Abstract
Jararaca GPIb-BP, a snake venom protein composed of alpha and beta subunits purified from Bothrops jararaca, binds to platelet glycoprotein (GP)Ib and functions as a receptor blocker for von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura, S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K., Taniuchi, Y., and Kawasaki, T. (1995) Thromb. Haemostasis 74, 743-750). We present here the entire 142- and 123-residue amino acid sequence of the respective alpha and beta subunits and also demonstrate that the platelet GPIb-binding site resides on the beta and not on the alpha subunit based on an enzyme-linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands. Sequences of the alpha and beta subunits were determined by analysis of the intact S-pyridylethylated proteins and their peptides generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl ketone-trypsin. A 38-39% identity of amino acid sequence between the alpha and beta subunits of jararaca GPIb-BP was observed, as well as a high degree of sequence identities (38-64%) with the respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., and Titani, K (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 928-932) and the beta-chain of echicetin (Peng, M., Holt, J. C., and Niewiarowski, S. (1994) Biochem. Biophys. Res. Commun. 205, 68-72).
Highlights
The first step in primary hemostasis is thought to be platelet adhesion to exposed subendothelium at the site of vascular injury
We present here the entire 142- and 123-residue amino acid sequences of the respective ␣ and  subunits and demonstrate that the platelet GPIb-binding site resides on the  and not on the ␣ subunit based on an enzyme-linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands
We determined the complete amino acid sequence of jararaca GPIb-BP isolated from the venom of Bothrops jararaca
Summary
Materials—All reagents used were of analytical grade. Jararaca GPIb-BP (Japanese patent name “Yoshitobin”) was purified from the crude venom of B. jararaca (Sigma) as described recently (6).  subunits of jararaca GPIb-BP were separated by RP-HPLC on a SynChropak RP-8 column (4.1 ϫ 250 mm; Synchrom, Lafayette, IN) with a gradient of acetonitrile into 0.1% aqueous trifluoroacetic acid (12). Each digest was separated by RP-HPLC on a SynChropak RP-8, RP-18 column or Cosmosil 5C8 –300 column (4.6 ϫ 100 mm, Nacalai Tesque, Kyoto, Japan) with a gradient of acetonitrile into 0.1% aqueous trifluoroacetic acid. The amount of biotin-labeled jararaca GPIb-BP bound to the immobilized platelets was measured by an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled streptoavidin (Dakopatts, Denmark) and O-phenylenediamine as a substrate and spectrometry at 492 nm. A mixture (125 l) of biotinylated jararaca GPIb-BP (1 g/ml, final) and various concentrations of competing ligands was incubated for 15 min at room temperature, followed by the addition of 50 l of the mixture to the wells.
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