Release of a fluorescent probe as an indicator of lysosomal granule secretion by thrombin-stimulated human platelets

Abstract

Investigations in this laboratory have demonstrated that thrombin induces dose-dependent changes in the transmembrane electrical potential of gel-filtered human platelets. This change is monitored with the fluorescent lipophilic cation, 3,3′-dipropylthiodicarbocyanine (diS-C 3-(5)), whose rapid release from the platelet (maximal within 30 s) correlates with a rapid, dose-dependent influx of sodium, a depolarization, and an increase in the intracellular pH. There is also a later release of this probe, detectable only 60 s after activation by thrombin. It is shown that this latter probe release is also thrombin dose dependent, and correlates in time course and extent with the secretion of β-glucuronidase from the platelet's lysosomal granules, implying that it corresponds to probe sequestered in these granules in the resting platelet. Such a conclusion is corroborated by the fact that both the thrombin-induced secondary release of diS-C 3-(5) and the secretion of the lysosomal enzyme, β-glucuronidase, are inhibitable to the same extent by pretreatment of the probe-equilibrated platelets with valinomycin, a K + ionophore, are partially inhibited to a comparable extent when thrombin is removed from the platelet membrane by an excess of hirudin within 15 s of activation, and are unaffected by amiloride, a Na + blocking agent. We suggest therefore that some of the membrane potential probe diS-C 3-(5) is accumulated by the platelet lysosomal granules and is secreted when the platelets are stimulated by the high doses of thrombin which induce lysosomal enzyme secretion. This secondary dye release is linearly proportional to, and can be used as a continuous and quantitative indicator of, the thrombin-induced lysosomal enzyme secretion by human platelets.