GPIbalpha shedding and platelet clearance



 DESCRIPTION (provided by applicant): The objective of this project is to define the role of ectodomain shedding of glycoprotein (GP)Iba in the clearance of platelets that are stored at room temperature (RT) or 4°C. Although platelets have been used for transfusion for more than 50 years, human platelets can only be stored at RT for up to 5 days, making improving platelet storage a major challenge in blood banking. Recent studies have suggested that ectodomain shedding of GP1bα, a membrane receptor abundantly and exclusively expressed on the surface of platelets, as a key step in mediating platelet clearance. However, it has not been possible to determine whether shedding of GP1bα is an inconsequential indicator for the to-be-cleared platelet or actually the trigger for its clearance. Such ambiguity is largely due to the usage of metalloprotease inhibitors in previous studies that nonspecifically block ectodomain shedding of all receptors. Recently novel monoclonal antibodies (MAbs) have been developed to specifically inhibit shedding of human GP1bα in the platelet. Preliminary studies of a prototypical MAb designated 5G6 showed that it exhibited no detectable effect on normal platelet functions and it did not cause acute thrombocytopenia in a transgenic hTg mouse line expressing only human GP1bα. In addition, recent identification of a juxtamembrane mechano-sensitive domain in GP1bα has greatly advanced our understanding of GPIb- IX-mediated signaling and suggested a novel mechanism linking GPIba shedding to desialylation and platelet clearance. This project seeks to address the role of GP1bα shedding in platelet clearance by utilizing the novel MAb 5G6 and its derivatives. Specific Aim 1 is to characterize the association of 5G6 and its F(ab')2 and Fab fragments with platelets and to elucidate their effects on platelet functions at the molecular level, including the clustering of GP1bα. Specific Aim 2 is to test whether treatment of 5G6 or its derivative during storage can mitigate the storage lesion and improve the post-transfusion survival of RT-stored platelets. Specific Aim 3 is to explore the effects of 5G6 treatment, or the combined treatment of 5G6 with other storage methods, on the post-transfusion survival and hemostatic functions of cold- stored platelets. For Aims 2 and 3, two novel animal models have been developed to monitor the survival of human leukoreduced apheresis-derived platelets in the immunodeficient mice and that of hTg murine platelets in appropriate recipient mice. Analysis and comparison of the results obtained from studies on both human and murine platelets will generate unique mechanistic insights into the various changes the platelet undertake during the storage at RT and 4°C. Completion of the proposed studies will define the role of GPIba shedding in storage and clearance of RT- and cold-stored platelets, and provide proof-of- concept evidence regarding the prospect of specific inhibition of GP1bα shedding as a viable approach to improve platelet storage for the transfusion treatment.