Platelet activating factor-induced aggregation of calf platelets: Apparent positive cooperativity in the kinetics and non competitive inhibition by diltiazem

Aggregation of calf platelets by platelet activating factor was characterized by a spectrophotometric method. The aggregation kinetics of both platelet-rich plasma and purified platelets showed concave up double-reciprocal plots and linear Hill plots withh > 1 (1.7 ± 02) consistent with positive cooperativity. Comparable values of maximum rates of aggregation(R) were obtained with platelet-rich plasma (0.25 ± 0.08) and purified platelets (0.28 ± 0.18) but the half-maximal saturation concentration (S_0.5) differed greatly between platelet-rich plasma (6 ± 3 nM) and purified platelets (0.28 ± 0.18 nM). An Arrhenius activation energy of 21 ±2 kcal/mol was found for aggregation of purified platelets. Diltiazem was inhibitory with half-maximal inhibitory concentration (I_0.5) of 4 M but the inhibition was not competitive. Diltiazem inhibited rates but not the extent of shape-change. The receptor-antagonist and sulphydryl reagent N-ethylmaleimide and the platelet antagonistic omega-3-fatty acid, 5,8,11,14,17-eicosa pentaenoic acid, inhibited both rates and extent of shape-change reactions and inhibited aggregation competitively (I_0.5 ∼ 5 M). Eicosa pentaenoic acid at > 25 M could abolish shape-change reactions and at 50 M served as an activator of platelets and the activation was enhanced by aspirin (1 mM). Although N-ethylmaleimide at > 20 M could also induce platelet activation it failed to induce aggregation and aspirin had no effect on the shape-change reactions induced by it.