Progesterone metabolites rapidly stimulate calcium influx in human platelets by a src-dependent pathway

Abstract

The effects of several steroids and their metabolites were examined for their ability to rapidly alter intracellular free calcium ([Ca 2+] i) in the anucleate human platelet. Earlier studies suggested that steroids had direct and rapid non-genomic effects to alter platelet physiology. The rationale for performing this study was to investigate the signal transduction events being activated by steroids. Super-physiologic concentrations (1.0–10.0 μM) of β-estradiol and several estradiol metabolites and analogs potentiated (approximately twofold) the action of thrombin to elevate [Ca 2+] i in platelets, whereas 10.0 μM progesterone inhibited the action of thrombin by 10–15%. Progesterone and β-estradiol by themselves did not affect [Ca 2+] i. Progesterone metabolites can achieve high blood concentrations. Some progesterone metabolites, particularly those in the β-conformation, were potent stimulators of Ca 2+ influx and intracellular Ca 2+ mobilization in platelets. They activated phospholipase C because their ability to increase [Ca 2+] i was inhibited by the phospholipase C inhibitor U-73122. The ability of pregnanediol and collagen to increase [Ca 2+] i was inhibited by the src tyrosine kinase inhibitor PP1, whereas the actions of thrombin and thapsigargin to increase [Ca 2+] i were not affected by PP1. The effects of progesterone metabolites to increase [Ca 2+] i were observed with concentrations as low as 0.1 μM. Pregnanolone synergized with thrombin to increase [Ca 2+] i. It is hypothesized that human platelets possess receptors for progesterone metabolites. These receptors when stimulated will activate platelets by causing a rapid increase in [Ca 2+] i. Pregnanolone, isopregnanediol and pregnanediol were the most effective stimulators of this newly identified src-dependent signal transduction system in platelets. Progesterone metabolites may regulate platelet aggregation and hence thrombosis in vivo.