The Ca2+-mobilizing potency of alpha-thrombin and thrombin-receptor-activating peptide on human platelets - Concentration and time effects of thrombin-induced Ca2+ signaling

In single platelets and in suspensions of platelets, alpha-thrombin evokes dose-dependent, transient increases in cytosolic Ca2+ concentration, [Ca2+](i), which are more prolonged than the [Ca2+](i) transients evoked by other platelet agonists such as the thrombin-receptor-activating hexapeptide SFLLRN, thromboxane A(2) analog U46619, and ADP. As a quantity taking into account both the magnitude and length of the Ca2+ response, we defined the Ca2+-mobilizing potency (CMP) of an agonist as the integrated rise in [Ca2+](i) during the time of the Ca2+ signal. It was observed that: (a) the CMP increased with the agonist concentration in a saturating way, its maximal value being about four-times higher with alpha-thrombin than with SFLLRN; (b) the high CMP of alpha-thrombin was for only a small part due to endogenous production of ADP or thromboxane, and was mainly a consequence of prolonged influx of external Ca2+; (c) the CMP declined when alpha-thrombin was inactivated during the course of the Ca2+ signal; (d) CMP values increased with the agonist concentration upon sequential addition of increasing amounts of alpha-thrombin or SFLLRN; (e) when alpha-thrombin was gradually added to the platelets or formed by an in sill reconstituted prothrombinase system (with factor Xa, factor Va, and prothrombin), integrated Ca2+ responses were a function of the product of the alpha-thrombin concentration and the time of its presence. However, in these cases, the final CMP values were independent of the rate of alpha-thrombin addition or formation. We conclude that alpha-thrombin-induced Ca2+ signals in platelets rely largely upon Ca2+ influx, are not, or only slightly, subjected to homologous desensitization, and reflect the enzymatic capacity of alpha-thrombin to cleave protease-activated receptors. Thus, the high and prolonged Ca2+ signal induced by alpha-thrombin is due to continuous receptor cleavage without desensitizing effects of previously cleaved receptors.