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?

Most proteolytic enzymes that cleave glycoprotein lb (GPlb) also cleave oth er glycoproteins or receptors on the surface of platelets. We have used an O-sialoglycoprotein endoprotease from Pasteurella haemolytica that selectiv ely cleaves the heavily O-glycosylated CPlb, but does not cleave N-linked g lycoproteins or unglycosylated proteins. Isolated, [C-14]serotonin-labeled platelets in Tyrode-albumin solution were incubated with 10 mu g/mL endopro tease for 60 minutes at 37 degrees C, These platelets did not release [C-14 ]serotonin, had no detectable GPIb, and were unresponsive to ristocetin/von Willebrand factor. Compared with control platelets, aggregation and releas e of [C-14]serotonin by the endoprotease-pretreated platelets were inhibite d in response to low concentrations of thrombin, SFLLBN (the PAR-1-activati ng 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, primar y ADP-induced aggregation was scarcely affected by pretreatment with the en doprotease. Thus, the PAR-1 receptor for thrombin, and receptors for collag en, thromboxane A(2), fibrinogen (GPIIb/IIIa), and ADP appear to function n ormally on the endoprotease-pretreated platelets. Since only GPIb is cleave d by the endoprotease, these platelets seem to provide potential surrogates for Bernard-Soulier syndrome platelets for further studies of platelet fun ctions in this condition. (C) 2000 Elsevier Science Ltd. All rights reserve d.