Coagulation Activity Of Gln49-PLA₂ From Gloydius Ussuriensis Snake Venom In Vitro

Phospholipase A₂ homogue with Gin at 49 sites, named Gln49-PLA₂, is isolated andpurified from Gloydius ussurensis snake venom by Hitrap SP cation exchange and Superdex75 gel filtration chromatography. It is the single component by SDS-PAGE. The molecularmass of Gln49-PLA₂ is 13880.79, which shows myotoxicity, neurotoxicity and cytotoxicactivity, but lacks the phospholipase A₂ activity.The coagulant effects of Gln49-PLA₂ are investigated on human citrated plasma andfibrinogen. The results indicate as follows: Gln49-PLA₂ clots human plasma and purifiedfibrinogen solution dose-dependently from 139.5±4.56 min to 12.87±0.12 min and180.67±1.86s to 19.00±0.58s respectivily. Moreover, Gln49-PLA₂ reduces re-calcificationtime from 7.46±1.17 to 0.75±0.33 min dose-dependently.The effects of Gln49-PLA₂ on four coagulation parameters—prothrombin time (PT),activated partial thromplastin time (APTT), thrombin time (TT) and fibrinogen concentration(Fib) are studied. The results show that Gln49-PLA₂ has no effect on TT and Fib. However itcan reduce prothrombin time from 12.4±0.2% to 6.95±0.20s, and prolong the activated partialthromboplastin time from 39.22±0.88s to 44.47±0.47s. Gln49-PLA₂ could not activate factorâ…©â…¢, and the pro-coagulant activity is not inhibited by heparin and anti-HI. The specificclotting activity of Gln49-PLA₂ is equivalent to 1100 NIH thrombin U/mg on humanfibrinogen, and the specific arginine esterase activity on substrate BAEE is 1747U/mg, soGln49-PLA₂ is a thrombin-like.Gln49-PLA₂ hydrolyzes fibrinogen Aαchain to release fibrinopeptide A preferentially,with the time prolonging (more than 10 hours), fibrinogen Bβcan be hydrolyzed to releasefibrinopeptide B, and the fibrinongenlytic ability is inhibited by serine protease inhibitorPMSF, but not by metalloprotease inhibitor EDTA. However, Gln49-PLA₂ is different fromthrombin enzyme in coagulation cascade. Gln49-PLA₂ can not induce the fibrin cross-link.This finding demonstrates that Gln49-PLA₂ is consistent with thrombin-tike properties,should be a new thrombin-like serine protease.