Proteases from <i>Vipera lebetina </i>Venom Affecting Coagulation and Fibrinolysis

Abstract

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants in the same venom. We showed that V. lebetina venom contains: (1) proteases that degrade fibrinogen, but not fibrin; (2) fibrinolytic enzyme (lebetase); (3) factor X activator (VLFXA); (4) factor V activator (VLFVA). Fibrinolytic enzyme and VLFXA are metalloproteases; the other studied enzymes are serine proteases. α-Fibrinogenase has no homolog among known serine proteases. β-Fibrinogenase is a typical thermostable arginine esterase that hydrolyzes esters and amides of arginine and attacks the β-chain of fibrinogen. Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins. We used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique for the recovery and identification of peptides released by protease hydrolysis and for the detection of human factor X cleavage products after VLFXA hydrolysis. VLFXA cleaves the Arg⁵²-Ile⁵³bond in the heavy chain of human factor X and the Arg²²⁶-Val²²⁷ bond in human factor IX precursor; VLFVA cleaves Arg¹⁵⁴⁵-Ser¹⁵⁴⁶ in factor V.

A Bipartite Autoinhibitory Region within the B-domain Suppresses Function in Factor V

“…However, cleavage at Arg 1545 , which is adjacent to the AR, must have a significant impact. Support for this comes from the longstanding observation in the field that cleavage of FV by a protease in Russell's viper venom at Arg 1545 results in full activation (7,13,(15)(16)(17)(18)(19)(20). Due to the nature of how the PRR inhibits cofactor function, it is likely that there are multiple ways in which FV could be activated.…”

“…Broadly, these studies found that variable amounts of cofactor activity are observed depending on which region of the B-domain is cleaved and which assay is used to evaluate activity. A clear consensus is that maximal activity correlates with cleavage at Arg 1545 (7,13,(15)(16)(17)(18)(19)(20)(21)(22). Although these studies have advanced the field, such correlative studies have not provided mechanistic insight into how FV activation unfolds.…”

Vipera lebetina Venom Contains Two Disintegrins Inhibiting Laminin-binding β1 Integrins

“…However, in most of them, proteolytic enzymes, phospholipase activities, and other factors may account for their destructive effects on muscle tissue (13,14), as only one of 33 tested venoms entirely inhibited interaction of ␣ 7 ␤ 1 integrin with its laminin ligands. In addition to abundant phospholipase activity and numerous proteases (14,17), the venom of V. lebetina contains two disintegrins (lebein-2 and lebein-1) that inhibit ␣ 7 ␤ 1 integrin binding to its laminin ligands. In this study, we have established and optimized their purification from the crude toxin and have characterized their integrin-inhibiting function.…”

“…Being abundant in most snake venoms, phospholipase A 2 is mainly responsible for myotoxic effects (13,14). Various proteases of snake venoms specifically cleave blood-clotting factors or components of the ECM, thus resulting in bleeding dysfunctions and tissue necrosis (15)(16)(17).…”

Unusual visual loss after snakebite

“…While there are enzymes that cause hypofibrinogenemia, hypoprothrombinemia, thrombocytopenia and fibrinolysis; at the same time, there are potent proteases (e.g. arginine esterase and hydrolase) acting as activators of coagulation factors X and V, thereby promoting coagulation (7). In addition, enzymes -such as hyaluronidase -damage connective tissues, leading to enhanced toxin dissemination.…”