Reduction of Surface Negative Charge in Human Platelets by Ristocetin

Abstract

The effect of ristocetin on the platelet surface charge was examined. One part of citrated PRP or platelet suspensions was mixed with nine parts of lOμM Tris-buf f ered saline (pH 7.4) and the mixtures were incubated at 25°C with or without ristocetin. These mixtures were applied for a cell electrophoresis microscope to determine the electrophoretic mobility(EM), from which the platelet surface charge was estimated. A significant reduction in surface negative charge of platelets was observed when ristocetin was added to the mixtures. EM of non-treated and risto-cetin(lmg/ml)-treated platelets was −1.269±0.045 and −0.870±0.040 μ/sec/V/cm, respectively. A dose-response relationship was observed between platelet EM change and ristocetin concentra-tion(0.1~1.2mg/ml). The change in EM reached to the maximum within seconds. Similar reduction of EM was observed when formalin-fixed platelets were incubated with ristocetin. Heparin at concentrations(10u~100u/ml) inhibited, but protamine sulfate(0.05mg/ml) enhanced the change of EM and platelet aggregation by ristocetin. Both the ristocetin-induced aggregation and the reduction of EM were optimally observed at pH 7.3 and were inhibited at pH above 8.0. The presence of plasma component was found to be essential in the reaction mixture for this reduction of EM. EM was decreased in the presence of ristocetin proportionally with the increase in per cent of plasma in the mixture up to 10(v/v)%. The ristocetin-induced reduction in EM of platelets was not observed when normal platelets were suspended in plasma from a patient with Willebrands’ disease(EM= −1.278±0.070 μ/sec/V/cm, with ristocetin, 1mg/ml).These results strongly suggest the involvement of decreased surface negative charge in the mechanism of platelet aggregation induced by ristocetin.

Electrochemical Properties of Platelets: Clinical and Pharmacological Applications

“…32 We ourselves have confirmed this result in two patients. However, Coller and Muraki et al 33 have shown that ristocetin reduced platelet charge at doses of 0.5 and 1 mg/ml, but no change was observed when normal platelets were suspended in plasma taken from a patient with Willebrands disease. According to this result, which should be confirmed and studied in further detail, it might be assumed that positively charged ristocetin is adsorbed on the platelet membrane by forming a complex with the Willebrand factor.…”