rACLF, a recombinant snake venom metalloprotease, activates endothelial cells in vitro

Snake venom metalloproteases (SVMPs) comprise a family of snake venom toxins responsible for most of local and systemic effects observed during envenomation by snakes from the Viperidae family. The vascular system and more specifically the endothelium seem to be the preferential targets of these proteins. This work describes the effects of rACLF, a recombinant SVMP from Agkistrodon contortrix laticinctus on human umbilical vein endothelial cells (HUVECs) in vitro. Our results showed that rACLF activates HUVECs by the release of mediators involved in inflammation and hemostasis such as prostacyclin and interleukin-8. We also demonstrated that rACLF increased the expression of ICAM-I and decay accelerating factor (DAF). Moreover, rACLF protects the HUVECs against apoptosis induced by serum deprivation. These results suggest that the endothelial cell activation induced by SVMPs may have a significant role in the development of the local inflammatory lesion observed in Viperidae envenomation.

Insularinase A, a prothrombin activator from Bothrops insularis venom, is a metalloprotease derived from a gene encoding protease and disintegrin domains

The first low-molecular-mass metalloprotease presenting prothrombin activating activity was purified from Bothrops insularis venom and named insularinase A. It is a single-chain protease with a molecular mass of 22 639 Da. cDNA sequence analysis revealed that the disintegrin domain of the precursor protein is post-translationally processed, producing the mature insularinase A. Analysis of its deduced amino acid sequence showed a high similarity with several fibrin(ogen)olytic metalloproteases and only a moderate similarity with prothrombin activators. However, SDS-PAGE of prothrombin after activation by insularinase A showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin independently of the prothrombinase complex. In addition, insularinase A activates factor X and hydrolyses fibrinogen and fibrin. Chelating agents fully inhibit all insularinase A activities. Insularinase A induced neither detachment nor apoptosis of human endothelial cells and was also not able to trigger an endothelial proinflammatory cell response. Nitric oxide and prostacyclin levels released by endothelial cells were significantly increased after treatment with insularinase A. Our results show that, although its primary structure is related to class P-I fibrin(ogen)olytic metalloproteases, insularinase A is functionally similar to group A prothrombin activators.

A prothrombin activator serine protease from the Lonomia obliqua caterpillar venom (Lopap) biochemical characterization.

Lonomia obliqua venom causes a severe consumptive coagulopathy, which can lead to a hemorrhagic syndrome. The crude bristles extract displays a procoagulant activity due to a Factor X and to a prothrombin activating activity. Here, we describe a 69 kDa prothrombin activator serine protease purified from L. obliqua caterpillar bristle extract using gel filtration (Sephadex G 75) and HPLC (C(4) column). The purified protein was able to activate prothrombin in a dose-dependent manner, and calcium ions increased this activity. The prothrombin-derived fluorogenic peptide (Abz-YQTFFNPRTGSQ-EDDnp) had its main cleavage site at the Arg-Thr bond. The kinetic parameters obtained for this substrate were Kmapp of 4.5 microM, kcat of 5.32 s(-1), and a kcat/Kmapp of 1.2 x 10(6) M(-1) s(-1). The prothrombin fragments generated by the purified enzyme corresponded to the molecular masses of prethrombin 2, fragment 1, fragment 2, and thrombin as seen in SDS-PAGE. The thrombin generated was able to clot purified fibrinogen. The partial amino acid sequence of the purified protein, named Lopap (L. obliqua prothrombin activator protease), showed no similarity to any known prothrombin activator.

Effects of lopap on human endothelial cells and platelets.

Severe consumption coagulopathy has been detected in rats after Lopap (a prothrombin activator from Lonomia obliqua caterpillar bristles) infusion and in humans after accidental contact with L. obliqua bristles. However, platelet count and antithrombin (AT) levels were only modestly affected, suggesting that a different form of blood coagulation activation may be involved in this hemorrhagic syndrome. Here we describe that Lopap had no effect on aggregation of washed human platelets induced by several agonists, suggesting that it might not impair platelet function in vivo. AT was able to inhibit the amidolytic activity of thrombin generated by incubation of Lopap with prothrombin in a purified system, which may be different from that generated by the prothrombinase complex in vivo. The surface expression of both ICAM-1 and E-selectin but not of VCAM-1 was upregulated by Lopap in cultured HUVEC, suggesting that it may behave differently from other mediators, such as thrombin and tumor necrosis factor-alpha.

Species- and subtype-specific recognition by antibody WF6 of a sequence segment forming an alpha-bungarotoxin binding site on the nicotinic acetylcholine receptor alpha subunit.

The monoclonal antibody WF6 competes with acetylcholine and alpha-bungarotoxin (alpha-BGT) for binding to the Torpedo nicotinic acetylcholine receptor (nAChR) alpha 1 subunit. Using synthetic peptides corresponding to the complete Torpedo nAChR alpha 1 subunit, we previously mapped a continuous epitope recognized by WF6, and the prototope for alpha-BGT, to the sequence segment alpha 1(181-200). Single amino acid substitution analogs have been used as an initial approach to determine the critical amino acids for WF6 and alpha-BGT binding. In the present study, we continue our analysis of the structural features of the WF6 epitope by comparing its cross-reactivity with synthetic peptides corresponding to the alpha 1 subunits from the muscle nAChRs of different species, the rat brain alpha 2, alpha 3, alpha 4 and alpha 5 nAChR subtypes, and the chick brain alpha-BGT binding protein subunits, alpha BGTBP alpha 1 and alpha BGTBP alpha 2. Our results indicate that WF6 is able to cross-react with the muscle alpha 1 subunits of different species by virtue of conservation of several critical amino acid residues between positions 190-198 of the alpha 1 subunit. These studies further define the essential structural features of the sequence segment alpha 1(181-200) required to form the epitope for WF6.