Purification, characterization, and cDNA sequence of halysetin, a disintegrin-like/cysteine-rich protein from the venom of Agkistrodon halys Pallas.

By means of DEAE-Sepharose CL-6B column chromatography, gel filtration on Sephadex G-75 and Superose 12 FPLC, halysetin, an antiplatelet protein, was purified from the venom of Agkistrodon halys Pallas with molecular mass of 29 kDa on SDS-PAGE and 23,168 Da by mass spectrometry. The p1 was about 5.0. Halysetin was devoid of phospholipase A2, fibrino-(geno)lytic, esterase, hemorrhagenic activities. Halysetin dose-dependently inhibited the aggregation of human platelet, which was stimulated by collagen with IC50 of 420 nM, but not that stimulated by ADP. The N-and C-terminal sequences of halysetin were characterized. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. It encoded a protein of 212-amino-acid residues with disintegrin-like/cysteine-rich domains and was highly homologous with SYMPs (snake venom metalloprotease).