Mocarhagin, a Novel Cobra Venom Metalloproteinase, Cleaves the Platelet von Willebrand Factor Receptor Glycoprotein Ibα. Identification of the Sulfated Tyrosine/Anionic Sequence Tyr-276−Glu-282 of Glycoprotein Ibα as a Binding Site for von Willebrand Factor and α-Thrombin

Publisher Website
Google Scholar
Abstract
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 α-chain of GP Ib contains binding sites for both vWF and α-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 metalloproteinase, 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 Ibα (glycocalicin) in inhibiting botrocetin-dependent binding of vWF to washed platelets (IC50 ∼0.3 μM), whereas His-1−Leu-275 was an order of magnitude less effective (IC50 ∼3 μM). 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 were O-sulfated to at least 90%, whereas Tyr-276 was only ∼50% O-sulfated. Four anti-GP Ibα 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 Ibα 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 Ibα residues Tyr-276−Glu-282 are important for the binding of thrombin and the botrocetin-dependent binding of vWF, but that vWF also interacts with residues within His-1−Leu-275.