长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化方法研究

目的:研究长白山白眉蝮蛇蛇毒中类凝血酶的简单分离纯化方法。方法:采用DEAE-Sephadex A-25及Sephadex G-25层析的方法,比较二者对长白山白眉蝮蛇蛇毒中类凝血酶简单分离纯化的效果。结果:从长白山白眉蝮蛇蛇毒中分离出类凝血酶,SDS-PAGE电泳显示为一条带,分子量大约为35.5kDa,达到电泳纯。理化性质研究表明,此类凝血酶具有体外凝血活性,体外凝血酶比活力为12.57IU.mg-1,用N-苯甲酰-L-精氨酸乙酯盐酸盐测得该酶的精氨酸酯酶比活力为137.65IU.mg-1。用蛋白酶抑制剂和乙二胺四乙酸对该酶进行抑制实验,结果表明该酶属于丝氨酸蛋白酶,而不是金属蛋白酶。结论:本方法可用于长白山白眉蝮蛇蛇毒中类凝血酶的分离纯化。     

Local and systemic pathophysiological alterations induced by a serine proteinase from the venom of the snake Bothrops jararacussu.

The local and systemic pathophysiological alterations induced by BjussuSP-I, a thrombin-like serine proteinase from the venom of the snake Bothrops jararacussu, were assessed in mice. BjussuSP-I induced a mild edema but no local myonecrosis or hemorrhage. It did not induce any microvascular alteration in the cremaster muscle. Intramuscular injection of BjussuSP-I promoted an increase in the expression of proMMP-9, but it did not induce the activation of proMMP-2 or proMMP-9 synthesized in muscle tissue injected with a myotoxic phospholipase A(2) homolog. BjussuSP-I induced defibrin(ogen)ation upon intravenous and intramuscular injections, with reduction in plasma fibrinogen concentration and increments in the levels of fibrin degradation products and D-dimer. When compared with animals having normal coagulation, mice defibrin(ogen)ated by BjussuSP-I developed a slightly larger hemorrhagic lesion in the skin when injected with metalloproteinase BaP1. Intravenous injection of sublethal doses of BjussuSP-I promoted a series of behavioral and motor changes similar to those previously described for 'gyroxin', i.e. opisthotonus and a circular body movement along the longitudinal axis.     

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.     

Agacutase体外水解牛纤维蛋白原机制的研究(英文)

Agacutase是自尖吻蝮蛇蛇毒中分离出的新的具有止血功能的蛇毒类凝血酶,它能够将纤维蛋白原转化为纤维蛋白单体。通过SDS-PAGE和ELISA方法,我们研究了Agacutase体外水解牛纤维蛋白原的分子机制。实验结果显示,Agacutase仅仅水解牛纤维蛋白原的α亚基并释放血纤肽A,而对牛纤维蛋白原的β和/或γ亚基没有影响。研究表明Agacutase属于血纤肽A类(FP-A类型)的类凝血酶。     

Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom

A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708 bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 °C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aα-chain, apparently not degrading the Bβ and γ-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 °C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.     

Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis

The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95 Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5 Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BAρNA, Michaelis-Menten behavior with K_m 2.3 × 10⁻¹ M and the V_max 0.52 × 10⁻¹ nmoles ρ-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause α- and β-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity.     

一种类凝血酶制剂的人体药动学研究

目的：研究单次静注注射用尖吻蛇毒血凝酶（HCA）的人体药代动力学规律。方法：30例男性健康志愿者随机分组至1 U、2 U、4 U 3个剂量组,每组10例,采用放射免疫分析法对受试血清检测HCA的含量,用DAS软件计算其药代动力学参数。结果：受试者给药后,药-时曲线符合二室模型,主要药代动力学参数：t1/2α为34～55 min;t1/2β为301～351 min;CL分别为0.001 1、0.001 2、0.000 9 L/（min.kg）,Vd分别为1.357 0、0.593 4、0.410 1 L/kg,AUC0-tn分别为16 518.38、22 405.67和29 531.41μU/（min.ml）,呈明显的剂量依赖关系。结论：单次静注注射用HCA体内药代动力学特征符合一级线性动力学二室模型。     

Primary structure of a thrombin-like serine protease, kangshuanmei, from the venom of Agkistrodon halys brevicaudus stejneger.

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.     

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

In the present study, a thrombin-like enzyme named BpSP-I was isolated from Bothrops pauloensis snake venom and its biochemical, enzymatic and pharmacological characteristics were determined. BpSP-I is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with M_r = 34,000 under reducing conditions and pI  6.4. The N-terminal sequence of the enzyme (VIGGDECDINEHPFL) showed high similarity with other thrombin-like enzymes from snake venoms. BpSP-I showed high clotting activity upon bovine and human plasma and was inhibited by PMSF, benzamidine and leupeptin. Moreover, this enzyme showed stability when examined at different temperatures (−70 to 37 °C), pH values (3–9) or in the presence of divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺ and Mn²⁺). BpSP-I showed high catalytic activity upon substrates, such as fibrinogen, TAME, S-2238 and S-2288. It also showed kallikrein-like activity, but was unable to act upon factor Xa and plasmin substrates. Indeed, the enzyme did not induce hemorrhage, myotoxicity or edema. Taken together, our data showed that BpSP-I is in fact a thrombin-like enzyme isoform isolated from Bothrops pauloensis snake venom.     

Purification and biological activity of the thrombin-like substance isolated from Bothrops insularis venom.

The venom of Bothrops insularis snake, known in Brazil as jararaca ilhoa, contains a variety of proteolytic enzymes such as a thrombin-like substance that is responsible for various pharmacological effects. B. insularis venom chromatography profile showed an elution of seven main fractions. The thrombin-like activity was detected in fractions I and III, the latter being subjected to two other chromatographic procedures, so to say DEAE and Hi Trap Benzamidine. The purity degree of this fraction was confirmed by analytical reverse phase HPLC, which displayed only one main fraction confirmed by SDS-PAGE constituting fraction III. About 5 microg of fraction III protein potentiated the secretion of insulin induced by 2.8 mM of glucose in rats isolated pancreatic beta-cells treated; the increase being around 3-fold higher than its respective control. B. insularis lectin (BiLec; 10 microg/mL) was also studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. BiLec increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, the thrombin-like substance isolated from B. insularis venom induced an increase in insulin secretion, in vitro, and transiently altered vascular, glomerular and tubular parameters in the isolated rat kidney.