Venom Proteases Research Articles

Effects of Aqueous Stem Extract of <i>Achyranthes aspera</i> on <i>Bitis arietans</i> Venom Protease and Phospholipase A<sub>2</sub> Activity

Aqueous stem extract of <i>Achyranthes aspera</i> was investigated for inhibitory activity against Bitis arietans venom protease and phospholipase A₂ activity. The elemental analysis and phytochemical screening of the plant extract were carried out. The activities of protease and phospholipase A₂ (V_o) of the crude <i>Bitis arietans</i> venom was determined and the data obtained was used to estimate K_M, V_max and K_cat. Inhibition studies were carried out using the same procedure except that different concentrations of the extracts (5%, 10%, 15% for protease assay and 0.5%, 0.75%, 10%, 1.25% and 1.5% for phospholipase A₂ assay) were added to the reaction mixture. The result showed that the <i>Bitis arietans</i> venom protease had a V_max of 0.062 ± 0.013 µmol/min, K_M of 0.496 ± 0.095mg/ml and a K_cat of 0.125 ± 0.001min^-1. The result also indicates that the Bitis arietans phospholipase A₂ had a V_max of 3.27 ± 0.030min^-1, K_M of 8.358 ± 0.050 mg/ml and K_cat of 0.391 ± 0.002min^-. The aqueous stem extract produced a statistically significant (P&lt;0.05) decrease in the V_max, K_M and K_cat of the <i>Bitis arietans</i> venom phospholipase A₂ in a dose dependent manner and a statistically significant (P&lt;0.05) increase in the V_max, K_M and K_cat of <i>Bitis arietans</i> protease in a dose dependent manner. The phytochemical screening revealed the presence of flavonoids, tannins, steroids, saponins and terpenoids in the extract while the elemental analysis revealed the presence of Zn, Cr, Ni, Cd, Mn, Fe and Na. The result suggests that aqueous stem extract of <i>Achyranthes aspera</i> inhibited the <i>Bitis arietans</i> venom phospholipase A₂ in an uncompetitive manner while the protease activity was stimulated by the extracts. It was observed that the use of the stem of <i>Achyranthes aspera</i> may be important in the treatment of snake bites.

Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase.

The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension.

Activation-resistant homozygous protein C R229W mutation causing familial perinatal intracranial hemorrhage and delayed onset of thrombosis

IntroductionWe describe a family with two first-degree cousins who presented with similar phenotypes characterized by neonatal intracranial hemorrhage and subsequent onset of thrombosis. Patients/methodsWe enrolled the two affected patients, five unaffected family members and fifty-five normal controls. Clinical, laboratory, and radiological characteristics of patients were obtained. Exome sequencing was performed for the older affected child. PROC c.811 C>T was genotyped by PCR in patients, family members, and controls. Protein C amidolytic activity and antigen were measured using the STACHROM® protein C kit and ELISAs. To define functional abnormalities caused by the patients' mutation, recombinant wildtype protein C and its mutants R229W, R229Q and R229A were studied. ResultsFor the two cousins, protein C amidolytic activity was 61% and 59% and antigen was 57% and 73% (nl 70–140%), respectively. Exome sequencing revealed a homozygous variant in exon 9 of the protein C (PROC) gene c.811 C>T (R229W). The R229W mutation is located in the calcium binding loop of protein C's protease domain that mediates thrombomodulin interactions. Recombinant R229W-protein C mutant was strikingly defective in rate of activation by thrombin: thrombomodulin, suggesting an in vivo deficit in these children for generation of activated protein C. ConclusionsThese cases emphasize that protein C and activated protein C are important in maintaining the integrity of the brain vascular endothelium in humans. Moreover, routine protein C assays utilizing snake venom protease fail to detect protein C mutants that are resistant to thrombin:thrombomodulin activation.

Moojenactivase, a novel pro-coagulant PIIId metalloprotease isolated from Bothrops moojeni snake venom, activates coagulation factors II and X and induces tissue factor up-regulation in leukocytes.

Coagulopathies following snakebite are triggered by pro-coagulant venom toxins, in which metalloproteases play a major role in envenomation-induced coagulation disorders by acting on coagulation cascade, platelet function and fibrinolysis. Considering this relevance, here we describe the isolation and biochemical characterization of moojenactivase (MooA), a metalloprotease from Bothrops moojeni snake venom, and investigate its involvement in hemostasis in vitro. MooA is a glycoprotein of 85,746.22Da, member of the PIIId group of snake venom metalloproteases, composed of three linked disulfide-bonded chains: an N-glycosylated heavy chain, and two light chains. The venom protease induced human plasma clotting in vitro by activating on both blood coagulation factors II (prothrombin) and X, which in turn generated α-thrombin and factor Xa, respectively. Additionally, MooA induced expression of tissue factor (TF) on the membrane surface of peripheral blood mononuclear cells (PBMC), which led these cells to adopt pro-coagulant characteristics. MooA was also shown to be involved with production of the inflammatory mediators TNF-α, IL-8 and MCP-1, suggesting an association between MooA pro-inflammatory stimulation of PBMC and TF up-regulation. We also observed aggregation of washed platelets when in presence of MooA; however, the protease had no effect on fibrinolysis. Our findings show that MooA is a novel hemostatically active metalloprotease, which may lead to the development of coagulopathies during B. moojeni envenomation. Moreover, the metalloprotease may contribute to the development of new diagnostic tools and pharmacological approaches applied to hemostatic disorders.

Protective effect of Euphorbia hirta and its components against snake venom induced lethality

Ethnopharmacological relevanceDespite the use of snake anti-venom therapy, herbal medicine is still in practice to treat snakebites. Euphorbia hirta is traditionally used as antidote for snakebites and also for numerous other ailments. However, the scientific evidence for its anti-snake venom property is still lacking. Materials and methodsMethanolic extract of E. hirta was evaluated for anti-venom activity under in vitro and ex vivo conditions. Histopathological changes in the vital organs of the mice were also monitored. UHPLC-SRM/MS was used to estimate the phenolic constituents whereas GC–MS analysis was performed to analyze the volatile metabolites present. The major compound was further evaluated for its contribution to the overall inhibitory potential of the extract. ResultsMethanolic extract of E. hirta completely inhibited the venom enzymes under in vitro and reduced the edema ratio. The extract increased the survival time (>24h) of mice which was further evidenced by histopathological analysis of vital organs. Phytochemical analysis revealed higher content of phenolic (144mg/g extract) compounds in the extract. UHPLC-SRM/MS demonstrated that ellagic acid, gallic acid and quinic acid are the major phenolics whereas GC–MS analysis revealed pyrogallol as the major constituent (60.07%) among the volatile components of the extract. It was also shown that pyrogallol has the ability to differentially inhibit venom protease but not phospholipase A2. ConclusionThe present study confirmed that E. hirta methanolic extract was able to completely inhibit Naja naja venom induced toxicity under in vitro as well as ex vivo conditions, thus providing scientific evidence to its traditional use.

Functional analysis of the venom of mealybug parasitoid Aenasius bambawalei (Hymenoptera: Encyrtidae)

Aenasius bambawalei (Hymenoptera: Encyrtidae) is a koinobiont nymphal endoparasitoid of cotton mealybug, Phenacoccus solenopsis (Hemiptera: Pseudocccidae). Functional analysis of the venom of the wasp was performed by artificial microinjections of both crude and treated venom (heat and proteinase) of the wasp containing 0.3 and 0.5 μl in non-parasitized and synchronized adult hosts (mealybugs) and the mortality data were recorded 24, 48, 72 and 96 hours post injecton while mealybugs receiving saline injections were acted as control. The main effects for artificially envenomated mealybugs were observed on their mortality and survival. The biological activity of crude venom was also evaluated by heat and protease treatment. Here, we demonstrate that maximum mortality (82 ± 2.0%) was achieved by microinjections containing higher volume (0.5 μl) of crude venom while lower mortality (68 ± 4.0%) was achieved with lower volume of venom (0.3 μl). On the other hand, heat and proteinase K treated venom did not show any significant effect on mortality of the host insect. Our findings suggest that bioactive components of the crude venom are proteins which lost their activity upon heat and protease treatment. This basic information regarding the functional role of the venom of A. bambawalei serves as a starting point for comprehensive analysis of the role of the venom of the parasitoid on the regulation processes in its host.

Validation of new venom protease assays for translational research (LB589)

Snake envenomation is an understudied significant world health problem, designated an orphan disease by the WHO. In the U.S. lives are usually saved, but significant tissue is often lost. Overseas without hospitalization and antivenin, lives are forfeit. For most American snake venoms the major pathologies of tissue loss and coagulopathies are due to proteolytic degradation of host tissues and plasma cascades. For HTS leading to therapeutics including drug repurposing, sensitive new substrates may finally allow direct comparisons of venom activities, but only if the reptile enzymes are active upon them, which has been not previously been published. Here I show direct comparisons of representative American venoms strong protease and collagenase, but weak elastase activities using the new technology. Assay CV range 1‐5%. Caseinolytic activity is inhibited by the nonspecific protease inhibitor 1,10‐phenanthroline and by EDTA. So is collagenase activity, consistent with the action of matrix metalloproteases. The Km measured shows that high throughput assays could be done at substrate levels well below the Km for the reaction. Such a system will be invaluable for the testing leading to the repurposing of drugs to treat envenomations.Grant Funding Source: Supported by CHS‐OSU

Preliminary assessment of Hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by Bothrops and Lachesis snake venoms.

BackgroundThe search for new inhibitors of snake venom toxins is essential to complement or even replace traditional antivenom therapy, especially in relation to compounds that neutralize the local effects of envenomations. Besides their possible use as alternative to traditional antivenom therapy, some plant species possess bioactive secondary metabolites including essential oils, which can be extracted from weeds that are considered substantial problems for agriculture, such as Hedychium coronarium.MethodsThe essential oils of leaves and rhizomes from H. coronarium were extracted by hydrodistillation, and their potential inhibitory effects on the coagulant and fibrinogenolytic activities induced by the venoms of Lachesis muta, Bothrops atrox and Bothrops moojeni were analyzed. Citrated human plasma was used to evaluate the clotting time whereas changes in fibrinogen molecules were visualized by electrophoresis in polyacrylamide gel. The experimental design used for testing coagulation inhibition was randomized in a 3 × 2 factorial arrangement (concentration × essential oils), with three replications. The essential oils were compared since they were extracted from different organs of the same botanical species, H. coronarium.ResultsThe results suggest that the oils interact with venom proteases and plasma constituents, since all oils evaluated, when previously incubated with venoms, were able to inhibit the clotting effect, with less inhibition when oils and plasma were preincubated prior to the addition of venoms.ConclusionsThus, after extensive characterization of their pharmacological and toxicological effects, the essential oils can be used as an alternative to complement serum therapy, especially considering that these plant metabolites generally do not require specific formulations and may be used topically immediately after extraction.

Activation-Resistant Homozygous Protein C R229W Mutation Causing Familial Perinatal Intracranial Hemorrhage

Two first-degree cousins presented with similar phenotype characterized by neonatal intracranial hemorrhage and subsequent onset of thrombosis at the age of four months. One girl, now 9 yr old, was a full term baby presenting at 13 days of age with seizures and brain CT showing intracranial hemorrhage. At 4 months of age, she presented with multiple thrombotic lesions over both hands and feet that required extensive debridement and improved with anticoagulation and plasma replacement. She has been on enoxaparin with no new lesions afterward. At 9 years of age, protein C amidolytic activity (STACHROM® protein C) was 61% (nl 70-140%) and protein C antigen (ELISA) was 57% (nl 70-140%). Thrombophilia work up was otherwise normal. The second child is a 1 yr old boy who was delivered by cesarean section because of fetal distress. The mother gave history of decreased fetal movement for few weeks prior to his birth and was noted to be hypotonic. Brain MRI in the first week of life showed massive subacute hemorrhage in the brain parenchyma and upper spinal canal. Bleeding stopped with appropriate supportive care. He did not have purpura fulminans or any evidence of thrombosis during the neonatal period. At 4 months of age, a ventriculoperitoneal shunt was placed because of hydrocephalus and post operatively he developed thrombotic lesion in his left foot that resolved after starting enoxaparin and plasma replacement. He remains on enoxaparin with no new significant lesions. At 10 months of age, protein C amidolytic activity was 59% and protein C antigen was 73%. Both patients suffer from severe permanent neurological deficit and blindness. Dx of protein C homozygous deficiency was established after exome sequencing of the older child revealed a homozygous variant in exon 9 of PROC gene c.811 C>T (R271W equivalent to R229W in mature protein C numbering). Sanger sequencing validated this mutation. Both patients were homozygous (T/T) while tested parents and two siblings were heterozygous (C/T), and 55 controls were normal (C/C). No family history of thrombosis among family members who are heterozygous for this mutation was found. Complete exome sequence analysis also showed no mutations for ATIII, protein S, factors V or VIII, prothrombin, plasminogen, protein Z, or any ADAMTS genes. The R229W mutation is located in the calcium binding loop of protein C's protease domain that mediates thrombomodulin (TM) interactions. To define functional abnormalities caused by the patients' mutation, recombinant protein C mutants R229W, R229Q and R229A were studied. Each was strikingly defective in rate of activation by thrombin:TM, showing an activation rate that was only 1%, 2% and 4%, respectively, compared to wildtype protein C. Once activated, each activated protein C (APC) mutant was moderately affected, showing a 50% reduction in anticoagulant activity and in rate of cleavage of purified factor Va at Arg506. The amidolytic activities were less affected and close to wildtype APC. The plasma half-lives were similar to wildtype APC. These properties of recombinant R229W protein C strongly suggest a severe in vivo deficit in these children for generation of APC, implying a severe deficiency of APC in spite of half-normal levels of protein C zymogen. As routine coag assays utilize snake venom protease, they fail to detect TM-activation-resistant protein C mutants. In summary, homozygous protein C R229W mutation in two related cousins was associated with significant leaky brain blood vessels in the perinatal period and significant neurological insult even prior to the first evidence of clinical thrombosis. This highlights the importance of protein C and APC in maintaining the integrity of the brain vascular endothelium in humans. Disclosures:No relevant conflicts of interest to declare.

Inhibition of venom serine proteinase and metalloproteinase activities by Renealmia alpinia (Zingiberaceae) extracts: Comparison of wild and in vitro propagated plants

Ethnopharmacological relevanceThe plant Renealmia alpinia has been used in folk medicine to treat snakebites in the northwest region of Colombia. In addition, it has been shown to neutralize edema-forming, hemorrhagic, lethal, and defibrin(ogen)ating activities of Bothrops asper venom. In this work, extracts of Renealmia alpinia obtained by micropropagation (in vitro) and from specimens collected in the wild were tested and compared in their capacity to inhibit enzymatic and toxic activities of a snake venom metalloproteinase isolated from Bothrops atrox (Batx-I) venom and a serine proteinase (Cdc SII) from Crotalus durissus cumanensis venom. Materials and methodsWe have investigated the inhibition capacity of Renealmia alpinia extracts on enzymatic and toxic actions of isolated toxins, a metalloproteinase and a serine proteinase. The protocols investigated included inhibition of proteolytic activity on azocasein, inhibition of proteolytic activity on fibrinogen, inhibition of pro-coagulant activity, inhibition of hemorrhagic activity and inhibition of edema-forming activity. ResultsColorimetric assays detected the presence of terpenoids, flavonoids, tannins and coumarins in Renealmia alpinia extracts. Renealmia alpinia extracts inhibited the enzymatic, hemorrhagic and fibrinogenolytic activities of Batx-I. Extracts also inhibited coagulant, defibrin(ogen)ating and edema-forming activities of Cdc SII. Results highlight that Renealmia alpinia in vitro extract displayed comparable inhibitory capacity on venom proteinases that Renealmia alpinia wild extract. No alteration was observed in the electrophoretic pattern of venom proteinases after incubation with Renealmia alpinia extracts, thus excluding proteolytic degradation or protein denaturation/precipitation as a mechanism of inhibition. ConclusionsOur results showed that Renealmia alpinia wild and in vitro extracts contain compounds that neutralize metallo- and serine proteinases present in snake venoms. The mechanism of inhibition is not related to proteolytic degradation of the enzymes nor protein aggregation, but is likely to depend on molecular interactions of secondary metabolites in the plant with these venom proteinases.

Expression and characterization of recombinant ecarin.

The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37 °C after death of the host cells. Maximal ecarin activity was reached 7 days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250 nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a K m value of 0.4 μM prethrombin-2 was determined but only a rough estimate could be made of the K m for prothrombin of 0.9 μM. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.

Snake Venom Proteases, Structure-Function Relationship and Effects on Blood Coagulation

Snake venoms are probably the most highly concentrated secretion products found in vertebrate. Furthermore, proteases are in a unique position in snake venoms, especially in Crotalid and Viperid venoms. Biologically, proteolytic enzymes may contribute in two ways: the immobilization of the prey and the digestion of prey organism. They are responsible for the hemorrhage, shock, or disorder of blood coagulation after envenomation. They act, by activating, inactivating, or other converting effects, on almost all the components of hemostatic and fibrinolytic systems. Biochemically, they can be mainly classified into two groups: serine proteases, which are inhibited by phenylmethanesulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP). Their sequences are homologous to trypsin-kallikrein serine proteases. They share a common active site geometry and enzymatic mechanism but variation of primary sequences out of active center results in the differences of substrate specificities and further the difference of biological and pharmacological activities. Metalloproteinases, which are inhibited by metal chelating reagent (EDTA), are zinc-dependent. Up to now, sequence data indicate that they belong to a new metalloproteinase subfamily which has no significant sequence similarities with any other know metalloproteinases except for a conservative zinc-chelating sequence His-Glu-X-X-His. Because of their common and unique properties compared to their physiological corresponding factors, snake venom proteases have find a position uniquely in hemostasis and thrombosis, both in research and application.

Scyphozoan jellyfish venom metalloproteinases and their role in the cytotoxicity

The present study, for the first time, comparatively investigated the enzymatic activities (proteases and hyaluronidases) in the venoms of four Scyphozoan jellyfish species, including Nemopilema nomurai, Rhopilema esculenta, Cyanea nozakii, and Aurelia aurita. For this, various zymographic analyses were performed using assay specific substrates. Interestingly, all the four jellyfish venoms showed gelatinolytic, caseinolytic, and fibrinolytic activities, each of which contains a multitude of enzyme components with molecular weights between 17 and 130 kDa. These four jellyfish venoms demonstrated a huge variation in their proteolytic activities in quantitative and qualitative manner depending on the species. Most of these enzymatic activities were disappeared by the treatment of 1,10-phenanthroline, suggesting they might be belonged to metalloproteinases. Toxicological significance of these venom proteases was examined by comparing their proteolytic activity and the cytotoxicity in NIH 3T3 cells. The relative cytotoxic potency was C. nozakii > N. nomurai > A. aurita > R. esculenta. The cytotoxicity of jellyfish venom shows a positive correlation with its overall proteolytic activity. The metalloproteinases appear to play an important role in the induction of jellyfish venom toxicities. In conclusion, the present report proposes a novel finding of Scyphozoan jellyfish venom metalloproteinases and their potential role in the cytotoxicity.

Molecular Mechanism Underlying the Need for Three Cleavage Sites within the B-Domain to Activate Factor V by Thrombin

AbstractAbstract 2219Blood coagulation factor V (FV) is a multi-domain protein (A1-A2-B-A3-C1-C2) with little or no procoagulant activity and circulates in blood as a procofactor. Recent data indicate that specific sequences within the B-domain (963-1008) play a predominant role in keeping FV inactive. Removing these sequences via deletion or mutagenesis drives the activation of FV. In normal physiological situations, proteolytic processing of FV by thrombin within the B-domain (710-1545) ultimately removes these inhibitory sequences. Thrombin cleaves FV at three sites in a kinetically preferred order at Arg709, Arg1018 followed by Arg1545 to ultimately generate the active cofactor species, FVa consisting of a heavy and light chain. The need for three thrombin cleavage sites to generate an active cofactor species is not entirely obvious considering that RVV-V, a snake venom protease, activates FV following a single cleavage at Arg1545. Here we uncover new structure/function insights into how specific B-domain sequences restrict thrombin recognition of FV thereby forcing the need for three cleavage sites. To investigate this, we generated a panel of recombinant FV derivatives in BHK cells with different regions of the B-domain eliminated and combined functional measurements with SDS-PAGE to examine FV activation. As a control, we generated FV-1033 (residues 1034–1491 deleted) which is functionally equivalent to full-length FV and retains all three thrombin cleavage sites. Like FV, incubation of FV-1033 with thrombin resulted in rapid cleavage at Arg709, to generate the heavy chain (105 kDa) followed by cleavage at Arg1018 and Arg1545 to produce the light chain (74 kDa). In contrast to these results, elimination of the Arg1018 cleavage site via mutagenesis (FV-1033R1018Q) or deletion (FV-1015; 1016–1491 deleted) dramatically delayed cleavage at Arg1545 while proteolysis at Arg709 was unaltered compared to FV-1033. Control experiments with RVV-V revealed that FV-1033R1018Q and FV-1015 were rapidly cleaved at Arg1545 similar to FV-1033. These data are consistent with prior mutagenesis studies with full length FV and collectively suggest that the middle cleavage site somehow potentiates cleavage at 1545. However, further elimination of the B-domain using FV-902 (903-1491 deleted; Arg1018 not present) surprisingly restored rapid cleavage at Arg1545. These data suggest that sequences N-terminal to the 1018 site (902-1017) are involved in regulating thrombin cleavage at Arg1545 and that the purpose of cleavage at Arg1018 is to remove these constraints. In support of this, FV derivatives in which these sequences are exchanged with non-homologous regions of the FVIII B-domain were rapidly cleaved at Arg1545 whether or not Arg1018 was present. Furthermore, we were able to demonstrate that residues 964–1007 are the principal determinants in restricting thrombin cleavage at Arg1545. Functional measurements, including clotting assays, thrombin generation assays, and purified component systems revealed that FV derivatives which exhibited delayed cleavage at Arg1545 all have a markedly reduced ability to generate thrombin compared to FV-1033 or FV. Collectively these data demonstrate that FV cleavage site preferences are not only influenced by the geometric constraints imposed on thrombin but are also influenced in a dramatic way by specific B-domain sequences adjacent to Arg1018. These sequences, which conformationally restrict the ability of thrombin to access Arg1545, affect the rate and order of bond cleavage. Thus proteolysis at Arg1018 is required to alleviate these constraints allowing for accelerated cleavage at Arg1545 by thrombin. Furthermore, initial cleavage at Arg709 appears unaltered by any region of the B-domain as each of FV derivatives studied show equivalent processing at this site. These data provide new structure/function insights into the relationship between the removal of B-domain constraints and accessibility of thrombin cleavage sites which lead to the activation of FV. Disclosures:Camire: Pfizer: Patents & Royalties, Research Funding.

Effects of snake venom proteases on human fibrinogen chains.

Proteomic approach is an effective method to study changes in human plasma proteome. Coagulopathies are commonly encountered in victims of viper envenomation which were treated with an administration of immunoglobulin. Unfortunately, this treatment shows significant risk to the patient due to an anaphylactic reaction. Since Echis carinatus Venom (EV) toxins mainly acts both directly and indirectly on fibrinogen, we planned to establish a suitable analysis of its beta (FIBB) e gamma (FIBG) chains. This study will help us to understand the mechanism of envenomation and to find alternative treatments other than the common treatment with the administration of IgG. We evaluated the EV proteolytic activity on whole human plasma proteome from the blood of an healthy volunteer. Two-dimensional electrophoresis (2-DE) using mini-gel was performed to analyse EV effects on the differents fibrinogen chains. Changes in whole plasma proteome were focused on fibrinogen beta and gamma chains after EV incubation. Protein spots were detected and analyzed using ImageMaster 2D Platinum software. Results were represented as mean +/- standard deviation (mean+/-SD) with p<0.05 as a statistically significant value. 2-DE gel analysis showed that some spots of FIBB disappeared and some spots of FIBG decreased. We found that the proteomic approach is a valid method in studying in-depth causes of different diseases, in particular those are involved in coagulopathies linked with proteins like fibrinogen from victims of viper envenomation.

Enzymatic analysis of Hemiscorpius lepturus scorpion venom using zymography and venom-specific antivenin

Hemiscorpius lepturus envenomation exhibits various pathological changes in the affected tissues, including skin, blood cells, cardiovascular and central nervous systems. The enzymatic activity and protein component of the venom have not been described previously. In the present study, the electrophoretic profile of H. lepturus venom was determined by SDS-PAGE (12 and 15%), resulting in major protein bands at 3.5–5, 30–35 and 50–60 kDa. The enzymatic activities of the venom was, for the first time, investigated using various zymography techniques, which showed the gelatinolytic, caseinolytic, and hyaluronidase activities mainly at around 50–60 kDa, 30–40 kDa, and 40–50 kDa, respectively. Among these, the proteolytic activities was almost completely disappeared in the presence of a matrix metalloproteinase inhibitor, 1, 10-phenanthroline. Antigen-antibody interactions between the venom and its Iranian antivenin was observed by Western blotting, and it showed several antigenic proteins in the range of 30–160 kDa. This strong antigen–antibody reaction was also demonstrated through an enzyme-linked immunosorbent assay (ELISA). The gelatinase activity of the venom was suppressed by Razi institute polyvalent antivenin, suggesting the inhibitory effect of the antivenin against H. lepturus venom protease activities. Prudently, more extensive clinical studies are necessary for validation of its use in envenomed patients.

Separation and analysis of peptides and proteins from Vipera lebetina snake venom

Our studies of Levantine viper venom have demonstrated that the venom is a rich source of biomedically important proteins and peptides. The venom contains metalloproteases: thrombolytic, fibrin-degrading lebetase, an endothelial cell apoptosis inducing metalloprotease (VLAIP), factor X activator (VLFXA); serine proteases: factor V activator, bradykinin-releasing serine proteases, β-fibrinogenase, α-fibrinogenase and chymotrypsin-like protease and different other enzymes such as phosphodiesterase, 5`-nucleotidase, ribonuclease, phospholipase A2s and L-amino acid oxidase. Among nonenzymatic components venom contains: nerve growth factor, vascular endothelial growth factor, disintegrins, C-type lectins.Here we report the isolation and characterization of proteins and peptides from Vipera lebetina venom using size exclusion, ion exchange, hydrophobic interaction and affinity chromatography, HPLC, UPLC and MALDI-TOF MS methods. N-terminal sequences and internal sequences of tryptic peptides of different proteins were determined using Edman sequencing and LC-ESI-MS/MS techniques. On the basis of fragmental sequences of proteins the oligonucleotides were designed and used as primers for cDNA cloning. Using cDNA library of the venom gland of a single snake the cDNAs coding proteins were cloned and sequenced. Protein sequences were deduced from cDNA sequences.The substrate specificity of venom proteases against insulin B-chain, bradykinin, substance P, and 6-10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α2-macroglobulin bait region and pregnancy zone protein were studied using MALDI-TOF mass spectrometry technique.

An Analysis of Cleavage of the Factor IX Activation Sites by Factor XIa

During blood coagulation, the plasma zymogen factor IX (fIX) is converted to the active protease factor Ixaβ (fIXaβ). The severe bleeding disorder associated with deficiency of fIX (hemophilia B) attests to the importance of this protein in hemostasis. Conversion of fIX to fIXaβ requires two proteolytic cleavages after Arg145 and Arg180, releasing an activation peptide. This process is mediated by the proteases factor VIIa (fVIIa) and factor XIa (fXIa). FVIIa in complex with tissue factor initially cleaves fIX after Arg145 forming an intermediate, factor IXα (fIXα), which is then cleaved after Arg180 to form fIXaβ. Western blots of activation time courses demonstrate fIXα accumulation during this process, indicating cleavage at Arg180 is rate limiting. In contrast, little intermediate accumulation occurs during fIX activation by fXIa. Previously, we showed that fXIa also cleaves fIX initially after Arg145, generating fIXα (Smith et al., J. Biol. Chem. 283;6696: 2008). To account for the lack of intermediate accumulation, then, the subsequent cleavage after Arg180 must occur at least as rapidly as the initial cleavage. We examined the relative rates of conversion of fIX, fIXα, and the alternative intermediate factor IXaα (fIXaα - cleaved after Arg180) to fIXaβ by fXIa. FIXα or fIXaα were prepared from tritium-labeled fIX by incubation with fXIa-Pro192 (discussed below) or Russell's Viper Venom protease, respectively, and purified by anion exchange chromatography. Conversion to fIXaβ was determined by measuring release of the tritiated activation peptide. FXIa converted fIX to fIXaβ with a kcat of 29.4 ± 0.4/min, a value reflecting cleavage at both activation sites. Kcat for conversion of fIXα and fIXaα to fIXaβ were 29.9 ± 0.5 and 30.0 ± 1.0/min, respectively. The rate of conversion of fIX to fIXα, estimated by measuring tritiated activation products separated by SDS-PAGE, was 30.0 ± 0.4/min. Recently, we showed that amino acid substitutions in fXIa for the conserved active site residue Gly193 (chymotrypsin numbering) decreased kcat for fIX activation 7–1000 fold, with residues with long branched side-chains having the greatest effect (Schmidt et al. Biochemistry 47;1326: 2008). Gly193 substitutions had a modestly larger detrimental effect (1.2–1.5 fold) on cleavage of fIX after Arg180 compared to Arg145 that was associated with varying degrees of fIXα accumulation. Similar effects were noted with substitutions for the adjacent residue Lys192. FXIa with Pro192 cleaved fIX after Arg180 >10-fold more slowly than after Arg145, generating fIXα with little subsequent conversion to fIXaβ. Cumulatively, these data support the premise that the rates for the two sequential reactions required for normal fIX activation by fXIa are comparable. Therefore, perturbations causing a greater effect on cleavage after Arg180 compared to Arg145, even if relatively small, result in fIXα accumulation. Initial recognition of fIX by fXIa involves substrate binding exosites distinct from the enzyme active site. At least one exosite appears to be located in the fXIa third apple (A3) domain, and may interact with an epitope on the fIX Ca2+-binding Gla-domain. The rate of fIX activation to fIXaβ by fXIa was significantly reduced in the presence of an antibody to the fXIa A3 domain or by mutations in the A3 domain. Similarly, rates of activation were decreased in the absence of Ca2+, in the presence of an antibody to the fIX Gla-domain, or when fIX with a decarboxylated Gla-domain was the substrate. In all cases, significant fIXα accumulation was noted in time courses, indicating that interfering with this particular substrate-exosite interaction has a significantly greater effect on cleavage after Arg180 than after Arg145. These findings raise the possibility that the exosite on fXIa A3 plays a larger role in conversion of fIXα to fIXaβ than in initial fIX conversion to fIXα, and are consistent with the possibility, recently proposed by Sinha et al. (Biochemistry 46;9830: 2007), that a second fIX binding exosite is present on fXIa.

Enzymatic activities and functional characterization of a novel recombinant snake venom proteinase from Agkistrodon Acutus

Snake venom from Agkistrodon acutus consists of a number of compounds which may potentially be used as drugs. However, it is hard to obtain enough pure protein for drug development. Recently, we reported expression and purification of a novel recombinant fibrinogenase which was named rFII. Here we reported for the first time the enzymatic activities and functional characterization of rFII. Circular dichroism spectra showed the gross conformation of FIIa and rFII to be notably similar. It is an alkaline proteinase and the amino acid sequence exhibits a high degree of sequence identity with other snake venom metalloproteinases. rFII also exhibits amidase activity against N-(p-Tosyl)-Gly-Pro-Lys-p-nitroanilide, which is specified synthetic substrate for plasmin. Functional characterization showed that rFII possesses both fibronectin and type IV collagen cleaving activities. In addition, rFII preferentially cleaved the Aalpha-chain of fibrinogen, followed by the Bbeta-chain and finally, the gamma(γ) chain was affected. Furthermore, rFII was also capable of cleaving fibrin without plasminogen activation and suppressing ADP-induced platelet aggregation. The proteolytic activity of rFII was inhibited completely by PMSF and mostly by EDTA. The cations Ca 2+, Mg 2+, Na +, K + didn't affect its proteolytic activity, while Cu 2+ and Zn 2+ slightly inhibited this activity. Study of hydrolysis of oxidized insulin B-chain reveals that rFII preferentially cleaved oxidized insulin B-chain at the site of Val 12-Glu 13, Leu 15-Tyr 16, and Phe 24-Phe 25.

Variability in expression of Bothrops insularis snake venom proteases: An ontogenetic approach

Bothrops insularis is a threatened snake endemic to Queimada Grande Island, southern coast of São Paulo, Brazil, and the occurrence of sexual abnormalities in males, females and intersexes (females with functional ovaries and rudimentary hemipenis) has been reported in this population. The aim of this study was to identify ontogenetic shifts in protease expression of offspring of captive-bred B. insularis. Three neonates from a single litter were maintained at the facilities of Laboratory of Herpetology, Institute Butantan, for 41 months. The snakes were individually milked and venoms were analyzed both by SDS–PAGE, under reducing conditions, and for biochemical activities. The venoms from the mother and from a pool of adult specimens were used as references. In regard to the electrophoretic patterns, common bands were identified mainly between 14 and 50 kDa among snakes. The occurrence of proteolytic activity was noticed predominantly between 27 and 45 kDa in zymograms. Inhibitory assays with 1,10-phenantroline (10 mM) and PMSF (5 mM) showed that venoms possessed both metalloproteases and serine proteases. Venoms of young specimens showed a higher coagulant activity than those of adults, especially upon factors X and II. All venoms presented fibrino(geno)lytic activity, degrading Aα and Bβ chains of fibrinogen, and lysing fibrin plate. These findings can reflect important individual, ontogenetic and sexual differences on venom composition and are likely correlated with diet habits of this species.