Agkistrodon Contortrix Venom Research Articles

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.

Genome wide association study for plasma levels of natural anticoagulant inhibitors and protein C anticoagulant pathway: the MARTHA project

Deficiencies of antithrombin (AT), protein C (PC) and protein S (PS) or an impaired PC anticoagulant pathway increase the risk of venous thrombosis (VT). By conducting a genome-wide association study (GWAS) on two independent samples of VT patients totalling 951 subjects typed for 472 173 single nucleotide polymorphisms (SNPs), we observed that common SNPs explain 21% and 27% of the genetic variance of plasma AT and PS levels, even though no SNP reached genome-wide significance. For PC, we showed that two PROCR SNPs, rs867186 (Ser219Gly) and rs6060278, additionally explained c. 20% (P = 1·19 × 10(-31)) of the variance of plasma PC levels. We also observed that c. 40% of the remaining genetic variance of PC levels could be due to yet unidentified common SNPs. The PROCR locus was also found to explain c. 8% (P < 10(-10)) of agkistrodon contortrix venom (ACV) (exploring the PC pathway) variability which was under the main control of the F5 and F2 loci that further explained about 40% and 10%, respectively. We presented here the first GWAS for plasma AT and free-PS levels and ACV in Caucasian samples. We identified three independent loci associated with ACV (F2, F5 and PROCR) and replicated two independent effects on plasma PC levels at the PROCR locus.

Anticoagulant Response to Agkistrodon Contortrix Venom (ACV test): a New Global Test to Screen for Defects in the Anticoagulant Protein C Pathway

As specific assays used to identify defects in the protein C (PC) anticoagulant pathway are laborious and expensive, we describe here a global test to screen for these defects. This assay is expressed as the ratio of two activated partial thromboplastin times, one in the absence and one in the presence of 0,125 U/ml of the PC activator of Agkistrodon contortrix venom (ACV). Eight of the 168 healthy volunteers of the control group exhibited an ACV ratio below the lower normal limit of 3.37 [6 subjects with the mutation Arg 506 to Gln in their factor V gene (FV R506Q) and one with PS deficiency]. 128 patients who have had at least one episode of deep-vein thrombosis were retrospectively studied. All patients carrying FV Q506R (n = 48), PC deficiency (n = 14) or combined defects, i.e. FV Q506R and PC deficiency (n = 4) or FV Q506R and PS deficiency (n = 3), had ACV ratios < 3.37. PS deficient plasmas (n = 20) exhibited ACV ratios which overlapped normal range. ACV ratios of one out of seven patients with antithrombin deficiency, and 10% of patients without identified defect in the PC anticoagulant pathway (n = 30) were < 3.37. An ACV ratio raised to 3.70 could lead to a test identifying all patients with a defect in the PC anticoagulant pathway.

Skin necrosis associated with acquired protein C deficiency in patients with renal failure and calciphylaxis

purpose: To determine if the natural anticoagulant protein C plays a role in the pathogenesis of systemic calciphylaxis, a syndrome characterized by extensive vascular and soft tissue calcification and skin necrosis, which is similar to that seen in warfarin-induced skin necrosis. patients and methods: The study population included five patients with end-stage renal disease and systemic calciphylaxis undergoing hemodialysis, 12 patients without evidence of calciphylaxis undergoing dialysis, eight patients with nephrotic syndrome, and eight normal healthy volunteers. Protein C antigen levels were measured by rocket immunoelectrophoresis, and functional activity was quantitated by a chromogenic assay and an anticoagulant assay utilizing the venom of Agkistrodon contortrix. results: Skin biopsy specimens of involved areas in three patients showed thrombotic occlusion of venules identical to that seen in warfarin-induced skin necrosis. Protein C antigen levels were normal in all groups. However, protein C activity was significantly reduced as measured by chromogenic (p<0.01) or anticoagulant assays (p<0.01) in patients with calciphylaxis compared with the other three groups. conclusion: These findings suggest that hypercoagulability due to functional protein C deficiency may contribute to thrombosis, resulting in skin necrosis and digital gangrene in systemic calciphylaxis.

心臓人工弁置換術あるいは末梢血管再建術後のＷａｒｆａｒｉｎ投与患者におけるＰｒｏｔｅｉｎ Ｃ活性の検討

Changes in protein C activity, protein C antigen, Vitamin K-dependent coagulation factors (II, VII, IX, X) and protein C induced vitamin K abscense of factor II (PIVKA-II) were investigated in 30 patients treated with Warfarin for long periods. Those included 23 patients with prosthetic heart valves and 7 patients reconstructed peripheral vessels. Protein C activity was measured by rapid assays. amidolytic and clotting time assays using a protein C activator (PROTAC®) from the venom of Agkistrodon contortrix. Protein C levels measured by amidolytic assay correlated significantly with the immunological assay (ELISA) (r=0.718, p<0.001), but the clotting time assay gave lower protein C values than the amidolytic and immunological assays. Furthermore, the ratio of protein C activity (clotting time assay) to protein C antigen tended to decrease against the increase in PIVKA-II. The comparison of the three PC assays to thrombotest indicated that highly significant correlation was found between clotting time assay and thrombotest (r=0.483, p<0.01), furthermore, protein C values measured by the clotting time assay correlated with prothrombin activity (r=0.598, p<0.001). However a decrease in the ratio of protein C activity (clotting time assay) to prothrombin activity was observed in three cases with prosthetic heart valves (ratios of 0.25, 0.28, 0.34 and the prothrombin activities of these cases were beyond 30%). These results indicate that the protein C activity measured by clotting time assay evaluates the in vivo function of protein C, and that protein C function as well as prothrombin activity is inhibited in patients on stable oral anticoagulant therapy.

Conversion of fibrinogen to fibrin induced by preferential release of fibrinopeptide B

Fibrin clot-promoting enzyme preferentially releasing fibrinopeptide B from fibrinogen was isolated from the crude venom of Agkistrodon contortrix and its mode of action was studied in detail. A purification procedure involving affinity chromatographies on immobilized lectin and arginine removed plasmin-like and kallikrein-like activities towards low-molecular-weight chromogenic substrates. Fibrin-promoting enzyme cleaved off only fibrinopeptides A and B from fibrinogen. The initial relative rate of release of fibrinopeptide B/fibrinopeptide A depended strongly on the presence of Ca2+. In the absence of Ca2+ the amount of fibrinopeptides released by fibrin-promoting enzyme at the gel point was greater. Fibrinopeptide B was no longer released before fibrinopeptide A from the non-polymerizing N-terminal disulphide knot of fibrinogen. Catalyzed by activated factor XIII, complete gamma-dimer and alpha-polymer formation was observed in fibrin from which only 23% of fibrinopeptide A, but 100% of fibrinopeptide B was released by fibrin-promoting enzyme. gamma-dimer formation markedly preceded alpha-polymer formation. These results strongly imply a similar overall arrangement of monomer molecules in this fibrin when compared with a thrombin-induced fibrin in which fibrinopeptide A is released before fibrinopeptide B. These observations support the concept that on fibrinopeptide B release from fibrinogen polymerization sites are exposed which reinforce the sites exposed on the release of fibrinopeptide A.

Rapid amidolytic assay of protein C in whole plasma using an activator from the venom of Agkistrodon contortrix.

A rapid assay for protein C activity in whole plasma using a protein C activator from the venom of Agkistrodon contortrix is described. Plasma samples are incubated with venom activator in the presence and absence of anti-protein C antibody for 8 minutes at 37 degrees C in microtiter plate wells, followed by addition of a chromogenic substrate for activated protein C and measurement of the absorbance at 405 nm after 10-20 minutes incubation at 37 degrees C. The difference in absorbance between plasma assayed in the presence and absence of anti-protein C antibody is used to calculate the protein C activity by reference to a dilution curve of normal plasma. With this assay, mean protein C activity in 33 normal subjects was 87%. Thirty-eight patients receiving warfarin had a mean protein C of 34%. Twenty-four patients with disseminated intravascular coagulation had a mean protein C of 24%. The venom activator appears to preferentially activate gamma-carboxylated protein C, indicating specificity for the physiologically active form of the molecule.

蛇毒を用いたプロテインＣ活性の測定 先天性プロテインＣ欠乏症，播種性血管内凝固症候群，血栓性血小板減少性紫斑病，糖尿病，肝障害およびワーファリン服用患者におけるプロテインＣ抗原との比較

Functional protein C (PC) activity (both anticoagulant and amidolytic) was measured using a specific PC activator extracted from the venom of Agkistrodon contortrix in 4 patients with hereditary PC deficiency, 37 with disseminated intravascular coagulation (DIC), 9 with thrombotic thrombocytopenic purpura (TTP), 8 with diabetes mellitus (DM), 8 with liver injury and 30 under long-term warfarin therapy. The results were compared with those obtained by an immunological assay (ELISA). In patients with hereditary PC deficiency, DIC and TTP, PC activity was closely related with PC antigen levels. Both functional and immunological PC were decreased in many patients with DIC, while most TTP patients had normal PC levels. Some DM patients had high PC antigen levels, and their PC activity was somewhat lower than the antigen levels. PC activity and antigen were decreased in patients with hepatic injury, but in some of them functional assays (especially anticoagulant assay) gave lower PC values than the immunoassay. During stabilized warfarin treatment, PC antigen and amidolytic activity were decreased in parallel, but a decrease in anticoagulant activity was more pronounced. In addition, only anticoagulant activity correlated significantly with thrombotest values.These results indicate that the measurement of PC activity is valuable for the assessment of the behavior of PC in various disease states. Only anticoagulat PC assay can evaluate the PC function in patients under oral anticoagulant therapy and in some patients with hepatic injury.

Distribution and pathology of copperhead ( Agkistrodon contortrix) venom

Rats injected i.p. or i.m. with varying doses of tagged Agkistrodon contortrix venom showed the largest amounts of venom in the kidney and liver at 24 hr, with a slightly different distribution for other organs, and the smallest amounts in the brain and spinal cord. However, the rats injected i.m. showed the highest level at the site of the injection. Edema was seen in both the s.c. and muscle tissues of the rats given the venom i.m., and focal hemorrhage was observed in rats receiving 25 mg/kg or more of venom.