosin/G04 and analogues for efficacious refrigerated long-term platelet storage in mouse models and non-human primates. The combination of drug discovery, medicinal chemistry and structural biology, cell biology, and platelet transfusion expertise as demonstrated by the co-PIs led to this highly innovative, outside-the-box method for intervention in platelet storage technology. Small molecule targeting of RhoA GTPase through the interference of protein-protein interaction is a revolutionary approach allowing for reversible inhibition of RhoA and help preserve cold stored platelet function. Our innovation through the preclinical drug discovery effort will benefit cancer patient care.
Cancer patients after radiation or chemotherapies often require platelet transfusion. The development of a method to prevent platelet damage upon refrigeration is a dream in transfusion medicine and it would revolutionize the current method of platelet storage. The proposal intends to understand the mechanism of action of a lead drug in preventing cold storage lesion in platelets and develop a method of platelet cold storage by using small molecule inhibitors of RhoA. The studies will provide a breakthrough in our understanding and translational application of a new technology towards the development of long-term and safer platelet products for cancer care.
