Different regions of the class P-III snake venom metalloproteinase jararhagin are involved in binding to α2β1 integrin and collagen

SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to α2β1 integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to α2β1 integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344–421), showed a significant binding to recombinant α2β1 integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant α2β1 integrin nor to cell-surface-exposed α2β1 integrin (α2-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and α2β1 integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.