蝮蛇抗栓酶对不稳定性心绞痛患者血中部分凝血及纤溶指标的影响

测定３８例不稳定性心绞痛（ＵＡＰ）患者血中纤维蛋白原（Ｆｇ）含量、组织型纤溶酶原激活物（ｔ－ＰＡ）活性、血管性假血友病因子（ｖＷＦ）活性和血小板聚集率水平（ＰＡｇＴ）并与２１名正常人对比。患者中１８例用蝮蛇抗栓酶治疗,２０例行常规治疗。结果显示：ＵＡＰ患者血中ＰＡＩ－１、ｖＷＦ以及ＰＡｇＴ水平明显增高,ｔ－ＰＡ水平明显降低;蝮蛇抗栓酶组治疗后血中Ｆｇ、ＰＡＩ－１、ＰＡｇＴ水平均明显下降,使ｔ－ＰＡ水平明显升高;常规治疗组治疗前后各项指标无明显变化。认为蝮蛇抗栓酶对ＵＡＰ有可靠疗效     

精制溶栓酶对家兔血浆t－PA和PAI的影响

精制溶栓酶对家兔血浆ｔ－ＰＡ和ＰＡＩ的影响雷丹青广西医科大学蛇毒研究所南宁５３００２１纤溶系统是机体阻止血栓形成的屏障,ｔ－ＰＡ（纤溶酶原激活剂）是纤溶系统的关键物质,ｔ－ＰＡ对纤维蛋白原有极高的亲和力,它能选择性的激活血凝块中的纤溶酶原生成纤溶酶,...     

Batroxobin治疗脑血栓急性期15例疗效观察（摘要）

Ｂａｔｒｏｘｏｂｉｎ治疗脑血栓急性期１５例疗效观察（摘要）李永昌，余海，秦淑梅，冯大刚Ｂａｔｒｏｘｏｂｉｎ（商品名：东菱精制克栓酶）系南美蛇毒中采用生物技术提取的单成份溶血栓微循环治疗剂，能够专一性很强地分解血纤维蛋白原、诱发ｔ－ＰＡ释放、增强ｔ－Ｐ...     

蛇毒降纤酶的主要药理作用

蛇毒中 “凝血酶样酶”（ Ｔｈｒｏｍ ｂｉｎｌｉｋｅ Ｅｎｚｙｍ ｅ, Ｔ Ｌ Ｅ） 由中国卫生部定名为降纤酶 （ Ｄｅｆｉｂｒａｓｅ, Ｄｆ）。类同生理凝血酶 （ Ｔｈｒｏｍ ｂｉｎ, Ｔｂ） 能降解血浆纤维蛋白原 （ Ｆｉｂｒｉｎｏｇｅｎ, Ｆｇ）, 但只解离 Ｆｇ 结构的肽链 Ａ, 而不象生理凝血酶能同时解离肽链 Ａ 和肽链 Ｂ; 经 Ｄｆ降解的 “脱肽链 Ａ 纤维蛋白”是可溶性单体, 不接受血中稳定因子 （第十三因子, ⅩⅢ Ｆ） 的催化交联, 易被血中溶纤酶 （ Ｐｌａｓｍ ｉｎ, Ｐ１） 迅速消溶并被吞噬细胞所消除, 不产生 Ｄ Ｉ Ｃ现象; 蛇毒 Ｄｆ的直接效果是降低血浆纤维蛋白原在血中的含量, 从而降低血液粘度, 得以疏畅血流, 用于防治血液高粘滞伴发的血管闭塞性疾病; 蛇毒 Ｄｆ能激发组织溶纤酶原激活物 （ｔ Ｐ Ａ） 的释放, 促使溶纤酶原转变为有活性的溶纤酶, 能使纤维蛋白降解产物 （ Ｆ Ｄ Ｐ） 得以消除, 使血栓崩解。蛇毒 Ｄｆ能阻抑血小板的聚集从而延缓血栓的形成。     

凝血酶激活的单链尿激酶型纤溶酶原激活剂的构建、表达、纯化和性质研究

用Ｋｕｎｋｅｌ突变法,将单链尿激酶型纤溶酶原激活剂（ｓｃｕ－ＰＡ）ｃＤＮＡ基因中编码Ｐｒｏ１５５—Ｌｙｓ１５８的片段定点突变,并将此突变的ｓｃｕ－ＰＡ（ｔｓｃｕ－ＰＡ）的ｃＤＮＡ克隆到表达载体ｐＣＭ－β－ｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞．获得的稳定表达株在无血清培养基中２４ｈ的表达量为６２０ＩＵ／１０６细胞．经锌离子螯合Ｓｅｐｈａｒｏｓｅ亲和层析得到ｔｓｃｕ－ＰＡ纯品．ＳＤＳ－ＰＡＧＥ显示ｔｓｃｕ－ＰＡ分子量为５３ｋＤ左右,与预期的结果相符．ｔｓｃｕ－ＰＡ是由凝血酶激活而不是由纤溶酶激活,但激活后也能转变为双链分子（ｔｃｕ－ＰＡ）．ｔｓｃｕ－ＰＡ仍保持了ｓｃｕ－ＰＡ的血纤维蛋白亲和性．酶动力学研究表明,激活后的ｔｓｃｕ－ＰＡ水解Ｓ２４４４的Ｋｍ和Ｋｃａｔ值与高分子量尿激酶（ＨＵＫ）相似．体外溶栓实验结果表明,ｔｓｃｕ－ＰＡ可以选择性地溶解富含凝血酶的血凝块,对贫凝血酶的血凝块作用不大     

L—精氨酸和LDL对血管内皮细胞合成t—PA和PAI的影响

本实验采用酶联免疫分析法检测猪主协脉内皮细胞培养液中ｔ－ＰＡ抗原,ｔ－ＰＡ活性和ＰＡＩ活性,并观察了ＬＤＬ和Ｌ－精氨酸对ｔ－ＰＡ和ＰＡ泊影响。结果表明：ＬＤＬ能明显降低,ｔ－ＰＡ抗原含量。同时伴有ｔ－ＰＡ活性的降低和ＰＡＩ活性增高,Ｌ－到能增加ｔ－ＰＡ抗原含量伴ｔ－ＰＡ活性增高,但对ＰＡＩ活性无明显影响。     

蛇毒中凝血酶样酶的研究

蛇毒中凝血酶样酶的研究韦世秀广西医科大学蛇毒研究所南宁５３００２１许多蝮亚科蛇毒中都含有一种氨基酸酯酶［１］,它催化纤维蛋白原分子特定部位Ａｒｇ－Ｇｌｙ肽键的裂解,释出血纤肽而转换为纤维蛋白。其作用与血浆凝血酶十分相似,因而称之为凝血酶样酶（Ｔｈｒｏ...     

t—PA S165W突变体的分子设计及其生物活性的初步分析

为得到对纤维蛋白的亲和力提高的ｔ－ＰＡ突变体,采用计算机分子技术提出对纤维蛋白亲和力提高的ｔ－ＰＡ结构改造方案（ｔ－ＰＡＳ１６５Ｗ）,并将共在ＣＨＯ细胞中进行表达,对表达产物进行生物活性及与纤维蛋白亲和力的分析表明;ｔ－ＰＡ Ｓ１６５Ｗ突变体与野生型ｔ－ＰＡ对蛋白的亲和力没有明显的变化,说明单一的Ｓ１６５Ｗ的点 变并不能导致ｔ－ＰＡ对蛋白亲和力的提高。     

蝮蛇抗栓酶和酚妥拉明治疗肺心病疗效观察

１３例肺心病急性发作期的患者,应用蝮蛇抗栓酶和酚妥拉明治疗,分别在一个疗程开始前和结束后观察心率、ＰａＯ２、ＰａＣＯ２、全血比粘度,血浆比粘度、红细胞压积、纤维蛋白原。其结果是心率减慢、ＰａＯ２增高,ＰａＣＯ２降低,全血比粘度、血浆比粘度、红细胞压积,纤维蛋白原均降低,经ｔ检验Ｐ＜０．０１。症状明显改善,心肺功能好转。文章对蝮蛇抗栓酶和酚妥拉明的作用机理进行了分析和探讨。     

清栓酶作用机理研究

本文应用动物DIC模型进行清栓酶作用机理的实验研究,观察指标包括应用清栓酶前后血浆中6-K-PGF_2α,TXB_2,全血粘度等的变化以及脑、肺、肾等脏器的不同病理改变。实验结果表明,A组血中TXB_2增高明显,全血粘度增高,病理改变以血栓形成、血小板聚集,血管内纤维蛋白原沉积为主,B组血中6-K-PGF_1α增高明显、全血粘度下降,病理改变以血管扩张为著,程度淤血,未见透明血栓。从而推测清栓酶之所以具有去纤、溶栓、扩血管、改善微循环等作用与血中前列腺毒的增高等因素有关。     

Molecular mechanisms of fibrinolysis and their application to fibrin-specific thrombolytic therapy

The fibrinolytic system comprises a proenzyme, plasminogen, which can be converted to the active enzyme, plasmin, which degrades fibrin. Plasminogen activation is mediated by plasminogen activators, which are classified as either tissue-type plasminogen activators (t-PA) or urokinase-type plasminogen activators (u-PA). Inhibition of the fibrinolytic system may occur at the level of the activators or at the level of generated plasmin. Plasmin has a low substrate specificity, and when circulating freely in the blood it degrades several proteins including fibrinogen, factor V, and factor VIII. Plasma does, however, contain a fast-acting plasmin inhibitor, alpha 2-antiplasmin, which inhibits free plasmin extremely rapidly but which reacts much slower with plasmin bound to fibrin. A "systemic fibrinolytic state" may, however, occur by extensive activation of plasminogen and depletion of alpha 2-antiplasmin. Clot-specific thrombolysis therefore requires plasminogen activation restricted to the vicinity of the fibrin. Two physiological plasminogen activators, t-PA and single-chain u-PA (scu-PA) induce clot-specific thrombolysis, via entirely different mechanisms, however. t-PA is relatively inactive in the absence of fibrin, but fibrin strikingly enhances the activation rate of plasminogen by t-PA. This is explained by an increased affinity of fibrin-bound t-PA for plasminogen and not by alteration of the catalytic rate constant of the enzyme. The high affinity of t-PA for plasminogen in the presence of fibrin thus allows efficient activation on the fibrin clot, while no significant plasminogen activation by t-PA occurs in plasma. scu-PA has a high affinity for plasminogen (Km = 0.3 microM) but a low catalytic rate constant (kcat = 0.02 sec-1). However, scu-PA does not activate plasminogen in plasma in the absence of a fibrin clot, owing to the presence of (a) competitive inhibitor(s). Fibrin-specific thrombolysis appears to be due to the fact that fibrin reverses the competitive inhibition. The thrombolytic efficacy and fibrin specificity of natural and recombinant t-PA has been demonstrated in animal models of pulmonary embolism, venous thrombosis, and coronary artery thrombosis. In all these studies intravenous infusion of t-PA at sufficiently high rates caused efficient thrombolysis in the absence of systemic fibrinolytic activation. The efficacy and relative fibrinogen-sparing effect of t-PA was recently confirmed in three multicenter clinical trials in patients with acute myocardial infarction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tissue-type plasminogen activator inhibits aggregation of platelets in vitro

The effects of tissue-type plasminogen activator (t-PA) on the platelet aggregation were studied using citrated whole blood and platelet-rich plasma (PRP) obtained from human donors. t-PA suppressed adenosine 5'-diphosphate (ADP)- or collagen-induced platelet aggregation in a dose-dependent manner. The 50% inhibitory concentration (IC50) for t-PA was lower by one order of magnitude than that for urokinase (UK) in whole blood and PRP. The suppression of platelet aggregation was not completely inhibited by alpha-2-antiplasmin. t-PA did not cause the degradation of fibrinogen or fibrin in PRP, whereas UK caused the reduction of fibrinogen and fibrin, and the increase of fibrinogen- and fibrin-degradation products (FDP). These results suggest that the mode of action of t-PA in inhibiting platelet aggregation may be different from that of UK.     

Identification of the mechanism responsible for the increased fibrin specificity of TNK-tissue plasminogen activator relative to tissue plasminogen activator

TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA.     

Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.     

Plasminogen activation by tissue plasminogen activator on fibrin clots with different surface structures

Transformation of fibrinogen into fibrin with consequent formation of the fibrin clot trimeric structure is one of the final steps in the blood coagulation system. The plasminogen activation by the tissue plasminogen activator (t-PA) is one of the fibrinolysis system key reactions. The effect of different factors on transformation of plasminogen into plasmin is capable to change essentially the equilibrium between coagulation and fibrinolytic sections of haemostasis system. We have studied the plasminogen activation by tissue plasminogen activator on fibrin clots surface formed on the interface between two phases and in presence of one phase. The t-PA plasminogen activation rate on fibrin clots both with film and without it the latter has been analyzed. These data allow to assume that the changes of fibrin clot structure depend on its formations, as well as are capable to influence essentially on plasminogen activation process by means of its tissue activating agent.     

A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators.

The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (KM) of the reaction, whereas the catalytic rate constant (kcat) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the KM values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The kcat however, was lower with "KHRR" t-PA than with wild-type t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic studies on the effect of heparin and fibrin on plasminogen activators.

1. Possible interactions between fibrin(ogen) and heparin in the control of plasminogen activation were studied in model systems using the thrombolytic agents tissue-type plasminogen activator (t-PA), urokinase and streptokinase.plasminogen activator complex and the substrates Glu- and Lys-plasminogen. 2. Both t-PA and urokinase activities were promoted by heparin and by pentosan polysulphate, but not by chondroitin sulphate or hyaluronic acid. The effect was on Km. 3. In the presence of soluble fibrin (and its mimic, CNBr-digested fibrinogen) the effect of heparin on t-PA was attenuated, although not abolished. In studies using a monoclonal antibody and 6-aminohexanoic acid, it was found that heparin and fibrin did not seem to share a binding site on t-PA. 4. The activity of t-PA B-chain was unaffected by heparin, so the binding site is located on the A-chain of t-PA (and urokinase). 5. Fibrin potentiated the activity of heparin on urokinase. The activity of streptokinase.plasminogen was unaffected by heparin whether or not fibrin was present. 6. If these influences of heparin and fibrin also occur in vivo, then, in the presence of heparin, the relative fibrin enhancement of t-PA will be diminished and the likelihood of systemic activation by t-PA is increased.     

Interaction of one-chain and two-chain tissue plasminogen activator with intact and plasmin-degraded fibrin

Tissue-type plasminogen activator (t-PA) plays a central role in fibrinolysis in vivo. Although it is known to bind to fibrin, the dissociation constant (Kd) and number of moles bound per mole of fibrin monomer (n) have never been measured directly. In this study, the binding of both the one-chain form and the two-chain form of recombinant, human t-PA to fibrin was measured. Although more one-chain t-PA than two-chain t-PA is bound to fibrin, the Kd's and n's were within experimental error of each other. Significantly more t-PA is bound to clots made from fibrinogen which has been digested with plasmin than to clots made from intact fibrinogen. The additional binding was shown to be due to the formation of new set(s) of binding site(s) with dissociation constants that are 2-4 orders of magnitude tighter than the binding site present on clots made from intact fibrinogen. epsilon-Aminocaproic acid was capable of competing for the loose binding site present on both intact and degraded fibrin but had little effect on the binding of t-PA to the new site(s) formed by plasmin digestion. This increase in binding caused by plasmin-mediated proteolysis of fibrin suggests a possible mechanism for a positive regulation capable of accelerating fibrinolysis.     

Characterization of functional domains in human tissue-type plasminogen activator with the use of monoclonal antibodies

Two murine monoclonal antibodies (MA-2G6 and MA-1C8), secreted by hybridomas obtained by fusion of myeloma cells with spleen cells from mice immunized with human tissue-type plasminogen activator (t-PA), inhibited the activity of t-PA on fibrin plates. MA-2G6 inhibited the amidolytic activity of t-PA and did not react with t-PA in which the active-site serine was blocked with diisopropylfluorophosphate nor with t-PA in which the active-site histidine was alkylated by reaction with D-Ile-Pro-Arg-CH2Cl. This indicated that MA-2G6 is directed against an epitope covering the active site of t-PA. MA-1C8 did not inhibit the amidolytic activity of t-PA, but abolished both the binding of t-PA to fibrin and the stimulatory effect of fibrin on the activation of plasminogen by t-PA. Thus MA-1C8 is directed against an epitope which covers the fibrin-binding site of t-PA. The A and B chains of partially reduced two-chain t-PA were separated by immunoadsorption on immobilized MA-1C8 and MA-2G6. The purified B chain reacted with MA-2G6 but not with MA-1C8 and activated plasminogen following Michaelis-Menten kinetics with kinetic constants similar to those of intact t-PA (Km = 100 microM and kcat = 0.02 s-1). However, fibrin or CNBr-digested fibrinogen did not stimulate the activation of plasminogen by the B chain. The purified A chain reacted with MA-1C8 but not with MA-2G6. It bound to fibrin with an affinity similar to that of intact t-PA but did not activate plasminogen. It is concluded that the active center of t-PA is located in the B chain and the fibrin-binding site in the A-chain. Both functional domains are required for the regulation by fibrin of the t-PA-mediated activation of plasminogen.     

Fibrin structures during tissue-type plasminogen activator-mediated fibrinolysis studied by laser light scattering: relation to fibrin enhancement of plasminogen activation

The aim was to relate fibrin structure and the stimulatory effect of fibrin on plasminogen activation during t-PA-mediated fibrinolysis using Lys⁷⁸-plasminogen as activator substrate. Structural studies were undertaken by static and dynamic laser light scattering, cryo transmission electron microscopy and by the measurement of conversion of fibrin to X-, Y- and D-fragments. The kinetics of plasmin formation were monitored by measurement of the rate of pNA-release from Val-LeuLys-pNA. The process of fibrin formation and degradation comprised three phases. In the first phase, protofibrils with an average length of about 10 times that of fibrinogen were formed. The duration of this phase decreased with increasing t-PA concentration. The second phase was characterized by a sudden elongation and lateral aggregation of fibrin fibers, most pronounced at low levels of t-PA, and by formation of fragment X-polymer. The third phase was dominated by fragmentation of fibers and by formation of Y- and D-fragments: Plasmin degraded the fibers from within, resulting in the formation of long loose bundles, which subsequently disintegrated into thin filaments with a length of less than 10 and a mass per length close to one relative to fibrinogen. Plasmin generation at high t-PA concentrations sets in just prior to (and at low t-PA concentrations shortly after) the onset of the rapid second phase of elongation and lateral aggregation of fibrin fibers. The maximal rate of plasmin formation per mol t-PA was the same at all concentrations of activator and was achieved close to the time of the peak level of fragment X-polymer. Plasmin formation ceased after formation of substantial amounts of Y- and D-fragments. At this stage the length was between 300 and 3 and the mass per length close to 1, both relative to fibrinogen. In conclusion our results indicate that (1) formation of short fibrin protofibrils is the minimal requirement for the onset of the stimulatory effect of fibrin on plasminogen activation by t-PA, (2) formation of fragment X protofibrils is sufficient to induce optimal stimulation of plasminogen activation, and (3) plasmin degrades laterally aggregated fibrin fibers from within, resulting in the conversion of the fibers into long loose bundles, which later disintegrate into thin filaments.Abbreviations t-PA tissue-type plasminogen activator - Lys⁷⁸-plasminogen plasmin-modified plasminogen, mainly with NH₂-terminal lysine (residues 78-791, residue numbering according to Forsgren et al. 1987) - Val-Leu-Lys-pNA H-D-valyl-L-leucyl-L-lysine-4-nitroanilide - Phe-Pip-Arg-pNA H-D-phenylalanyl-L-arginine-4-nitroanilide - pNA p-nitroanilide - SDS sodium dodecyl sulphate The present work has been supported by the Danish Natural Science Research Council and the Danish Agricultural and Veterinary Research CouncilDeceased on August 2, 1991 Correspondence to: R. Bauer