Snake venom metalloproteinases, crotarhagin and alborhagin, induce ectodomain shedding of the platelet collagen receptor, glycoprotein VI.
Glycoprotein (GP)VI, that binds collagen, together with GPIb-IX-V which binds vonWillebrand factor, forms an adheso-signalling complex on platelets that initiates thrombus formation in haemostasis and thrombosis. In this study, we show that two snake venom metalloproteinases, crotarhagin and alborhagin, induce ectodomain shedding of GPVI by a mechanism that involves activation of endogenous platelet metalloproteinases. Alborhagin is a viper venom metalloproteinase from Trimeresurus albolabris, while crotarhagin is a previously undescribed toxin from the rattlesnake Crotalus horridus horridus ( approximately 60-kDa non-reduced and reduced). Like alborhagin, crotarhagin induces aggregation in human platelet-rich plasma (maximal activity, approximately 0.3 microg/ml). Aggregation of washed platelets was inhibited by soluble GPVI ectodomain expressed as an Fc-fusion protein, confirming crotarhagin targeted GPVI. Treating washed platelets with crotarhagin or alborhagin resulted in time-dependent loss of surface GPVI and the appearance of an approximately 55-kDa soluble GPVI fragment in supernatants. Crotarhagin also induced shedding in GPVItransfected RBL-2H3 cells. Crotarhagin-induced shedding was metalloproteinase-dependent (inhibited by EDTA), but also blocked by inhibitors of GPVI signalling (Src kinase inhibitors, PP1 or PP2, or Syk inhibitor, piceatannol), indicating shedding required GPVI-dependent platelet activation. Together, the data suggest that the rattlesnake metalloproteinase, crotarhagin, and the viper toxin alborhagin, induce GPVI shedding by a mechanism involving activation of endogenous platelet metalloproteinases rather than direct cleavage of GPVI.
Department of Biochemistry and Molecular Biology, Monash University, Alfred Medical Research & Education Precinct, Melbourne, Victoria, Australia.
Lakshmi C
LC
Wijeyewickrema
Elizabeth E
EE
Gardiner
Masaaki
M
Moroi
Michael C
MC
Berndt
Robert K
RK
Andrews
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D002460:Cell Line; D003435:Crotalid Venoms / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008666:Metalloendopeptidases / Q000633:toxicity*; D045726:Metalloproteases / Q000633:toxicity*; D010446:Peptide Fragments / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects*; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D038981:Receptors, Collagen / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013267:Stilbenes / Q000494:pharmacology; D000072377:Syk Kinase; D013997:Time Factors; D014162:Transfection; D014757:Viper Venoms / Q000633:toxicity*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; C412373:AG 1879; D003435:Crotalid Venoms; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011480:Protease Inhibitors; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D038981:Receptors, Collagen; D011993:Recombinant Fusion Proteins; D013267:Stilbenes; D014757:Viper Venoms; C098341:platelet membrane glycoprotein VI; C041525:3,3',4,5'-tetrahydroxystilbene; D004492:Edetic Acid; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606898:Syk protein, rat; D019061:src-Family Kinases; D045726:Metalloproteases; D008666:Metalloendopeptidases; C433245:alborhagin; C526671:crotarhagin, Crotalus horridus horridus