Cerastotin, a Serine Protease from Cerastes Cerastes Venom, with Platelet‐Aggregating and Agglutinating Properties

Abstract

Cerastotin, a thrombin-like enzyme from the venom of the desert viper Cerastes cerastes, has been purified by gel filtration on Sephadex G-75 and two ion-exchange chromatographies on Mono S columns. It is a neutral glycoprotein (PI = 6.6), present as a single polypeptide chain of 40 kDa. Its N-terminal sequence shows strong similarity with those of other thrombin-like enzymes from snake venoms. Cerastotin possesses esterase and amidolytic activities measured with W-tosyl-L-arginine methyl ester and the thrombin chromogenic substrate D-phenylalanyl-L-pipecolyl-L-arginine p-nitroanilide, respectively. The amidolytic activity is inhibited by phenylmethylsulfonyl fluoride, W-tosyl-L-lysine chloromethane, N"-tosyl-L-phenylalanyl chloromethane, D-phenylalanyl-L-prolyl-L-arginyl chloromethane and benzamidine, suggesting that cerastotin is a serine protease. Cerastotin efficiently clots human plasma and cleaves preferentially the a chain of fibrinogen. Cerastotin did not induce aggregation of washed normal platelets, but did aggregate platelets in the presence of exogenous fibrinogen. A monoclonal antibody directed against glycoprotein (GPIb), which specifically inhibits induced agglutination by ristocetin also completely blocks platelet aggregation induced by cerastotin. However, another anti-GPIb monoclonal antibody, which specifically inhibits a-thrombin binding to GPlb, did not prevent this aggregation. Furthermore, platelets which were desensitised by a-thrombin still aggregate in the presence of cerastotin, but not a-thrombin. Similarly a monoclonal antibody, anti-GPIIb-IIIa, which blocks fibrinogen binding, did not inhibit cerastotin-induced platelet aggregation. This activity is abolished in the presence of 1 mM phenylmethylsulfonyl fluoride and/or 10 mM EDTA. Cerastotin also agglutinates formalin-fixed and washed platelets, only in the simultaneous presence of fibrinogen and of Von Willebrand factor.

Snake venom thrombin-like enzymes: from reptilase to now

“…Its activity is not inhibited by a monoclonal antibody antiGPIIb-IIIa that blocks fibrinogen binding, but by a monoclonal antibody directed against glycoprotein Ib, which also specifically inhibits induced agglutination by ristocetin ( fig. 4) [99]. On the other hand, some SVTLEs do not need fibrinogen to aggregate platelets.…”

Grape seed extract neutralizes the effects of Cerastes cerastes cerastes post-synaptic neurotoxin in mouse diaphragm

“…Although many papers on the effect of C. cerastes venom fractions have been published (Daoud et al, 1986;Basheer et al, 1995;Laraba-Djebari et al, 1995;Marrakchi et al, 1995Marrakchi et al, , 1997Abu-Sinna et al, 2003;Boukhalfa-Abib et al, 2009;Chérifi et al, 2010), yet post-synaptic neurotoxins are among the poorly-studied venom fractions of vipers. To our knowledge, the current study reports for the first time the ultrastructural toxic effects produced in the diaphragm of sub-lethally intoxicated mice, by the post-synaptic neurotoxin isolated from the Egyptian sand viper, C. cerastes cerastes.…”

A Brief Appraisal on Russell’s Viper Venom (Daboia russelii russelii) Proteinases

“…Russelobin was found to be significantly inhibited by thrombin inhibitors such as heparin and weakly inhibited by antithrombin-III unlike other thrombin-like snake venom proteinases such as batroxobin, cerastocytin, cerastotin, and contortrixobin (Mukherjee and Mackessy 2013). The difference in the active site of Russelobin compared to other SVSPs was more prominent due to the fact that Russelobin was not inhibited by tosyl phenylalanyl chloromethyl ketone (TPCK) and tosyl lysine chloromethyl ketone (TLCK) (Mukherjee and Mackessy 2013), whereas other SVSPs were inhibited by TPCK as well as TLCK (Marrakchi et al 1997;Amiconi et al 2000). Russelobin demonstrated a high degree of selectivity toward fibrinogen without detectable proteolysis of other plasma proteins, suggesting that fibrinogen is the primary physiological substrate for Russelobin.…”