Inhibition of Human Platelet Aggregation and Calcium-Dependent, Phospholipase A₂ Activity by Calcium Antagonists: Evidence for Intracellular Effects of Calcium Slow Channel Blockers

Abstract

Recent studies demonstrate that calcium antagonists, such as bepridil and verapamil, enter and accumulate in muscle cells, and suggest that these drugs may have intracellular effects in addition to their role as inhibitors of the slow inward Ca²⁺ current (1,2). Therefore, we have tested a group of calcium “slow channel” blockers for their ability to inhibit human platelet aggregation, which requires extracellular Ca²⁺, and the activity of human platelet phospholipase A₂,an enzyme that is calcium-dependent and is associated with intracellular membranes (3,4). All the calcium antagonists tested, except nifedipine, inhibited ADP-induced aggregation of human platelets. The extent of inhibition by a given concentration of bepridil increased with the time of preincubation with drug. D-600 and diltiazem were the most potent inhibitors of aggregation. Human platelet phospholipase A₂ activity was maximally active at pH 7.0 and had an absolute requirement for, and was extremely sensitive to, low concentrations of CaCl₂. Activity was stimulated 220% by 10^-7 M D-600, was inhibited to different degrees by bepridil, nitrendipine, and verapamil, and was essentially unaffected by nifedipine, mesudipine, and higher concentrations of D-600. Inhibition of phospholipase A₂ activity by bepridil was relieved by increasing concentrations of CaCl₉. Bepridil, verapamil, and propranolol displaced ⁴⁵Ca from the membranes of autoclaved E. coli. The differential effects of these calcium antagonists on human platelet phospholipase A2 activity suggest that these drugs may produce secondary effects inside the cell.