Studies were performed to define the role(s) for protein tyrosine phosphorylation and cytochrome p450 in control of platelet calcium influx and platelet activation. Influx of extracellular calcium, which contributes significantly to the rise of cytosolic calcium in activated platelets, is regulated in part, by the level of calcium in intracellular calcium stores. Presence or absence of calcium in the internal stores regulates tyrosine phosphorylation of a set of platelet proteins. These tyrosine phosphorylated proteins maybe the controlling link between calcium in the stores and plasma membrane permeability to calcium. One of the most intensely tyrosine phosphorylated proteins under calcium control was identified as vinculin based on molecular mass, isoelectric focusing and immunoprecipitation with monoclonal antibodies. The functional role of tyrosine phosphorylation in platelet calcium influx was investigated with a series of tyrosine kinase inhibitors called tyrphostins. These compounds also inhibited store- regulate calcium influx with similar IC50 as for tyrosine phosphorylation. Neither inhibitors of the serine/threonine protein kinase C nor an analogue of tyrphostins which was inactive towards tyrosine kinases inhibited calcium influx. These observations suggest that tyrosine phosphorylation of platelet proteins is functionally linked to store-regulated calcium influx. Cytochrome p450 has recently been suggested as a mediator of the store- regulated calcium influx. We tested the effects of a cytochrome p450 inhibitors econazole and carbon monoxide (CO) store-regulated calcium influx. Inhibition of calcium influx by econazole was not specific for cytochrome p450 since a more specific inhibitor carbon monoxide inhibited p450 but did not block calcium influx. These observations indicate that 1) cytochrome p450 does not mediate store-regulated calcium influx and 2) econazole likely inhibits store-regulated calcium influx by an alternate mechanism such as interacting with plasma membrane calcium channels. The significance of this project lies in the identification of mechanisms which control activation of platelets by elevation of cytosolic calcium as could occur during platelet storage. Inhibitors of these mechanisms will be used to optimize storage conditions for platelets.
