Snake venom proteinases as tools in hemostasis studies: structure-function relationship of a plasminogen activator purified from Trimeresurus stejnegeri venom.

Snake venom serine proteinases affect many steps of the blood coagulation cascade. Each of them usually acts selectively on one coagulation factor. They are therefore potentially useful components to study the mechanisms of action, the regulation and the structure-function relationships of human serine proteinase coagulation factors. This strategy is illustrated for a plasminogen activator purified from Trimeresurus stejnegeri venom.

Snake venom proteins acting on hemostasis.

The venoms of Viperidae and Crotalidae snakes are a rich source of proteins with activity against various factors involved in coagulation and fibrinolysis. These proteins are very specific for their molecular targets, resistant to physiological inhibitors and stable in vitro and in vivo. They have therefore proved to be useful for diagnostic tests. Based on sequence similarities, these snake venom proteins have been classified into various families, such as serine proteinases, metalloproteinases, C-type lectins, disintegrins and phospholipases A(2). The various members of a given family, although structurally similar, act selectively on different blood coagulation factors. This opens up the possibility of characterizing the structural elements involved in target molecule recognition. Thus, snake venom proteins provide excellent models for studies of structure-function relationships.

Activation of rabbit blood platelets by anandamide through its cleavage into arachidonic acid.

Anandamide (ANA), a cannabinoid receptor ligand, stimulated platelet aggregation at concentrations similar to those of arachidonic acid (AA). The aggregating effect of ANA was inhibited by aspirin but not by SR-141716, a cannabinoid receptor antagonist. In addition, HU-210, a cannabinoid receptor agonist, failed to induce platelet activation. Radiolabelling experiments showed that exogenous ANA was cleaved by platelets into AA through a phenylmethylsulfonyl fluoride (PMSF)-sensitive pathway. In agreement, PMSF was shown to abolish the aggregating effect of ANA. In conclusion, ANA is able to induce platelet activation via its cleavage by a PMSF-sensitive amidase activity, leading to the release of AA which in turn activates platelets.

The contribution of residues 192 and 193 to the specificity of snake venom serine proteinases.

Snake venom serine proteinases, which belong to the subfamily of trypsin-like serine proteinases, exhibit a high degree of sequence identity (60-66%). Their stringent macromolecular substrate specificity contrasts with that of the less specific enzyme trypsin. One of them, the plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA), which shares 63% sequence identity with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom, specifically activates plasminogen to plasmin like tissue-type plasminogen activator (t-PA), even though it exhibits only 23% sequence identity with t-PA. This study shows that TSV-PA, t-PA, and batroxobin are quite different in their specificity toward small chromogenic substrates, TSV-PA being less selective than t-PA, and batroxobin not being efficient at all. The specificity of TSV-PA, with respect to t-PA and batroxobin, was investigated further by site-directed mutagenesis in the 189-195 segment, which forms the basement of the S(1) pocket of TSV-PA and presents a His at position 192 and a unique Phe at position 193. This study demonstrates that Phe(193) plays a more significant role than His(192) in determining substrate specificity and inhibition resistance. Interestingly, the TSV-PA variant F193G possesses a 8-9-fold increased activity for plasminogen and becomes sensitive to bovine pancreatic trypsin inhibitor.

Alteration of the actin-based cytoskeleton by rabies virus.

We investigated the effect of rabies virus infection on the actin cytoskeleton using various techniques. Confocal microscopic examination of rabies virus-infected neuroblastoma cells at late stages of infection revealed a dramatic decrease in F-actin staining. The results of a fluorimetric assay with pyrenylated actin indicated that purified rabies virus nucleocapsid has no direct action on the kinetics of actin polymerization and only a weak effect on the final extent of polymerization. Video-microscopy experiments with purified components showed that rabies virus nucleocapsid inhibits the actin-bundling effect induced by dephospho-synapsin I, a neuron-specific protein which is known to exert a control on the actin-based cytoskeleton. Thus, the observed decrease in F-actin staining in infected cells might be ascribed to an indirect action of rabies nucleocapsid on the effects of actin-binding proteins such as synapsin I.