Agkistrodon Acutus Snake Venom Research Articles

Biochemical and Hemostatic Mechanism of A Novel Thrombin-Like Enzyme

Thrombin-like enzyme (TLE) plays a significant role in vessel injury hemostasis. A novel snake venom TLE (Agacutin) was purified from Agkistrodon Acutus snake venom. Structural analysis indicated that Agacutin is a heterodimer that has a MW of 29,402 Da, a pI value of 5.39, and optimum activity at 35 °C and pH 7.5. The N-terminal 15 amino acid sequences of Agacutin are DSSGWSSYEGHEYYV (small subunit) and DCSSGWSSYEEHQYY (large subunit). In vitro studies indicated that the coagulation activity of Agacutin was activated by Ca +2 or inhibited by phenylmethanesulfonyl fluoride, but not influenced by heparin or hirudin. The arginine esterase activity and fibrinogen hydrolysis result showed that Agacutin only cleaves α-subunit and releases fibrinopeptide A. In vivo studies indicated that Agacutin iv (0.01-0.05 U/kg) shortened 30.2-49% of the rabbit blood clotting time, or ip (0.5-2.0 U/kg) shortened 29.7-73.1% of the mouse tail bleeding time. Agacutin does not influence APTT, platelet or euglobulin clotting time, and activate Factor II or XIII. It converts fibrinogen into the soluble fibrin that accelerates hemostasis at wound.

Determination of the structure form of the fourth ligand of zinc in Acutolysin A using combined quantum mechanical and molecular mechanical simulation.

Acutolysin A, which is isolated from the snake venom of Agkistrodon acutus, is a member of the SVMPs subfamily of the metzincin family, and it is a snake venom zinc metalloproteinase possessing only one catalytic domain. The catalytic zinc ion, in the active site, is coordinated in a tetrahedral manner with three imidazole nitrogen atoms of histidine and one oxygen atom. It is uncertain whether this oxygen atom is a water molecule or a hydroxide ion just from the three-dimensional X-ray crystal structure. The identity of the fourth ligand of zinc is theoretically determined for the first time by performing both combined quantum mechanical and molecular mechanical (QM/MM) simulation and high-level quantum mechanical calculations. All of the results obtained indicate that the fourth ligand in the active site of the reported X-ray crystal structure is a water molecule rather than a hydroxide anion. On the basis of these theoretical results, we note that the experimental observed pH dependence of the proteolytic and hemorrhagic activity of Acutolysin A can be attributed to the deprotonation of the zinc-bound water to yield a better nucleophile, the hydroxide ion. Structural analyses revealed structural details useful for the understanding of acutolysin catalytic mechanism.

Isolation and characterization of ACTX-6: a cytotoxic L-amino acid oxidase from Agkistrodon acutus snake venom

The characterization of an L-amino acid oxidase purified from Agkistrodon acutus snake venom was investigated. An L-amino acid oxidase (LAAO) was purified from A. acutus snake venom through DEAE Sepharose F.F. and Source 30 S chromatography. The molecular mass of this enzyme was determined by SDS-PAGE, size exclusion chromatography, and mass spectrometry. Substrate specificity, cytotoxicity, antitumor activity in vivo, and apoptosis-inducing activity were assayed. The LAAO purified from A. acutus snake venom was designated as ACTX-6. It is a covalently bound homodimer and its molecular mass is about 96 kDa. This enzyme preferred to oxidize hydrophobic L-amino acids; the best substrates were L-Met, L-Leu, L-Trp, and L-Phe. ACTX-6 demonstrated cytotoxicity in vitro and could inhibit tumor growth in vivo. Flow cytometry analysis showed that it could markedly increase accumulation of sub-G1 phase, which suggested that this enzyme could induce apoptosis. ACTX-6 could effectively inhibit tumor growth and it is a potential substance to develop into an antitumor drug.

A cytotoxin isolated from Agkistrodon acutus snake venom induces apoptosis via Fas pathway in A549 cells

ACTX-6 is a protein isolated from Agkistrodon acutus snake venom and demonstrated cytotoxic activity to various cancer cells in vitro. In this paper the exact mechanism in ACTX-6-induced cell death was investigated and it was found that ACTX-6 could induce cell apoptosis. The results of Western blot and RT-PCR showed that ACTX-6 could induce Fas and FasL protein expression. When Fas signaling pathway was blocked by neutralizing antibodies to Fas or FasL, ACTX-6-induced apoptosis was inhibited. DISC formation was also detected by immunoprecipitation. These results suggested that Fas pathway was involved in ACTX-6-induced apoptosis. The activities of caspase-3, 8 and 9 were assayed and the activation of caspase-9 demonstrated that mitochondrial pathway was also involved in ACTX-6-induced apoptosis. Bid cleavage and dissipation of mitochondrial membrane potential (Δ ψ m) verified the involvement of mitochondria. ACTX-6 is an l-amino acid oxidase and can oxidize l-amino acid to generate hydrogen peroxide. The production of ROS in ACTX-6-treated cells was detected and the ROS scavenger catalase could inhibit ACTX-6-induced apoptosis. Western blot analysis showed that JNK was phosphorylated in ACTX-6-treated cells and c-Jun was also activated. JNK inhibitor SP600125 could inhibit ACTX-6-induced apoptosis and catalase could inhibit JNK and c-Jun phosphorylation. It could be concluded that JNK pathway was necessary in ACTX-6-induced apoptosis and the oxidative stress generated by ACTX-6 was responsible for the activation of JNK.

ACTX-8, a cytotoxic l-amino acid oxidase isolated from Agkistrodon acutus snake venom, induces apoptosis in Hela cervical cancer cells

ACTX-8 is a protein isolated from Agkistrodon acutus snake venom in our laboratory. It demonstrates cytotoxic activity on various carcinoma cell lines in vitro. However, the mechanism by which ACTX-8 inhibits cell proliferation remains poorly understood. In this study the influence of ACTX-8 on the activation of apoptotic pathway in Hela cells was investigated. We demonstrated that cell death induced by ACTX-8 was concentration- and time-dependent. Apoptotic changes such as phosphatidyl serine externalization and DNA fragmentation were detected in ACTX-8-treated cells. Caspase activation and reactive oxygen species (ROS) production were involved in ACTX-8-induced apoptosis, but pan caspase inhibitor, z-VAD-fmk, could not inhibit cell death induced by ACTX-8 completely, which proved the existence of another pathway for ACTX-8-induced cell death. We found cytochrome c release into cytosol and mitochondrial membrane potential (MMP) dissipation in ACTX-8-treated cells, which indicated that mitochondrial pathway played a role in ACTX-8-induced cell apoptosis. The ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members was not changed by ACTX-8 treatment. However Bad and Bax were translocated from cytosol into mitochondria, and the coimmunoprecipitation result indicated that in mitochondria Bak and Bcl-xL dissociation was followed by the binding of Bad and Bcl-xL. Taken together, the study indicated mitochondrial pathway played an important role in the ACTX-8-induced apoptosis, which was regulated by Bcl-2 family members.

Crystal structure of a non-hemorrhagic fibrin(ogen)olytic metalloproteinase complexed with a novel natural tri-peptide inhibitor from venom of Agkistrodon acutus

Thrombotic occlusive diseases pose a great threat to human health. Thrombolytic agents are in widespread use for the dissolution of arterial and venous pathologic thrombi in these kinds of diseases. Snake venom metalloproteinases (SVMPs) can act directly on fibrin/fibrinogen and are therefore potential candidates for therapeutic use against thrombotic occlusive diseases. In this study, we have determined the crystal structure of FII, a novel non-hemorrhagic SVMP isolated from Anhui Agkistrodon acutus snake venom by molecular replacement. The structure reveals that FII is a member of the P-I class SVMPs. The Zn 2+ ion essential for hydrolytic activity is found in the active site and is tetrahedrally co-ordinated by three histidine residues and water molecule. Unambiguous electron density for a tri-peptide with sequence KNL is also found located near the active site. Biochemical evidences show that the tri-peptide KNL can inhibit the enzymatic activity of FII.

A Novel Tetrameric Venom Protein, Agglucetin from Agkistrodon acutus , Acts as a Glycoprotein Ib Agonist

A novel platelet agglutination inducer, agglucetin, was purified from the Formosan Agkistrodon acutus snake venom. It migrated as a single band with an apparent molecular mass of 58.8 kDa and two distinct bands of 16.2/14.5 kDa under non-reducing and reducing conditions by SDS-PAGE, respectively. Further confirmed by FPLC, electrospray ionization mass spectrometry and 2D-PAGE, native agglucetin exists as a tetramer composed of disulfide-linked alpha1, alpha2, beta1 and beta2 subunits. Partial N-terminal sequence of agglucetin subunit showed a high degree of homology to those of C-type lectin-like glycoprotein (GP) Ib binding proteins. Functional studies showed that agglucetin. in the absence of von Willebrand factor (vWF), dose-dependently induced platelet agglutination and caused a negligible elevation of intracellular Ca+2 mobilization and thromboxane B, formation in human platelet suspensions. Anti-GP Ib monoclonal antibodies (mAbs), AP1 or LJ-Ib1, specifically inhibited agglucetin-induced platelet agglutination in a dose-dependent manner. However, EDTA, arietin (a long chain RGD-containing disintegrin), 7E3 (an anti-GP IIb/IIIa mAb), heparin, hirudin, PGE1, or indomethacin exhibited no inhibitory effect on agglucetin-induced platelet agglutination. Furthermore, flow cytometric analysis revealed that FITC-agglucetin dose-dependently bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by anti-GP Ib mAb. It is concluded that agglucetin, acts specifically on an epitope of platelet membrane GP Ib overlapping with that of API, causing platelet agglutination in a Ca+2- and GP IIb/IIIa-independent manner.

Purification and characterization of two fibrinogen-clotting enzymes from five-pace snake ( Agkistrodon acutus) venom

From the snake venom of Agkistrodon acutus, two proteases, acuthrombin-A and acuthrombin-C, were isolated and purified to homogeneity. They can cleave the human fibrinogen to release the fibrinopeptide A and fibrinopeptide B with specific activity of 120 and 370 NIH units/mg, respectively; the fibrinogen-clotting activity can be inhibited distinctly by PMSF or DFP or EDTA, but not by heparin. The two proteases show also arginine-esterase activity hydrolyzing some synthetic substrates such as TAME and BAEE. Additionally, they are glycoproteins with an average content of 2.4% (acuthrombin-A) and 2.1% (acuthrombin-C) neutral carbohydrates, respectively. Acuthrombin-A has a MW of 13,900 as estimated by SDS-PAGE under reduced or nonreduced conditions and 28,000 as determined by gel filtration. For acuthrombin-C, there were two protein bands corresponding to MW of 13,900 and 14,800 on SDS-PAGE with different darkness under reduced or nonreduced conditions, while its MW was estimated to be 69,000 by gel filtration. The isoelectric points were 7.5 for acuthrombin-A and 5.0 for acuthrombin-C by isoelectric focusing. Neither acuthrombin-A nor acuthrombin-C has haemorrhagic or lethal activity. Acuthrombin-A has also a small amount of activity to activate the Factor XIII.

Crystal structures of acutolysin A, a three-disulfide hemorrhagic zinc metalloproteinase from the snake venom of Agkistrodon acutus

Acutolysin A alias AaHI, a 22 kDa hemorrhagic toxin isolated from the snake venom of Agkistrodon acutus, is a member of the adamalysin subfamily of the metzincin family and is a snake venom zinc metalloproteinase possessing only one catalytic domain. Acutolysin A was found to have a high-activity and a low-activity under weakly alkaline and acidic conditions, respectively. With the adamalysin II structure as the initial trial-and-error model, the crystal structures were solved to the final crystallographic R-factors of 0.168 and 0.171, against the diffraction data of crystals grown under pH 5.0 and pH 7.5 conditions to 1.9 Å and 1.95 Å resolution, respectively. One zinc ion, binding in the active-site, one structural calcium ion and some water molecules were localized in both of the structures. The catalytic zinc ion is coordinated in a tetrahedral manner with one catalytic water molecule anchoring to an intermediate glutamic acid residue (Glu143) and three imidazole N ε2 atoms of His142, His146 and His152 in the highly conserved sequence H 142E 143XXH 146XXGXXH 152. There are two new disulfide bridges (Cys157-Cys181 and Cys159-Cys164) in acutolysin A in addition to the highly conserved disulfide bridge Cys117-Cys197. The calcium ion occurs on the molecular surface. The superposition showed that there was no significant conformational changes between the two structures except for a few slight changes of some flexible residue side-chains on the molecular surface, terminal residues and the active-site cleft. The average contact distance between the catalytic water molecule and oxygen atoms of the Glu143 carboxylate group in the weakly alkaline structure was also found to be closer than that in the weakly acidic structure. By comparing the available structural information of the members of the adamalysin subfamily, it seems that, when lowering the pH value, the polarization capability of the Glu143 carboxylate group to the catalytic water molecule become weaker, which might be the structural reason why the snake venom metalloproteinases are inactive or have a low activity under acidic conditions.

Crystal structure determination of alkaline haemorrhagin AaH III from snake venom ofAgkistrodon acutus

The haemorrhagin AaH III isolated from the snake venom of Agkistrodon acutus is one of the few alkaline ones in snake venoms. Its crystals belong to space group P2(1)2(1)2(1) with a = 9. 573 4 nm, b = 4. 996 7 nm and c = 4.728 8 nm. Its crystal structure was determined by the molecular replacement method according to the model of metalloproteinase Adamalysin II from eastern rattlesnake venom. The AaH III structure has been refined by PROLSQ. The final R factor was 0.254 and the RMS deviations of bond lengths and angles were 0.001 8 nm and 1.5 degrees . The structure comparison suggested that AaH III has a similar structure to other snake venom zinc-metalloproteinases. They all belong to matrix metalloproteinases super-family.

Purification, crystallization and preliminary X-ray diffraction analysis of haemorrhagin IV from the snake venom of Agkistrodon acutus.

Haemorrhagin IV, a medium molecular weight haemorrhagin from the snake venom of Agkistrodon acutus (AaHIV), has been purified and crystallized. The molecular weight and isoelectric point of AaHIV are 44 kDa and pI 5.0, respectively. The crystal belongs to space group C222(1) with unit-cell dimensions of a = 124.2, b = 114.5, c = 98.4 A, and could o diffract X-rays to 3.0 A, resolution. There are one or two molecules in the crystallographic asymmetric unit.

Crystallization and preliminary X-ray diffraction studies of haemorrhagin I from the snake venom of Agkistrodon acutus.

Haemorrhagin I from the snake venom of Agkistrodon acutus (AaHI) has been crystallized using the hanging-drop vapour diffusion method. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 with unit-cell dimensions a = b = 63.61 and c = 95.69 A. There is one molecule in the asymmetric unit. Data to 2.35 A resolution have been collected using a single-crystal.

Separation and identification of proteins obtained from Agkistrodon acutus snake venom by capillary zone electrophoresis and laser desorption/ionization mass monitoring.

Fractions of seven protein principles with fibrinolytic or thrombin-like activities obtained from Agkistrodon acutus snake venom purified by two steps of normal pressure chromatography were separated further by capillary zone electrophoresis (CZE). Mass determination for these fractions were achieved by performing laser desorption/ionization mass monitoring (LDIM). The comparative study between CZE and LDIM on the separation of these fractions was made.

Platelet aggregation inhibitors from Agkistrodon acutus snake venom

C. Ouyang and T.-F. Huang. Platelet aggregation inhibitors from Agkistrodon acutus snake venom. Toxicon 24, 1099 – 1106, 1986. — Among all the purified components from A. acutus venom, including ADPase, 5′-nucleotidase, phospholipase A 2 and fibrinogenases, only the venom ADPase (50 – 100 μg/ml) shows marked inhibitory action on ADP(10 μM)-, collagen(10 μg/ml)- and sodium arachidonate(100 μM)-induced platelet aggregations of rabbit platelet-rich plasma. The venom 5′-nucleotidase (100 μg/ml) inhibited ADP-induced platelet aggregation by 31±4% ( n=4, P<0.05). Fibrinogenolytic enzymes (fractions I and IX, 100 μg/ml) did not significantly inhibit platelet aggregation induced by ADP (10 μM), collagen (10 μg/ml) or sodium arachidonate (100 μM). However, when the fibrinogenase (fraction IX, 100 μg/ml) was preincubated with platelet-rich plasma for 30 min it inhibited collagen(20 μg/ml)- and ADP(10 μM)-induced platelet aggregations by 34±9% ( n=4, P<0.05) and 35±6% ( n-4, P<0.05), respectively. The phospholipase A 2 (100 μg/ml) did not affect platelet aggregation. The venom ADPase is a single chain polypeptide with a molecular weight of 94,000. The specific ADPase activity is estimated to be 4.3 μmoles Pi/min/mg of protein. It also possesses phosphodiesterase and weak 5′-nucleotidase activities.

Agkistrodon acutus snake venom inhibits prothrombinase complex formation

The purified anticoagulant principle AAV-XaI isolated from the snake venom of Agkistrodon acutus binds to purified bovine Factor Xa in the presence of calcium ions. The complex has amidolytic activity with S-2222. AAV-XaI inhibits the conversion of prothrombin to thrombin by the prothrombinase complex, but has no influence on the amidolytic activity of Factor Xa. AAV-XaI binds to Factor X more effectively than to Factor Xa. The complex of Factor X ·Ca 2 +· AAV-XaI yields only amidolytic activity after activation with RVV-X. In 25% sodium citrate solution AAV-XaI does not modify the generation of thrombin from prethrombin 1 by Factor Xa. β-Factor Xa-E is a form of Factor Xa with only amidolytic activity and is not inhibited or bound by AAV-XaI. The venom inhibitor covers binding sites on the light chain of Factor Xa that are necessary for phospholipid function.

Production of Factor X and Factor Xa variants with thrombin, acutin and by autolysis

Research in several laboratories established that β-Factor X, as compared with α-Factor X, has 17 less amino acids and less carbohydrate at the C-terminal end of the heavy chain. Using the purified thrombin-like enzyme acutin, isolated from the snake venom of Agkistrodon acutus , β-Factor X was digested to obtain γ-Factor X and δ-Factor X by shortening the heavy chain from the N-terminal end. β, γ, and δ-Factor X were purified and each one was converted to the same active β-Factor Xa by the purified activator of Russell's viper venom (RVV-X). β-Factor Xa converted to β-Factor Xa-E by autolysis and had esterase activity, but did not function in prothrombin activation. δ-Factor X converted to ε-Factor X by autolysis or with α-chymotrypsin. In the conversion the light chain was reduced in length. The active form obtained with RVV-X was β-Factor Xa-E. By SDS polyacrylamide gel electrophoresis δ-Factor X could not be distinguished from β-Factor Xa.

Production of prothrombin fragment 1–44 with acutin and some effects on thrombin generation

The thrombin-like enzyme from Agkistrodon acutus snake venom is called acutin. It cleaves three peptide bonds in bovine prothrombin; namely, Lys 44-Tyr 45; Arg 156-Ser 157, and Arg 274-IIe 275. By digesting prothrombin fragment I with acutin, peptides 1–44 and 45–156 were obtained and purified. Peptide 1–44 contains all ten γ-carboxyglutamic acid residues of prothrombin. Working with the prothrombinase system, thrombin generation was inhibited by peptide 1–44 when the substrate was prothrombin, accelerated when the substrate was prethrombin 1, and there was no effect with prethrombin 2 even if peptide 45–156 and prothrombin fragment 2 were also present. When peptide 1–44 was covalently bound as in prothrombin fragment 1 it did function with fragment 2 in prethrombin 2 activation. Peptide 45–156 thus serves a function in thrombin generation.

The properties of the purified fibrinolytic principle from Agkistrodon Acutus snake venom

The properties of the purified fibrinolytic principle from Agkistrodon acutus snake venom. Toxicon 15, 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 effects of the purified thrombin-like and anticoagulant principles of Agkistrodon acutus venom on blood coagulation in vivo

The effects of the purified thrombin-like and anticoagulant principles of the snake venom of Agkistrodon acutus on blood coagulation of rabbits in vivo were studied. The thrombin-like principle caused a marked prolongations of whole blood coagulation time and one-stage plasma prothrombin time and a marked decrease of the fibrinogen concentration, while no significant change in the two-stage plasma prothrombin level was detected. It is concluded that the retardation of blood clotting by the thrombin-like principle was chiefly due to the decrease of plasma fibrinogen level. The anticoagulant principle caused a marked, but transient prolongation of whole blood coagulation time and one-stage plasma prothrombin time with no significant change in the two-stage plasma prothrombin level or plasma fibrinogen level. Combining these results with our previous in vitro findings, it is concluded that the retardation of blood clotting by the anticoagulant principle might be due to the interference in the interaction between prothrombin and its activation factors.