Snake Venom Thrombin-like Enzymes Research Articles

Evaluation of the Inhibitory Potential of Synthetic Peptides Homologous to CDR3 Regions of a Monoclonal Antibody against Bothropic Venom Serine Proteases.

Snakebite accidents, neglected tropical diseases per the WHO, pose a significant public health threat due to their severity and frequency. Envenomation by Bothrops genus snakes leads to severe manifestations due to proteolytic enzymes. While the antibothropic serum produced by the Butantan Institute saves lives, its efficacy is limited as it fails to neutralize certain serine proteases. Hence, developing new-generation antivenoms, like monoclonal antibodies, is crucial. This study aimed to explore the inhibitory potential of synthetic peptides homologous to the CDR3 regions of a monoclonal antibody targeting a snake venom thrombin-like enzyme (SVTLE) from B. atrox venom. Five synthetic peptides were studied, all stable against hydrolysis by venoms and serine proteases. Impressively, four peptides demonstrated uncompetitive SVTLE inhibition, with Ki values ranging from 10-6 to 10-7 M. These findings underscore the potential of short peptides homologous to CDR3 regions in blocking snake venom toxins, suggesting their promise as the basis for new-generation antivenoms. Thus, this study offers potential advancements in combatting snakebites, addressing a critical public health challenge in tropical and subtropical regions.

Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Determination of the species origin and thrombin-like enzyme content of Bothrops atrox venom by ultra-high performance liquid chromatography-tandem mass spectrometry based on marker peptide

蛇毒血凝酶类药物是以蝮蛇蛇毒为原料制备的止血药,主要活性成分为蛇毒类凝血酶(svTLEs)。不同蛇种来源的svTLEs结构不同,止血机制不同,药理作用也存在差异,因此准确鉴别蛇毒种属来源和svTLEs含量对于保障该类产品的质量至关重要。研究基于蛋白质组学技术,筛选出了具有种属特异性的矛头蝮蛇svTLE特征肽,并建立了基于特征肽的超高效液相色谱-串联质谱(UHPLC-MS/MS)检测矛头蝮蛇蛇毒种属来源及类凝血酶含量的方法。采用胰蛋白酶对纯化的矛头蝮蛇svTLE进行酶解,利用纳升液相色谱-四极杆/静电场轨道阱高分辨质谱(Nano LC-Q-Exactive-MS)和Proteome Discoverer 2.2软件分别进行多肽的检测和鉴定,通过BLAST搜索与Uniprot数据库对比分析,筛选出具有种属特异性的矛头蝮蛇svTLEs特征肽“EAYNGLPAK”。针对该特征肽对酶解温度、酶解时间和酶用量等样品前处理方法进行了优化,利用超高效液相色谱-串联质谱,以m/z 481.9>315.2和481.9>485.2作为检测离子对,采用ESI+模式进行了多反应监测(MRM)定性定量分析。结果显示,特征肽在2.5~30 ng/mL范围内线性关系良好,相关系数(r)大于0.9996,多水平加标回收率范围为95.5%~101.9%,各水平平行测定结果的相对标准偏差(RSD)为1.1%~3.2%,完全能够满足实际样品检测需求。方法简便快捷,灵敏度高,专属性强,可用于矛头蝮蛇蛇毒种属鉴别及svTLE含量测定,从源头保证血凝酶类产品的质量,并可为其他蛇毒类产品的质量控制提供参考。

A Species-Specific Strategy for the Identification of Hemocoagulase Agkistrodon halys pallas Based on LC-MS/MS-MRM

Hemocoagulase Agkistrodon halys pallas is a complex mixture composed of snake venom thrombin-like enzymes (svTLEs) and small amounts of thrombokinase-like enzymes. It has been widely used as a hemostatic with rapidly growing marketing due to its advantage of localized clotting fibrinogen other than systemic coagulation. However, svTLEs from different species have various structures, functions, and hemostatic mechanisms. To ensure the efficacy and safety of Hemocoagulase Agkistrodon halys pallas, an exclusive and sensitive method has been developed to identify specific marker peptides based on liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS-MRM) mode. By combining transcriptomics and proteomics, a series of species-specific peptides of Agkistrodon halys pallas were predicted and examined by LC-MS/MS. After reduction, alkylation, and tryptic digestion were performed on Hemocoagulase Agkistrodon halys pallas, a target peptide TLCAGVMEGGIDTCNR was analyzed by LC-MS/MS-MRM. It offers a new and effective approach for the quality control of Hemocoagulase Agkistrodon halys pallas products. This method is superior to the current assays in terms of sensitivity, specificity, precision, accuracy, and throughput. The strategy can also be applied in studying other important protein-based medicines.

Differential sialic acid content in adult and neonatal fibrinogen mediates differences in clot polymerization dynamics

AbstractNeonates possess a molecular variant of fibrinogen, known as fetal fibrinogen, characterized by increased sialic acid, a greater negative charge, and decreased activity compared with adults. Despite these differences, adult fibrinogen is used for the treatment of bleeding in neonates, with mixed efficacy. To determine safe and efficacious bleeding protocols for neonates, more information on neonatal fibrin clot formation and the influence of sialic acid on these processes is needed. Here, we examine the influence of sialic acid on neonatal fibrin polymerization. We hypothesized that the increased sialic acid content of neonatal fibrinogen promotes fibrin B:b knob-hole interactions and consequently influences the structure and function of the neonatal fibrin matrix. We explored this hypothesis through analysis of structural properties and knob:hole polymerization dynamics of normal and desialylated neonatal fibrin networks and compared them with those formed with adult fibrinogen. We then characterized normal neonatal fibrin knob:hole interactions by forming neonatal and adult clots with either thrombin or snake-venom thrombin-like enzymes that preferentially cleave fibrinopeptide A or B. Sialic acid content of neonatal fibrinogen was determined to be a key determinant of resulting clot properties. Experiments analyzing knob:hole dynamics indicated that typical neonatal fibrin clots are formed with the release of more fibrinopeptide B and less fibrinopeptide A than adults. After the removal of sialic acid, fibrinopeptide release was roughly equivalent between adults and neonates, indicating the influence of sialic acid on fibrin neonatal fibrin polymerization mechanisms. These results could inform future studies developing neonatal-specific treatments of bleeding.

Towards toxin PEGylation: The example of rCollinein-1, a snake venom thrombin-like enzyme, as a PEGylated biopharmaceutical prototype

PEGylation was firstly described around 50 years ago and has been used for more than 30 years as a strategy to improve the drugability of biopharmaceuticals. However, it remains poorly employed in toxinology, even though it may be a promising strategy to empower these compounds in therapeutics. This work reports the PEGylation of rCollinein-1, a recombinant snake venom serine protease (SVSP), able to degrade fibrinogen and inhibit the hEAG1 potassium channel. We compared the functional, structural, and immunogenic properties of the non-PEGylated (rCollinein-1) and PEGylated (PEG-rCollinein-1) forms. PEG-rCollinein-1 shares similar kinetic parameters with rCollinein-1, maintaining its capability of degrading fibrinogen, but with reduced activity on hEAG1 channel. CD analysis revealed the maintenance of protein conformation after PEGylation, and thermal shift assays demonstrated similar thermostability. Both forms of the enzyme showed to be non-toxic to peripheral blood mononuclear cells (PBMC). In silico epitope prediction indicated three putative immunogenic peptides. However, immune response on mice showed PEG-rCollinein-1 was devoid of immunogenicity. PEGylation directed rCollinein-1 activity towards hemostasis control, broadening its possibilities to be employed as a defibrinogenant agent.

The absence of thrombin-like activity in Bothrops erythromelas venom is due to the deletion of the snake venom thrombin-like enzyme gene.

Snake venom thrombin-like enzymes (SVTLEs) are serine proteinases that clot fibrinogen. SVTLEs are distributed mainly in venoms from snakes of the Viperidae family, comprising venomous pit viper snakes. Bothrops snakes are distributed throughout Central and South American and are responsible for most venomous snakebites. Most Bothrops snakes display thrombin-like activity in their venoms, but it has been shown that some species do not present it. In this work, to understand SVTLE polymorphism in Bothrops snake venoms, we studied individual samples from two species of medical importance in Brazil: Bothrops jararaca, distributed in Southeastern Brazil, which displays coagulant activity on plasma and fibrinogen, and Bothrops erythromelas, found in Northeastern Brazil, which lacks direct fibrinogen coagulant activity but shows plasma coagulant activity. We tested the coagulant activity of venoms and the presence of SVTLE genes by a PCR approach. The SVTLE gene structure in B. jararaca is similar to the Bothrops atrox snake, comprising five exons. We could not amplify SVTLE sequences from B. erythromelas DNA, except for a partial pseudogene. These genes underwent a positive selection in some sites, leading to an amino acid sequence diversification, mostly in exon 2. The phylogenetic tree constructed using SVTLE coding sequences confirms that they are related to the chymotrypsin/kallikrein family. Interestingly, we found a B. jararaca specimen whose venom lacked thrombin-like activity, and its gene sequence was a pseudogene with SVTLE structure, presenting nonsense and frameshift mutations. Our results indicate an association of the lack of thrombin-like activity in B. jararaca and B. erythromelas venoms with mutations and deletions of snake venom thrombin-like enzyme genes.

Differential Sialic Acid Content in Adult and Neonatal Fibrinogen Mediate Differences in Clot Polymerization

Recent studies have identified several major qualitative and quantitative differences in important hemostatic proteins between adult and neonatal humans, including the primary coagulation protein fibrinogen.Despite these differences, neonates with post-operative bleeding from procedures requiring cardiopulmonary bypass (CPB) or extracorporeal membrane oxygenation (ECMO) are treated with transfusions of adult blood products, namely adult fibrinogen. The effectiveness of such transfusions is inconsistent in neonates and often results in a deficient fibrin matrix structure which may not be sufficient for mitigating bleeding. Our recent studies have also identified differences at the bulk clot level between purified neonatal and adult fibrin clots, including major structural and functional distinctions, which could contribute to these outcomes. Notably, adult fibrinogen degrades slower than neonatal clots, therefore transfusion of adult blood products to neonatal patients could contribute to thrombotic complications. Given the inconsistent results and potential complications from the transfusion of adult blood products to neonatal patients, there is a critical need to better understand the mechanistic differences in hemostatic processes between adults and neonates. Neonates possess a molecular variant of fibrinogen known as fetal fibrinogen. Increased sialic acid concentration compared to adults has been identified in many neonatal glycoproteins across physiological systems, including fetal fibrinogen. Studies have shown that fibrin clot properties are influenced by fibrin polymerization mechanisms and post translational modifications (e.g. sialic acid). For example, an increased sialic acid content in the dysfibrinogenemia associated with liver disease has been associated with an altered fibrin clot structure. Additionally, recent work from our group has found that the increased sialic acid in neonatal fibrin networks results in significantly greater fibroblast attachment than adult networks. Therefore, we hypothesized that differences in neonatal and adult fibrin clot properties are due to mechanistic differences in fibrin polymerization between neonates and adults owing to altered sialic acid concentrations. The activation of fibrinogen and conversion into fibrin by the proteolytic enzyme thrombin is essential for the formation of a stable blood clot and the cessation of bleeding. Thrombin converts soluble fibrinogen to insoluble fibrin via cleavage of fibrinopeptides A and B, exposing fibrin knobs A and B. Fibrin protofibrils are then formed from the noncovalent binding of fibrin knobs to complementary fibrin holes a and b on adjacent proteins. In adults, fibrin A:a knob:hole interactions are critical for polymerization. However, these mechanisms have not been explored in neonates. Therefore, we characterized the influence of sialic acid on the knob:hole interactions in neonatal fibrin polymerization. We first investigated the influence of sialic acid concentration on neonatal fibrin polymerization by removing sialic acid via enzymatic digestion and performing structural and functional analysis on desialylated fibrinogen. Desialylated adult and neonatal fibrinogen had roughly equivalent structure, polymerization kinetics, and clottability results. These results indicate that differential sialylation may at least partially explain functional differences been adult and neonatal clots. Additionally, we investigated the role that sialylation plays on neonatal fibrin polymerization dynamics by comparing neonatal or adult fibrin clots formed with snake venom thrombin-like enzymes (svTLE) that preferentially cleave either A or B fibrinogen fibrinopeptides or thrombin. Structural, mechanical, fibrinolytic, and polymerization assays were conducted. Quantitative release of fibrinopeptides was determined via ELISA. Results indicate neonatal fibrin polymerization mechanisms are more dependent on B:b knob:hole interactions than adult fibrin. Results from this study provide insight into the mechanism of the neonatal clotting process and are a critical contribution for the development of neonatal-specific treatments for bleeding and thrombosis. Disclosures Brown: Selsym Biotech, Inc.: Other: Founder and CEO.

Coagulant Effect and Tolerability of Yeast-Produced Recombinant Batroxobin in Healthy Adult Subjects.

Batroxobin, a snake venom thrombin-like enzyme, converts fibrinogen into fibrin by cleaving fibrinopeptide A. It is used for hemostasis; however, the supply of native batroxobin is limited. Therefore, we developed a recombinant batroxobin (r-batroxobin) from Pichia pastoris and evaluated its pharmacodynamics and safety in humans. A randomized, double-blind, placebo-controlled, single ascending-dose study was performed. Eight healthy subjects were enrolled in each r-batroxobin dose group (2.5, 5.0, or 10.0 BU/2.0mL administered intravenously), and randomized to receive r-batroxobin (n = 6) or matching placebo (n = 2). Safety was evaluated during the study, and pharmacodynamics was assessed using prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen level. All subjects in each cohort completed the study. No significant changes in PT or aPTT occurred after intravenous r-batroxobin administration. Compared with the placebo group, the fibrinogen level in all r-batroxobin dose groups decreased significantly to 8.68-33.57% from the baseline within 12h (p ≤ 0.05). The TT in the 5.0 and 10.0 BU/2.0mL groups significantly increased to 7.53-18.48% from baseline within 12h compared with that of the placebo group (p ≤ 0.05), whereas that of the 2.5 BU/2.0mL group exhibited non-significant changes compared with the placebo group. No serious adverse events occurred. A single intravenous injection of r-batroxobin within a dose range of 2.5-10.0 BU/2.0mL was well tolerated and resulted in a significant decrease in fibrinogen and prolongation of TT. This study is registered at the Clinical Research Information Service (CRIS, http://cris.nih.go.kr ), number KCT0002518.

Agkihpin, a Distinct SVTLE from the Venom of Gloydius halys Pallas: Purification, Characterization and Structure-Activity Determination.

Blood clots produced by snake-venom thrombin-like enzymes (SVTLEs) are cleared rapidly, which makes SVTLEs attractive as potential candidates for antithrombotic therapy. We isolated a SVTLE, agkihpin, from the venom of Gloydius halys Pallas. Agkihpin was confirmed to a single-chain TLE with molecular mass of 25.5 kD, pI of 7.43, optimal pH of 8.0 (hydrolyzing TAME), linked carbohydrate absent, and weak fibrinogen clotting activity. It was also found that (i) G. halys might be the latest species in SVTLEs phylogenetic tree; (ii) different level of conservation was shown among the SVTLEs from the Viperidae snakes. Some of those site may account for different activities exhibited by those SVTLEs, especially position 181, at which a fibrinogenolytic activity increase was found when a basic and larger amino acid substituted by a neutral and smaller one; (iii) an extra α-helix constructed with a 'Pro + acidic amino acid + aromatic amino acid' pattern was found in the SVTLEs from Gloydius and Agkistrodon snakes, although it does not necessarily imply an effect on the fibrinogenolytic activity of the SVTLEs. This study provided some new insight into the activity of SVTLE.

Acute and repeated dose (28 days) toxicity studies in rats and dogs of recombinant batroxobin, a snake venom thrombin-like enzyme expressed from Pichia pastoris

Recombinant batroxobin is a thrombin-like enzyme of Bothrops atrox moojeni venom. To evaluate its toxicological effect, it was highly expressed in Pichia pastorisand successfully purified to homogeneity from culture broth supernatant following Good Manufacturing Practice (GMP). The maximum tolerated dose of the recombinant batroxobin was examined in Sprague-Dawley (SD) rat and Beagle dogs following Good Laboratory Practice (GLP) regulations. The approximate lethal dose of recombinant batroxobin was 10 National Institute of Health (NIH) u/kg in male and female rats. Slight test substance-related effects were clearly in male and female dogs at more than 10 NIH u/kg. The maximum tolerated dose (MTD) was considered to be greater than 30 NIH u/kg in dogs. To investigate the repeated dose toxicity of batroxobin, the test item was intravenously administered to groups of SD rat and Beagle dog every day for 4 weeks. We observed that all animals survived the duration of the study without any effects on their mortality. There were no effects in both rats and dogs regarding their clinical signs, body weight, food consumption, ophthalmological examination, urinalysis, hematology, clinical chemistry, organ weightand gross post mortem examinations. The no adverse effect level (NOAEL) of recombinant batroxobin for both males and females is considered to be greater than 2.5 NIH u/kgin rats and 1 NIH u/kg in dogs, respectively. No toxic effects were noted in target organs. In conclusion, these results show a favorable preclinical profile and may contribute clinical development of recombinant batroxobin.

Agkihpin, a novel SVTLE from Gloydius halys Pallas, promotes platelet aggregation in vitro and inhibits thrombus formation in vivo in murine models of thrombosis

In previous work, a snake venom arginine esterase (SVAE), agkihpin from the venom of Gloydius halys Pallas, was isolated and its biochemical data including Mr, PI, amino acid components and sugar content was collected. Here, the agkihpin was cloned and further characterized and we found that agkihpin could promote ADP-induced platelets aggregation, hydrolyze fibrin, cleave Aα and Bβ chains of fibrinogen and reduce the thrombosis induced by thrombin. Moreover, agkihpin hydrolyzed TAME with optimum temperatures at 30 °C–45 °C, and the hydrolysis was inhibited by EDTA, PMSF, DTT and promoted by Ca2+, Fe3+, Mg2+, Zn2+. The sequence features of agkihpin were detected as follows: the N-terminal residues was determined as I(V)L(Y)GDDECNINE by protein sequencing; the ORF was determined as 705 bp, and the deduced amino acid sequence was identified by peptide mass fingerprinting; the cysteines, cleavage sites, active sites and substrate binding sites of snake venom thrombin-like enzyme (SVTLE), were all conserved in amino acid sequence of agkihpin; 2 Leu(Tyr), 4 Asn and 121 Ile in amino acid sequence of agkihpin were first found in the amino acid sequences of SVTLEs. These findings indicated that agkihpin is a novel SVTLE. What's more, due to its several advantages of fibrino(gen)olytic and thrombosis-reduced activities, and devoid of bleeding risk, agkihpin may be developed into a thrombolytic drug in the future.

The structure-function relationship of thrombin-like enzymes from the green pit viper (Trimeresurus albolabris).

Pit viper venoms can decrease fibrinogen levels in snakebite patients. Studies have shown that the hypofibrinogenemia is a consequence of snake venom thrombin-like enzymes (TLEs), the serine proteases that have the potential to be both diagnostic and therapeutic agents. Exosites of thrombin are the molecular regions that determine the substrate specificities, but its presence and significance in TLEs are unclear. Therefore, the putative exosites of recombinant TLEs derived from Green pit viper (Trimeresurus albolabris), GPV-TL1 and GPV-TL2, were mutated in a Pichia pastoris system. In a previous report, GPV-TL1 showed a strong fibrinogenolytic activity on the Aα and Bβ chains of fibrinogen, as well as a plasma clotting activity. Compared with GPV-TL1, the GPV-TL1m mutated in the putative exosite (TRN to RRR at residues 60-62) showed a weaker fibrinogenolytic activity with a similar clotting activity of 207.1 thrombin units/mg. GPV-TL2 contained two-residue differences from GPV-TL1 in the putative exosite (N73M and V74Y). GPV-TL2 selectively cleaved only the Aα chain of fibrinogen without detectable clotting activity. The mutated GPV-TL2 (GPV-TL2m) showed a weaker fibrinogenolytic activity compared with that of the wild type. These results support the important roles of the putative exosite in snake venom TLE activities. This information is helpful for future protein engineering.

In vitro Dissolution of Calcium Oxalate Stones with Ethylenediaminetetraacetic Acid and Snake Venom Thrombin-Like Enzyme

Objective: The aim of this study was to determine the feasibility of using snake venom thrombin-like enzyme (SVTLE) and/or ethylenediaminetetraacetic acid (EDTA) to dissolve calcium oxalate stones in vitro. Methods: Seven calcium oxalate stones were incubated with various chemolytic agents [EDTA, Tris-HCl/EDTA (TE) buffer or SVTLE diluted in TE buffer]. The pH, calcium concentration, stone weight and stone surface integrity were recorded, as well as related pathological changes to bladder mucosae. Results: Compared to all other solutions, those containing SVTLE and buffered EDTA had higher concentrations of mobilized calcium and caused significantly more stone weight loss, stone fragility and gaps in the calcium crystals. Also, there were no adverse pathological effects on rabbit bladder mucosae from any of the solutions. Conclusions: The data indicate that buffered EDTA and SVTLE can be used to dissolve calcium oxalate stones and, at the concentrations used here, do not damage tissue.

Biochemical properties and comparative pharmacology of a coagulant from Deinagkistrodon acutus snake venom

A number of snake venom thrombin-like enzymes (TLEs) have already been characterized. Some TLEs play significant roles in vessel injury hemostasis. A novel TLE (Agacutase) was purified from Deinagkistrodon acutus snake venom by the means of Sephadex G-75, DEAE-Sepharose FF, and Sephadex G-25 column chromatography. Structural analysis indicated that Agacutase is a single-chain glycoprotein with a molecular mass of 31,084Da, isoelectric point of 4.38, optimal activity at 37°C and pH 6.6, sugar content of 7.6%. Its N-terminal 44 amino acid sequence was determined to be VIGGNECDTNEHRFLAAFFTSRPWIFQCAGTLIHEEWVLAAAHC, showing maximum identity of 80% with that of Dav-X protease. The Agacutase-induced clotting activity was not influenced by heparin, hirudin, or Dextran 40, but activated by Ca2+ and inhibited by PMSF or lactose, which suggests that Agacutase is a serine protease and the coagulation activity is independent of Thrombin. Agacutase with arginine esterase activity specifically cleaves the α-chain of fibrinogen. Agacutase iv (0.03–0.12U/kg) shortened 16–68% of the rabbit blood clotting time. No significant influence was indicated on platelet, Factor II and XIII, or fibrinolytic system. It converts fibrinogen into the soluble fibrin that accelerates hemostasis at wound. Pharmacological comparison showed the hemostatic effect of Agacutase lasted 24h while Reptilase did 8h. Its maximum tolerated, abnormal toxicity, allergic, and hemorrhagin doses were 80U/kg, 1U, 2U, and 50U, respectively, whereas those of Reptilase or Agacutin were 35U/kg, 0.25U, 0.25U, and 0.2U, respectively. The results indicated that Agacutase may be a predominant coagulant.

Biochemical and biological characterization of two serine proteinases from Colombian Crotalus durissus cumanensis snake venom

Two clotting serine proteinases, named Cdc SI and Cdc SII, were isolated and characterized for the first time from Colombian Crotalus durissus cumanensis snake venom. The enzymes were purified using two chromatographic steps: molecular exclusion on Sephacryl S-200 and RP-HPLC on C8 Column. The molecular masses of the proteins, determined by MALDI-TOF mass spectrometry, were 28,561.4 and 28,799.2 Da for Cdc SI and Cdc SII, respectively. The aim of the present study was to evaluate enzymatic, coagulant and toxic properties of the two enzymes. The serine proteinases hydrolyzed specific chromogenic substrate (BaPNA) and exhibited a Michaelis–Menten behavior. Cdc SI had Vmax of 0.038 ± 0.003 nmol/min and KM of 0.034 ± 0.017 mM, while Cdc SII displayed values of Vmax of 0.267 ± 0.011 nmol/min and KM of 0.145 ± 0.023 mM. N-terminal sequences were VIGGDEXNIN and VIGGDICNINEHNFLVALYE for Cdc SI and Cdc SII, respectively. Molecular masses, N-terminal sequences, inhibition assays, and enzymatic profile suggest that Cdc SI and Cdc SII belong to the family of snake venom thrombin-like enzymes. These serine proteinases differed in their clotting activity on human plasma, showing a minimum coagulant dose of 25 μg and 0.571 μg for Cdc SI and Cdc SII, respectively. Enzymes also showed coagulant activity on bovine fibrinogen and degraded chain α of this protein. Toxins lack hemorrhagic and myotoxic activities, but are capable to induce defibrin(ogen)ation, moderate edema, and an increase in vascular permeability. These serine proteinases may contribute indirectly to the local hemorrhage induced by metalloproteinases, by causing blood clotting disturbances, and might also contribute to cardiovascular alterations characteristic of patients envenomed by C. d. cumanensis in Colombia.

Crystal structure and activating effect on RyRs of AhV_TL-I, a glycosylated thrombin-like enzyme from Agkistrodon halys snake venom

A snake venom thrombin-like enzyme (SVTLE) from Agkistrodon halys pallas venom was isolated by means of a two-step chromatographic procedure. The purified enzyme, named AhV_TL-I, showed fibrinogenolytic activity against both the Aα and Bβ chains of bovine fibrinogen. Unlike the other SVTLEs, AhV_TL-I has poor esterolytic activity upon BAEE substrate. The N-terminal sequence of AhV_TL-I was determined to be IIGGDEXNINEHRFLVALYT, and the molecular mass was confirmed to 29389.533Da by MALDI-TOF mass spectrometry. Its complete cDNA and derived amino acid sequence were obtained by RT-PCR. The crystal structure of AhV_TL-I was determined at a resolution of 1.75 Å. A disaccharide was clearly mapped in the structure, which involved in regulating the esterolytic activity of AhV_TL-I. The presence of the N-glycan deformed the 99-loop, and the resulting steric hindrances hindered the substrates to access the active site. Furthermore, with the carbohydrate moiety, AhV_TL-I could induce mouse thoracic aortic ring contraction with the EC(50) of 147nmol/L. Besides, the vasoconstrictor effects of AhV_TL-I were also independent of the enzymatic activity. The results of [Ca(2+)](i) measurement showed that the vasoconstrictor effects of AhV_TL-I were attributed to Ca(2+) releasing from Ca(2+) store. Further studies showed that it was related to the activation of ryanodine receptors (RyRs). These offer new insights into the snake SVTLEs functions and provide a novel pathogenesis of A. halys pallas venom.

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen>dog fibrinogen≈human fibrinogen>goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.

Structure of saxthrombin, a thrombin-like enzyme fromGloydius saxatilis

Snake-venom thrombin-like enzymes (SVTLEs) are serine proteases that are widely distributed in snakes from the Crotalinae subfamily of the Viperidae. In contrast to other snake-venom serine proteases, they have a biochemical activity similar to that of thrombin and play an important role in the process of blood coagulation. However, SVTLEs cannot activate factor VIII, which is essential in blood-clot stabilization. Consequently, blood clots produced by SVTLEs are not stable and are cleared rapidly. This characteristic makes SVTLEs attractive as potential candidates for antithrombotic therapy. Saxthrombin, an SVTLE from Gloydius saxatilis, was purified and crystallized to obtain a high-quality crystal, from which data were acquired to 1.43 Å resolution. Preliminary X-ray diffraction analysis showed that the crystal belonged to space group C2, with unit-cell parameters a = 94.2, b = 52.2, c = 50.1 Å, β = 96.7°. The crystal structure was determined by molecular replacement and the final R factor was 18.69%; the R(free) was 20.01%. This is the first report of a crystal structure of an SVTLE. Saxthrombin belongs to the typical α/β-hydrolase fold of serine proteases. Its structure was compared with those of thrombin and other snake-venom serine proteases. The observed differences in the amino-acid composition of the loops surrounding the active site appear to contribute to different surface-charge distributions and thus alter the shape of the active-site cleft, which may explain the differences in substrate affinity.

Molecular mechanism analysis of Gloydius shedaoensis venom gloshedobin interaction with inhibitors by homology modeling

Snake venom thrombin-like enzymes (SVTLEs) are widely applied in the treatment of thrombotic diseases, however, the molecular mechanism of its inhibition by synthetic and natural proteinaceous inhibitors is not yet understood. Here we investigated effects of protease inhibitors including phenylmethylsulfonil fluoride (PMSF), benzamidine (BMD) and its derivates on the activity of recombinant gloshedobin, a SVTLE from the snake Gloydius shedaoensis. The molecular inhibition mechanism was postulated by separately docking inhibitors into three-dimensional model of gloshedobin using protein C activator from Agkistrodon contortrix contortrix venom (ACC-C, which bear 78% identity with gloshedobin) as template. The analysis indicated that the strongest inhibitor, PMSF, was via a covalent bond with the catalytic Ser195, while other inhibitors showing weaker inhibitory activity were via hydrogen bond with Ser195 or non-catalytic residues.