Rhodocytin induces platelet aggregation by interacting with glycoprotein Ia/IIa (GPIa/IIa, Integrin alpha 2beta 1). Involvement of GPIa/IIa-associated src and protein tyrosine phosphorylation.
Although glycoprotein Ia/IIa (GPIa/IIa, integrin alpha(2)beta(1)) has established its role as a collagen receptor, it remains unclear whether GPIa/IIa mediates activation signals. In this study, we show that rhodocytin, purified from the Calloselasma rhodostoma venom, induces platelet aggregation, which can be blocked by anti-GPIa monoclonal antibodies. Studies with rhodocytin-coupled beads and liposomes loaded with recombinant GPIa/IIa demonstrated that rhodocytin directly binds to GPIa/IIa independently of divalent cations. In vitro kinase assays and Western blotting of GPIa immunoprecipitates revealed that Src and Lyn constitutively associate with GPIa/IIa and that Src activity increases transiently after rhodocytin stimulation. Src specifically associates with p130 Crk-associated substrate (Cas) in a manner dependent upon Cas phosphorylation, suggesting that Src is responsible for Cas tyrosine phosphorylation. While all these phenomena occur early after rhodocytin stimulation in a cAMP-resistant manner, tyrosine phosphorylation of Syk and phospholipase Cgamma2, intracellular Ca(2+) mobilization, and platelet aggregation occur later in a cAMP-sensitive manner. Cytochalasin D, which interferes with actin polymerization and blocks receptor clustering, inhibits all the rhodocytin-mediated signals we examined in this study. We suggest that rhodocytin, by clustering GPIa/IIa, activates GPIa/IIa-associated Src, which then mediates downstream activation signals.
Suzuki-Inoue
K
K
Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Yamanashi 409-3898, Japan.
Ozaki
Y
Y
Kainoh
M
M
Shin
Y
Y
Wu
Y
Y
Yatomi
Y
Y
Ohmori
T
T
Tanaka
T
T
Satoh
K
K
Morita
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D001798:Blood Proteins / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D016023:Integrins / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology*; D037181:Lectins, C-Type; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D010973:Platelet Adhesiveness / Q000502:physiology; D010974:Platelet Aggregation / Q000187:drug effects / Q000502:physiology*; D038981:Receptors, Collagen; D017452:Receptors, IgG / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D014443:Tyrosine / Q000097:blood; D014757:Viper Venoms; D017819:Viperidae
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D016023:Integrins; D037102:Lectins; D037181:Lectins, C-Type; D038981:Receptors, Collagen; D017452:Receptors, IgG; D014757:Viper Venoms; C112292:rhodocytin protein, Calloselasma rhodostoma; D019000:Phosphotyrosine; D014443:Tyrosine; D015232:Dinoprostone