Inhibition of Thrombin-Induced Platelet Aggregation by a Derivative of Wheat Germ Agglutinin. Evidence for a Physiologic Receptor of Thrombin in Human Platelets

Abstract

We heve prepered a nonagglutinating derivative of wheat germ agglutinin by cyanogen bromide treatment of the lectin and used it as a probe to study the thrombin-platelet interaction. The lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by collagen, ristocetin, adenosine diphosphate, wheat germ agglutinin, or trypsin was not significantly affected. Under similar conditions, the secretion of serotonin by thrombin was blocked by the derivative. The inhibitory action of the derivative on thrombin-induced platelet aggregation could be overcome by increasing the thrombin concentration. A Schild plot of these data yielded a slope of 1.0 and an apparent dissociation constant of 1.0 µM. Thus, the inhibition of thrombin-induced platelet aggregation by the derivative fits a model of competitive inhibition. Control experiments showed that the lectin derivative acted on platelets and not on thrombin. However, the binding of [125I]thrombin to platelets was not affected. Isolated platelet membranes were solubilized and passed through a column of the lectin derivative coupled to Sepharose. After extensive washing, the material bound to the column was eluted with N-acetyl-d-glucosamine. This membrane isolate inhibited platelet aggregation by thrombin while aggregation induced by adenosine diphosphate, trypsin or ristocetin was not significantly affected. It also blocked thrombin-induced serotonin secretion from platelets. Gel electrophoresis followed by autoradiography of the membrane isolate revealed a prominent glycoprotein of apparent molecular weight of 74,000 that contained inhibitory properties. The electrophoretic mobility or the intensity of this band was not significantly affected following incubation of the glycoprotein isolate with physiologic concentrations of thrombin. The glycoprotein also retained its inhibitory activity on thrombin-induced platelet aggregation following incubation with 20 µg/ml of trypsin or chymotrypsin for 10 min. These results suggest that (A) the thrombin receptor in human platelets is different from the ristocetin or ristocetin-von Willebrand factor receptor and (B) the 74,000 dalton glycoprotein may be a physiologic receptor of thrombin in human platelets.