Isolation by preparative isoelectric focusing of a direct acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix (southern copperhead)

Fibrolase, a blood clot-lysing enzyme, was isolated from the venom of the snake Agkistrodon contortrix contortrix using preparative scale isoelectric focusing in the recycling isoelectric focusing (RIEF) apparatus. Two sequential purifications, beginning with 1.0 g of whole, dried venom, were employed. A pH 6-8 range gradient effected the first separation. While 100% of the enzyme was recovered in three fractions, 43% (one fraction) had 70% purity. The second run was a refractionation of three, pooled fractions from the first run, in a 0.7 pH range gradient. Of the fibrolase in the venom, 63% was recovered in four fractions. One of these represented 29% of venom fibrolase, with 97% purity. Gel filtration chromatography removed most of the remaining, higher molecular weight contaminants of the RIEF-purified enzyme.     

Fibrinopeptide-releasing enzymes in the venom from the southern copperhead snake (Agkistrodon contortrix contortrix)

Protein fractionation techniques utilizing the different properties of the sample (size, charge, sugar moiety) were employed to characterize the crude A. c. contortrix venom. Gel filtration chromatography resolved about six to eight peaks, high performance liquid ion exchange chromatography and chromatofocusing about 12-14 peaks exhibiting hydrolytic activity. Two fibrin clot-promoting enzymes--both releasing fibrinopeptides A and fibrinopeptides B, but with different fibrinopeptide A/fibrinopeptide B relative rates were observed. The two enzymes (or enzyme isoforms) were serine proteinases with essentially the same hydrolytic activity towards low molecular chromogenic substrate for thrombin. They were of approximately the same size (one peak of 68,000 relative mol.wt on gel filtration chromatography), had apparent isoelectric points of about 6.4 and 5.4, respectively, and were glycoproteins.     

Purification and characterization of fibrolase isoforms from venom of individual southern copperhead (Agkistrodon contortrix contortrix) snakes

Fibrolase, a zinc metalloproteinase possessing direct-acting fibrinolytic activity, has been previously purified from southern copperhead (Agkistrodon contortrix contortix) snake venom. We recently reported that a pool of southern copperhead venom from different geographical locations possesses two isoforms of fibrolase (fib1 and fib2) [ , S. L. et al. (1994) J. Chromat. B, in press]. We now report that venom from individual southern copperhead snakes contains the two isoforms which can be separated by a three-step high performance liquid chromatography (HPLC) procedure consisting of hydrophobic interaction chromatography, hydroxylapatite chromatography and weak cation exchange chromatography. Utilizing mass spectrometry we determined that fib1 has a molecular mass of 22,879 atomic mass units (amu) compared to 22,753 amu for fib2. These results support earlier observations during amino acid sequence analysis that a truncated version of the enzyme is produced which is missing the amino-terminal amino acid (vs. the intact enzyme 50 values (concentration of enzyme required to degrade 50% of fibrin in a micro-fibrin plate assay) are 6.4 (±1.0) μM and 5.2 (±0.8) μM for fib 1 and fib2, respectively. Therefore, loss of the amino-terminal amino acid does not appear to influence enzymatic activity. We conclude that the two isoforms of fibrolase arise from variations in the molecular processing of the enzyme by the snake venom gland rather than being caused by the pooling of southern copperhead venoms from different geographical locations.     

Characterization of the protein C activator Protac from the venom of the southern copperhead (Agkistrodon contortrix) snake

A single chain glycopeptide with a molecular weight of approximately 37,000, an isoelectric point of 3.0 +/- 0.2 and a carbohydrate content of approximately 20% was isolated from the venom of the southern copperhead Agkistrodon contortrix contortrix. It was capable of converting zymogen protein C in plasma of man and various vertebrates into its activated form, a serine proteinase which exerts an anticoagulant effect. Conversion of the zymogen protein C into the active proteinase was demonstrated by measuring the prolongation of the activated partial thromboplastin time due to proteolytic degradation of factors Va and VIIIa by the activation product, as well as by direct measurement of the generated enzyme activity by means of a synthetic chromogenic substrate. Intravenous injection of the venom protein C activator into rabbits caused prolonged activated partial thromboplastin time. Repeated subcutaneous injections led to formation of an antibody which formed, with purified protein C activator as well as with crude A. contortrix venom, a precipitating complex devoid of protein C activator potency. As revealed by activity measurements and by immunodiffusion experiments, the venoms of various A. contortrix, A. bilineatus subspecies contain similar protein C activators.     

Yield of venom from the Osage copperhead,Agkistrodon contortrix phaeogaster

Venom was extracted from eighteen Osage copperheads, Agkistrodon contortrix phaeogaster, by electrical stimulation. Mean venom yield was 43.4 mg/snake and mean body length was 57.5 cm (snout-vent). Solid content was 28.3 mg dry venom/100 ml fresh venom. These yields were comparable to those obtained for three other subspecies.     

Inhibition of the protein C activator Protac, a serine proteinase from the venom of the southern copperhead snake Agkistrodon contortrix contortrix

ProtacR, the single chain glycoprotein in the venom of Agkistrodon contortrix snake subspecies and related pit vipers has found broad application as a protein C activator in blood coagulation research and diagnosis. Inhibition studies were performed to further elucidate the nature of this serine proteinase. Benzamidine derivatives are poor inhibitors of ProtacR. Only a few 4-amidinoanilides of N alpha-substituted omega-phenyl-alpha-aminoalkylcarboxylic acids exert potent inhibitory activities. The active site of ProtacR appears to be accessible only for inhibitors with well-fitting structural features.     

Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the x-ray structure of adamalysin II and atrolysin C

The fibrinolytic enzyme from southern copperhead snake venom, fibrolase, contains 1 mole of zine per mole of protein, belongs to the major family of metalloproteinases known as the metzincins, and has been shown to degrade fibrin clots in vitro and in vivo. The purpose of this study was to develop a 3-dimensional model of fibrolase to investigate the geometry of conserved and variable sequences between members of the snake venom metalloproteinases. When compared to atrolysin C (form D) or adamalysin II (metzincins with completely different substrate specificity), fibrolase has approximately 60% overall sequence identity and nearly 100% sequence similarity in the active site. We used the crystal structure of adamalysin II to build a 3-dimensional homology model of fibrolase. Three disulfide bonds were constructed (the highly conserved disulfide bond [118–198] was maintained from the adamalysin II structure and 2 new disulfide bonds were introduced between residues 158–182 and 160–165). We used Sculpt 2.5 and HyperChem 5.0 to “dock” a substrate fragment octapeptide (HTEKLVTS), and a water molecule into the active site cleft. We calculated the differential average homology profile for fibrolase compared to 8 hemorrhagic and 5 nonhemorrhagic metzincins. We then determined the sequence regions that might be responsible for their substrate specificity. Our 3-dimensional homology model shows that the variable sequences lie on the periphery of the identified active site region containing the His triangle; this indicates that substrate specificity may depend on surface residues that are not directly associated with the active site.     

Venom from southern copperhead snake (Agkistrodon contortrix contortrix). I. Characterization of a protease that preferentially releases fibrinopeptide B

Using gel permeation chromatography with high performance liquid chromatograph (HPLC), a highly purified preparation of a protease has been obtained from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix). Both gel permeation chromatography with HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that it had an apparent Mr of 60,000-64,000. It consisted of a single polypeptide chain. The activity was inhibited by dithiothreitol. It neither induced platelet aggregation nor activated plasma factor XIII. It cleaved fibrinopeptide B at a rate much faster than fibrinopeptide A from fibrinogen. This specificity was steadily lowered when the incubation temperature was elevated from 0 degrees C to 45 degrees C. Fibrinopeptides were released only at neutral pH.     

Venom from southern copperhead snake (Agkistrodon contortrix contortrix). II. A unique phospholipase A2 that induces platelet aggregation

A platelet aggregation factor was purified from the venom of southern copperhead snake (Agkistrodon contortrix contortrix) by DEAE-cellulose ion-exchange chromatography, precipitation with ammonium sulfate, affinity chromatography using bovine serum albumin as ligand, and gel filtration on Cellulofine GCL-2000. It had molecular weights of 11,000 and 14,000, as determined by gel filtration chromatography and sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), respectively. It consists of a single polypeptide, and was identified as a phospholipase A2. It was quite resistant to heat and various denaturing reagents including urea and SDS. It lost both phospholipase A2 activity and platelet aggregating activity upon modification of histidine residue(s) with p-bromophenacyl bromide. Its specificity towards the beta-position of phospholipid in esterolytic reaction was confirmed by gas-liquid chromatography using a pure synthetic phosphatidylcholine. Platelet aggregation by this phospholipase A2 was completely inhibited by prostacyclin, but was little inhibited by aspirin which indicates almost no direct participation of released arachidonic acid in the aggregation mechanism.     

The action of a fibrin-promoting enzyme from the venom of Agkistrodon contortrix contortrix on rat fibrinogen and plasma

The action of a fibrin-promoting enzyme isolated from the venom of A.c.contortrix was investigated. The ratio of fibrinopeptides A and B released was similar in isolated human and rat fibrinogen and human plasma, fibrinopeptide B always being released preferentially. However, no clotting occurred in rat plasma, and no fibrinopeptides were released even after prolonged incubation. The results suggest strong, fast-acting irreversible neutralization of the enzyme activity in rat plasma.     

Isoelectric focusing of Protac, the protein C activator from copperhead (Agkistrodon contortrix) venom: a note on experimental problems

The isoelectric point of Protac was recently estimated to be at pH 3. However, further investigations using different experimental procedures revealed that Protac, due to its particular binding properties, is able to form complexes with carrier ampholytes. Thus, the actual isoelectric point of Protac was found to be in the basic region.     

Enzymological characterization of FII(a), a fibrinolytic enzyme from Agkistrodon acutus venom

AIM: To study the enzymological characterization of a fibrinolytic enzyme (FII(a)) from Agkistrodon acutus venom. METHODS: The fibrinogenolytic effect and the influences of several protease inhibitors, chelating agents, and metal ions on fibrinogenolytic activity were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The metal content of FII(a) was determined by atomic absorption spectroscopy. RESULTS: After incubation with FII(a) (0.25 g/L), Aalpha-, Bbeta- and gamma-chains of fibrinogen disappeared within 5 min, 30 min, and 8 h , respectively. The molecular weights of major degradation products were 45,000 and 41,000, which were different from those bands produced by plasmin. The fibrinogenolytic activity of FIIa was strongly inhibited by ethylenediamine tetraacetic acid (EDTA), ethyleneglycol tetraacetic acid (EGTA), dithiothreitol and cysteine, but not by phenylmethyl-sulfonyl fluoride and soybean trypsin inhibitor. Zinc (3171+/-25 mg/kg), potassium (489+/-17 mg/kg) and calcium (319+/-13 mg/kg) were found in FIIa. Zn2+, Ca2+ and Mg2+ could recover the fibrinogenolytic activity of FIIa, which was inhibited by EDTA. Only Ca2+ could recover the fibrinogenolytic activity inhibited by EGTA. CONCLUSION: FIIa can degrade the Aalpha-, Bbeta- and gamma-chains of fibrinogen. FII(a) is a metalloproteinase, and Zn2+, Ca2+, and disulfide bonds are necessary for its fibrinogenolytic activity.     

Resistance of the prairie vole (Microtus ochrogaster) and the woodrat (Neotoma floridana), in Kansas, to venom of the osage copperhead (Agkistrodon contortrix phaeogaster)

Prairie vole (Microtus ochrogaster) serum has no anti-lysing or antibody activity against Osage copperhead (Agkistrodon contortrix phaeogaster) venom. However, the serum has an anti-hemorrhagic component, which significantly reduces the size of hemorrhage produced by the minimal hemorrhagic dose of venom and which blocks the minimal hemorrhagic dose at a dilution of 1/8. Woodrat (Neotoma floridana) serum also has an antihemorrhagic component which blocks the minimal hemorrhagic dose at a dilution of 1/32. When compared to similar sized rodents previously tested in Texas, the anti-hemorrhagic activity in the serum of the Kansas rodents is similar. This suggests that venom resistance in prey items of venomous snakes may be more common than thought previously.     

Hemorrhagic activity and mechanism of FⅡa′a fibrinolytic enzyme from Agkistrodon acutus venom

AIM: To study the local hemorrhagic activity of a fibrinolytic enzyme (FⅡa) from Agkistrodon acutus venom and its mechanism. METHODS: The local hemorrhagic activity was determined by subcutaneous injection on the back of mouse. The effects of FⅡa on factor X, prothrombin, gelatin, and collagen were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Platelet aggregation assays were performed in rat platelet-rich plasma (PRP). Human umbilical vein endothelial cells (HUVEC) were cultured and passaged in complete M 199 medium. Cell viability and nuclear morphology change were determined by fluorescein diacetate (FDA) staining and Hoechst 33258 staining, respectively. RESULTS: The minimum hemorrhagic dose (MHD) of FⅡa was 89 μg.In vitro, FⅡa (0.25 g/L) degraded factor X, prothrombin, collagen, and gelatin, and dose-dependently (0.25, 0.50,0.75, and 1.00 g/L) inhibited the platelet aggregation induced by ADP in rat PRP. When HUVEC in culture treated with FⅡa, HUVEC showed detachment in a dose-dependent manner, but no apoptosis sign was observed.CONCLUSION: FⅡa had local hemorrhagic activity, and the mechanism was related to the degradation of factor X,prothrombin, gelatin, and collagen, the inhibition of ADP-induced platelet aggregation, and inducement of HUVEC detachment.     

Biochemical characterization of a thrombin-like enzyme and a fibrinolytic serine protease from snake (Agkistrodon saxatilis) venom.

A thrombin-like enzyme and a fibrinolytic serine protease were purified to homogeneity from the venom of a Korean snake Agkistrodon saxatilis emelianov. Both the purified enzymes migrated as a single protein band corresponding to 39 kDa in SDS-PAGE. However, the molecular mass was reduced to 28 kDa by enzymatic removal of the N-linked carbohydrates in those two different enzyme species. Although the thrombin-like enzyme and the fibrinolytic protease show homologous features in their molecular sizes and N-terminal amino acid sequences, yet they can be clearly distinguished from each other in terms of substrate specificity, susceptibility to inhibitors and fibrinogen degradation. It is postulated that these two enzymes are capable of functioning in a cooperative manner to effectively remove fibrinogen and consequently to reduce the blood viscosity.     

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom.

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom. Toxicon15, 161–167, 1977.—In addition to fibrinolytic, fibrinogenolytic and caseinolytic activities, the purified fibrinolytic principle of Agkistrodon acutus venom possessed hemorrhagic activity. Trasylol had a much higher inhibitory action on the fibrinolytic activity of the fibrinolytic principle of the venom than did ε-aminocaproic acid. Thus, the fibrinolytic action of the fibrinolytic principle was chiefly due-to a direct action on fibrin. Both EDTA (5 × 10^-4 M) and cysteine (5 × 10^-3 M) completely inhibited the fibrinolytic, fibrinogenolytic, hemorrhagic and caseinolytic activities of the fibrinolytic principle of the venom. Disulfide bonds might be essential for the biological activities of the fibrinolytic principle.     

The effect of the fibrinolytic enzyme FIIa from Agkistrodon acutus venom on acute pulmonary thromboembolism

Aim:

To evaluate the effects of the fibrinolytic enzyme FII_a from Agkistrodon acutus venom on acute pulmonary thromboembolism (APT) in animal models.

Methods:

Both rabbit and dog APT models were used. For the rabbit APT model, the thrombi weight before and after administration was measured. Central venous pressure (CVP) and mean arterial pressure (MAP) were measured before and 15, 30, 60, and 120 min after the injection of the blood clot. Partial thromboplastin time (APTT), prothrombin time (PT), platelet count, and fibrinogen concentration were measured using auto analyzers. Plasminogen activity was measured based on chromogenic substrates. In the dog APT model, pulmonary blood flow was recorded using pulmonary angiography.

Results:

Intravenous administration of FIIa (0.1–5.0 mg/kg) improved the APT-induced hemodynamic derangements and reduced thrombi weight. The angiography evidence also showed that the pulmonary emboli had almost disappeared after FII_a infusion. FII_a (0.1, 0.5, or 1.0 mg/kg) did not impair the coagulation pathways, although very high doses of FII_a (5.0 mg/kg) could stimulate the production of plasminogen and result in impairment of the pathways.

Conclusion:

FII_a could effectively protect against APT via degradation of thrombi with less activation of plasminogen, and may provide a novel fibrinolytic enzyme for targeting the main pathological processes of the disease.     

Purification and biochemical characterization of F II(a), a fibrinolytic enzyme from Agkistrodon acutus venom.

A fibrinolytic enzyme, F II(a), was isolated from Agkistrodon acutus venom by ion-exchange chromatography and gel filtration. F II(a) consisted of a single polypeptide chain with a molecular weight of 26,000 and an isoelectric point of 4.6. F II(a) was shown to solubilize fibrin and fibrinogen. F II(a) cleaved, primarily, the alpha chain of fibrinogen and fibrin followed by the beta chain, while the gamma chain was minimally affected. Thus, the enzyme was an alpha,beta-fibrinogenase. The cleavage pattern of fibrinogen clearly varied from plasmin cleavage of the same molecule. In vivo, F II(a) had no influence on the rat's tissue-type plasminogen activator and plasminogen activator inhibitor-1 activities in plasma. At the dosage of 5mg/kg, histological examination of heart, liver and lung tissue showed no hemorrhage. F II(a) is an enzyme that hydrolyzed fibrin directly without hemorrhagic activity.     

Snake venom NAD nucleosidase: its occurrence in the venoms from the genus Agkistrodon and purification and properties of the enzyme from the venom of A. halys blomhoffii.

Nicotinamide adenine dinucleotide nucleosidase (NAD glycohydrolase, EC 3.2.2.5) was demonstrated in venoms of various snakes. Among the venoms from 37 species of Viperidae, Crotalidae and Elapidae, venom of snakes in the genera Bungarus and Agkistrodon showed the highest activities. No NAD nucleosidase activity was detected in venoms of the Naja genus.A NAD nucleosidase found in the venom of Agkistrodon halys blomhoffii was purified by successive column chromatographies on DEAE-cellulose and DEAE-Sephadex A-50 and gel-filtration on Sepharose 6B. Final purification was over 25-fold with 20 per cent yield. The purified enzyme showed maximal activity at pH 7.5 and hydrolysed β-NAD⁺, NADP⁺ and 3-acetylpyridine adenine dinucleotide. No hydrolysis could be observed on α-NAD⁺, NADH, NADPH and NMN⁺. The Km values for β-NAD⁺, NADP⁺ and 3-acetylpyridine adenine dinucleotide were 8.3 × 10^?4 M and 7.4 × 10^?4 M, respectively. Over 60 per cent inhibition of β-NAD⁺ hydrolysis was observed in the presence of 10^?3 M of HgCl₂ and cysteine.