De Novo Venom Gland Transcriptome Assembly and Characterization for Calloselasma rhodostoma (Kuhl, 1824), the Malayan Pit Viper from Malaysia: Unravelling Toxin Gene Diversity in a Medically Important Basal Crotaline.

In Southeast Asia, the Malayan Pit Viper (Calloselasma rhodostoma) is a venomous snake species of medical importance and bioprospecting potential. To unveil the diversity of its toxin genes, this study de novo assembled and analyzed the venom gland transcriptome of C. rhodostoma from Malaysia. The expression of toxin genes dominates the gland transcriptome by 53.78% of total transcript abundance (based on overall FPKM, Fragments Per Kilobase Million), in which 92 non-redundant transcripts belonging to 16 toxin families were identified. Snake venom metalloproteinase (SVMP, PI > PII > PIII) is the most dominant family (37.84% of all toxin FPKM), followed by phospholipase A2 (29.02%), bradykinin/angiotensin-converting enzyme inhibitor-C-type natriuretic peptide (16.30%), C-type lectin (CTL, 10.01%), snake venom serine protease (SVSP, 2.81%), L-amino acid oxidase (2.25%), and others (1.78%). The expressions of SVMP, CTL, and SVSP correlate with hemorrhagic, anti-platelet, and coagulopathic effects in envenoming. The SVMP metalloproteinase domains encode hemorrhagins (kistomin and rhodostoxin), while disintegrin (rhodostomin from P-II) acts by inhibiting platelet aggregation. CTL gene homologues uncovered include rhodocytin (platelet aggregators) and rhodocetin (platelet inhibitors), which contribute to thrombocytopenia and platelet dysfunction. The major SVSP is a thrombin-like enzyme (an ancrod homolog) responsible for defibrination in consumptive coagulopathy. The findings provide insight into the venom complexity of C. rhodostoma and the pathophysiology of envenoming.

[Agkistrodon halys venom antitumor component-I inhibits vasculogenic mimicry in triple-negative breast cancer cells in vitro by down-regulating MMP2].

OBJECTIVE: To investigate the inhibitory effect of agkistrodon halys venom antitumor component-I (AHVAC-I) on vasculogenic mimicry (VM) formation in triple-negative breast cancer MDA-MB-231 cells and explore its possible mechanism. METHODS: CCK8 assay was used to determine the optimal concentration of AHVAC-I for cell treatment based on its halfinhibitory concentration (IC50). MDA-MB-231 cells were treated with different concentrations of AHVAC-I or 5-Fu, and the changes in vasomimetic capacity of the cells were examined using Matrigel assay. The expression levels of matrix metalloproteinase-2 (MMP2) and MMP9 in the treated cells were detected using quantitative PCR and Western blotting. RESULTS: Compared with the control treatment with culture medium, treatment with 5, 10 and 20 µg/mL AHVAC-I significantly reduced vasomimetic ability of MDA-MB-231 cells in a dose-dependent manner (P < 0.01). MMP2 supplementation obviously restored the vasomimetic ability of the cells inhibited by AHVAC-I. CONCLUSION: AHVAC-I inhibits VM formation in triplenegative breast cancer cells in vitro by down-regulating MMP2 production.

Use of Crotalidae equine immune F(ab')2 antivenom for treatment of an Agkistrodon envenomation.

INTRODUCTION: Anavip (F(ab')2AV) is a lyophilized F(ab')2 immunoglobulin fragment derived from horses immunized with venom from Bothrops asper and Crotalus durissus. It was approved by the FDA in 2015 for treatment of North American rattlesnake envenomation but not for Agkistrodon envenomation. Published data regarding the efficacy and safety of Anavip in treating Agkistrodon envenomations is limited. We present a case of a patient treated with Anavip after confirmed Agkistrodon laticinctus envenomation. CASE DETAILS: A 77 year-old man was bitten on his fifth finger by a captive A. laticinctus. He was taken to a local emergency department where he received a 10 vial initial dose of F(ab')2AV for pain and swelling and was transferred. At the receiving facility, his pain had improved and his swelling had not progressed. Over the next 30 h, his platelets declined to 132,000/mm3 and he received an additional 4 vials of F(ab')2AV. The remainder of his course was unremarkable with complete recovery by 3 months. DISCUSSION: This case provides an additional published datapoint on the use of this F(ab')2AV in the treatment of envenomation by Agkistrodon.

Structural, enzymatic and pharmacological profiles of AplTX-II - A basic sPLA2 (D49) isolated from the Agkistrodon piscivorus leucostoma snake venom.

A basic sPLA2 (D49) from the venom of snake Agkistrodon piscivorus leucostoma (AplTX-II) was isolated, purified and characterized. We determined the enzymatic and pharmacological profiles of this toxin. AplTX-II was isolated with a high level of purity through reverse phase chromatography and molecular exclusion. The enzyme showed pI 9.48 and molecular weight of 14,003 Da. The enzymatic activity of the AplTX-II depended on Ca2+ pH and temperature. The comparison of the primary structure with other sPLA2s revealed that AplTX-II presented all the structural reasons expected for a basic sPLA2s. Additionally, we have resolved its structure with the docked synthetic substrate NOBA (4-nitro-3-octanoyloxy benzoic acid) by homology modeling, and performed MD simulations with explicit solvent. Structural similarities were found between the enzyme's modeled structure and other snake sPLA2 X-Ray structures, available in the PDB database. NOBA and active-site water molecules spontaneously adopted stable positions and established interactions in full agreement with the reaction mechanism, proposed for the physiological substrate, suggesting that NOBA hydrolysis is an excellent model to study phospholipid hydrolysis.

Purification and characterization of Protein C activator from Agkistrodon acutus Venom.

Protein C (PC) plays an important role in the balance of coagulation and anticoagulation. Thus, the detection of PC activity is diagnostically significant for patients with cardiovascular diseases. Presently, the key methods to detect PC activity are the chromogenic assay and activated partial thromboplastin time (APTT) test. PROTAC used in the chromogenic assay is isolated from Agkistrodon contortrix venom as protein C activator (PCA). However, the use of the chromogenic assay is limited because of the high price of PROTAC. In this study, PCA was successfully purified from Agkistrodon acutus venom (AAV) by ion-exchange and gel chromatography. PCA from AAV has a relative molecular mass of 24 kD, calculated from the measurement of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The components of PCA were identified by MALDI-TOF/TOF-MS and mascot searches revealed that the coverage rate between PCA and zinc metalloproteinase AaPA from AAV was 21%. The chromogenic assay and APTT test were used to measure the enzymatic activity of PCA, and the results showed that PCA from AAV could specifically activate PC. In summary, the chromogenic assay described herein is highly sensitive and easy to perform.

Potential use of 13-mer peptides based on phospholipase and oligoarginine as leishmanicidal agents.

Phospholipase A2 toxins present in snake venoms interact with biological membranes and serve as structural models for the design of small peptides with anticancer, antibacterial and antiparasitic properties. Oligoarginine peptides are capable of increasing cell membrane permeability (cell penetrating peptides), and for this reason are interesting delivery systems for compounds of pharmacological interest. Inspired by these two families of bioactive molecules, we have synthesized two 13-mer peptides as potential antileishmanial leads gaining insights into structural features useful for the future design of more potent peptides. The peptides included p-Acl, reproducing a natural segment of a Lys49 PLA2 from Agkistrodon contortrix laticinctus snake venom, and its p-AclR7 analogue where all seven lysine residues were replaced by arginines. Both peptides were active against promastigote and amastigote forms of Leishmania (L.) amazonensis and L. (L.) infantum, while displaying low cytotoxicity for primary murine macrophages. Spectrofluorimetric studies suggest that permeabilization of the parasite's cell membrane is the probable mechanism of action of these biomolecules. Relevantly, the engineered peptide p-AclR7 was more active in both life stages of Leishmania and induced higher rates of ethidium bromide incorporation than its native template p-Acl. Taken together, the results suggest that short peptides based on phospholipase toxins are potential scaffolds for development of antileishmanial candidates. Moreover, specific amino acid substitutions, such those herein employed, may enhance the antiparasitic action of these cationic peptides, encouraging their future biomedical applications.

The extract from Agkistrodon halys venom protects against lipopolysaccharide (LPS)-induced myocardial injury.

BACKGROUND: Snake venoms contain various bioactive constituents which possess potential therapeutic effects. The aim of this work was to investigate the effect of the extract from Agkistrodon halys venom on lipopolysaccharide (LPS)-induced myocardial injury. METHODS: Thirty male Sprague-Dawley rats were randomly assigned to three groups (10 rats per group): control group, LPS group and LPS + extract group. Rats in control and the LPS groups were intravenously injected with sterile saline solution, and rats in the LPS + extract group with the extract. After 2 h, rats of the control group were intraperitoneally injected sterile saline solution, and rats in the LPS and the LPS + extract groups were treated with LPS (20 mg per kg body weight). Levels of creatine kinase (CK) and lactate dehydrogenase (LDH) in serum were determined. Anti-inflammation of the extract was analyzed via determination of TNF-α and IL-6 in serum, and expression of TNF-α, IL-6, COX-2 and p-ERK protein in hearts. Heme oxygenase-1 (HO-1) and p-NF-κB protein expression in hearts, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in serum were used to evaluate the anti-oxidative properties of the extract. RESULTS: Extract pretreatment significantly decreased the level of serum CK and LDH, reduced the generation of inflammatory cytokines such as TNF-α and IL-6, and also reduced serum level of MDA in the LPS + extract group compared with the LPS group. In addition, the extract increased SOD activity in serum, HO-1 protein expression in hearts, and decreased TNF-α, IL-6, COX-2, p-NF-κB and p-ERK1/2 protein expression. CONCLUSION: Our results suggested that beneficial effect of this extract might be associated with an improved anti-oxidation and anti-inflammatory effect via downregulation of NF-κB/COX-2 signaling by activating HO-1/CO in hearts.

Snake Venom Extracellular vesicles (SVEVs) reveal wide molecular and functional proteome diversity.

Proteins constitute almost 95% of snake venom's dry weight and are produced and released by venom glands in a solubilized form during a snake bite. These proteins are responsible for inducing several pharmacological effects aiming to immobilize and initiate the pre-digestion of the prey. This study shows that proteins can be secreted and confined in snake venom extracellular vesicles (SVEVs) presenting a size distribution between 50 nm and 500 nm. SVEVs isolated from lyophilized venoms collected from four different species of snakes (Agkistrodon contortrix contortrix, Crotalus atrox, Crotalus viridis and Crotalus cerberus oreganus) were analyzed by mass spectrometry-based proteomic, which allowed the identification of proteins belonging to eight main functional protein classes such as SVMPs, serine proteinases, PLA2, LAAO, 5'nucleotidase, C-type lectin, CRISP and Disintegrin. Biochemical assays indicated that SVEVs are functionally active, showing high metalloproteinase and fibrinogenolytic activity besides being cytotoxic against HUVEC cells. Overall, this study comprehensively depicts the protein composition of SVEVs for the first time. In addition, the molecular function of some of the described proteins suggests a central role for SVEVs in the cytotoxicity of the snake venom and sheds new light in the envenomation process.

Effects of hemocoagulase agkistrodon on the coagulation factors and its procoagulant activities.

OBJECTIVE: Hemocoagulase agkistrodon (HCA), a thrombin-like enzyme (TLE) from the venom of the Chinese moccasin snake (Deinagkistrodon acutus), has been used in clinical practice as a hemostatic compound. The aim of this study was to further investigate the pharmacological properties of HCA. MATERIALS AND METHODS: Sodium dodecyl sulfate or native polyacrylamide gel electrophoresis (SDS- or N-PAGE) as well as enzyme linked immunosorbent assays (ELISAs) were conducted to study the effects of HCA on the human plasma fibrinogen and prothrombin levels, as well as its in vitro interactions with some coagulation factors. In addition, the bleeding time effects of HCA in the mouse tail-bleeding model as well as its effects on the fibrinogen levels in rabbits were determined in vivo. RESULTS: In vitro results revealed that HCA exerts its procoagulant activities by hydrolyzing fibrinogen into segments that are easier to be absorbed, reducing the risk of thrombus formation. Besides, HCA could significantly inhibit the activation of prothrombin at the concentration of 0.3 µM. Unexpectedly, we also found that HCA was able to strongly bind to factor X/Xa (in a ratio of 1:1) and thus inhibit the acceleration of active factor X to tissue plasminogen activator-catalyzed plasminogen activation, demonstrating that it could be less likely to lead to thrombus formation. Finally, in vivo results indicated that HCA could significantly shorten the bleeding time in the mouse tail-bleeding model and had no effect on the fibrinogen levels in rabbits. CONCLUSION: In summary, HCA, a unique and new family member of TLEs, may become a new clinical drug for the prevention and treatment of hemorrhage due to its unique and complex interactions with the blood system. Clarification of these features will enable us to further understand the mechanism of action of HCA and then promote its further application in clinical practice as a therapeutic drug.

The Alapahoochee watershed microgeographic structure and its potential influence on metal concentrations and genetic structure in the Florida cottonmouth, Agkistrodon piscivorus conanti, within the watershed.

This study examines the microgeographic structure of the Alapahoochee watershed, part of the Suwannee River basin, south-central GA, USA, and relates it to variations in liver metal concentrations and genetic structure of the Florida cottonmouth, Agkistrodon piscivorus conanti. One objective was to determine if liver metal concentrations in A. piscivorus differ between Grand Bay and Mud creeks, which form the watershed's upper portion. Grand Bay Creek is relatively pristine, whereas Mud Creek is polluted with various metals. Genetic analyses were used to assess possible migration patterns between the creeks indicating whether the basin possesses a single population or two populations. Collections occurred in 2008 and 2009. Specimens were captured, euthanized, or collected as road kills, and liver metal concentrations were analyzed and DNA extracted. No differences in metal concentrations were detected between the creeks, except for nickel in females. Metal concentrations in A. piscivorus were not significantly different between males and females nor show a relationship to body size. Genetic analyses were limited to three primer sets, which amplified informative loci. Locus, CH4B, was highly divergent between the putative populations and particularly informative. Genetic structure indicates potential population isolation within the two creeks. Results suggest that two distinct A. piscivorus populations were present and those populations did not differ in their liver metal concentrations (exception being nickel), despite the differences in environmental metal concentrations in the areas. These findings provide new insight into metal accumulation and detoxification in these animals.

Pharmacokinetics of nebulized and subcutaneously implanted terbinafine in cottonmouths (Agkistrodon piscivorus).

Ophidiomyces ophiodiicola, the causative agent of snake fungal disease, is proposed as a serious threat to the conservation of several snake populations. The objective of this study was to determine the pharmacokinetic parameters of terbinafine administered through nebulization and a sustained subcutaneous implant as potential treatments of Ophidiomyces in reptiles. Seven adult cottonmouths (Agkistrodon piscivorus) were used in single-dose trials. Each snake was nebulized with terbinafine (2 mg/ml) for 30 min and had blood collected before nebulization and up to 12 hr after nebulization. Following a 5-month washout, the same snakes were administered a subcutaneous implant containing 24.5 mg terbinafine; blood was collected at baseline, 1 day postimplant placement, and then once weekly for 9 weeks. Plasma for both studies was analyzed by high-performance liquid chromatography. The mean plasma concentrations of nebulized terbinafine peaked between 0.5 and 4 hr. The subcutaneously implanted terbinafine reached therapeutic concentrations on day 1 and maintained therapeutic for over 6 weeks. These methods and doses are recommended as potential treatment options for snake fungal disease in reptiles.

The characterization of trans-pecos copperhead (Agkistrodon contortrix pictigaster) venom and isolation of two new dimeric disintegrins.

The vast amounts of toxins within the venom of snakes, while known to cause medical emergencies, display various biological functions. Trans-pecos copperhead (Agkistrodon contortrix pictigaster) crude venom separated by cation-exchange chromatography showed several fractions with fibrinolytic, hemorrhagic, gelatinase and platelet activities. Venom fractions 1, 2, 4, 5, and 12-17 contained fibrinolytic activity. Venom fractions 1, 2, 5 and 12-14 had hemorrhagic activity. Fractions 1, 2, 12, 13 and 17 contained gelatinase activity. Reverse-Phase C18 High Performance Liquid Chromatography was also used to purify and isolated disintegrins from this venom. Anti-platelet aggregation activity of the C18 fractions collected and performed on whole human blood showed that they inhibited platelet aggregation in presence of several agonists. Results from both SDS-PAGE and N-terminal sequencing determined that pictistatin 1 obtained from the Trans-Pecos copperhead venom was a dimeric disintegrin, and pictistatin 2 was a heterodimeric disintegrin. The molecules with anti-platelet activity could be considered in the development of more effective drugs, for numerous blood-related diseases such as stroke, heart attacks, thrombosis, and other medical conditions. In this study, we are presenting the first report of the purification, isolation, and partial characterization of two new dimeric disintegrins isolated from the venom of trans-pecos copperhead.

Effects of agkistrodon in different dosage forms on collagen-induced arthritis in rats.

OBJECTIVE: To determine the effective dosage and formulation of agkistrodon in collagen-induced arthritis (CIA) rats. METHODS: CIA was induced by injection of collagen in complete/incomplete Freund's adjuvant. Agkistrodon decoction, agkistrodon powder, and agkistrodon wine were administered daily starting from the onset of arthritis. Paw swelling degree was measured by using a volume-measuring instrument every 7 days after primary immunization. Arthritis index was measured and calculated using the "five scoring method" every 7 days. The levels of serum interleukin-1ß (IL-1ß) and type II collagen IgG antibodies were detected by enzyme-linked immunosorbent assay. Finally, all ankles were removed, and X-ray radiography was performed with In-vivo Imaging System FX. Samples were counterstained with hematoxylin and eosin for analysis. RESULTS: Among the various dosage formulations of agkistrodon, high-dose powder, which was equivalent to an amount of 6 g/day in adults, showed better effects on the inhibition of joint swelling and reduction of arthritis index score. The relatively low levels of serum IL-1 and anti-type II collagen IgG antibodies, as well as the X-ray radiography and pathology results, further proved the superiority of the high-dose powder over the other formulations. The effect of decoction on inhibiting joint swelling was inversely proportional to the dosage. Other effects, such as reduction of arthritis index score and the levels of serum IL-1 and anti-type II collagen IgG antibodies, were directly proportional to the dosage. While the use of large dose agkistrodon wine led to negative effects. CONCLUSION: These data highlight the potential function of high-dose agkistrodon powder, which was equivalent to an amount of 6 g/day in adults. The powder can quickly relieve the symptoms of rheumatoid arthritis and prevent aggravation of disease, especially during the early period.

Balancing the Expression and Production of a Heterodimeric Protein: Recombinant Agkisacutacin as a Novel Antithrombotic Drug Candidate.

Agkisacucetin extracted from the venom of Agkistrodon acutus has been demonstrated to be a promising antithrombotic drug candidate in clinical studies due to its function as a novel platelet membrane glycoprotein (GP) Ib inhibitor. Agkisacucetin is a heterodimeric protein composed of α- and ß-subunits with seven disulphide bonds. Both subunits form inactive homodimeric products, which cause difficulties for recombinant production. In this study, Agkisacucetin α- and ß-subunits were inserted sequentially into the chromosome of Pichia pastoris at the mutant histidinol dehydrogenase gene and ribosomal DNA repeat sites, respectively. By optimizing the gene copies and productivity of each subunit by drug screening, we successfully obtained a recombinant strain with balanced expression of the two subunits. Using this strain, a yield greater than 100 mg/L recombinant Agkisacucetin in fed-batch fermentation was reached. The recombinant Agkisacucetin possessed extremely similar binding affinity to recombinant GPIb and human platelets in in vitro assays, and its ristocetin-induced platelet aggregation activity ex vivo was identical to that of the extracted native Agkisacucetin, demonstrating that the yeast-derived Agkisacucetin could be an effective alternative to native Agkisacucetin. Moreover, this study provides an effective strategy for balancing the expression and production of heterodimeric proteins in P. pastoris.

rAdinbitor, a disintegrin from Agkistrodon halys brevicaudus stejneger, inhibits tumorigenicity of hepatocarcinoma via enhanced anti-angiogenesis and immunocompetence.

Adinbitor is a disintegrin previously obtained from Agkistrodon halys brevicaudus stejneger by our group. Here, we investigated the in vitro and in vivo anti-tumor activities of recombinant Adinbitor (rAdinbitor). rAdinbitor stimulation can inhibit the in vitro proliferation, migration and invasion capacities of murine hepatocarcinoma H22 and Hca-F cells. The administrations of rAdinbitor either by gavage or intraperitoneal injection suppress the tumor malignancy and prolong the survival rate and time of H22-transplanted mice. The number and size of formed blood vessels decreased dramatically in tumorous tissues in that the expression levels of vascular endothelial growth factor (VEGF) and cluster of differentiation 34 (CD34) were significantly decreased in responding to rAdinbitor treatment. The protein levels of IL-18 and IgG increased significantly in the serum of H22-transplanted tumor mice with rAdinbitor treatment. rAdinbitor shows in vitro and in vivo anti-tumor effects as an angiogenesis inhibitor and immunocompetence enhancer.

A Novel Direct Factor Xa Inhibitory Peptide with Anti-Platelet Aggregation Activity from Agkistrodon acutus Venom Hydrolysates.

Snake venom is a natural substance that contains numerous bioactive proteins and peptides, nearly all of which have been identified over the last several decades. In this study, we subjected snake venom to enzymatic hydrolysis to identify previously unreported bioactive peptides. The novel peptide ACH-11 with the sequence LTFPRIVFVLG was identified with both FXa inhibition and anti-platelet aggregation activities. ACH-11 inhibited the catalytic function of FXa towards its substrate S-2222 via a mixed model with a Ki value of 9.02 µM and inhibited platelet aggregation induced by ADP and U46619 in a dose-dependent manner. Furthermore, ACH-11 exhibited potent antithrombotic activity in vivo. It reduced paralysis and death in an acute pulmonary thrombosis model by 90% and attenuated thrombosis weight in an arterio-venous shunt thrombosis model by 57.91%, both at a dose of 3 mg/kg. Additionally, a tail cutting bleeding time assay revealed that ACH-11 did not prolong bleeding time in mice at a dose of 3 mg/kg. Together, our results reveal that ACH-11 is a novel antithrombotic peptide exhibiting both FXa inhibition and anti-platelet aggregation activities, with a low bleeding risk. We believe that it could be a candidate or lead compound for new antithrombotic drug development.

Venomics of New World pit vipers: genus-wide comparisons of venom proteomes across Agkistrodon.

We report a genus-wide comparison of venom proteome variation across New World pit vipers in the genus Agkistrodon. Despite the wide variety of habitats occupied by this genus and that all its taxa feed on diverse species of vertebrates and invertebrate prey, the venom proteomes of copperheads, cottonmouths, and cantils are remarkably similar, both in the type and relative abundance of their different toxin families. The venoms from all the eleven species and subspecies sampled showed relatively similar proteolytic and PLA2 activities. In contrast, quantitative differences were observed in hemorrhagic and myotoxic activities in mice. The highest myotoxic activity was observed with the venoms of A. b. bilineatus, followed by A. p. piscivorus, whereas the venoms of A. c. contortrix and A. p. leucostoma induced the lowest myotoxic activity. The venoms of Agkistrodon bilineatus subspecies showed the highest hemorrhagic activity and A. c. contortrix the lowest. Compositional and toxicological analyses agree with clinical observations of envenomations by Agkistrodon in the USA and Central America. A comparative analysis of Agkistrodon shows that venom divergence tracks phylogeny of this genus to a greater extent than in Sistrurus rattlesnakes, suggesting that the distinct natural histories of Agkistrodon and Sistrurus clades may have played a key role in molding the patterns of evolution of their venom protein genes. BIOLOGICAL SIGNIFICANCE: A deep understanding of the structural and functional profiles of venoms and of the principles governing the evolution of venomous systems is a goal of venomics. Isolated proteomics analyses have been conducted on venoms from many species of vipers and pit vipers. However, making sense of these large inventories of data requires the integration of this information across multiple species to identify evolutionary and ecological trends. Our genus-wide venomics study provides a comprehensive overview of the toxic arsenal across Agkistrodon and a ground for understanding the natural histories of, and clinical observations of envenomations by, species of this genus.

[Extraction, purification and identification of type II collagen from Agkistrodon acutus].

The object of the research was to extract, purify and identify the type II collagen of Agkistrodon acutus. Type II collagen of A. acutus was extracted by enzyme decomposition method, and purified by ion exchange column chromatography. It was characterized by SDS-PAGE gel electrophoresis, ultraviolet spectrophotometry, infrared absorption spectroscopy and mass spectroscopy. The results showed that the size of C II was about 130 kDa. It absorbed at 223 nm. IR spectrum obtained showed that the triple helical domains of amino-acid sequences were characterized by the repetition of triplets Gly-X-Y. The MS spectrum graphically stated that C II extracted from cow and A. acutus have the similar peptides. The C II of A. acutus was obtained by extraction and purification. Appraisal analysis by SDS-PAGE, UV, IR and MS, C II of A. acutus was consistent with the standard C II of cow. It was proved that the extracted protein was C II.

Anticoagulation factor I, a snaclec (snake C-type lectin) from Agkistrodon acutus venom binds to FIX as well as FX: Ca2+ induced binding data.

Anticoagulation factor I (ACF I), a snake C-type lectin (snaclec) from the venom of Agkistrodon acutus binds specifically with activated factor X (FXa) in a Ca2+-dependent manner and prolongs the blood-clotting time in vitro. In this study, the inhibition of the coagulation pathway by ACF I was measured in vivo by activated partial thromboplastin time and prothrombin time assays and the binding of ACF I to factor IX (FIX) was investigated by native PAGE and surface plasmon resonance. The results indicate that ACF I inhibits both intrinsic and extrinsic coagulation pathways, but does not inhibit thrombin activity. ACF I also binds FIX in a Ca2+-dependent manner and their maximal binding occurs at 0.25 mM Ca2+. ACF I has a higher binding-affinity to FIX than to FX. Ca2+ is required to maintain in vivo function of FIX Gla domain for its recognition of ACF I. However, Ca2+ at high concentrations (>0.25 mM) inhibits the binding of ACF I to FIX. Ca2+ functions as a switch for the binding between ACF I and FIX. The results suggest that the binding of ACF I with FIX may play a dominant role in the anticoagulation activity of ACF I in vivo.

Innate immune performance and steroid hormone profiles of pregnant versus nonpregnant cottonmouth snakes (Agkistrodon piscivorus).

Squamates (lizards and snakes) have independently evolved viviparity over 100 times, and exhibit a wide range of maternal investment in developing embryos from the extremes of lecithotrophic oviparity to matrotrophic viviparity. This group therefore provides excellent comparative opportunities for studying endocrine and immune involvement during pregnancy, and their possible interactions. We studied the cottonmouth (Agkistrodon piscivorus), since they exhibit limited placentation (e.g., ovoviviparity), allowing comparison with squamate species hypothesized to require considerable maternal immune modulation due to the presence of a more extensive placental connection. Furthermore, the cottonmouth's biennial reproductive cycle provides an opportunity for simultaneously comparing pregnant and non-pregnant females in the wild. We document significantly elevated concentrations of progesterone (P4) and significantly lower concentrations of estradiol (E2) in pregnant females relative to non-pregnant females. Pregnant females had lower plasma bacteria lysis capacity relative to non-pregnant females. This functional measure of innate immunity is a proxy for complement performance, and we also determined significant correlations between P4 and decreased complement performance in pregnant females. These findings are consistent with studies that have determined P4's role in complement modulation during pregnancy in mammals, and thus this study joins a growing number of studies that have demonstrated convergent and/or conserved physiological mechanisms regulating viviparous reproduction in vertebrates.