Suppression of severe lesions, myonecrosis and hemorrhage, caused by Protobothrops flavoviridis venom with its serum proteins

Protobothrops flavoviridis serum proteins precipitated with ammonium sulfate were chromatographed on a DEAE-Toyopearl 650M column at pH 7.5 with stepwise increase or with linear gradient of NaCl concentration. Peaks 3 and 4 serum proteins, obtained by linear gradient elution and named Frde3 and Frde4, contained Habu serum factors (HSF) and phospholipase A2 (PLA2) inhibitors (PfPLI), respectively. The serum proteins eluted at 0.2 M NaCl by stepwise elution, named Fr0.2NaCl, effectively suppressed myonecrosis and hemorrhage caused by P. flavoviridis venom in rat or mouse thigh muscles. The Fr0.2NaCl were fractionated by HPLC and the fractions, after SDS-PAGE, underwent far-western blot analysis with PLA2 ([Asp49]PLA2) and BPI ([Lys49]PLA2) as the probes. Four PfPLIs, namely, PfαPLI-A, PfαPLI-B, PfγPLI-A and PfγPLI-B, were identified together with their selective binding specificities to PLA2 species. In addition, a new 9 kDa protein, which is specifically bound to BPI, was found. Suppression of P. flavoviridis venom-induced severe lesions, such as myonecrosis, hemorrhage and edema, with its serum proteins was histopathologically observed in the present work for the first time. The cooperative use of P. flavoviridis antivenom and its serum proteins as medication for P. flavoviridis snake bites is discussed.