Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom

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Abstract
C. atrox venom contains agents that render human fibrinogen and plasma incoagulable by thrombin. To elucidate the mechanism of alteration of fibrinogen clotting function by the venom, 4 immunochemically different proteases, 1, 2, 3 and 4, were purified from the venom by anion-exchange chromatography and column gel filtration. All 4 proteases had anticoagulant activity rendering purified fibrinogen incoagulable. Proteases 1 and 4 did not affect fibrinogen in plasma but in purified fibrinogen cleaved the A.alpha. chain first and then the B.beta. and .gamma. chains. Both enzymes were metalloproteases containing a single polypeptide chain with 1 mol Zn, were inhibited by EDTA and human .alpha.2-macrogobulin and had an optimal temperature of 37.degree. C and an optimal pH of 7. Protease 1 had a MW of 20,000 and was the most cationic. Protease 4 had an MW of 46,000 and was the most anionic glycoprotein with 1 free SH group. Proteases 2 and 3 degraded purified fibrinogen and fibrinogen in plasma, cleaving only the B.beta. chain and leaving the A.alpha. and .gamma. chains intact. Both enzymes were alkaline serine proteases, cleaved chromogenic substrates at the COOH terminal of Arg or Lys, were inhibited by DFP and phenylmethanesulfonyl fluoride and had an optimal temperature of 50.degree.-65.degree. C. Protease 2 was a single polypeptide chain glycoprotein with an MW of 31,000. Protease 3 was a 2 polypeptide chain protein with an MW of 24,000, each of the 2 chains having an MW of 13,000; its activity was not affected by major protease inhibitors of human plasma. Proteases 2 and 3 were enzymes with unique and limited substrate specificity by cleaving only the B.beta. chain, releasing a peptide of MW 5000 and generating a fibrinogen derivative of MW 325,000, with intact A.alpha. and .gamma. chains and poor coagulability. Since the 2 enzymes were active in human plasma and serum, it was postulated that proteases 2 and 3 mediated anticoagulant effects in vivo after envenomation.