The Use Of Proteolytic Probes To Identify Platelet Membrane Receptors

One approach to the study of structure-function relationships with plasma membrane glycoproteins is to use proteases whose substrate specificity permit cleavage of only a single glycoprotein. We have employed this approach using two proteases, human platelet calcium-dependent protease and thrombin, as probes to investigate the identity of the platelet membrane von Willebrand Factor and thrombin receptors, respectively. Extensively purified calcium-dependent protease added exogenously to washed human platelets catalyzed the hydrolysis of only one membrane glycoprotein, glycoprotein Ib. The hydrolytic product of glycoprotein Ib as determined by immunoprecipitation and other criteria was glycocalicin. Correlation of the level of intact glycoprotein Ib with maximal agglutination rate to and binding of Factor VIII/von Willebrand Factor confirms previous observations consistent with glycoprotein Ib being the von Willebrand Factor receptor on human platelets. Since the catalytic integrity of thrombin is requisite for platelet response, we have sought to identify a thrombin substrate(s) on the platelet membrane surface. Glycoprotein V was the sole apparent thrombin substrate evident on O’Farrell gels of periodate-labeled control and thrombin-treated washed human platelets. We have recently purified this glycoprotein to homogeneity and confirmed it to be a thrombin substrate (Bemdt and Phillips (1981) J. Biol. Chem., 256: 59-65). Several lines of evidence will be discussed that are consistent with glycoprotein V being the probable functional thrombin receptor on human platelets.