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

OBJECTIVETo explore the clinical effect of Agkistrodon antithrombogenase (AAT) in the treatment of rheumatoid arthritis (RA) and its possible mechanism.

METHODSBesides the conventional non-steroid anti-inflammatory agents and disease-modifying anti-rheumatic drug, patients were treated supplementally with intravenous injection of AAT. The intracutaneous test showed allergic to AAT patients were treated with Salvia injection and taken as control group. Changes of related clinical indexes in the two groups were observed.

RESULTSAfter 3 weeks treatment, condition of patients in both groups were improved clinically in joint swollen index, joint tenderness index, morning stiffness time, pain assessment (VAS) and health assessment questionnaire (HAQ) on daily life activity as well as ESR level (P < 0.05 or P < 0.01), with the VAS, HAQ and fibrinogen levels more significantly improved than those of control (P < 0.05 or P < 0.01), and the total effective rate higher in the AAT treated group than those in the control group (P < 0.05).

CONCLUSIONAAT has good effect on easing clinical symptoms of RA possibly through anti-inflammation and improving the microcirculation with less toxic and adverse reaction, so is worthy of recommendation.

Adult ; Ancrod ; therapeutic use ; Anti-Inflammatory Agents, Non-Steroidal ; therapeutic use ; Arthritis, Rheumatoid ; drug therapy ; Crotalid Venoms ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies

Agkistrodon acutus is a traditional Chinese herb medicine which has immunological regulation,anti-tumor,anti-inflammatory and analgesic effects,which is mainly used for the treatment of rheumatoid arthritis,ankylosing spondylitis,sjogren's syndrome and tumors. In order to excavate more important functional genes from A. acutus,the transcriptome of the venom gland was sequenced by the Illumina Hi Seq 4000,and 32 862 unigenes were assembled. Among them,26 589 unigenes were mapped to least one public database. 2 695 unigenes were annotated and assigned to 62 TF families,and 5 920 SSR loci were identified. The majority of mapped unigenes was from Protobothrops mucrosquamatus in the NR database,which revealed their closest homology. Three secretory phospholipase A_2 with different amino acid sequences showed similar spatial structures and all had well-conserved active sites. The 3 D structural models of C-type lectin showed conserved glycosylation binding sites( Asn45). This study will lay the foundation for the further study of the function of snake venom protein,and promoting the development and utilization of genome resources from A. acutus.
Agkistrodon/genetics* ; Animals ; Crotalid Venoms ; Gene Expression Profiling ; Snake Venoms/genetics* ; Snakes ; Transcriptome

The infrared (IR) receptors in the pit organ of crotaline snakes are very sensitive to temperature. The sensitivity to IR radiation is much greater in crotaline snakes than in boid snakes because they have a thermosensitive membrane suspended in a pair of pits that comprise the pit organ. The vasculature of the pit membrane, which is located near IR-sensitive terminal nerve masses, the IR receptors, supplies the blood necessary to provide cooling and the energy and oxygen that the IR receptors require. The ophthalmic and maxillary branches of the trigeminal nerve innervate the pit membrane. In crotaline snakes, the trigeminal ganglion (TG) is divided into the ophthalmic and maxillomandibular ganglia; a prominent septum further separates the two divisions of the maxillomandibular ganglion. The TG neurons in the ophthalmic ganglion and the maxillary division of the maxillomandibular ganglion relay IR sensation to the brain. This article reviews the IR-sensitive pit organ and trigeminal sensory system structures in crotaline snakes.
Brain ; Crotalid Venoms ; Equipment and Supplies ; Ganglion Cysts ; Membranes ; Neurons ; Oxygen ; Sensation ; Snakes ; Trigeminal Ganglion ; Trigeminal Nerve

Snake venom proteins,particularly from the viper and elapid families, have been known to contain a number of platelet active components including what cause platelet aggregation or inhibit platelet aggregation. Some of them have potential clinical usefulness for the treatment of human hemorrhagic or thrombotic disease. Agkistrodon halys pallas belonging to viper family is only growing in China. The aim of this study was to purify a human platelet aggregation inhibitor from venom of Agkistrodon halys pallas and determine its biochemical character. Whether a component could inhibit human platelet aggregation was act as a method to follow the tracks of the protein. Crude venom of Agkistrodon halys pallas was loaded onto a DEAE-Sepharose CL-6B chromatography column could gain 6 peaks. A platelet inhibitor with molecular mass of 65 kD on SDS-PAGE, was purified from peak 2 by Sephadex G-75 gel filtration and SP-Sepharose, Mono Q on FPLC. It could inhibit human platelet aggregation induced by ADP, collagen without activities of phospholipase A2, esterase, fibrinogenolytic. It is concluded that a platelet inhibitor can be isolated and purified from venom of Agkistrodon halys pallas and its inhibition of platelet aggregation is does-dependent.
Crotalid Venoms ; analysis ; Humans ; Oligopeptides ; chemistry ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; isolation & purification

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

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

To investigate the effects of component I from Agkistrodon acutus venom (AAVC-I) on the biological features of chronic myeloid leukemia cells, K562/A02 leukemia cells were cultured in the presence of AAVC-I (6.25 - 100 µg/ml) and the proliferation status was assayed by CCK-8 method. Morphological changes were observed by inversed microscope after Giemsa and Hochest 33258 staining, and cell apoptosis was detected by flow cytometry. Caspase 3 activity was tested by using Chromogenic Activity Assay Kit. The results showed that AAVC-I inhibited the growth of K562/A02 cells in time- and concentration-dependant manners, and the IC(50) at 48 h was 30.988 µg/ml. Giemsa and Hochest 33258 staining showed the typical apoptotic features in K562/A02 cells after induction with AAVC-I for 48 h. Flow cytometric analysis revealed that the percentage of the apoptotic cells reached from 0.88 up to 53.66 as the treated concentration was elevated from 0 to 50 µg/ml. Compared with the control group, the expression of caspase 3 in the tested group was enhanced in a dose-dependent manner (P < 0.05). It is concluded that AAVC-I can effectively inhibit the growth and promote apoptosis of K562/A02 cells. Elevated expression of caspase-3 may be attributed to the apoptosis of K562/A02 cells.
Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Crotalid Venoms ; pharmacology ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; Leukemia ; metabolism ; pathology

To study the long-term toxicity of arginine esterase from Agkistrodon halys ussuriensis venom for the clinical application of arginine esterase in the future. Beagle dogs were used as experimental animals and were divided into control group, arginine esterase low dose group (0.06 u/kg), the middle dose group (0.18 u/kg) and high dose group (0.36 u/kg). Every group consisted of four dogs. The arginine esterase was given intravenously once a day for 180 days. Then three dogs in each group were sacrificed and the fourth one was fed without injecting arginine esterase for 15 days. The toxic reactions during treatment and recovery period were determined by evaluating and comparing the general criteria ( including locomotor activity, growth rate, appetite and death rate), clinical criteria (including blood test and urine test), pathological dissection and viscera coefficient of the treated animals and the control animals. There were no significant differences in general criteria. The clinical criteria of the treated animals were the same as those of the control animals except liver function. There were no significant differences in pathological dissection and viscera coefficient between the treated animals and the control animals except livers. The livers in high dose arginine esterase treated animals were swollen and vacuolated and there was significant difference in liver coefficient between them (P<0.05). The toxic symptom of liver disappeared after withdrawal of treatment. From these results, the non-toxic dose of arginine esterase for dogs was estimated to be 0.18 u/kg under the present study conditions and is about 15 times the clinical dosage for using the drug "Qingshuanmei" of which the main component is arginine esterase. The long-term toxicity test result indicates that the toxicity of pure arginine esterase is lower than that of "Qingshuanmei", suggesting that clinical use of the arginine esterase is safe.
Animals ; Anticoagulants ; toxicity ; Carboxylic Ester Hydrolases ; toxicity ; Crotalid Venoms ; enzymology ; toxicity ; Dogs ; Female ; Kidney ; drug effects ; pathology ; Liver ; drug effects ; pathology ; Male

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