Effect of proteins from the Agkistrodon blomhoffii ussuriensis snake venom on platelets in the hemostasis system

Influence of proteins from the Agkistrodon blomhoffii ussuriensis snake venom on platelet activation and aggregation was developed on different model systems in vitro. It was shown that novel disintegrin (Blomus-B) and phospholipase A2 (Blopholipase) from the venom, activated platelets and inhibited their aggregation. Fibrino(geno)lityc enzyme (Blomulyse) does not activate platelets and has no effect on their aggregation stimulated by collagen, but inhibit ADP and adrenalin-stimulated platelet aggregation. Thrombin-like enzyme (Ancistron-Bu) activates platelets but has no effect on their aggregation. Obtained proteins can be used under development of new antiplatelet agents and as instruments for detailed elaboration and deep investigation of processes which proceed with participation of platelets.     

Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.     

长白山白眉蝮蛇蛇毒酸性PLA_2质谱鉴定及其对血小板聚集的抑制作用

通过对纯化得到的长白山白眉蝮蛇蛇毒磷脂酶A2（ABUSV-PLA2）进行胶内胰蛋白酶酶解,产生的肽段经高效液相色谱-电喷雾串联质谱（HPLC-nESI-MS/MS）进行序列测定,蛋白鉴定采用SequestBioworks软件完成。ABUSV-PLA2与其它蛇毒来源PLA2的氨基酸序列比对表明：ABUSV-PLA2是一种新的蛇毒酸性磷脂酶A2。以ADP诱导的人血小板聚集实验结果表明：ABUSV-PLA2对ADP诱导的血小板聚集有轻微的解凝效应,但具有显著拮抗血小板的聚集作用,并呈现明显的剂量-效应关系,IC50为356nmol/L。     

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.     

Potent platelet aggregation inhibitor from Trimeresurus gramineus snake venom

Using DEAE-Sephadex A-50 column chromatography and gel filtration, a potent platelet aggregation inhibitor from Trimeresurus gramineus venom was purified. It was an acidic phospholipase a, rich in aspartic acid, glutamic acid and half-cystine, with an isoelectric point of 3.6. At a concentration of 10 μg/ml, the purified inhibitor showed a marked inhibitory effect on platelet aggregations induced by adenosine diphosphate, collagen, sodium arachidonate and ionophore A-23187 in rabbit platelet-rich plasma, washed platelet suspension, as well as in thrombin-degranulated platelet suspension. The ID₅₀ of this venom inhibitor was about 2.5–5 μg/ml in platelet aggregations induced by all these aggregation inducers. The action of this inhibitor could be partially antagonized by phosphatidylethanolamine. High concentration of Ca²⁺ (5 mM) did not reverse the inhibitory action even in the presence of ionophore A-238187. The [¹⁴C]serotonin release induced by sodium arachidonate and thrombin was unaffected. Malonic dialdehyde formation induced by these aggregation inducers remained unchanged. Basal and prostaglandin E₁-stimulated cAMP levels were not altered by this inhibitor. No lactate dehydrogenase was released even at a concentration of 62.5 μg/ml. Polylysine-induced platelet agglutination was not affected. β-Mercaptoethanol inactivated both its phospholiase A enzymatic and platelet inhibitory activities, while p-bromophenacyl bromide only inactivated the former activity. The possibility of acting on a common final step of platelet aggregation, i.e. the intercellular adhesion between the activated platelets, was proposed.     

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。     

Characterization of Ac3-proteinase from the venom of Agkistrodon acutus (hundred-pace snake)

Ac3-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. Ac3-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, dimethylcaseinolytic and hide powder azure hydrolytic activities. These activities were inhibited when Ac3-Proteinase was incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), tetraethylenepentamine (TEP), 1,10-phenanthroline, phosphoramidon or beta-mercaptoethanol. The toxin also hydrolyzed the oxidized A and B chains of both insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25). The A alpha chain of fibrinogen was digested first followed by hydrolysis of the B beta chain. Toxicological and biochemical properties of Ac3-Proteinase were investigated further and are reported in this paper.     

Characterization of a fibrinolytic enzyme (ussurenase) from Agkistrodon blomhoffii ussurensis snake venom: insights into the effects of Ca2+ on function and structure

Fibrino(geno)lytic enzymes from snake venoms have been identified as high quality therapeutic agents for treatment of blood clots and strokes. They act on fibrinogen and fibrin, leading to defibrinogenation of blood, lysis of fibrin, and a consequent decrease in blood viscosity. In this work, a fibrinolytic enzyme (ussurenase) from China Agkistrodon blomhoffii Ussurensis snake venom, was purified to homogeneity, identified as a stable 23,367.8 Da monomeric protein, and was identified as a new kind of snake venom metalloproteinase. Ussurenase reacts optimally with fibrin clots at pH 7.5-8.3 and a temperature of 33-41 degrees C. Although many fibrinolytic enzymes are known to be zinc-dependent, measurements from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveal that ussurenase is a Ca2+-containing protein with a molar ratio of 1:1 ([Ca2+]:[enzyme]). Ca2+ is crucial to the fibrin clot hydrolysis by ussurenase but also plays an important role in maintaining the structural integrity of the enzyme. The addition of Ca2+ to the apoenzyme induces a conformational change making the environments surrounding the Trp residues of the enzyme more hydrophobic. The presence of Ca2+ also increases the structural stability of ussurenase, so that higher concentrations of the denaturant guanidine hydrochloride are required to denature the native ussurenase compared to the apo-form. UV absorption and CD spectroscopy experiments show that Ca2+ increases the thermostability and changes the secondary structure of ussurenase. All these data suggest that Ca2+ is crucial for the correct folding and activity of ussurenase.     

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.     

Characterization of Ac1-Proteinase from the venom of Agkistrodon acutus (Hundred-pace snake) from Taiwan

1. Ac1-Proteinase from the venom of Agkistrodon acutus was isolated in a homogeneous form by a previously published method. 2. Ac1-Proteinase possessed lethal, hemorrhagic, caseinolytic, azocaseinolytic, azoalbumin hydrolytic and hide powder azure hydrolytic activities. 3. The toxin also hydrolyzed the oxidized B chain of insulin and fibrinogen. The cleavage sites in the oxidized B chain of insulin were identified as Ala(14)-Leu(15) and Tyr(16)-Leu(17). The A alpha chain of fibrinogen was digested. 4. Biological properties of Ac1-Proteinase were investigated further and are reported in this paper.     

Characterization of clotting factors from Agkistrodon acutus venom

1. Four clotting factors, Cf-1(C), Cf-2(C), Cf-1(T) and Cf-2(T) were isolated from Agkistrodon acutus (collected on mainland China and Taiwan) venom by Komori et al. (1987). It was reported that all factors possessed coagulant activity in the conversion of fibrinogen to fibrin, although they showed different chemical properties and antigenicities. 2. Their role in the clot formation system was clarified and compared with that of thrombin. Clotting factors from A. acutus venom released only fibrinopeptide A from the A alpha chain of fibrinogen, while thrombin released fibrinopeptide A and B from the A alpha and B beta chains. 3. Cf-1(C) and Cf-2(T), like thrombin, rapidly activated factor XIII in the presence of calcium ions, whereas Cf-2(C) and Cf-1(T) had little effect on factor XIII. These effects are shown by Cf-1(C) and Cf-2(T) forming a clot that remained insoluble in 8 M urea or 0.44 M monochloroacetic acid, whereas Cf-2(C) and Cf-1(T) formed a soluble clot in these agents.     

A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): purification and characterization

A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N(alpha)-tosyl-l-arginine-methyl ester (TAME) at approximately pH 7.5 and at 51 degrees C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn(2+)-containing protein with a stoichiometry of 1:1 [Zn(2+)]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn(2+) plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca(2+), Mg(2+), Cu(2+), Ni(2+), Mn(2+), and Co(2+), increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.     

Action mechanism of the platelet aggregation inducer and inhibitor from Echis carinatus snake venom

Platelet aggregation inducer and inhibitor were isolated from Echis carinatus snake venom. The venom inducer caused aggregation of washed rabbit platelets which could be inhibited completely by heparin or hirudin. The venom inducer also inhibit both the reversibility of platelet aggregation induced by ADP and the disaggregating effect of prostaglandin E₁ on the aggregation induced by collagen in the presence of heparin. The venom inhibitor decreased the platelet aggregation induced by collagen, thrombin, ionophore A23187, arachidonate, ADP and platelet-activating factor (PAF) with an IC₅₀ of around 10 μg/ml. It did not inhibit the agglutination of formaldehyde-treated platelets induced by polylysine. In the presence of indomethacin or in ADP-refractory platelets or thrombin-degranulated platelets, the venom inhibitor further inhibited the collagen-induced aggregation. Fibrinogen antagonized competitively the inhibitory action of the venom inhibitor in collagen-induced aggregation. In chymotrypsin-treated platelets, the venom inhibitor abolished the aggregation induced by fibrinogen. It was concluded that the venom inducer caused platelet aggregation indirectly by the conversion of prothrombin to thrombin, while the venom inhibitor inhibited platelet aggregation by interfering with the interaction between fibrinogen and platelets.     

Reevaluation of hemorrhagic toxin, HR-I, from Agkistrodon halys blomhoffii venom: proof of proteolytic enzyme

HR-I is a hemorrhagic toxin originally isolated from Agkistrodon halys blomhoffii (Mamushi) venom by Oshima et al. (1972). It was reported by the original investigators that it was nonproteolytic when casein was used as the substrate. HR-I was isolated again and proteolytic activity was tested using different substrates and assay methods. It is shown that HR-I is indeed a proteolytic enzyme hydrolyzing a number of peptide bonds. This present investigation suggests that more than one method should be used for proteolytic enzyme assay of hemorrhagic toxins. Toxicological and biochemical properties of HR-I were further investigated and are reported in this paper.