Losac, a factor X activator from Lonomia obliqua bristle extract: its role in the pathophysiological mechanisms and cell survival

Contact with the bristles of the caterpillar Lonomia obliqua can cause serious hemorrhage. Previously it was reported that a procoagulant protein (Lopap) in the bristle extract of L. obliqua increases cell longevity by inhibiting apoptosis. In this work, we purified from bristle extract a factor X activator that stimulates proliferation of endothelial cells. This protein, named Losac, was purified by ion exchange chromatography, followed by gel filtration chromatography and reverse-phase HPLC. Losac is a 45-kDa protein that activates factor X in a concentration-dependent manner and does not depend on calcium ions. In cultures of HUVECs, Losac increased cell proliferation and inhibited the apoptosis induced by starvation. HUVECs incubated with Losac (0.58microM for 1h) increased release of nitric oxide and tissue-plasminogen activator, which both may mediate anti-apoptosis. Losac also increased slightly the decay-accelerating factor (DAF=CD55), which protects cells from complement-mediated lysis. On the other hand, Losac did not alter the release or expression of von Willebrand factor, tissue factor, intercellular adhesion molecule-1, interleukin-8, and prostacyclin. These characteristics indicate that Losac, a protein with procoagulant activity, also functions as a growth stimulator and an inhibitor of cellular death for endothelial cells. Losac may have biotechnological applications, including the reduction of cell death and consequently increased productivity of animal cell cultures, and the use of hemolymph of L. obliqua for this purpose is already being explored. Further study is required to elucidate the mechanism for the inhibition of apoptosis by Losac.     

A prothrombin activator (Lopap) modulating inflammation, coagulation and cell survival mechanisms

A severe hemorrhagic syndrome produced by contact with Lonomia obliqua caterpillars has become epidemic in southern Brazil. A significant thrombin production with intense consumption of fibrinogen and high D-dimer production indicates a consumption coagulopathy and secondary fibrinolysis in patients. Lopap is a single-chain 69kDa serine protease isolated from the crude extract of L. obliqua bristles. Experiments in mice showed that the purified protein, similar to the crude extract, causes uncoagulable blood by fibrinogen depletion. In order to characterize the effects of Lopap on cells involved with hemostatic system, we performed experiments using human umbilical vein endothelial cells (HUVECs). Our results show that Lopap exerts a direct effect on endothelial cells by increasing the liberation of molecules involved in the regulation of vascular tone, inhibiting platelet activation and chemotaxis, apart from inducing the expression of cell adhesion molecules which participate in inflammatory responses. The release or new synthesis of mediators involved in coagulation as von Willebrand factor and tissue factor, or in fibrinolysis as tissue plasminogen activator, was not affected by Lopap. Also our results demonstrated that Lopap acts on cell survival of HUVECs, regulating the expression of molecules as NO and avoiding cell death.     

Lonomia genus caterpillar toxins: biochemical aspects

In 1967 we reported for the first time five cases of an acquired bleeding disorder in humans which developed after contact with saturnidae caterpillars. Since that time, other cases have been reported in Brazil, French Guyana, Peru, Paraguay and Argentina. The caterpillars have been identified as Lonomia achelous (LA) in Venezuela and northern Brazil and as Lonomia obliqua (LO) in southern Brazil. All patients present pain and a burning sensation at the site of contact. Within a few hours hematomas and hematuria are seen in combination with intracerebral and intraperitoneal hemorrhage (in some cases also renal failure). Hematological tests show: mild anemia with leucocytosis; prolonged PT, PTT and ThT; decreased fibrinogen, factor V, factor XIII, plasminogen and alpha2-antiplasmin levels; increased factor VIII:c, von Willebrand factor, and FDPs/D-dimers levels with normal ATIII and platelets. Factor VII, factor II and PC levels varied. Several activities similar to or directed against blood clotting factors have been identified in LA: fibrinolytic enzymes, which degrade fibrinogen producing abnormal FDPs; prothrombin activators: one direct and one factor Xa-like; a thermostable factor V activator; a thermolabile factor V inhibitor; a factor XIII proteolytic/urokinase-like activity; and a kallikrein-like activitiy. In LO three activities have been described: a prothrombin activator called 'Lonomia obliqua prothrombin activator protease' (LOPAP); a factor X activator; and a phospholipase A(2)-like activity called Lonomiatoxin. No fibrinolytic activity has been described in LO. Subcutaneous injection of crude hemolymph and some chromatographic fractions of LA induce a decrease in fibrinogen, plasminogen and factor XIII. Intravenous injection of factor XIII proteolytic/urokinase-like activity induce a dose-dependent thrombolysis with a decrease in plasmatic factor XIII without hemorrhagic manifestations. Intradermal injection of LO bristle extracts in rats and rabbits produce incoagulability whereas intravenous injection of LOPAP induced DIC in mice.     

Blood coagulation induced by Bothrops atrox venom: identification and properties of a factor X activator

The coagulating activity of Bothrops atrox venom was investigated in vitro with purified fibrinogen, with normal plasma and with plasma deficient in various factors of the coagulation cascade. This study indicated that Bothrops atrox venom possesses a thrombin-like action caused by one or several serine proteases sensitive to DFP treatment, but that its clotting action is in fact mainly due to components insensitive to DFP which activate prothrombin and factor X (Stuart factor). We partially purified the DFP insensitive activator of factor X from Bothrops atrox venom and found it to be a protein of molecular weight 77,000. Analysis of the purified fraction by electrophoresis on polyacrylamide gel in the presence of SDS showed that it is mainly composed of one heavy polypeptide chain (65,000) and one light doublet (12 - 13,000). This activator is calcium-dependent and catalyzes in vitro the conversion of purified factor X into factor X alpha. By its molecular properties, it resembles the factor X activator from Vipera russellii venom rather than physiological activators.     

Purification of a phospholipase A2 from Lonomia obliqua caterpillar bristle extract

Lonomia obliqua caterpillar bristle extract induces both direct and indirect hemolytic activity on human and rat washed erythrocytes, and provokes intravascular hemolysis in Wistar rats. Indirect hemolytic activity is assumed to be caused by a phospholipase A(2) (PLA(2)) present in this extract, and this investigation was initiated in order to characterize this enzyme. Phospholipase A(2) activity of crude extract was inhibited by both a PLA(2)-specific inhibitor (pBpb) and the metal ion chelator EDTA. L. obliqua PLA(2) was purified by liquid chromatography from the crude bristle extract and had a molecular mass of 15kDa and a pI of 5.9; its N-terminal sequence showed high homology to a sequence of a putative PLA(2) obtained from a cDNA library of L. obliqua bristles, and it is tentatively placed among Group III phospholipases A(2). This enzyme was stable at 4 degrees C, sensitive to higher temperatures, and its maximum catalytic activity was at pH 8.0. L. obliqua PLA(2) induced hemolysis only when incubated with exogenous lecithin. Thus, the PLA(2) purified herein appears to be responsible for the indirect hemolytic activity of the crude bristle extract.     

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.     

Sequence similarity-based proteomics in insects: characterization of the larvae venom of the Brazilian moth Cerodirphia speciosa

Using a combination of tandem mass spectrometric sequencing and sequence similarity searches, we characterized the larvae venom of the moth Cerodirphia speciosa, which belongs to the Saturniidae family of the Lepidoptera order. Despite the paucity of available database sequence resources, the approach enabled us to identify 48 out of 58 attempted spots on its two-dimensional gel electrophoresis map, which represented 37 unique proteins, whereas it was only possible to identify 13 proteins by conventional non-error tolerant database searching methods. The majority of cross-species hits were made to proteins from the phylogenetically related Lepidoptera organism, the silk worm Bombyx mori. The protein composition of the venom suggested that envenoming by C. speciosa toxins might proceed through the contact with its hemolymph, similarly to another toxic Lepidoptera organism, Lonomia obliqua.     

New insights into the functions and N-glycan structures of factor X activator from Russell's viper venom

The coagulation factor X activator from Russell's viper venom (RVV-X) is a heterotrimeric glycoprotein. In this study, its three subunits were cloned and sequenced from the venom gland cDNAs of Daboia siamensis. The deduced heavy chain sequence contained a C-terminal extension with four additional residues to that published previously. Both light chains showed 77-81% identity to those of a homologous factor X activator from Vipera lebetina venom. Far-western analyses revealed that RVV-X could strongly bind protein S, in addition to factors X and IX. This might inactivate protein S and potentiate the disseminated intravascular coagulation syndrome elicited by Russell's viper envenomation. The N-glycans released from each subunit were profiled and sequenced by MALDI-MS and MS/MS analyses of the permethyl derivatives. All the glycans, one on each light chain and four on the heavy chain, showed a heterogeneous pattern, with a combination of variable terminal fucosylation and sialylation on multiantennary complex-type sugars. Amongst the notable features were the presence of terminal Lewis and sialyl-Lewis epitopes, as confirmed by western blotting analyses. As these glyco-epitopes have specific receptors in the vascular system, they possibly contribute to the rapid homing of RVV-X to the vascular system, as supported by the observation that slower and fewer fibrinogen degradation products are released by desialylated RVV-X than by native RVV-X.     

Implications of hemolin glycosylation and Ca2+-binding on homophilic and cellular interactions.

Insects are useful models for the study of innate immune mechanisms because of their lack of antibodies and receptors involved in adaptive immune response. Nevertheless, hemolin cloned from moths is a soluble and membrane associated Ig-related molecule that is up-regulated during immune response [Lanz-Mendoza, H. & Faye, I. (1999) Dev. Comp. Immunol. 23, 359-374]. The hemolin monomeric form has four, pair-wise, interacting Ig-domains, forming a strongly bent horseshoe structure [Su, X.-D., Gastinel, L.N., Vaughn, D.E., Faye, I., Poon, P. & Bjorkman, P. (1998) Science 281, 991-995]. To elucidate the nature of its homophilic and cellular interactions, the glycosylation and Ca2+-binding properties of hemolin were investigated. We used Hyalophora cecropia hemolin isolated from hemolymph of bacteria-injected pupae, or produced as a recombinant protein in a baculovirus/insect cell system. Both types of hemolin contain N-acetylglucosamine and probably sialic acid, as indicated by peptide:N-glycosidase F and neuraminidase digestion and glycosylation detection by Western-blotting analysis. The N-acetylglucosamine residues on hemolin were confirmed with the use of specific lectins. In addition, hemolin was shown to specifically bind calcium when spotted onto nitrocellulose and treated as for 45Ca2+ autoradiography. Earlier studies demonstrated that hemolin can bind to hemocytes and this was tested for its dependence on calcium and carbohydrates, using hemolin-coated fluorescent microspheres. A greater level of attachment of microspheres occurred in the presence of calcium than if calcium was absent. Furthermore, this binding was inhibited by EGTA and N-acetylglucosamine or N-acetylneuraminic acid, implying that carbohydrates and calcium are crucial factors in homophilic binding and cell-adhesion events mediated by this Ig-superfamily molecule.     

Detection of specific proenzyme activators in snake venoms by a new immunoabsorbant-chromogenic substrate method

In separate experiments, antibodies to plasminogen, factor X and protein C were applied to microtitre trays as commonly used in enzyme-linked immunoassays. After incubation with dilute normal human plasma as a source of the corresponding proenzyme antigen, the wells were exposed to dilutions of various snake venoms. After thorough washing, the microtitre tray wells were tested overnight with chromogenic tripeptide substrates known to be relatively specific for the activated forms of the above factors, i.e., plasmin, factor Xa and activated protein C. The immunochromometric assay described detected two new activators of protein C in Agkistrodon piscivorus and Agkistrodon contortrix venoms and a new factor X activator in Agkistrodon rhodostoma venom. Gel filtration of the latter venom indicated that the factor X activator eluted with high molecular weight, was clearly distinct from the peak fibrinogen clotting activity (Ancrod) and appeared to have no procoagulant activity. Although several Bothrops venoms appeared to contain plasminogen activator by this technique, the observed strong chromogenic activity was observed in microtitre wells independently of plasminogen and represented nonspecific amidase activity.     

Structural models of the snake venom factor V activators from Daboia russelli and Daboia lebetina

Blood coagulation factor V (FV) is a multifunctional protein that circulates in human plasma as a precursor molecule which can be activated by thrombin or activated factor X (FXa) in order to express its cofactor activity in prothrombin activation. FV activation is achieved by limited proteolysis after Arg709, Arg1018, and Arg1545 in the FV molecule. The venoms of Daboia russelli and Daboia lebetina contain a serine protease that specifically activates FV by a single cleavage at Arg1545. We have predicted the three-dimensional structure of these enzymes using comparative protein modeling techniques. The plasminogen activator from Agkistrodon acutus, which shows a high degree of homology with the venom FV activators and for which a high-quality crystallographic structure is available, was used as the molecular template. The RVV-V and LVV-V models provide for the first time a detailed and accurate structure of a snake venom FV activator and explain the observed sensitivity or resistance toward a number of serine protease inhibitors. Finally, electrostatic potential calculations show that two positively charged surface patches are present on opposite sides of the active site. We propose that both FV activators achieve their exquisite substrate specificity for the Arg1545 site via interactions between these exosites and FV.     

Identification and characterization of a baculovirus from Lonomia obliqua (Lepidoptera: Saturniidae)

A baculovirus has been isolated from larvae of Lonomia obliqua, a Saturniidae of medical importance due to a potent toxin found in their spines. Electron Microscopy analysis of the occlusion body obtained from diseased larvae showed polyhedra of approximately 1 microm in diameter containing multiple nucleocapsids per envelope. This baculovirus was thus named Lonomia obliqua multicapsid nucleopolyhedrovirus (LoobMNPV). Restriction endonuclease profiles of viral DNA digested with three restriction enzymes were obtained and the genome size was estimated to be 95.52 +/- 2.3 kbp. The polyhedrin gene of LoobMNPV was identified and its DNA sequence was determined. Phylogenetic analysis of the polyhedrin gene showed that the LoobMNPV polyhedrin belongs to group I NPV and that it is closely related to the polyhedrin of the NPV of Amsacta albistriga.     

Allosteric Regulation of the Carbohydrate-binding Ability of a Novel Conger Eel Galectin by D-Mannoside

Conger eel has two galectins, termed congerins I and II (Con I and II), that function in mucus as biodefense molecules. Con I and II have acquired a novel protein fold via domain swapping and a new ligand-binding site by accelerated evolution, which enables recognition of some marine bacteria. In this study, we identified a new congerin isotype, congerin P (Con-P), from the peritoneal cells of conger eel. Although Con-P displayed obvious homology with galectins, we observed substitution of 7 out of 8 amino acid residues in the carbohydrate recognition domain that are conserved in all other known galectins. To understand the structure-function relationships of this unique galectin, recombinant Con-P was successfully expressed in Escherichia coli by using a Con II-tagged fusion protein system and subsequently characterized. In the presence of d-mannose, Con-P displayed 30-fold greater hemagglutinating activity than Con I; however, no activity was observed without mannose, indicating that d-mannoside can act as a modulator of Con-P. Frontal affinity chromatography analysis showed that activated Con-P, allosterically induced by mannose, displayed affinity for oligomannose-type sugars as well as N-acetyllactosamine-type β-galactosides. Thus, Con-P represents a new member of the galectin family with unique properties.     

Cloning and expression in COS-1 cells of a full-length cDNA encoding human coagulation factor X

A 1.5-kb cDNA (FX) encoding full-length human coagulation factor X was isolated from a human fetal liver cDNA library. The identity of the insert in a selected phage lambda clone was confirmed to be FX by nucleotide (nt) sequence analysis and restriction mapping. This FX cDNA clone contained 1467 bp of coding sequence, no 5'-untranslated sequence, a short 3'-untranslated sequence of 10 nt and a poly(A) tail at the 3'-end. The FX cDNA was inserted into a mammalian expression vector and transfected into COS-1 monkey kidney cells. Media from transfected cells showed evidence of factor X antigen and, following addition of Russel's viper venom factor X activator, enhanced amidolytic activity toward a synthetic peptide rho-nitroanilide substrate. Immunoprecipitation with an anti-factor X monoclonal antibody of [35S]methionine-labeled cell-conditioned media showed evidence of polypeptides of 74, 55, and 17 kDa, as determined by SDS-PAGE followed by autoradiography. Together, these results indicate that an active factor X can be successfully expressed in a recombinant DNA expression system. This approach will allow the systematic structure/function investigation of this important blood-clotting enzyme.     

Beta-hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. Its role in Ca2+ binding and biological activity

Protein C is a vitamin K-dependent regulator of blood coagulation. It has beta-hydroxyaspartic acid in position 71 which is in the first of its two domains that are homologous to epidermal growth factor (EGF). This region has recently been demonstrated to have a Ca2+ binding site with a Kd of approximately 100 microM. Recombinant human protein C, expressed in mammalian tissue culture, had full biological activity and contained beta-hydroxyaspartic acid. Furthermore, it had a Ca2+-dependent epitope in the EGF-like domain, recognized by a monoclonal antibody. In contrast, a mutant recombinant human protein C in which beta-hydroxyaspartic acid had been replaced with glutamic acid in position 71 did not have the Ca2+-dependent epitope, and its biological activity was reduced to about 10% of normal. Fab' fragments of this antibody inhibited the anticoagulant activity of plasma-derived activated protein C, apparently by interfering with the interaction between activated protein C and its cofactor, protein S. The latter contains four tandemly arranged EGF homology domains. We propose that beta-hydroxyaspartic acid is directly involved in Ca2+ binding in protein C and in related proteins and that protein C interacts with protein S by means of its EGF homology regions.     

Expression of recombinant rabbit tissue factor in Pichia pastoris,and its application in a prothrombin time reagent

Tissue factor (TF), or thromboplastin, is a cell membrane-associated glycoprotein composed, in full length, of cytoplasmic, transmembrane, and extracellular domains. It functions as a cofactor in a complex with factor VII (FVII), generating activated factor VII (FVIIa) and initiating blood coagulation. The prothrombin time (PT) assay uses TF as the in vitro activator of coagulation under defined conditions, and it is primarily used to diagnose and manage the extrinsic-pathway factor defficiencies. To overcome the limitations of natural-source TF, we have expressed the mature full-length recombinant rabbit TF (rRTF) protein in Pichia pastoris. Isolation, by purification by immobilized metal-affinity chromatography, of full-length rRTF was facilitated by engineering a (His)(6) tail on its C-terminus, which maximizes the selection of rRTF with intact transmembrane and cytoplasmic domains, critical for proper activity. A PT reagent that incorporates this purified rRTF has performance characteristics similar to those of PT reagents made with natural TF as indicated in method comparison studies, and shows lot-to-lot consistency and reproducibility.     

Blood coagulation profile of the Asian elephant (Elephas maximus)

The coagulation profile of seven Asian elephants was assessed using human reference plasma as a standard. The plasma values for the majority of the coagulation proteins evaluated, including Factors VII, IX, X, and XI, and antithrombin, were similar to that of human plasma. The average Factor VIII:C value was 1.95 units/ml, approximately twice that of the human value. Human recombinant tissue factor was effective as an activator of the tissue factor-factor VII pathway as measured by the prothrombin time assay. The elephant plasma effectively corrected the clotting defect of human Factor XI-deficient plasma but failed to do so with bovine Factor XI-deficient plasma. However, elephant plasma Factor XII was not readily activated by the commercial activated partial thromboplastin time (APTT) reagent formulated with a soluble activator, and consequently the activity of this protein could not be precisely determined. The average (±SD) APTT result of 65.6 ± 9.2 sec was twice as long as that of the human reference plasma. Despite the presence of relatively high levels of fibrinogen, 4.61 ± 0.49 gm/l, no fibrinolytic activity was detected in any of the elephant plasma samples using a standard fibrin plate assay system. © 1996 Wiley-Liss, Inc.     

Rat prostate tumors express cancer procoagulant, an activator of coagulation factor X

Two common procoagulant activities associated with tumors are tissue factor and cancer procoagulant (CP), an activator of coagulation factor X. We have identified a convenient source of CP in transplanted Lobund-Wistar rat PA3 prostate tumors. CP activity was purified from 5 independent transplanted prostate tumors by column chromatography. The protein activated factor X in the absence of TF and factor VII. An antihuman CP antibody recognized rat CP in an ELISA and inactivated CP activity in a chromogenic assay. Lobund-Wistar prostate tumors may provide a convenient animal model useful in determining the role of CP in cancer development.