THE THIENOPYRIDINE PCR 4099 INHIBITS THE ADP AGGREGATION PATHWAY OF HUMAN PLATELETS BY INTERFERING WITH THE BINDING OF FIBRINOGEN TO THE GLYCOPROTEIN IIb-IIIa COMPLEX
The thienopyridine, PCR 4099, is a synthetic structural analog of ticlopidine. After oral administration in man, it prolongs the bleeding time (BT) and inhibits ADP-induced aggregation. The aim of the study was to evaluate the effects of oral administration of 200 mg per day PCR 4099 to 10 human volunteers for 7 days on primary hemostasis and to study the mechanism of inhibition of the drug on the ADP-fibrinogen-GPIIb-IIIa pathway of aggregation. BT (measured by a Simplate device) was 4-8 min
... vice) was 4-8 min before treatment and 30 min after 7 days of treatment. Platelets were washed and resuspended in Tyrode's buffer containing apyrase and 0.35 % human albumin. Washed platelet suspensions were used at 37°C for aggregation and fibrinogen binding studies. Human fibrinogen was purified by successive ether precipitation and gelatin affinity chromatography to remove fibronectin. Fibrinogen was pure bv SDS-PAGE and > 95 % clottable by thrombin. It was labeled with 125I by the Iodogen method. The binding of 125I-fibrinogen to intact washed platelets exposed to ADP or thrombin was measured after centrifugation at 11 ,000 g for 1 min in the presence of 131I-human albumin as a space marker. The membrane GPIIb-IIIa complex was examined by crossed immunoelectrophoresis (CIE) in the presence of rabbit anti-human platelet antiserum. The prolonged administration of PCR 4099 inhibited almost completely platelet aggregation induced by 0.5 to 10 μM ADP. Although the effect of ADP on aggregation was blocked at high concentrations, PCR 4099 did not modify ADP-induced shape change. Only the effects of low concentrations of thrombin (< 0.05 U/ml) were inhibited by PCR 4099 administration. The binding of 125I-fibrin0gen was reduced by 50 to 90 % when platelets were stimulated by 5 μM ADP or by 0.05 U/ml thrombin. PCR 4099 did not modify the pattern of immunoprecipitates as revealed by CIE. In particular the GPIIb-IIIa complex was not dissociated and its electrophoretic mobility was not changed. In conclusion, PCR 4099, which is more potent than ticlopidine in man, inhibits specifically the ADP aggregation pathway by interfering with the binding of fibrinogen to the GPIIb-IIIa complex in platelets having undergone shape change.