Abstract 454: Mechanism of Alpha-Synuclein Inhibition of Platelet Granule Exocytosis
Alpha-synuclein is only expressed in the brain and platelets. The normal function of alpha-synuclein in the brain is not known, but studies indicate it is an inhibitor of exocytosis in platelets. Platelet granule exocytosis is an important step in stable thrombus formation and its regulatory mechanisms are poorly defined. We therefore sought to determine the mechanism of alpha-synuclein inhibition of exocytosis. Using platelets from alpha-synuclein knockout (syn−/−) mice we first confirmed that syn−/− mice have increased alpha-granule release in response to agonist stimulation as compared to wild-type (WT) control mice. Using a mesenteric intravital microscopy model, we have also found that syn−/− mice have shortened time to thrombus formation as compared to wild-type control mice. The granule exocytosis and thrombosis phenotypic changes are restored to normal in syn null mice made transgenic to express human alpha-synuclein, clearly demonstrating that alpha-synuclein is an inhibitor of exocytosis in platelets. We have also determined the mechanism by which alpha-synuclein inhibits platelet granule exocytosis. Alpha-synuclein interacts with the exocytosis associated molecule Rab3, a modulator of the ATPase molecule N-ethylmaleimide Sensitive Factor (NSF). NSF activity is necessary for exocytosis and is increased by Rab3 interactions. Using platelet lysates, addition of an excess of recombinant alpha-synuclein inhibits NSF and Rab3 co-immunoprecipitation. Furthermore, syn−/− mouse platelets have increased Rab3 immunoprecipitation with NSF as compared to WT controls, that is restored to normal in synuclein Tg mice. Using recombinant tagged alpha-synuclein, NSF, and Rab3 in pull down assays we have confirmed that alpha-synuclein inhibits NSF-Rab3 interactions, and furthermore, decreases NSF ATPase activity. In summary, we now demonstrate using in vitro, ex vivo, and in vivo models that alpha-synuclein is a negative regulator of platelet granule exocytosis by inhibiting NSF-Rab3 association and thus NSF ATPase activity.