Inhibition of collagen-induced platelet aggregation as the result of cleavage of alpha 2 beta 1-integrin by the snake venom metalloproteinase jararhagin.
Jararhagin is a high-molecular-mass (52 kDa) haemorrhagic metalloproteinase from Bothrops jararaca venom and a member of the metalloproteinase/disintegrin/cysteine-rich protein family. The disintegrin domain of jararhagin has been implicated in the inhibition of platelet responses to collagen by a mechanism that is not entirely known. The present investigation demonstrates that both active and 1,10-phenanthroline-inactivated jararhagin inhibit platelet aggregation by collagen with an IC50 of 40 and 140 nM respectively. The apparently higher inhibitory effect of the active enzyme clearly indicates that, in addition to the disintegrin region, the metalloproteinase domain of jararhagin also participates in this inhibition. As collagen interacts with platelets via alpha 2 beta 1-integrin, we investigated the effects of jararhagin on this integrin using selected function-blocking monoclonal antibodies against both of its subunits. Flow cytometry of platelets treated with native jararhagin and immunoprecipitation of platelet surface glycoproteins from lysates after jararhagin treatment showed an apparently selective reduction of alpha 2 beta 1-integrin immunoreactivity with both anti-alpha 2 and anti-beta 1 monoclonal antibodies. The loss of immunoreactivity was not due to integrin internalization, since it also took place in cytochalasin D-treated platelets. Here we show that jararhagin cleaved isolated alpha 2 beta 1-integrin resulting in the generation of a 115 kDa beta 1 fragment. We therefore propose that the inhibition by jararhagin of platelet response to collagen is mediated through the binding of jararhagin to platelet alpha 2-subunit via the disintegrin domain, followed by proteolysis of the beta 1-subunit with loss of the integrin structure (conformation) necessary for the binding of macromolecular ligands.
Kamiguti
A S
AS
Department of Haematology, Royal Liverpool University Hospital, University of Liverpool, Liverpool L69 3BX, U.K.
Hay
C R
CR
Zuzel
M
M
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002846:Chromatography, Affinity; D003094:Collagen / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D003435:Crotalid Venoms / Q000378:metabolism / Q000494:pharmacology*; D015638:Cytochalasin D / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D019012:Integrin beta1 / Q000502:physiology; D016023:Integrins / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011485:Protein Binding; D038981:Receptors, Collagen; D000097146:Bothrops jararaca Venom
D003094:Collagen; D003435:Crotalid Venoms; D015638:Cytochalasin D; D019012:Integrin beta1; D016023:Integrins; D008666:Metalloendopeptidases; D010975:Platelet Aggregation Inhibitors; D038981:Receptors, Collagen; D000097146:Bothrops jararaca Venom