Abstract

Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are detected with indo 1. They are extremely rapid and are maximimal by 1015 seconds. Suspension studies, which reflect an average value over 3 × 107 cells/ml, indicate a thrombin dose-dependent increase in cytoplasmic calcium. However, flow cytometry indicates that subpopulations of cells are responding differently. The responses of these populations seem to be dependent on thrombin concentration. A maximal overall response is found when more than 50 % of the cells respond. This may explain multiple stimulations of the same suspension of cells at low thrombin dosesElevated extracellular Ca++ increases the maximal cytoplasmic response and significantly retards its subsequent decrease. When extracellular Ca++ is removed via addition of EGTA just before stimulation, the initial response is halved and the subsequent slow decrease is more marked and returns to lower (near-basal) levels. Intracellular Ca++ can be chelated with s - (0-am inophenoxy) - e thane-N, N, N',N'-tetraacet ic acid (BAPTA), which has a twofold greater affinity far Ca++ than does indo, but which does not fluoresce (at indo sensitive wavelengths) upon binding of Ca++. Cells loaded with both indo and BAPTA exhibit no rise in cytosolic Ca++ or altered membrane potential when stimulated with αthrombin, unlike cells loaded with indo alone. When Ca++ (2 mM) is added back to the extracellular buffer, these cells will take up the Ca++ at a constant rate as seen by a rise in indo fluorescence. When cytosolic Ca levels reach those of the resting platelets loaded with indo alone, these cells recover the αthrombin-indue ed Ca response of control platelets. However, they no longer depolarize partially under these same Ca++ replenished conditions. This implies that some of the Ca++ required for the platelet thrombin response comes from non-replenishable internal stores.

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