A novel tetrameric venom protein, agglucetin from Agkistrodon acutus, acts as a glycoprotein Ib agonist.
A novel platelet agglutination inducer, agglucetin, was purified from the Formosan Agkistrodon acutus snake venom. It migrated as a single band with an apparent molecular mass of 58.8 kDa and two distinct bands of 16.2/14.5 kDa under non-reducing and reducing conditions by SDS-PAGE, respectively. Further confirmed by FPLC, electrospray ionization mass spectrometry and 2D-PAGE, native agglucetin exists as a tetramer composed of disulfide-linked alpha1, alpha2, beta1 and beta2 subunits. Partial N-terminal sequence of agglucetin subunit showed a high degree of homology to those of C-type lectin-like glycoprotein (GP) Ib binding proteins. Functional studies showed that agglucetin. in the absence of von Willebrand factor (vWF), dose-dependently induced platelet agglutination and caused a negligible elevation of intracellular Ca+2 mobilization and thromboxane B, formation in human platelet suspensions. Anti-GP Ib monoclonal antibodies (mAbs), AP1 or LJ-Ib1, specifically inhibited agglucetin-induced platelet agglutination in a dose-dependent manner. However, EDTA, arietin (a long chain RGD-containing disintegrin), 7E3 (an anti-GP IIb/IIIa mAb), heparin, hirudin, PGE1, or indomethacin exhibited no inhibitory effect on agglucetin-induced platelet agglutination. Furthermore, flow cytometric analysis revealed that FITC-agglucetin dose-dependently bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by anti-GP Ib mAb. It is concluded that agglucetin, acts specifically on an epitope of platelet membrane GP Ib overlapping with that of API, causing platelet agglutination in a Ca+2- and GP IIb/IIIa-independent manner.
Wang
W J
WJ
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei.
Huang
T F
TF
D017836:Agkistrodon / Q000378:metabolism*; D000527:Alprostadil / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001667:Binding, Competitive; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D003435:Crotalid Venoms / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000302:isolation & purification; D006493:Heparin / Q000494:pharmacology; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000037:antagonists & inhibitors* / Q000276:immunology; D011448:Prostaglandin Antagonists / Q000494:pharmacology; D011487:Protein Conformation; D021122:Protein Subunits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013929:Thromboxane B2 / Q000096:biosynthesis; D014841:von Willebrand Factor / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D003435:Crotalid Venoms; D000939:Epitopes; D005343:Fibrinolytic Agents; D006629:Hirudins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D019038:Platelet Glycoprotein GPIb-IX Complex; D011448:Prostaglandin Antagonists; D021122:Protein Subunits; C453260:agglucetin; D014841:von Willebrand Factor; C069270:arietin; D013929:Thromboxane B2; D006493:Heparin; D004492:Edetic Acid; D000527:Alprostadil; D007213:Indomethacin