Mocarhagin, a novel cobra venom metalloproteinase, cleaves the platelet von Willebrand factor receptor glycoprotein Ibalpha. Identification of the sulfated tyrosine/anionic sequence Tyr-276-Glu-282 of glycoprotein Ibalpha as a binding site for von Willebrand factor and alpha-thrombin.
Platelet adhesion to the subendothelium is the initiating event in hemostasis and thrombosis and involves the binding of von Willebrand factor (vWF) by the platelet membrane glycoprotein (GP) Ib-IX complex. The alpha-chain of GP Ib contains binding sites for both vWF and alpha-thrombin within a 45-kDa N-terminal tryptic fragment. In the present study, we have further delineated these sites using smaller proteolytic fragments and functional antibodies. Mocarhagin, a cobra venom metallaproteinase, generates the fragment His-1-Glu-282, while cathepsin G, a neutrophil granule serine protease, generates a slightly smaller fragment, His-1-Leu-275. His-1-Glu-282 was as effective as intact soluble GP Ibalpha (glycocalicin) in inhibiting botrocetin-dependent binding of vWF to washed platelets (IC50 approximately 0.3 microM) whereas His-1-Leu-275 was an order of magnitude less effective (IC50 approximately 3 microM). Residues Tyr-276-Glu-282 (YDYYPEE) are part of an anionic region homologous to thrombin-binding molecules such as hirudin. In ligand blot analysis, thrombin blotted the His-1-Glu-282 fragment, but not His-1-Leu-275. The three tyrosine residues within Tyr-276-Glu-282 meet the consensus criteria for O-sulfation. A method was developed to distinguish O-sulfated from nonsulfated tyrosine residues based on differences in the UV absorbance spectra. Residues Tyr-276-Glu-282 were isolated from glycocalicin by proteolysis with mocarhagin and cathepsin G. Ion spray mass spectrometry confirmed that Tyr-278 and Tyr-279 was only approximately 50% O-sulfated. Four anti-GP Ibalpha monoclonal antibodies (SZ2, ES85, C34 and VM16d) were found to be modulator-specific, strongly inhibiting botrocetin-dependent binding of vWF, but having less or no effect on ristocetin-dependent vWF binding. These antibodies also inhibited the binding of thrombin to fixed platelets. Immunoprecipitation with GP ibalpha fragments defined the epitopes for these antibodies as SZ2 (Tyr-276-Glu-282), ES85 (Asp-283-Arg-293), C34 (His-1-Glu-282), and VM16d (His-1-Leu-275). An antibody which inhibited ristocetin-dependent, as well as botrocetin-dependent, vWF binding but had no effect on thrombin binding (Ak2) had an epitope within His-1-Leu-275. These findings indicate that the sulfated tyrosine/anionic GP Ibalpha residues Tyr-276-Glu-282 are important for the binding of thrombin and botrocetin-dependent binding of thrombin and the botrocetin-dependent binding of vWF, but that vWF also interacts with residues within His-1-Leu-275.
Ward
C M
CM
Hazel and Pip Appel Vascular Biology Laboratory and Peptide Biology Laboratory, Baker Medical Research Institute, Prahran, Australia.
Andrews
R K
RK
Smith
A I
AI
Berndt
M C
MC
D000595:Amino Acid Sequence; D004546:Elapid Venoms / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D004546:Elapid Venoms; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; C023592:glycocalicin; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; D014443:Tyrosine; D013917:Thrombin; D008666:Metalloendopeptidases; C099092:mocarhagin