Bilinexin, a snake C-type lectin from Agkistrodon bilineatus venom agglutinates platelets via GPIb and alpha(2)beta(1)

A new snake protein, named bilinexin, has been purified from Agkistrodon bi lineatus venom by ion-exchange chromatography and,,el filtration chromatogr aphy. Under non-reducing conditions it has a mass of 110 kDa protein on SDS -PAGE. On reduction, it can be separated into five subunits with masses in the range 13-25 kDa. The N-terminal sequences of these subunits are very si milar to those of convulxin or the alboaggregins, identifying bilinexin as a new member of the snake C-type lectin family, unusual in having multiple subunits. Bilinexin agglutinates fixed platelets, washed platelets and plat elet rich plasma (PRP) without obvious activation (shape change) as confirm ed by light microscope examination. Both inhibitory and binding studies ind icate that antibodies against alpha (2)beta (1) inhibit not only platelet a gglutination induced by bilinexin, but also bilinexin binding to platelets. VM16d, a monoclonal anti-GPIb alpha antibody, completely inhibits platelet agglutination induced by bilinexin, and polyclonal antibodies against GPIb alpha prevent its binding to platelets, However, neither convulxin, polycl onal anti-GPVI antibodies, nor GPIIb/IIIa inhibitors affect its binding to and agglutination of platelets. Bilinexin neither activates GPIIb/IIIa inte grin on platelets nor induces tyrosine phosphorylation of platelet proteins , nor increases intracellular Ca2+ in platelets, Like alboaggregin B, bilin exin agglutinates platelets, which makes it a good tool to investigate the differences in mechanism between snake C-type lectins causing platelet aggl utination and those that induce full activation.