ABSTRACT
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.
Subject(s)
Prothrombin/metabolism , Serine Endopeptidases/pharmacology , Amino Acid Sequence , Animals , Arthropod Venoms/isolation & purification , Arthropod Venoms/pharmacology , Binding Sites , Blood Coagulation Tests , Calcium/pharmacology , Dose-Response Relationship, Drug , Factor X/metabolism , Fibrinogen/drug effects , Fibrinogen/metabolism , Fluorescent Dyes/metabolism , Humans , Kinetics , Larva , Lepidoptera , Molecular Sequence Data , Serine Endopeptidases/isolation & purificationABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Bungarotoxins , Muscles/immunology , Receptors, Cholinergic/immunology , Receptors, Nicotinic/immunology , Amino Acid Sequence , Animals , Antibody Specificity/immunology , Cattle , Cross Reactions/immunology , Humans , Mice , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/chemistry , Ranidae , Rats , Sequence Homology, Nucleic Acid , Snakes , Species Specificity , Torpedo , alpha7 Nicotinic Acetylcholine ReceptorABSTRACT
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.
Subject(s)
Blood Platelets/drug effects , Endothelium, Vascular/drug effects , Serine Endopeptidases/pharmacology , Animals , Arthropod Venoms/pharmacology , Cell Adhesion Molecules/drug effects , Cell Adhesion Molecules/metabolism , Endothelium, Vascular/cytology , Humans , Kinetics , Lepidoptera , Platelet Aggregation/drug effects , Prothrombin/drug effects , Prothrombin/metabolism , Umbilical Veins , Up-Regulation/drug effects , von Willebrand Factor/drug effects , von Willebrand Factor/metabolismABSTRACT
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.