Effects of the calmodulin antagonists fendiline and calmidazolium on aggregation, secretion of ATP, and internal calcium in washed human platelets

Abstract

Ca2(+)-calmodulin dependent phosphorylation of myosin is essential for the induction of platelet shape change and subsequent reactions. Therefore, we studied the effects of the calmodulin antagonists fendiline and calmidazolium on the thrombin-induced aggregation, secretion of ATP, and increases in the intracellular free calcium concentration ([Ca2+]i) in washed human platelets in the absence and presence of extracellular Ca2+. In Ca2+ free medium, fendiline (10-100 microM) and calmidazolium (3-30 microM) concentration-dependently inhibited aggregation. The effect of fendiline could be partly reversed by extracellular Ca2+ and higher thrombin concentrations. Furthermore, aggregations induced by the calcium ionophore ionomycin and by the protein kinase C-activator 4-beta-phorbol 12-myristate 13-acetate were inhibited by fendiline, although to a smaller degree than the thrombin-induced aggregation. Thrombin-induced secretion of ATP was attenuated by low concentrations of fendiline (1-3 microM) and calmidazolium (1 microM) but enhanced by higher concentrations (10-30 and 3-10 microM, respectively), independently of extracellular Ca2+. Fendiline (1-10 microM) did not affect [Ca2+]i in resting and thrombin-stimulated platelets. At higher concentrations (30-100 microM), it induced increases in [Ca2+]i in unstimulated platelets and attenuated the response to thrombin in Ca2+ free medium, whereas thrombin-induced Ca2+ influx was markedly enhanced. Similar results were obtained with calmidazolium (1-3 microM). These stimulating effects on ATP secretion and on [Ca2+]i of fendiline and calmidazolium may be attributed to interactions with platelet membranes by which the permeability of small cations is increased.(ABSTRACT TRUNCATED AT 250 WORDS)