Calcium-mediated tyrosine phosphorylation of the cytoskeletal protein vinculin and of other proteins is associated with increased store-regulated calcium (SOC) influx of extracellular calcium. We inhibited tyrosine phosphorylarion by preventing the cytosolic calcium rise induced by depletion of the calcium stores with the intracellular chelator BAPTA-AM. SOC calcium influx persisted in BAPTA-loaded platelets in the absence of tyrosine phosphorylation. Using tyrosine kinase inhibitors we found that their ability to inhibit detectable tyrosine phosphorylation does not directly correlate with their ability to inhibit SOC influx. Thus tyrosine phosphorylation does not appear to be required for SOC influx. ADP is an endogenous platelet agonist which is released with platelet activation and can be removed by the enzyme apyrase. Adding PGE1 before thrombin in apyrase-treated platelets selectively prevented thrombin-induced tyrosine phosphorylation of as the focal adhesion kinase (FAK) while other substrates were not affected. Conditions which prevented thrombin-induced FAK tyrosine phosphorylation also decreased fibrinogen binding to platelets. Thus, released endogenous ADP selectively prevents PGE1-mediated tyrosine dephosphorylation of platelet FAK most likely by stabilizing fibrinogen binding to platelets. Storage of platelets at 4 degrees C would decrease bacterial growth in contaninated platelet units and decrease the incidence of transfusion-associated sepsis. We have examined the differences in functional characteristics between platelets stored at room temperature and those stored at 4 degrees C. After 24 hours at 4 degrees C, the pH and mean platelet volume were determined while platelet aggregation, fibrinogen binding and P-selectin expression studies were performed with a panel of agonists. Our results demonstrate that cold-stored platelets exhibit a low degree of activation at the end of 24 hours at 4 degrees C and a potentiated response to a broad range of agonists. The significance of these projects lies identification of the various molecular mechanisms which control platelet activation by elevation of cytosolic calcium levels as could occur during platelet storage. Inhibitors of these mechanism will be used to optimize storage conditions for platelets.
