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Yuancong Zhou - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of platelet aggregation inhibitor component from venom of Agkistrodon Halys pallas
Journal of Experimental Hematology, 2004Co-Authors: Ping Liu, Qian Jin, Yiqing Wang, Hong Zhu, Yuancong ZhouAbstract: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.
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structures of cadmium binding acidic phospholipase a2 from the venom of Agkistrodon Halys pallas at 1 9a resolution
Biochemical and Biophysical Research Communications, 2003Co-Authors: Tao Jiang, Yuancong Zhou, Z LinAbstract:Abstract Phospholipase A2 coordinates Ca2+ ion through three carbonyl oxygen atoms of residues 28, 30, and 32, two carboxyl oxygen atoms of residue Asp49, and two (or one) water molecules, forming seven (or six) coordinate geometry of Ca2+ ligands. Two crystal structures of cadmium-binding acidic phospholipase A2 from the venom of Agkistrodon Halys Pallas (i.e., Agkistrodon blomhoffii brevicaudus) at different pH values (5.9 and 7.4) were determined to 1.9 A resolution by the isomorphous difference Fourier method. The well-refined structures revealed that a Cd2+ ion occupied the position expected for a Ca2+ ion, and that the substitution of Cd2+ for Ca2+ resulted in detectable changes in the metal-binding region: one of the carboxyl oxygen atoms from residue Asp49 was farther from the metal ion while the other one was closer and there were no water molecules coordinating to the metal ion. Thus the Cd2+-binding region appears to have four coordinating oxygen ligands. The cadmium binding to the enzyme induced no other significant conformational change in the enzyme molecule elsewhere. The mechanism for divalent cadmium cation to support substrate binding but not catalysis is discussed.
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functionally important residues for the anticoagulant activity of a basic phospholipase a2 from the Agkistrodon Halys pallas
Protein and Peptide Letters, 2002Co-Authors: Xiaoyan Zhong, Haomang Jiao, Liang Fan, Yuancong ZhouAbstract:To identify the anticoagulant region of the phospholipase A2 (PLA2) from the Agkistrodon Halys Pallas (class II), four mutants E53G, W70M, T56K, and D67K were produced according to the prediction from the crystal structure and the sequence comparison of the strong, weak and non-anticoagulant PLA2s. A test of blood clotting revealed that E53G and W70M had lost their effects on the blood clotting, while T56K and D67K had enhanced activity. The four residues are located on the same face in the tertiary structure of this enzyme. The result supported the prediction that there exists an anticoagulant region that is composed of some residues that are close to each other in tertiary structure to form a functional face.
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expression purification and biochemical characterization of a recombinant phospholipase a2 with anticoagulant activity from Agkistrodon Halys pallas
Journal of Natural Toxins, 2001Co-Authors: Xiaolan Zhong, J Liu, Yuancong ZhouAbstract:A cloned cDNA encoding a PLA2 from Agkistrodon Halys Pallas was found to have conservative residues Glu53 and Trp70 but with Lys56 and Lys67 substituted by Thr56 and Asp67, respectively, when compared with sequences of other class II PLA2 with anticoagulant activity. It was inserted into a temperature-sensitive bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies and recovered by centrifugation after enzyme digestion. By washing to partial purification, the expression product was refolded and was purified by FPLC superose 12 to appear as a single band in SDS-PAGE. The recombinant protein proved to have obvious enzymatic, anticoagulant and hemolytic activities, which were removed after modification by p-BPB. These findings suggest that the pharmacological activities of this recombinant PLA2 may be related to its catalytic activity and warrant further research on the structure-function relationships of the pharmacological site of the PLA2 from Agkistrodon Halys Pallas.
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purification characterization and cdna sequence of Halysetin a disintegrin like cysteine rich protein from the venom of Agkistrodon Halys pallas
Biochemical and Biophysical Research Communications, 2000Co-Authors: Jiewu Liu, Ping Liu, Xin Chen, Yuancong ZhouAbstract:Abstract By means of DEAE–Sepharose CL-6B column chromatography, gel filtration on Sephadex G-75 and Superose 12 FPLC, Halysetin, an antiplatelet protein, was purified from the venom of Agkistrodon Halys Pallas with molecular mass of 29 kDa on SDS–PAGE and 23,168 Da by mass spectrometry. The p I was about 5.0. Halysetin was devoid of phospholipase A2, fibrino(geno)lytic, esterase, hemorrhagenic activities. Halysetin dose-dependently inhibited the aggregation of human platelet, which was stimulated by collagen with IC 50 of 420 nM, but not that stimulated by ADP. The N- and C-terminal sequences of Halysetin were characterized. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. It encoded a protein of 212-amino-acid residues with disintegrin-like/cysteine-rich domains and was highly homologous with SVMPs (snake venom metalloprotease).
Doo-sik Kim - One of the best experts on this subject based on the ideXlab platform.
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Characterization and cDNA cloning of halyxin, a heterogeneous three-chain anticoagulant protein from the venom of Agkistrodon Halys brevicaudus.
Toxicon : official journal of the International Society on Toxinology, 2002Co-Authors: Bon-hun Koo, Young-doug Sohn, Ki-chul Hwang, Yangsoo Jang, Doo-sik Kim, Kwang-hoe ChungAbstract:Abstract We report upon the isolation, characterization, and cDNA cloning of an anticoagulant protein, halyxin from Agkistrodon Halys brevicaudus venom. The protein exists as a 29 kDa protein, and is separated into three chains on SDS–PAGE under reducing conditions. However, we cloned only two cDNAs encoding halyxin from the cDNA library of the snake venom gland, on the basis of the determined amino acid sequences. The complete amino acid sequences were deduced from their nucleotide sequences and named halyxin A (129 amino acid residues) and B chain (123 amino acid residues). The deduced amino acid sequence of halyxin A chain corresponds to the two smaller chains. Thus, it is considered that halyxin A chain could be synthesized as a single-chain protein that is subsequently cleaved to yield the mature two-chain protein. The amino acid sequence of halyxin is similar to that of other snake venom proteins of the C-type lectin superfamily, and prolongs plasma-clotting time. In the presence of Ca2+ ions, halyxin binds to coagulation factors IX, X, IXa, and Xa, but not to other vitamin K-dependent coagulation factors. It also inhibits factor Xa in a non-competitive manner but does not affect other activated coagulation factors.
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expression and characterization of a novel plasminogen activator from Agkistrodon Halys venom
Toxicon, 1998Co-Authors: Dongsu Park, Doo-sik Kim, Kwang-hoe Chung, Hakdai Kim, Yungdae YunAbstract:Abstract A venom gland cDNA library of Agkistrodon Halys was constructed and screened with a probe based on the consensus sequence of venomic serine proteases. Next, we determined the sequences of the entire open reading frames of two selected positives which were found to encode novel serine proteases of 234 and 233 amino acids in length and named as Haly-PA and Haly 2, respectively. Upon protein data base search, Haly-PA showed the highest similarity of 82% to the previously characterized plasminogen activator, TSV-PA (Zhang et al. 1995, J. Biol. Chem. , 270, 10246–10255). Haly 2 displayed a 78% similarity to β -fibrinogenase (Hung et al. 1994, B. B. R. C ., 205, 1707–1715). Haly-PA was successfully expressed using the baculovirus system and secreted into the culture media as a 32 kDa glycoprotein. In the western analysis of snake venom, anti-Haly-PA antibody detected the same size of band indicating that this enzyme is a component of snake venom. Recombinant Haly-PA was purified to homogeneity using the combination of anion exchange and gel filtration column. In the fibrino(geno)lytic assay, recombinant Haly-PA displayed an indirect fibrino(geno)lytic activity depending on the presence of plasminogen and cleaved the plasminogen to generate the active plasmin. These results indicate that Haly-PA is a plasminogen activator and displays fibrino(geno)lytic activity through conversion of plasminogen to plasmin.
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cloning and characterization of novel disintegrins from Agkistrodon Halys venom
Molecules and Cells, 1998Co-Authors: Dongsu Park, Doo-sik Kim, Kwang-hoe Chung, Incheol Kang, Hakdai Kim, Yungdae YunAbstract:Snake venom disintegrins act as potent inhibitors of platelet aggregation. In this report, we isolated genes encoding novel members of disintegrins through the screening of Agkistrodon Halys venom gland cDNA library. Subsequent characterization of positives revealed the presence of distinct disintegrins named salmosinl, 2, and 3, each containing a characteristic RGD/KGD sequence essential for the binding to integrins. Whereas salmosinl was identical to previously described salmosin purified from A. Halys venom, salmosin2 and salmosin3 were predicted to be a novel, 73 amino acid protein with a KGD sequence, and an 80 amino acid protein with an additional 7th disulfide bond, respectively. Taken together, this is the first report describing 3 unique disintegrins, namely, salmosinl with RGD, salmosin2 with KGD and salmosin3 with 7 disulfide bonds are found in a single species of venom. Subsequently, to compare the platelet aggregation inhibitory potential of the recombinant protein with that of natural protein, salmosinl was expressed in E. coli and purified to homogeneity. Recombinant and natural salmosin1 inhibited the binding of alphaIIbbeta3 to fibrinogen with an almost identical IC50 value of 2.2 nM and 4.5 nM respectively. Moreover, recombinant salmosinl displayed an IC50 value approximately 5-fold lower than flavoridin, which was previously described as the most potent venom disintegrin so far. In conclusion, we identified 3 disintegrins with distinct properties through the molecular cloning approach and found that the recombinant salmosinl retained one of the most potent alphaIIbbeta3 antagonist activity.
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purification and molecular cloning of a platelet aggregation inhibitor from the snake Agkistrodon Halys brevicaudus venom
Thrombosis Research, 1998Co-Authors: Incheol Kang, Kwang-hoe Chung, Yungdae Yun, Soo Jung Lee, Hongmo Moon, Doo-sik KimAbstract:A platelet glycoprotein IIb-IIIa (GP IIb-IIIa) antagonist, salmosin, was purified to homogeneity from Korean snake (Agkistrodon Halys brevicaudus) venom by means of chromatographic fractionations. We have isolated the cDNA encoding salmosin by using the cDNA library of the snake venom gland and analyzed its complete nucleotide sequence. The molecular identity was confirmed by comparison of the deduced amino acid sequence with the directly determined primary structure of salmosin. This protein is a single-chain polypeptide composed of 73 amino acids including 12 cysteines as well as the sequence Arg-Gly-Asp, a proposed recognition site of adhesive proteins. The primary sequence of salmosin shows considerable homology to previously described proteins of snake venom GP IIb-IIIa antagonist family. A molecular mass of 7474 for the protein was determined by matrix-assisted laser desorption ionization mass spectrometry. Salmosin inhibits GP IIb-IIIa binding to immobilized fibrinogen with an IC50 of 2.2 nM and ADP-induced platelet aggregation with an IC50 of 131 nM, respectively. This work demonstrates the purification, characterization, and cDNA cloning of salmosin, a platelet aggregation inhibitor that may have therapeutic potential as an antithrombotic agent.
Tadashi Okada - One of the best experts on this subject based on the ideXlab platform.
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structure of carbohydrate chain of a thrombin like protease from the venom of Agkistrodon Halys brevicaudus stejneger snake
Journal of Nagoya Gakuin University Humanities and natural sciences, 2009Co-Authors: 淳一 酒井, Ponlapat Roinuckarin, Panchalee Jangprasert, Takahiro Nakano, Kenji Saitou, Chikashi Yamamoto, Yutaka Murase, Tadashi OkadaAbstract:The structure of the carbohydrate chain of kangshuanmei, a thrombin-like serine protease isolated from Agkistrodon Halys brevicaudus stejneger snake venom, was determined. The carbohydrate content of the kangshuanmei was 18%. The sugar composition was analyzed by the acid hydrolysis followed by aminobenzoic ethyl ester labeling. Galactose, N-acetylglucosamin, mannose, and fucose were detected, indicating that the binding carbohydrate chain is asparagine-linked type oligosaccharides. N-Acetylneuraminic acid located at non-reduced terminal of the carbohydrate chain was identified by neuraminidase digestion. The carbohydrate chain moiety was separated from kangshuanmei by hydrazynolysis treatment followed by aminobenzoic octyl ester (ABOE) labeling. The isolated ABOEmodified carbohydrate chain was compared to the asparagine-linked type standard oligosaccharides. The carbohydrate chains were consisted of sialylated bi(39.4%)-, tri(50.4%)- and tetra(10.2%)antennary lactosamins complex containing fucose. The structure of the conjugated carbohydrate chain of kangshuanmei was significantly different from that of thrombin, which has a bisected antennary structure of oligosaccharide.
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primary structure of a thrombin like serine protease kangshuanmei from the venom of Agkistrodon Halys brevicaudus stejneger
Toxicon, 2006Co-Authors: Junichi Sakai, Shujuan Zhang, Hongmiao Chen, Fukiko Atsumi, Takuya Matsui, Hiroyuki Shiono, Susumu Sanada, Tadashi OkadaAbstract:The complete amino acid sequence of the thrombin-like serine protease, named kangshuanmei, isolated from the venom of a Chinese snake Agkistrodon Halys brevicaudus stejneger, was determined by Edman degradation. The serine protease was composed of 236 amino acid residues and conserved the catalytic triad as His43, Asp88 and Ser182. The protease had four sites of asparagine-linked glycosylation at 81, 99, 148 and 229, and contained fucose, N-acetylglucosamin, galactose, mannose and N-acetylneuraminic acid. The amino acid sequence exhibited considerable similarities with other thrombin-like proteases isolated from the snake venoms of the Viperidae family. However, the enzymatic characteristics of kangshuanmei distinct from that of thrombin and the other protease from the venom of Viperidae family may be derived from the structural difference of the sequence in the functional regions, especially corresponding to thrombin exosite 1, 2 and hydrophobic pocket.
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characterization of a thrombin like serine protease kangshuanmei isolated from the venom of a chinese snake Agkistrodon Halys brevicaudus stejneger
Journal of Natural Toxins, 2001Co-Authors: Shujuan Zhang, Junichi Sakai, Takuya Matsui, Hiroyuki Shiono, I Sugie, Tadashi OkadaAbstract:An enzyme, referred to as Kangshuanmei, was isolated from the venom of the Chinese snake Agkistrodon Halys brevicaudus stejneger by gel filtration chromatography followed by affinity chromatography. Kangshuanmei is composed of a single polypeptide chain with a molecular weight of approximately 34,000, estimated by SDS-PAGE. The enzyme hydrolyzed both benzoyl-arginine ethyl ester and H-D-Phe-Pip-Arg-p-nitroanilide, specific substrates for thrombin. The protease activity of Kangshuanmei was inhibited by 4-(2-aminoethyl)-benzensulfonyl fluoride, but was not affected by EDTA. The enzyme acted on human fibrinogen to form a fibrin clot and released three fragments. These fragments were shown to be fibrinopeptide A, fibrinopeptide B, and the Bbeta1-42 peptide of fibrinogen, respectively. These results indicate that Kangshuanmei is a thrombin-like serine protease with coagulant activity. However, the enzyme did not induce activation of blood coagulation factor XIII, unlike thrombin. Moreover, antithrombin-III, the specific thrombin inhibitor in plasma, had no inhibitory effect on the thrombin-like amidolytic activity of Kangshuanmei. The N-terminal amino acid sequence of the enzyme up to 50 residues was determined by a peptide sequencer. The N-terminal sequence of Kangshuanmei was highly homologous to most thrombin-like serine proteases from the venom of the snakes of the crotalidae family.
Z Lin - One of the best experts on this subject based on the ideXlab platform.
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structures of cadmium binding acidic phospholipase a2 from the venom of Agkistrodon Halys pallas at 1 9a resolution
Biochemical and Biophysical Research Communications, 2003Co-Authors: Tao Jiang, Yuancong Zhou, Z LinAbstract:Abstract Phospholipase A2 coordinates Ca2+ ion through three carbonyl oxygen atoms of residues 28, 30, and 32, two carboxyl oxygen atoms of residue Asp49, and two (or one) water molecules, forming seven (or six) coordinate geometry of Ca2+ ligands. Two crystal structures of cadmium-binding acidic phospholipase A2 from the venom of Agkistrodon Halys Pallas (i.e., Agkistrodon blomhoffii brevicaudus) at different pH values (5.9 and 7.4) were determined to 1.9 A resolution by the isomorphous difference Fourier method. The well-refined structures revealed that a Cd2+ ion occupied the position expected for a Ca2+ ion, and that the substitution of Cd2+ for Ca2+ resulted in detectable changes in the metal-binding region: one of the carboxyl oxygen atoms from residue Asp49 was farther from the metal ion while the other one was closer and there were no water molecules coordinating to the metal ion. Thus the Cd2+-binding region appears to have four coordinating oxygen ligands. The cadmium binding to the enzyme induced no other significant conformational change in the enzyme molecule elsewhere. The mechanism for divalent cadmium cation to support substrate binding but not catalysis is discussed.
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structure of basic phospholipase a2 from Agkistrodon Halys pallas implications for its association hemolytic and anticoagulant activities
Toxicon, 2000Co-Authors: Kehao Zhao, Yuancong Zhou, Z LinAbstract:Abstract The basic phospholipase A2 (PLA2) from the venom of Agkistrodon Halys Pallas is a potent hemolytic toxin and anticoagulant. Crystal structure of the enzyme complexed with detergent n-octyl β- d -glucopyranoside (β-OG) in monoclinic crystal form has been determined to 2.6 A resolution. β-OG molecules were found in the hydrophobic channels of the enzyme. SDS-PAGE and dynamic light scattering measurements showed that the enzyme had a strong tendency to dimerise in aqueous solution. In the crystal structure the enzyme molecules associate into a tetramer with pseudo 222 symmetry, and the interfacial recognition site linked dimers constituting the tetramer have intensive interface interactions, and may be stable in solution. The structure reveals a unique positively charged face at the C-terminal region and a characteristic non-cationic ‘anticoagulant’ region (53–77). The face is supposed to be the hemolytic site, and based on sequence and structure comparison residues Trp70 and Glu53 instead of the basic residues in ‘anticoagulant’ region might play an important role in the anticoagulant activity.
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structure of a basic phospholipase a2 from Agkistrodon Halys pallas at 2 13 a resolution
Acta Crystallographica Section D-biological Crystallography, 1998Co-Authors: Kehao Zhao, Z Lin, Shiying Song, Yuancong ZhouAbstract:The basic phospholipase A2 isolated from the venom of Agkistrodon Halys Pallas (Agkistrodon blomhoffii Brevicaudus) is a hemolytic toxin and one of the few PLA2's capable of hydrolyzing the phospholipids of E. coli membranes in the presence of a bactericidal/permeability-increasing protein (BPI) of neutrophils. The crystal structure has been determined and refined at 2.13 A to a R factor of 16.5% (F > 3\sigma) with excellent stereochemistry. A superposition of the two molecules in the asymmetric unit gives an r.m.s. deviation of 0.326 A for all Cα atoms. The refined structure allowed a detailed comparison with other PLA2 species of known structures. The overall architecture is similar to those of other PLA2's with a few significant differences. One of which is in the region connecting the N-terminal helix and the Ca2+-binding loop. Unexpectedly, the conformation of the peptide plane Cys29—Gly30 in the Ca2+-binding loop is very different to that of other PLA2's. The amide NH of Gly30 does not point toward the proposed site for stabilization of the tetrahedral intermediate oxyanion of the substrate analogue. The structure includes four residues which occur less frequently in other PLA2's. His1, Arg6 and Trp70 located at the interfacial recognition site may play an important role in the interaction with aggregated substrates, while Trp77 contributes to the hydrophobic interactions between the β-wing and the main body of the molecule. This structure analysis reveals that two clusters of basic residues are located at or near the interfacial recognition site, forming an asymmetric positively charge distribution. In contrast to the acidic isoform, the present enzyme is a dimer in the crystalline state. The special phospholipid hydrolysis behaviors are discussed in the light of the structure determined.
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crystal structure of ca 2 ion saturated acidic phospholipase a 2 from Agkistrodon Halys pallas
Acta Biochimica et Biophysica Sinica, 1997Co-Authors: Xiaoqiang Wang, Z Lin, Haiyan Zhao, Yuancong ZhouAbstract:The Ca(2+) ion is a cofactor for the catalysis of phospholipase A(2). The crystals of Ca(2+)-saturated acidic phospholipase A(2) from Agkistrodon Halys Pallas were obtained by adding CaCl(2) during the crystallization to ensure the complete binding of Ca(2+). The synchrotron diffraction data were collected at 1.6 Aring; resolution. The structure was determined by difference Fouriers methods. The refined structure of Ca(2+)-saturated acidicPLA(2) resembles closely that of the native acidicPLA(2) with, however, some small conformational differences in the Ca(2+)-binding site and the C-terminal loop. The pentagonal bipyramidal configuration consisting of seven oxygen ligands of Ca(2+) ion appears more regular than that of the native acidicPLA(2). The small conformational changes induced by Ca(2+) implies that the main role of Ca(2+) ion is to stabilize the oxyanion in the tetrahedral intermediate formed during the catalysis by electrophilic interaction.
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crystal structure determination of basic phospholipase a2 from venom of Agkistrodon Halys pallas by molecular replacement method
Science China-life Sciences, 1996Co-Authors: W Meng, Z Lin, Y ZhouAbstract:Basic phospholipase A2 (BPLA2) from the venom of Agkistrodon Halys pallas has a strong ability to hemolyze erythrocytes. The asymmetrical unit of P2(1)2(1)2(1) crystal of BPLA2 contains two molecules. Self-rotation function was used to study the orientation relationship of these two molecules. Cross-rotation and translation functions were then used to determine the orientations and positions of the two molecules in the unit cell. The model building and preliminary structure refinement were carried out. The result shows that the two molecules in the asymmetrical unit of orthorhombic crystal are related by a non-crystallographic 2-fold symmetry axis.
Yoshihiko Sakurai - One of the best experts on this subject based on the ideXlab platform.
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anticoagulant activity of m lao l amino acid oxidase purified from Agkistrodon Halys blomhoffii through selective inhibition of factor ix
Biochimica et Biophysica Acta, 2003Co-Authors: Yoshihiko Sakurai, Midori Shima, Tomoko Matsumoto, Hideo Takatsuka, Katsumi Nishiya, Shogo Kasuda, Yoshihiro Fujimura, Akira YoshiokaAbstract:One of haemorrhagic toxins present in snake venoms is L-amino acid oxidase (LAO), which catalyzes the oxidative deamination of L-amino acids with the generation of hydrogen peroxide. Although it is widely accepted that LAO alters platelet function, the effects of LAO on human blood coagulation remain largely unknown. The present study demonstrated, for the first time, that M-LAO, LAO purified from the venom of Agkistrodon Halys blomhoffii (Japanese mamushi), possesses an anticoagulant activity. Thrombelastography (TEG) showed that M-LAO significantly delayed the onset and the progress of the coagulation process. In addition, the enzyme prolonged the activated partial thromboplastin time (aPTT) dose-dependently, but had little effect on the prothrombin time (PT), suggesting that its principal activity was mediated in the intrinsic coagulation pathway. Furthermore, M-LAO reduced factor IX procoagulant activity in a dose-dependent manner and did not affect other coagulation factors. These results indicate that M-LAO has an anticoagulant activity that impairs the intrinsic clotting by inhibiting factor IX.
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molecular characterization of l amino acid oxidase from Agkistrodon Halys blomhoffii with special reference to platelet aggregation
Biochimica et Biophysica Acta, 2001Co-Authors: Hideo Takatsuka, Yoshihiko Sakurai, Akira Yoshioka, Tetsuro Kokubo, Yoshiko Usami, Masami Suzuki, Taei Matsui, Koiti Titani, Hideo Yagi, Masanori MatsumotoAbstract:L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon Halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 microM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.
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the cdna cloning and molecular characterization of a snake venom platelet glycoprotein ib binding protein mamushigin from Agkistrodon Halys blomhoffii venom
Thrombosis and Haemostasis, 1998Co-Authors: Yoshihiko Sakurai, Yoshihiro Fujimura, Tetsuro Kokubo, Masami Suzuki, Taei Matsui, Koiti Titani, Kouji Imamura, Tomihisa Kawasaki, Makoto Handa, Akira YoshiokaAbstract:The entire cDNA sequences of a novel snake venom platelet glycoprotein (GP) Ib-binding protein (BP) composed of an a/b heterodimeric structure, termed mamushigin, from Agkistrodon Halys blomhoffii were determined, that include the leader peptides (21/23 amino acid residues) and mature subunits (136/123 amino acid residues). The mature subunits of mamushigin are 37.5% identical, and showed a high degree of similarity (37.7-67.5% identity) with the respective subunits of group VII C-type lectins. The sequences of the leader peptides of the mamusigin subunits showed the highest similarity (a-73.9/ b-82.6%) with those of factor IX/X-BP from Trimeresurus flavoviridis, and the cleavage site residue in both proteins was the same Ala –1 . The GPIb-binding specificity of mamushigin is strongly supported by several lines of evidence, but mamushigin can directly aggregate normal platelets, similar to alboaggregin-B (AL-B). This differs from other GPIb-BP’s. In mamushigin-treated platelets, serotonin was not released, and flow cytometric analysis using a monoclonal antibody PAC-1 totally excluded platelet GPIIb/IIIa activation. Mamushigin enhanced platelet aggregation at low-shear stress, and this effect totally disappeared in the presence of GPIb-receptor blockers specific for von Willebrand factor binding, but not by GPIIb/IIIa-receptor blockers. At high-shear stress, mamushigin blocked platelet aggregation in a dose-dependent manner, as seen with other GPIb-BP’s. This paper, therefore, describes the cDNA cloning and molecular characterization of mamushigin which has a different effect on platelet aggregation under different shear stress.
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purification and amino acid sequence of Halystase from snake venom of Agkistrodon Halys blomhoffii a serine protease that cleaves specifically fibrinogen and kininogen
FEBS Journal, 1998Co-Authors: Taei Matsui, Yoshihiko Sakurai, Yoshihiro Fujimura, Masami Suzuki, Izumi Hayashi, Sachiko Ohishi, Jiharu Hamako, Yoshinobu Yamamoto, Junko Yamazaki, Michiko KinoshitaAbstract:We have isolated a serine protease, Halystase, from Agkistrodon Halys blomhoffii venom by chromatography on DEAE-Sepharose, heparin-Sepharose and Q-Sepharose columns, and have determined the complete amino acid sequence by Edman degradation and by mass spectral analysis of peptides generated by enzymatic and chemical cleavage. The 238-residue sequence of Halystase, containing N-linked carbohydrates (about 13 %) at two sites showed significant similarity to other thrombin-like snake venom serine proteases (66−72 %), mammalian tissue kallikrein (42 %) and thrombin (26 %). Halystase contained the tentative catalytic triad of His43, Asp88 and Ser184 common to all serine proteases and Asp178 in the primary substrate-binding site. Although Halystase contained an RGD sequence at residues 181−183, it did not inhibit platelet aggregation induced by ADP or collagen. It hydrolyzed most efficiently a tissue-kallikrein substrate, prolylphenylalanylarginyl-4-methyl-coumaryl-7-amide, and released bradykinin from bovine kininogen. Halystase did not coagulate human plasma, but it cleaved the fibrinogen B β chain at the carboxyl side of Arg42 and cleaved slowly the fibrogen A α chain. Fibrinogen thus treated gradually became insensitive to thrombin. The proteolytic activity was inhibited with diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride or leupeptin. These results indicate that Halystase is a serine protease structurally similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves fibrinogen at sites different from thrombin without inducing fibrin clotting, and hydrolyzes kininogen to produce bradykinin, resulting in the reduction of blood pressure.