Isolation and characterization of EMS16, a C-lectin type protein from Echis multisquamatus venom, a potent and selective inhibitor of the alpha 2 beta 1 integrin

We have isolated and characterized EMS16, a potent and selective inhibitor of the alpha 2 beta 1 integrin, from Echis multisquamatus venom. It belongs to the family of C-lectin type of proteins (CLPs), and its amino acid sequ ence is homologous with other members of this protein family occurring in s nake venoms. EMS 16 (M-r similar to 33K) is a heterodimer composed of two d istinct subunits linked by S-S bonds. K562 cells transfected with alpha 2 i ntegrin selectively adhere to immobilized EMS 16, but not to two other snak e venom-derived CLPs, echicetin and alboaggregin B. EMS16 inhibits adhesion of alpha 2 beta 1-expressing cells to immobilized collagen I at picomolar concentrations, and the platelet/collagen I interaction in solution at nano molar concentrations. EMS16 inhibits binding of isolated, recombinant I dom ain of a2 integrin to collagen in an ELISA assay, but not the interaction o f isolated I domain of al integrin with collagen TV. Studies with monoclona l antibodies suggested that EMS 16 binds to the alpha 2 subunit of the inte grin. EMS 16 inhibits collagen-induced platelet aggregation but has no effe ct on aggregation induced by other agonists such as ADP, thromboxane analog ue (U46619), TRAP: or convulxin. EMS16 also inhibits collagen-induced, but not convulxin-induced, platelet cytosolic Ca2+ mobilization. In addition, E MS16 inhibits HUVEC migration in collagen I gel. In conclusion, we report a new, potent viper venom-derived inhibitor of alpha 2 beta 1 integrin, whic h does not belong to the disintegrin family.