A novel alpha-type fibrinogenase from Agkistrodon rhodostoma snake venom.

By means of CM-Sephadex C-50 column chromatography, gel-filtration on sephadex G-75 and Sephacryl S-200 columns, a purified fibrinogenase, kistomin, was obtained from venom of Agkistrodon rhodostoma. It was a single peptide-chain with a molecular mass of about 21,800 Da containing about 202 amino-acid residues as revealed by amino acid analysis. Kistomin preferentially cleaved A alpha- and subsequently the gamma-chain of fibrinogen, leaving the B beta-chain unaffected. Its fibrinogenolytic activity was estimated to be 36.6 +/- 4.5 mg/min per mg protein and was inhibited by the pretreatment of EDTA, suggesting that it is a metalloproteinase. Its fibrinogenolytic activity in platelet-poor plasma is much less potent as compared to that in purified fibrinogen solution. It inhibited ristocetin-induced aggregation of human platelets in a dose-dependent manner in the presence of von Willebrand factor.     

Purification, gene cloning and expression of an acidic phospholipase A2 from Agkistrodon shedaoensis Zhao

A protein with the activity of phospholipase A2 named asAPLA2 was purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao through DEAE-Sepharose CL-6B anion exchange column, Source S and Mono Q FPLC. Its molecular weight was estimated as 19 kD by SDS-PAGE and its pI was about 3.5 by IEF analysis. It inhibits the platelet aggregation that was induced by 1 μmol/ L ADP, and the IC50 was determined to be 6 μmol/L. Degenerate primer was designed and synthesized according to the N-terminal amino acid sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. According to the deduced amino acid sequence, its molecular weight and pI are determined to be 13,649 and 4.39 respectively as calculated by DNAclub and DNAstar softwares. The gene was then cloned into the expression plasmid pET-40b( ) and expressed in E. coli BL21(DE3). Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the native     

A novel P-I class metalloproteinase with broad substrate-cleaving activity, agkislysin, from Agkistrodon acutus venom

The venom of Viperdae is a rich source of metalloproteinases, which have potential clinical applications for lowering plasma fibrinogen or dissolving thrombi. Recently, we purified a novel proteinase from Formosan Agkistrodon acutus venom using DEAE-Sephadex A-50 and Mono-Q HR 5/5 column chromatography. The purified getatinolytic component, agkislysin, is a 22kDa-monomeric protein without Asn-linked sugar. Functional characterization showed that agkislysin possessed both fibronectin- and type IV collagen-cleaving activities. In addition, agkislysin preferentially cleaved the Aalpha chain of fibrinogen, followed by the Bbeta chain, while the gamma chain was finally affected. Furthermore, agkislysin was also capable of cleaving prothrombin into various fragments, as well as suppressing ristocetin-induced platelet aggregation by hydrolyzing von Willebrand factor. However, the proteolytic activity of agkislysin was slightly enhanced by divalent metal ions and completely inhibited by chelating agents, but not serine proteinase inhibitor. These findings suggest that agkislysin is a P-I class metalloproteinase with unique biological properties.     

Purification and functional characterization of AAV1, a novel P-III metalloproteinase, from Formosan Agkistrodon acutus venom

AAV1, an alkaline glycoprotein (GP), was purified from Agkistrodon acutus venom by two chromatographic steps on successive DEAE-Sephadex A-50 and Superdex 75 FPLC columns. AAV1 on SDS-PAGE under non-reducing conditions migrated as a monomeric and a polymeric forms with apparent molecular mass of 57 and 180 kDa, respectively. Upon reduction, it appeared as a single broad band with a mass of 50.3 kDa corresponding to the size of a typical P-III metalloproteinase acurhagin. The N-terminal sequence of an autoproteolytical 30 kDa-fragment of AAV1 showed a high homology to that of venom proteins with Metalloproteinase, Disintegrin-like, and Cysteine-rich (MDC) domains. Although it was devoid of cleaving activity toward gelatin, fibronectin and prothrombin, AAV1 preferentially digested the Aalpha chain of fibrinogen and followed by the Bbeta chain, leading to the inhibition of fibrinogen-induced platelet aggregation in elastase-treated human platelets. However, the proteolytic activity of AAV1 was completely inactivated by the chelating agent but not serine proteinase inhibitor. Furthermore, AAV1 could concentration-dependently inhibit platelet aggregation and suppress tyrosine phosphorylation of intracellular proteins in collagen- and convulxin-stimulated platelets, respectively. The interaction of MDC domains in AAV1 molecule with platelet GPVI was responsible for the inhibitory effect of AAV1 on collagen- and convulxin-induced platelet aggregation. Taken together, these pieces of evidence suggest that AAV1 from Formosan viper venom belongs to a new member of high-molecular mass metalloproteinase family and functions as a GPVI antagonist.     

Primary structure and antiplatelet mechanism of a snake venom metalloproteinase, acurhagin, from Agkistrodon acutus venom

Acurhagin has been characterized as a P-III hemorrhagic metalloproteinase. We herein report the complete sequence of acurhagin by molecular cloning. Analysis of the cDNA-predicted amino acid sequence encoding acurhagin precursor revealed that this mosaic Asn-linked glycoprotein possesses a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domains (189/205/102/114 residues), with an overall 87% identity to that of jararhagin, an integrin alpha2beta1-cleaving metalloproteinase. Acurhagin has a Ser-Glu-Cys-Asp sequence in the disintegrin-like domain instead of the typical Arg-Gly-Asp motif. In contrast to inhibiting fibrinogen-integrin alphaIIbbeta3 interaction by disintegrins, acurhagin selectively showed a dose-dependent inhibition on platelet aggregation induced by collagen, and suppression on tyrosine phosphorylation of several signaling proteins in convulxin-stimulated platelets. Although the immobilized acurhagin was shown to bind platelet GPVI and collagen in a primary structure- and steric conformation-dependent manner, respectively, the mechanism of acurhagin under short incubation is mainly through its binding to GPVI and collagen, instead of binding to alpha2beta1, or cleaving platelet membrane glycoproteins. Moreover, the molecular conformation maintained by divalent cations is required for the proteolytic activity of acurhagin toward extracellular matrix fibronectin. Taken together, these results suggest that all the three domains in mature acurhagin may cooperatively contribute to its biological function.     

Characterization of a fibrinogenase from northern copperhead (Agkistrodon contortrix mokasen) venom

One of the fractions obtained by the carboxymethylcellulose ion-exchange chromatography of northern copperhead (Agkistrodon contortrix mokasen) venom prevented the thrombin-induced clotting of fibrinogen by proteolytically degrading the fibrinogen. The active component has been further purified to apparent electrophoretic homogeneity by molecular sieve chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated a molecular weight of 22 900 +/- 600 for the purified enzyme. In addition to its fibrinogenase activity, it catalyzed the hydrolysis of hide power azure and had an intraperitoneal LD50 value in mice of less than 5.1 microgram/g body weight. The enzyme rapidly destroyed fibrinogen's ability to form clots. Electrophoresis of fibrinogen which had been incubated only a few minutes with the fibrinogenase revealed the rapid disappearance of the alpha-chain and the appearance of lower molecular weight fragments. The neutral pH optimum and ethylenediamine-tetraacetic acid (EDTA) and dithiothreitol sensitivity indicated that this enzyme belonged to the class metalloproteinases. Atomic absorption studies have revealed one zinc atom per molecule of protein. The apoenzyme's activity was restored by incubation with ZnCl2.     

Recombinant production of fibrinogenase IV from Agkistrodon acutus venom and its preliminary evaluation

A novel metalloproteinase, recombinant fibrinogenase IV (rFIV_a), was expressed and purified from Agkistrodon acutus venom. It is a single-chain protein with an apparent molecular weight of 27 kDa. Western blot showed that it had a good immunological reaction against anti-FIV_a rabbit serum. The kinetic parameters K_m and K_cat of rFIV_a on the substrate T6140 were 7.471×10⁻⁴ mol/l and 5.103×10⁻⁵ s⁻¹. RFIV_a cleaved preferentially the α-chain, and the β- and γ-chains of fibrinogen were also cleaved when the incubation time was prolonged. The administration of rFIV_a (1.8 and 5.4 mg/kg) to animals with acute blood-stasis model produced a decrease in fibrinogen to control values. To our knowledge, this is the first report of the expression, purification, and evaluation of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from A. acutus venom.     

Purification and characterization of jararassin-I, A thrombin-like enzyme from Bothrops jararaca snake venom

Snake venoms of the Viperidae family contain a numberof proteins that cause hemostatic disturbances. Enveno-mation of this family is characterized by hemorrhage,edema, local tissue damage, myonecrosis, fibrinolytic andkinin releasing activities [1]. In southeastern Brazil, theviper Bothrops jararaca (Viperidae) is responsible for 90%of snakebite accidents [2]. The enzymes that have proteolytic, coagulate andhemorraghic activities can activate or interfere withthe process of coagulation, and…     

日本蝮蛇蛇毒碱性磷脂酶A2同源物的分离及鉴定

We purified and characterizated a phospholipase A2 homologue from Agkistrodon blomhoffii ussurensis snake venom. We used Hitrap SP cation exchange and Superdex 75 columns chromatography to obtain a basic protein, used SDS-PAGE to analyse molecular mass, and IEF (Isoelectric focusing electrophoresis) IEF to identify isoelectric point. The molecular mass was 16 kDa, and the isoelectric point was 8.56. We detected its phospholipase A2 activity on egg yolk phospholipids, hemolytic activity on washed erythrocytes, and anticoagulant effect on pig platelet-rich plasma, as well as the N-terminal sequence with protein sequencer. The results showed that it had no phospholipase A2 activity and hemolytic activity, but had obvious anticoagulant effect on in witro. The N terminal sequence (21 amino acid residues) compared with other phospholipases A2 demonstrated that the protein was homogenous with BPLA2s from Agkistrodon halys Palls.     

日本蝮蛇蛇毒磷脂酶A2(Gln49-PLA2)的细胞毒性

从日本蝮蛇(Agkistrodonblomhoffiiussurens)蛇毒中分离得到PLA2同源物Gln49PLA2,该蛋白具有抗凝血活性、肌肉毒性,缺乏磷脂酶A2活性。分析了该蛋白质对不同培养细胞生长的影响,探讨其细胞毒性。结果表明:贴壁细胞的Gln49PLA2半致死量(LD50)明显低于悬浮细胞,肝素可以明显抑制Gln49PLA2的细胞毒性。将其作用于K562细胞,随着Gln49PLA2用量的增加乳酸脱氢酶(LDH)释放量增加,细胞凋亡率增大,线粒体膜电位下降,但Bcl2蛋白的表达量无明显变化。     

Enzymatic activities and functional characterization of a novel recombinant snake venom proteinase from Agkistrodon Acutus

Snake venom from Agkistrodon acutus consists of a number of compounds which may potentially be used as drugs. However, it is hard to obtain enough pure protein for drug development. Recently, we reported expression and purification of a novel recombinant fibrinogenase which was named rFII. Here we reported for the first time the enzymatic activities and functional characterization of rFII. Circular dichroism spectra showed the gross conformation of FIIa and rFII to be notably similar. It is an alkaline proteinase and the amino acid sequence exhibits a high degree of sequence identity with other snake venom metalloproteinases. rFII also exhibits amidase activity against N-(p-Tosyl)-Gly-Pro-Lys-p-nitroanilide, which is specified synthetic substrate for plasmin. Functional characterization showed that rFII possesses both fibronectin and type IV collagen cleaving activities. In addition, rFII preferentially cleaved the Aalpha-chain of fibrinogen, followed by the Bbeta-chain and finally, the gamma(γ) chain was affected. Furthermore, rFII was also capable of cleaving fibrin without plasminogen activation and suppressing ADP-induced platelet aggregation. The proteolytic activity of rFII was inhibited completely by PMSF and mostly by EDTA. The cations Ca²⁺, Mg²⁺, Na⁺, K⁺ didn't affect its proteolytic activity, while Cu²⁺ and Zn²⁺ slightly inhibited this activity. Study of hydrolysis of oxidized insulin B-chain reveals that rFII preferentially cleaved oxidized insulin B-chain at the site of Val¹²-Glu¹³, Leu¹⁵-Tyr¹⁶, and Phe²⁴-Phe²⁵.     

蕲蛇蛇毒中一种新型金属蛋白酶AA-MP-I的纯化和表征

本研究通过嗜硫色谱、Sephadex G-75、蓝胶和POROS HQ20离子交换色谱,从蕲蛇蛇毒中分离得到一种新组分AA-MP-I。该酶为分子量22.9kDa的单体蛋白,等电点为5.55,不含中性糖基,N端序列为STE-FQRYMEIVIVVDHSMVK,结果表明其为新型P-I型金属蛋白酶,对温度敏感,具有抗凝血活性,40℃下抗凝血活性最强,具有出血毒性,无磷脂酶A2活性。     

蝮蛇毒致小鼠肾损伤模型的建立

目的探索一种建立蝮蛇毒致肾损伤动物模型的方法。方法将清洁级12周龄雄性昆明小鼠72只,随机分为9组,每组8只,A、B、C组为不同时间的对照组（A-3h,B-3d,C-5d）;D、E、F组为蝮蛇毒1mg／kg肌注组（D-3h,E-3d,F-5d）;G、H、I组为蝮蛇毒2mg／kg肌注组（G-3h,H-3d,I-5d）。按照分组要求计算每组小鼠中每只小鼠需肌注的蝮蛇毒的总量．配制2种不同浓度的蝮蛇毒液．D、E、F、G、H、I组小鼠分别肌注不同浓度的蝮蛇毒液0．15ml,A、B、C组小鼠右臀部注射生理盐水0．15ml以进行对照。结果肌注蝮蛇毒后的小鼠均出现中毒症状,但各组小鼠均无死亡。E、F、H、I组小鼠的肾功能与正常对照组比较有明显差异（P〈0．05）。D、E、F、G、H、I组小鼠均有如下病理学改变：肾被膜下出血．肾皮质充血．肾小球充血。肾小囊扩大．肾小管周围毛细血管充血．肾小管肿胀。小管细胞变性等。结论肌注蝮蛇毒是一种建立蛇毒致小鼠肾损伤动物模型的较好方法。     

A novel high molecular weight fibrinogenase from the venom of Bitis arietans

A fibrinogenase (Ba100) with an apparent molecular mass of 100 kDa under non-reducing conditions and a pI of 5.4 was purified from the venom of the African puff adder (Bitis arietans) by fibrinogen affinity chromatography. Under reducing conditions the protease dissociates into subunits of 21 kDa and 16 kDa. N-Terminal amino acid sequencing showed these two chains to have 66.7% homology and homology to C-type lectins. The fibrinogenase activity of Ba100 cleaves the Aalpha and Bbeta chain of fibrinogen rendering the molecule unable to polymerise into fibrin clots. Ba100 inhibited platelet aggregation in platelet rich plasma, and clot formation in whole blood, in a concentration dependent manner.     

蝮蛇毒诱导小鼠急性肾损伤的研究

目的观察蝮蛇毒诱导小鼠急性肾损伤肾脏病理变化特点,为肾损伤的防治提供理论依据。方法将小鼠分为N组（正常对照组）、L组（蛇毒低剂量组,0.50 mg/kg）、H组（蛇毒高剂量组,2.00 mg/kg）,每组按时相点又分为3、6、244、8 h四个亚组。96只小鼠随机分配到各亚组,采取肌注蝮蛇毒的方式建立动物模型。结果 SCr值,L组24 h升高（P〈0.05）,48 h下降（P〈0.05）;H组持续升高（P〈0.05）。光镜下肾损伤病理评分,L组6 h开始升高（P〈0.05）,48 h下降（P〈0.05）;H组3 h开始持续升高（P〈0.05）。SCr与光镜下,肾损伤病理评分呈正相关（L组：r=0.883,P〈0.01;H组：r=0.891,P〈0.01）。结论 SCr在一定条件下能反映肾损伤的严重程度,但急性肾损伤的发生早于SCr升高;急性肾损伤的严重程度与蝮蛇毒的剂量呈正相关。     

Identification of an unusual AT(D)Pase-like activity in multifunctional NAD glycohydrolase from the venom of Agkistrodon acutus

NAD-glycohydrolases (NADases) are ubiquitous enzymes that possess NAD glycohydrolase, ADPR cyclase or cADPR hydrolase activity. All these activities are attributed to the NADase-catalyzed cleavage of C-N glycosyl bond. AA-NADase purified from the venom of Agkistrodon acutus is different from the known NADases, for it consists of two chains linked with disulfide-bond(s) and contains one Cu(2+) ion. Here, we show that AA-NADase is not only able to cleave the C-N glycosyl bond of NAD to produce ADPR and nicotinamide, but also able to cleave the phosphoanhydride linkages of ATP, ADP and AMP-PNP to yield AMP. AA-NADase selectively cleaves the P-O-P bond of ATP, ADP and AMP-PNP without the cleavage of P-O-P bond of NAD. The hydrolysis reactions of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. ATP is the excellent substrate for AA-NADase with the highest specificity constant k(cat)/K(m) of 293+/-7mM(-1)s(-1). AA-NADase catalyzes the hydrolysis of ATP to produce AMP with an intermediate ADP. AA-NADase binds with one AMP with high affinity determined by isothermal titration calorimetry (ITC). AMP is an efficient inhibitor against NAD. AA-NADase has so far been identified as the first unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities.     

蝮蛇毒蛋白C激活物对凝血系统的影响

目的研究蝮蛇毒纯化蛋白C激活物(PCA)的抗凝机制。方法测定PCA对正常人混浆KPTT、PT、TT的影响,对血液凝血活酶生成试验(BTGT)及PA二期法的影响以及对白兔的KPTT和Fgn的影响。结果PCA在最终浓度为0.025mg/L时对正常人混浆KPTT可明显延长,但PT和TT不受影响;当它的浓度增加到50mg/L,PT也可延长,但TT依然无明显变化;最终浓度为0.0125mg/L时,血液凝血活酶生成明显受抑制;当它的浓度增加到2.5mg/L,凝血酶生成显著减少,但抗凝血酶时间始终无明显变化;PCA可明显延长白兔的KPTT。结论PCA在低浓度时首先抑制凝血系统第一阶段内凝途径,在高浓度时也妨碍外凝途径或共同途径,BTGT和PA二期法比KPTT和PT敏感;PCA具有体内抗凝作用。     

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 Zn2+ 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.     

Metal-ion- and pH-induced conformational changes of acutolysin D from Agkistrodon acutus venom probed by fluorescent spectroscopy

Acutolysin D isolated from the venom of Agkistrodon acutus is a protein of 44 kDa with marked hemorrhagic and proteolytic activities. The metal-ion- and pH-induced conformational changes of acutolysin D have been studied by following fluorescence and activity measurements. Here we provide evidence for the fact that native holo-acutolysin D adopts two different conformations, native state a, stable in the weak acidic pH range from 5.5 to 7.0 with low activity, and native state b, stable in the weak alkaline pH range from 8.0 to 9.0 with high activity. Holo-acutolysin D has an optimum pH of 9.0 for caseinolytic activity and a maximum fluorescence at pH 9.0. The protein adopts the most stable conformation at pH 9.0. The addition of 1 mM Zn(2+) shifts both the alkali-induced unfolding transition curve and the alkali-induced inactivation curve toward higher pH value but has little effect on the acid-induced unfolding transition curve. No obvious effects on the pH-induced unfolding transition curve and the pH-dependent activity curve have been observed after the addition of 1 mM Ca(2+) to holo-acutolysin D. The results indicate that Zn(2+) is essential for its CA, while Ca(2+) is not essential for its CA. Removal of Ca(2+) and Zn(2+) from the protein enhances its sensitivity to pH and significantly reduces its overall stability during acid-induced denaturation. The kinetic results of the demetalization of holo-acutolysin D show that the demetalization rate constant K(1) for a slower reaction linearly decreases with the pH increase from 5.0 to 9.0, while K(2) for the faster reaction linearly increases with the pH change from 5.0 to 7.0. It is also evident from the present work that the free Zn(2+)-induced inactivation in the pH range from 8.0 to 9.0 should be attributed to the effect of Zn(OH)(2) precipitation on the protein.     

蝮蛇毒蛋白C激活物对内毒素性离体大鼠心脏功能的影响

目的研究蝮蛇毒蛋白C激活物(PCA)组分对大鼠内毒素(LPS)性心肌损伤作用的影响。方法取SD雄性大鼠32只随机分成正常对照组、LPS组、PCA组和PCA LPS组,用Krebs-Henseleit(K-H)液对大鼠离体心脏行主动脉逆灌。在相应时点记录HR、LVSP、LVEDP、LVDP、 dp/dtmax、-dp/dtmax的变化,并测定冠脉流出液中的超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量。结果PCA明显减轻LPS诱导的心功能改变并抑制心肌SOD活性降低和MDA的升高(P<0.01)。结论PCA对内毒素诱导的心肌损伤改善作用明显,其机制可能是通过保护血管内皮功能,改善微循环,稳定心肌酶活性和膜相结构等途径有关。