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Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are very rapid, are maximal by 10-15 s, and can be detected with probes such as Indo-1. Suspension studies using spectrofluorometry, which reflect a value which is the average of 3 x lo7 cells per ml, indicate a thrombin dosedependent increase in cytoplasmic calcium at doses up to 0.025 units per ml. We show here, using flow cytometry, that at less than halfsaturating thrombin doses only subpopulations of platelets rather than the entire sample are responding. The extent of these responses, however, still depends on thrombin concentration. When the thrombin doses are between half and fully saturating, one subpopulation responds fully (i.e., its extent of increase in cytoplasmic calcium concentration, [Ca++]i, is 100% of that seen at saturating thrombin concentrations) while the remaining platelets respond partially or not at all. There is thus evidence of positive cooperativity leading to disproportionate thrombin receptor occupancy on different subpopulations when platelets are subjected to subsaturating doses of thrombin. The existence of responding subpopulations may explain how the reported multiple stimulations of the same suspension of platelets at low thrombin doses occur.

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.