Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom

Abstract

Crotalus 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, four immunochemically different proteases, I, II, III, and IV, were purified from the venom by anion-exchange chromatography and column gel filtration. All four proteases had anticoagulant activity rendering purified fibrinogen incoagulable. Proteases I and IV do not affect fibrinogen in plasma but in purified fibrinogen cleave the A alpha chain first and then the B beta and gamma chains. Both enzymes are metalloproteases containing a single polypeptide chain with 1 mol of zinc, are inhibited by (ethylenedinitrilo)tetraacetate and human alpha 2-macroglobulin, and have an optimal temperature of 37 degrees C and an optimal pH of 7. Protease I has a molecular weight (Mr) of 20 000 and is the most cationic. Protease IV has an Mr of 46 000 and is the most anionic glycoprotein with one free sulfhydryl group. Proteases II and III degrade both purified fibrinogen and fibrinogen in plasma, cleaving only the B beta chain and leaving the A alpha and gamma chains intact. Both enzymes are alkaline serine proteases, cleave chromogenic substrates at the COOH terminal of arginine or lysine, are inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride, and have an optimal temperature of 50-65 degrees C. Protease II is a single polypeptide chain glycoprotein with an Mr of 31 000. Protease III is a two polypeptide chain protein with an Mr of 24 000, each of the two chains having an Mr of 13 000; its activity is not affected by major protease inhibitors of human plasma. Proteases II and III are enzymes with unique and limited substrate specificity by cleaving only the B beta chain, releasing a peptide of Mr 5000 and generating a fibrinogen derivative of Mr 325 000, with intact A alpha and gamma chains and poor coagulability. Since the two enzymes are active in human plasma and serum, it is postulated that proteases II and III can mediate anticoagulant effects in vivo after envenomation.

Endothelial Cell VE-cadherin Functions as a Receptor for the β15–42 Sequence of Fibrin

“…Purified NDSK was treated with thrombin (3 units/ml for 3 h at 37°C) to generate NDSK II (NDSK lacking fibrinopeptides A and B). NDSK I (NDSK minus fibrinopeptide A) was prepared by treating NDSK with Atroxin (2 g/ml for 2 h at 37°C), and NDSK 325, which lacks B␤1-42, was prepared by treatment of NDSK with protease III (8 g/ml for 3 h at 37°C) from C. atrox venom (29). The identities of the NDSK derivatives were assessed by SDS-PAGE.…”

The Role of Putative Fibrinogen Aα-, Bβ-, and γA-chain Integrin Binding Sites in Endothelial Cell-mediated Clot Retraction

“…Fibrinogens-The following fibrinogens were used in this study to identify the binding sites utilized by endothelial cells to support clot retraction: peak 1 fibrinogen (␥A␥A) (6), peak 1 fibrinogen fraction I-9 (20), peak 2 fibrinogen fraction I-9 (6), and fibrinogen 325, which lacks the first 42 amino acids (amino terminus) of the B␤-chains (28,29). The fibrinogens used and the important binding sites present or absent on each form are listed in Table I.…”

“…Chromatographic separation into peak 1 and peak 2 subfractions was verified by DEAE-cellulose ion exchange chromatography using the gradient elution system described by Siebenlist et al (32). Fibrinogen 325 (des-B␤1-42 fibrinogen) that lacks the first 42 amino acids of B␤-chains (33) was produced from fraction I-2 fibrinogen as described by Pandya et al (28) and Pandya and Budzynski (29). Its structure was verified by SDS-polyacrylamide gel electrophoresis.…”

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

“…Thrombin cleaves fibrinogen at argl6-glyl7 of the Aa chain and argl4-glyl 5 19,20). The polypeptide chain composition offibrinogen and fibrin was assessed by 7% SDS PAGE after disulfide bond reduction as described (23).…”