Responses to Aggregating Agents after Cleavage of GPIb of Human Platelets by the O-Sialoglycoprotein Endoprotease from Pasteurella haemolytica— Potential Surrogates for Bernard-Soulier Platelets? ☆

Abstract

Most proteolytic enzymes that cleave glycoprotein lb (GPlb) also cleave other glycoproteins or receptors on the surface of platelets. We have used an O-sialoglycoprotein endoprotease from Pasteurella haemolytica that selectively cleaves the heavily O-glycosylated GPlb, but does not cleave N-linked glycoproteins or unglycosylated proteins. Isolated, [ 14C]serotonin-labeled platelets in Tyrode-albumin solution were incubated with 10 μg/mL endoprotease for 60 minutes at 37°C. These platelets did not release [ 14C]serotonin, had no detectable GPIb, and were unresponsive to ristocetin/von Willebrand factor. Compared with control platelets, aggregation and release of [ 14C]serotonin by the endoprotease-pretreated platelets were inhibited in response to low concentrations of thrombin, SFLLRN (the PAR-1-activating peptide), collagen, and U46619 (a thromboxane A 2 mimetic); aggregates were smaller in size. The presence of fibrinogen overcame the inhibition of responses induced by SFLLRN, collagen, and U46619. With fibrinogen, primary ADP-induced aggregation was scarcely affected by pretreatment with the endoprotease. Thus, the PAR-1 receptor for thrombin, and receptors for collagen, thromboxane A 2, fibrinogen (GPIIb/IIIa), and ADP appear to function normally on the endoprotease-pretreated platelets. Since only GPIb is cleaved by the endoprotease, these platelets seem to provide potential surrogates for Bernard-Soulier syndrome platelets for further studies of platelet functions in this condition.