title: Binding of a novel 50-kilodalton alboaggregin from Trimeresurus albolabris and related viper venom proteins to the platelet membrane glycoprotein Ib-IX-V complex. Effect on platelet aggregation and glycoprotein Ib-mediated platelet activation.
abstract: Binding of the multimeric adhesive glycoprotein, von Willebrand Factor (vWF), to the platelet membrane glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion and initiates signal transduction leading to platelet activation. Recently described viper venom proteins that bind to the GP Ib alpha-chain and inhibit vWF binding provide novel probes for studying receptor function. We have purified a 50-kDa form of alboaggregin from the white-lipped tree viper (Trimeresurus albolabris) and two 25-kDa proteins, CHH-A and CHH-B, from the timber rattlesnake (Crotalus horridus horridus) in addition to a previously described 25-kDa alboaggregin and echicetin. Complete or partial amino acid sequencing of CHH-A, CHH-B, and 50-kDa alboaggregin and cross-reactivity of these proteins with an anti-botrocetin antiserum confirmed that they were disulfide-linked heterodimers or higher multimers of the C-type lectin protein family. These proteins, together with 25-kDa alboaggregin and echicetin, specifically bound to GP Ib alpha within the N-terminal peptide domain, His-1-Glu-282, and inhibited vWF binding with comparable IC50 values (approximately 0.2 microgram/mL). However, cross-blocking studies between these structurally related proteins and anti-GP Ib alpha monoclonal antibodies demonstrated that the venom protein binding sites were not congruent. Further, the 50-kDa alboaggregin, but not the other venom proteins, potently induced platelet activation as assessed by dense granule serotonin release or elevation of cytosolic ionized calcium. Treatment of platelets with the 50-kDa alboaggregin was associated with activation of protein kinase C and tyrosine kinase(s), resulting in a platelet protein phosphorylation profile similar to that seen on shear-stress-induced vWF binding to platelets. These results suggest that the 50-kDa alboaggregin induces cytoplasmic signaling coincident with its binding to the GP Ib-IX-V complex and provides a potentially useful probe for studying the mechanism of vWF-dependent platelet activation.
authors.affiliation: Hazel and Pip Appel Vascular Biology Laboratory, Baker Medical Research Institute, Victoria, Australia.
authors.forename: R K
authors.initials: RK
authors.lastname: Andrews
authors.forename: M H
authors.initials: MH
authors.lastname: Kroll
authors.forename: C M
authors.initials: CM
authors.lastname: Ward
authors.forename: J W
authors.initials: JW
authors.lastname: Rose
authors.forename: R M
authors.initials: RM
authors.lastname: Scarborough
authors.forename: A I
authors.initials: AI
authors.lastname: Smith
authors.forename: J A
authors.initials: JA
authors.lastname: López
authors.forename: M C
authors.initials: MC
authors.lastname: Berndt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins; D003435:Crotalid Venoms / Q000378:metabolism* / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation; D010974:Platelet Aggregation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism* / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014757:Viper Venoms / Q000378:metabolism / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D003435:Crotalid Venoms; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; C029748:Trimeresurus venoms; D014757:Viper Venoms; D014841:von Willebrand Factor; C080995:echicetin; D012701:Serotonin; D010450:Endopeptidases; D013917:Thrombin