Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aalpha-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity.
Agkistrodon ; Animals ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Serine Endopeptidases ; biosynthesis ; genetics ; Thrombin ; biosynthesis ; genetics ; Viper Venoms ; enzymology

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.
Agkistrodon/metabolism* ; Animals ; Cell Line, Tumor ; Healthy Life Expectancy ; Humans ; Matrix Metalloproteinase 2/metabolism* ; Neovascularization, Pathologic/metabolism* ; Triple Negative Breast Neoplasms/metabolism* ; Venoms

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.
Agkistrodon ; metabolism ; Animals ; Collagen Type II ; chemistry ; isolation & purification ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Mass Spectrometry ; Reptilian Proteins ; chemistry ; isolation & purification ; metabolism

This study was purpose to investigate apoptosis pathway of leukemia K562 cells induced by anticoagulant fraction from Agkistrodon acutus venom (AVVC-1). The mitochondrial transmembrane potential (ΔΨm) of leukemia K562 cells was detected by flow cytometry with JC-1 single staining. The expression of cytochrome C in the mitochondrial of leukemia K562 cells was analyzed by Western blot after AVVC-1 treatment. The distribution of cytochrome C in leukemia K562 cells was measured by immuno-fluorescence test. The results showed that the potential of mitochondrial membrane decreased after treatment with different concentrations of AVVC-1 (12.5, 25, 50, 100 µg/ml) for 6 h (P < 0.01). The expression level of cytochrome C protein in mitochondria obviously declined after treatment with 30 µg/ml AVVC-1 for 48 h, and the fluorescent intensity of cytochrome C in cytosol was enhanced at the same time. It is concluded that AVVC-1-induced K562 cell apoptosis is related with mitochondrial damage, and cytochrome C may be a useful agent for investigating human leukemia therapy by using AVVC-1.
Agkistrodon ; Animals ; Apoptosis ; drug effects ; Cytochromes c ; metabolism ; Humans ; K562 Cells ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; metabolism ; Snake Venoms ; pharmacology

This study was aimed to investigate the hemostatic effects of hemocoagulase agkistrodon (HCA) and its mechanism. The procoagulative and hemostatic effects of HCA were evaluated by using rabbit blood coagulatin time and mouse tail bleeding time; the mechanisms of HCA hemostatic effect were analyzed by using rabbit blood clot lysis and fibrinogen lysis. The results showed that HCA shortened the rabbit blood coagulation time and the mouse tail bleeding time significantly. The effects are nearly similar to that of positive control (reptilase). HCA also induced rabbit blood clot lysis and directly hydrolysed the alpha-chain of fibrinogen. It is concluded that HCA exert its hemostatic effects by hydrolysing the alpha-chain of fibrinogen, but it is not able to induce production of XIII factor.
Agkistrodon ; Animals ; Batroxobin ; isolation & purification ; pharmacology ; Bleeding Time ; Blood Coagulation ; drug effects ; Crotalid Venoms ; enzymology ; Fibrinogen ; metabolism ; Hemostatics ; pharmacology ; Mice ; Rabbits ; Random Allocation

The study was purposed to investigate the effect of extract of Agkistrodon Halys venom on proliferation and apoptosis of K562 cells. The inhibition of K562 cell proliferation was measured by MTT assay; The morphologic changes of K562 cells was observed by microscopy; the apoptosis of K562 cells was measured by flow cytometry; the activity of extracellular signal-regulated kinase (ERK) in K562 cells was detected by Western blot. The results showed that when K562 cells were treated with 0, 1, 10, 20 microg/ml of the extraction for 48 hours, the apoptosis rates were 2.1%, 21.3%, 49.7%, 70.1%, respectively. The proliferation of K562 cells was obviously inhibited in dose-dependent manner. Typical morphologic changes significantly appeared in the extract-treated K562 cells. The extract obviously inhibited the activity of ERK in K562 cells. It is concluded that the extract of Agkistrodon Halys' venom can inhibit the proliferation of K562 cells and induce apoptosis of K562 cells.
Agkistrodon ; Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Complex Mixtures ; pharmacology ; Crotalid Venoms ; chemistry ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; K562 Cells

BACKGROUNDSnake venom contains a number of components with different pharmacological and biological activities, especially in cancer therapy, and has increasingly become a research focus. This study was designed to isolate and purify a novel anti-clotting protein component from the venom of Agkistrodon acutus, and to explore its physico-chemical properties and biological activity.

METHODSThe venom of Agkistrodon was isolated and purified by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sepharose Fast Flow, molecular sieve filtration through Sephadex G75, SP-Sepharose Fast Flow and molecular sieve filtration through Sephadex G50. We detected the activated partial thromboplastin time (APTT) of the eluant to select the anti-clotting protein component of interest. The molecular weight was determined by sodium dodecyl sulfate-polyacrylamid gel electrphoresis (SDS-PAGE) and liquid chromatography. Its protein content was detected by bicinchoninic acid (BCA).

RESULTSSDS-PAGE vertical gel electrophoresis showed that the anticoagulant factor is a tripolymer composed of three proteins whose molecular weights are 25 KDa, 30 KDa and 50 KDa. The factor contains about 65% percent protein.

CONCLUSIONSA novel anti-clotting protein component was purified by ion-exchange chromatography and molecular sieve filtration from the venom of Agkistrodon acutus and was found to be composed of three kinds of proteins.

Agkistrodon ; metabolism ; Animals ; Anticoagulants ; chemistry ; isolation & purification ; Chromatography, High Pressure Liquid ; Chromatography, Ion Exchange ; Crotalid Venoms ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Proteins ; chemistry ; isolation & purification

A novel serine protease with high purity was extracted from the venom of Agkistrodon hlays Pallas using monoclonal antibody affinity chromatography. This protease releases bradykinin and has arginine esterase activity without being activated. After purification, its hydrolytic activity exceeded 800 U/mg, far higher than its counterparts from mammalian sources. The purity of the kininogenase could exceed 95%. The acute toxicity and the long-term toxicity of this kallikrein was studied for its potential clinical application. The maximum tolerance dose in adult was 150,000 times greater than the maximum applied dose, and long-term administration at the dose 50 times of allowed clinical dose did not obviously after the animals' body weight, survival condition, liver function, renal function, or blood routines, suggesting the extremely low toxicity of the kallidrein.
Agkistrodon ; Animals ; Bradykinin ; metabolism ; Crotalid Venoms ; toxicity ; Kallikreins ; toxicity ; Maximum Tolerated Dose ; Mice ; Mice, Inbred BALB C ; Serine Endopeptidases ; toxicity ; Toxicity Tests, Acute

OBJECTIVETo evaluate the efficacy and safety of hemocoagulase acutus for injection and determine its curative dose.

METHODSForty-five patients on abdominal surgeries were randomly allocated into 2 study groups and 1 control group. Thirty minutes before the operation, the patients in the study groups received intravenous hemocoagulase acutus at 1 U and 2 U, respectively, and control group had no treatment. The hemostatic time, hemorrhagic volume, and hemoagglutination were observed in all the groups.

RESULTSThe average hemorrhagic volume and hemorrhagic volume per square were significantly lower in the two study groups than in the control group (P<0.05), and the average hemorrhagic volume per square were significantly lower in study group 2 U than in the 1 U group (P<0.05). No significant differences were found in adverse effects between the 3 groups.

CONCLUSIONHemocoagulase acutus for injection has good hemostatic effect for controlling capillary hemorrhage at the abdominal incisions and can be safely used in the surgical patients.

Abdomen ; surgery ; Adolescent ; Adult ; Aged ; Agkistrodon ; metabolism ; Animals ; Batroxobin ; administration & dosage ; therapeutic use ; Blood Coagulation ; drug effects ; Blood Loss, Surgical ; prevention & control ; Hemostasis, Surgical ; methods ; Hemostatics ; administration & dosage ; therapeutic use ; Humans ; Injections, Intravenous ; Male ; Middle Aged ; Treatment Outcome ; Young Adult

OBJECTIVETo determine the effective dosage and formulation of agkistrodon in collagen-induced arthritis (CIA) rats.

METHODSCIA 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.

RESULTSAmong 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.

CONCLUSIONThese 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.

Agkistrodon ; metabolism ; Animals ; Antibodies ; blood ; Arthritis, Experimental ; blood ; chemically induced ; drug therapy ; Collagen Type II ; immunology ; Dosage Forms ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Extremities ; diagnostic imaging ; pathology ; Female ; Interleukin-1beta ; blood ; Medicine, Chinese Traditional ; Rats, Wistar