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 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 microg/mL endoprotease for 60 minutes at 37 degrees 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.
Kinlough-Rathbone
R L
RL
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada.
Perry
D W
DW
Rand
M L
ML
Packham
M A
MA
D000371:Agglutination / Q000187:drug effects; D001606:Bernard-Soulier Syndrome / Q000097:blood*; D001792:Blood Platelets / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002250:Carbon Radioisotopes; D003029:Coagulants / Q000494:pharmacology*; D005340:Fibrinogen / Q000494:pharmacology; D006801:Humans; D016978:Mannheimia haemolytica / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000378:metabolism*; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism / Q000502:physiology*; D012310:Ristocetin / Q000494:pharmacology; D012701:Serotonin / Q000378:metabolism / Q000493:pharmacokinetics
D002250:Carbon Radioisotopes; D003029:Coagulants; D019038:Platelet Glycoprotein GPIb-IX Complex; D012310:Ristocetin; D012701:Serotonin; D005340:Fibrinogen; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase