Experimental Studies on the Effects of Antiplatelet Agents, Especially of Drugs That Affect Ca2+ Response, on Phospholipid Metabolism and Ca2+ Mobilization in Human Platelets

Abstract

The effects of antiplatelet agents on agonist-induced cytosolic Ca2± response and lipid metabolism, phosphatidylinositol turnover (PI turnover) and arachidonic acid metabolism, in human platelets were investigated. In aequorin-loaded platelets, verapamil (Ca2+ channel blocker), TMB-8 (intracellular Ca2± antagonist) and W-7 (calmodulin inhibitor) suppressed 0.05U/ml thrombin-induced elevation of cytosolic Ca2+ concentration. They, however, had no effect on 0.25U/ml thrombin-induced Ca2± change. Their specific inhibitory effects on Ca2+ influ x from extracellular fluid or intracellular Ca2+ mobilization, on which verapamil and TMB-8are considered to have their primary actions, were not evident. Thromboxane A2 (TxA2), an arachidonate metabolite, or Inositol 1,4,5-trisphosphate (IP3) derived from PI turnover is a main mediator of Ca2± mobilization in platelets stimulated by 0.05 or 0.25U/ml thrombin, respectively. These drugs seem to affect the production of the mediator rather than the Ca2±movement itself. They significantly suppressed arachidonic acid release from 14C-AA-labeled platelets stimulated by thrombin. Therefore, these drugs are considered to reduce TxA2 production by inhibiting the release of arachidonic acid from membrane phospholipids and, thus, to prevent the increase in cytosolic Ca2+ concentration under 0.05U/ml thrombin stimulation. In contrast, these drugs did not have any effect on the PI turnover estimated by production of phosphatidic acid (PA) and the Ca2±change in platelets stimulated by 0.25U/m1 thrombin. It has been assumed that cAMPproducing agents have direct action on intracellular Ca2± sequestration, but this Ca2± pump has not been identified yet. From the result of suppression of PA production, PGE1 and Forskolin seem to influence the PI turnover and subsequent Ca2+mobilization. In conclusion, these antiplatelet agents reduce agonist-induced Ca2± response in part by effect on phospholipid metabolism which is closely associated with Ca2+ mobilization.