Abstract
The two major pathways for Ca 2+ entry into cells are potential-sensitive channels and receptor-operated channels. The main object of this investigation was to identify which mechanism regulates Ca 2+ entry into human platelets. Platelet stimulation with thrombin, adenosine diphosphate, platelet activating factor and arachidonic acid resulted in a concentration-dependent 2.5-3-fold increase in cytoplasmic free calcium concentration over the basal levels (140 ± 32 nM or 104 ± 21 respectively) as measured with the fluorescent dyes Quin-2 and Fura-2. Adrenaline and collagen had no effect in promoting intracellular Ca 2+ increase as measured with Quin-2 and little effect when measured with Fura-2. Incubation of Quin-2-loaded platelets with the calcium antagonists verapamil and diltiazem, which are known to inhibit Ca 2+ entry from voltage-gated channels in many types of cells, over the concentration range 10 −8−10 −4 M did not alter significantly either the resting or the cytoplasmic free Ca 2+ after stimulation of platelets by several agonists. Moreover, the calcium antagonists exhibited little or no effect on aggregation and 5-hydroxytryptamine secretion induced by platelet activating factor, adenosine diphosphate, collagen or arachidonic acid in whole blood, platelet-rich plasma or washed platelets when employed at concentration ranges as above. Similar results were obtained in washed thrombin-stimulated platelets. High doses of verapamil (but not diltiazem) inhibited platelet aggregation and secretion in response to adrenaline. Direct radioligand binding studies with (−)[ 3H]desmethoxyverapamil showed that platelet membranes have no receptors for this drug, suggesting that Ca 2+ entry occurs in human platelets via a pathway different from potential-sensitive Ca 2+ channels. In contrast to other authors, who used concentrations of Ca 2+ antagonists higher than 500 μM, we suggest that verapamil and diltiazem do not have any antiaggregatory effect when employed at therapeutic concentrations.
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