Xylarinase: a novel clot busting enzyme from an endophytic fungus Xylaria curta

Xylarinase is a bi-functional fibrinolytic metalloprotease isolated from the culture filtrate of endophytic fungus Xylaria curta which is monomeric with a molecular mass of ～33.76?kDa. The enzyme displayed both plasmin and tissue plasminogen activator like activity under in vitro conditions. It hydrolyses Aα and Bβ chains of the fibrinogen. Optimal fibrinolytic activity of xylarinase is observed at 35?°C, pH 8. Ca²⁺ stimulated the fibrinolytic activity of xylarinase while Fe²⁺ and Zn²⁺ inhibited suggesting it to be a metalloprotease. The K_m and V_max values of xylarinase were 240.9?μM and 1.10?U/ml for fibrinogen and 246?μM and 1.22?U/ml for fibrin, respectively. Xylarinase was found to prolong the activated partial thromboplastin time and prothrombin time. The N-terminal sequence of xylarinase (SNGPLPGGVVWAG) did not show any homology with previously known fibrinolytic enzymes. Thus xylarinase is a novel fibrinolytic metalloprotease which could be possibly used as a new clot busting enzyme.     

Purification and partial characterization of a fibrinolytic enzyme from the fruiting body of the medicinal and edible mushroom Pleurotus ferulae

A fibrinolytic metalloprotease with in vitro fibrinolytic effects was purified from the edible mushroom Pleurotus ferulae using several chromatography steps including anion and ion exchange, gel filtration, and fast protein liquid chromatography columns. The molecular mass of the enzyme was estimated to be 20.0?kDa, as determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin zymography. The protease was active at 50°C, and pH 4.0, 5.0, and 8.0. The fibrinolytic activity of the enzyme was inhibited by ethyleneglycol-bis-(2-aminoethyl)-N,N,N′,N′ tetraacetic acid and strongly inhibited by two metal ions, Cu and Mg. In vitro assays evaluating fibrinolytic activity on a fibrin plate, fibrin turbidity, and thrombolytic activity on fibrin clots using human fibrinogen and human thrombin revealed that the enzyme could hydrolyze fibrin polymers directly and inhibit the formation of fibrin clots. In activated partial thromboplastin time (APTT) and prothrombin time assays, the enzyme strongly prolonged the APTT, which detects an activity of intrinsic and common pathways. The enzyme showed strong in vivo protective effect against mortality/paralysis from epinephrine plus collagen-induced acute thromboembolism in in vivo model. Our findings suggest that the enzyme may have a potential for treatment and prevention of thrombosis-relative diseases.     

Simultaneous activation of complement and coagulation by MBL-associated serine protease 2

The complement system is an important immune mechanism mediating both recognition and elimination of foreign bodies. The lectin pathway is one pathway of three by which the complement system is activated. The characteristic protease of this pathway is Mannan-binding lectin (MBL)-associated serine protease 2 (MASP2), which cleaves complement proteins C2 and C4. We present a novel and alternative role of MASP2 in the innate immune system. We have shown that MASP2 is capable of promoting fibrinogen turnover by cleavage of prothrombin, generating thrombin. By using a truncated active form of MASP2 as well as full-length MASP2 in complex with MBL, we have shown that the thrombin generated is active and can cleave both factor XIII and fibrinogen, forming cross-linked fibrin. To explore the biological significance of these findings we showed that fibrin was covalently bound on a bacterial surface to which MBL/MASP2 complexes were bound. These findings suggest that, as has been proposed for invertebrates, limited clotting may contribute to the innate immune response.     

Monoclonal antibody light chain with prothrombinase activity

Prothrombin is the precursor of thrombin, a central enzyme in coagulation. Autoantibodies to prothrombin are associated with thromboembolism, but the mechanisms by which the antibodies modulate the coagulation processes are not understood. We screened a panel of 34 monoclonal antibody light chains isolated from patients with multiple myeloma for prothrombinase activity by an electrophoresis method. Two light chains with the activity were identified, and one of the light chains was characterized further. The prothrombinase activity eluted from a gel-filtration column run in denaturing solvent (6 M guanidine hydrochloride) at the characteristic positions of the light chain dimer and monomer. A constant level of catalytic activity was observed across the width of the light chain monomer peak, assessed as the cleavage of IEGR-methylcoumarinamide, a peptide substrate corresponding to residues 268-271 of prothrombin. Hydrolysis of this peptide by the light chain was saturable and consistent with Michaelis-Menten-Henri kinetics (K(m) 103 microM; k(cat) of 2.62 x 10(-)(2)/min). Four cleavage sites in prothrombin were identified by N-terminal sequencing of the fragments: Arg(155)-Ser(156), Arg(271)-Thr(272), Arg(284)-Thr(285), and Arg(393)-Ser(394). The light chain did not cleave radiolabeled albumin, thyroglobulin, and annexin V under conditions that readily permitted detectable prothrombin cleavage. Two prothrombin fragments (M(r) 55 000 and 38 000), were isolated by anion-exchange chromatography and were observed to cleave a thrombin substrate, tosyl-GPR-nitroanilide. Conversion of fibrinogen to fibrin was accelerated by the prothrombin fragments generated by the light chain. These finding suggest a novel mechanism whereby antibodies can induce a procoagulant state, i.e., prothrombin activation via cleavage of the molecule.     

Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel β-strands with a unique fold that has a compact β-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe³⁺). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates in iron uptake from iron-withholding proteins of the host cell during infection.     

The effect of fibrin structure on fibrinolysis.

Fibrin structure contributes to the regulation of the fibrinolytic rate. As the fibrin fiber size is decreased, the fibrinolytic rate also decreases. Fibrin structure was altered by either changing the ratio of thrombin to fibrinogen, i.e. altering the assembly rate or by adding a fibrin assembly inhibitor, iopamidol. Changes in the fibrinolytic rate were followed by measuring the time dependence of the decrease in the fiber mass/length ratio during fibrinolysis. A measure of the overall fibrinolytic rate was determined from the decrease in the mass/length ratio versus time. An 8-fold reduction in the fibrinolytic rate was seen on decreasing the mass/length ratio from 2.7 x 10(12) daltons/cm to 0.5 x 10(12) daltons/cm. It is shown that thin fibrin fibers have a decreased rate of conversion of plasminogen to plasmin by tissue plasminogen activator and that thin fibrin fibers are lysed more slowly than thick fibrin fibers.     

Pleiotropic effects of a vibrio extracellular protease on the activation of contact system

Many proteases secreted by pathogenic bacteria can affect seriously on hemostatic system. We have reported that an extracellular zinc metalloprotease (named vEP-45) from Vibrio vulnificus ATCC29307 activates prothrombin to active thrombin, leading the formation of fibrin clot. In this study, the effects of vEP-45 on the intrinsic pathway of coagulation and the kallikrein/kinin system were examined. The protease could activate proteolytically clotting factor zymogens, including FXII, FXI, FX, and prothrombin, to their functional enzymes in vitro and plasma milieu. In addition, it could cleave plasma prekallikrein (PPK) to form an active kallikrein as well as actively digest high-molecular weight kininogen (HK), probably producing bradykinin. In fact, vEP-45 could induce a vascular permeability in a dose-dependent manner in vivo. Taken together, the results demonstrate that vEP-45 can activate plasma contact system by cleaving key zymogen molecules, participating in the intrinsic pathway of coagulation and the kallikrein/kinin system.     

Identification and characterization of novel salivary thrombin inhibitors from the ixodidae tick, Haemaphysalis longicornis.

Novel antithrombin molecules were identified from the ixodidae tick, Haemaphysalis longicornis. These molecules, named madanin 1 and 2, are 7-kDa proteins and show no significant similarities to any previously identified proteins. Assays using human plasma showed that madanin 1 and 2 dose-dependently prolonged both activated partial thromboplastin time and prothrombin time, indicating that they inhibit both the intrinsic and extrinsic pathways. Direct binding assay by surface plasmon resonance measurement demonstrated that madanin 1 and 2 specifically interacted with thrombin. Furthermore, it was clearly shown that madanin 1 and 2 inhibited conversion of fibrinogen into fibrin by thrombin, thrombin-catalyzed activation of factor V and factor VIII, and thrombin-induced aggregation of platelets without affecting thrombin amidolytic activity. These results suggest that madanin 1 and 2 bind to the anion-binding exosite 1 on the thrombin molecule, but not to the active cleft, and interfere with the association of fibrinogen, factor V, factor VIII and thrombin receptor on platelets with an anion-binding exosite 1. They appear to be exosite 1-directed competitive inhibitors.     

Formation of a ternary complex between thrombin, fibrin monomer, and heparin influences the action of thrombin on its substrates

The consequences of the combined effects of fibrin II monomer (FnIIm) and heparin (H) on the hydrolysis of peptidyl p-nitroanilide substrates by thrombin (IIa), the cleavage of prothrombin by thrombin and the thrombin-catalyzed release of fibrinopeptides from fibrinogen have been studied at pH 7.4 and I 0.15. The effects of fibrin II monomer and heparin on chromogenic substrate hydrolysis can be described by a hyperbolic mixed inhibition model in which substrate can interact with four possible enzyme species (IIa, IIa.H, IIa.FnIIm, and IIa.FnIIm.H) that arise as a result of random formation of a ternary complex among thrombin, fibrin II monomer, and heparin (Hogg, P. J. and Jackson, C. M. (1990) J. Biol. Chem. 265, 241-247). The formation of the ternary IIa.FnIIm.H complex results in an increase in the Km values of 7.03 +/- 1.17-fold (1.37-9.65 microM) and 1.94 +/- 0.60-fold (38.1-73.9 microM) for H-D-Ile-Pro-Arg-pNA and Cbz-Gly-Pro-Arg-pNA hydrolysis, respectively, and a decrease in the kc values of 0.45 +/- 0.08-fold (49.5-22.3 s-1) and 0.52 +/- 0.05-fold (93.1-48.4 s-1). Fibrin II monomer and heparin in combination also decrease the efficiency (kc/Km) with which thrombin cleaves prothrombin to produce Fragment 1 and Prethrombin 1 by 2.3-fold from 607 +/- 30 to 264 +/- 13 M-1 s-1. In contrast to the effects of fibrin II monomer and heparin on thrombin hydrolysis of chromogenic substrates, its proteolysis of prothrombin and its inactivation by antithrombin III (Hogg, P. J., and Jackson, C. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3619-3623), these components have no discernible influence on the ability of thrombin to cleave fibrinogen. These observations indicate that the substrate specificity of thrombin is altered when it is bound in a complex with fibrin II monomer and heparin and suggest that the catalytic efficiency of thrombin for its physiological substrates will be affected differentially by these interactions. Such ternary complex formation involving thrombin, fibrin II monomer, and heparin may provide a mechanism for selectively regulating thrombin action.     

Expression, isolation, and characterization of an active site (serine 528----alanine) mutant of recombinant bovine prothrombin

An active site mutant bovine prothrombin cDNA (Ser528----Ala) has been constructed, subcloned, and expressed in Chinese hamster ovary cells. The recombinant mutant prothrombin, expressed at the level of 1.5-2.0 micrograms/ml of cell medium, was fully carboxylated (9.9 +/- 0.4 mol of gamma-carboxyglutamic acid/mol of prothrombin). The mutant prothrombin could be activated to thrombin by Taipan snake venom and activated to meizothrombin by ecarin in a manner comparable to native bovine prothrombin or recombinant wild-type bovine prothrombin. The mutant meizothrombin thus formed was stable and did not autolyze. The initial rate of cleavage of mutant prothrombin catalyzed by the full prothrombinase was only 28% of the rate of cleavage of native prothrombin, while recombinant wild-type prothrombin was cleaved at the same rate as the native molecule. The mutant thrombin, obtained from the mutant prothrombin in situ by prothrombinase or Taipan snake venom activation, showed no enzymatic activity toward either fibrinogen or a synthetic chromogenic substrate, D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride (S2238). The mutant thrombin also bound dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, a specific fluorescent inhibitor of the thrombin active site, with a weaker binding affinity (kd = 5.4 x 10(-8) M) than did native thrombin (kd = 1.7 x 10(-8) M). These results indicate that the mutant recombinant prothrombin described here is a useful tool for the study of meizothrombin or thrombin without the complications arising from the proteolytic activities of these molecules. Study of the activation of this mutant has already revealed a functional link between the site of initial cleavage by the prothrombinase and the conformation at the nascent active site of prothrombin.     

Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation

The conversion of prothrombin into thrombin by Factor Xa requires the cleavage of two peptide bonds in prothrombin. Dependent on the order of cleavage, prethrombin 2 or meizothrombin occurs as intermediate. Since prethrombin 2 has as yet been the only observed intermediate, prothrombin activation is generally considered to proceed via prethrombin 2. In this paper we present new methods that allow differentiation between meizothrombin and thrombin formed during the initial phase of prothrombin activation. These methods, which make use of the different reactivities of meizothrombin and thrombin toward fibrinogen and antithrombin III plus heparin, enabled us to show the generation of considerable amounts of meizothrombin during Factor Xa-catalyzed prothrombin activation. Both meizothrombin and thrombin incorporated the active site-directed fluorescent chloromethyl ketone 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-CH2Cl. Gel electrophoretic analysis of chloromethyl ketone-treated aliquots of prothrombin activation mixtures confirmed meizothrombin formation. These observations demonstrate that prothrombin may also be converted into thrombin via meizothrombin.     

The N-terminal propeptide of Vibrio vulnificus extracellular metalloprotease is both an inhibitor of and a substrate for the enzyme

Vibrio vulnificus, a marine bacterium capable of causing wound infection and septicemia, secretes a 45-kDa metalloprotease (vEP) with many biological activities. The precursor of vEP consists of four regions: a signal peptide, an N-terminal propeptide (nPP), a C-terminal propeptide, and the mature protease. Two forms of vEP-vEP-45, which contains the mature protease plus the C-terminal propeptide, and vEP-34, which contains only the mature protease-were expressed in Escherichia coli and purified. vEP-45 and vEP-34 had similar activities with azocasein as a substrate, but vEP-34 had reduced activity toward insoluble proteins. The nPP of vEP was expressed as a His tag fusion protein, and its effect on vEP activity was investigated. nPP inhibited the activities of both vEP-45 and vEP-34 but not that of thermolysin, a different but related zinc-dependent protease. The inhibition of vEP by nPP was further examined using vEP-34 as a representative enzyme. The inhibition could be completely reversed under conditions of low enzyme and propeptide concentrations and with prolonged incubation, which resulted from the degradation of nPP by vEP. However, even at high nPP and vEP concentrations, inhibition of vEP by nPP at high temperatures was not effective, resulting in the degradation of both nPP and vEP. These results demonstrate that the nPP of vEP could bind to vEP and inhibit its activity, resulting in the degradation of the propeptide.     

SCM, a novel M-like protein from Streptococcus canis, binds (mini)-plasminogen with high affinity and facilitates bacterial transmigration

Streptococcus canis is an important zoonotic pathogen capable of causing serious invasive diseases in domestic animals and humans. In the present paper we report the binding of human plasminogen to S. canis and the recruitment of proteolytically active plasmin on its surface. The binding receptor for plasminogen was identified as a novel M-like protein designated SCM (S. canis M-like protein). SPR (surface plasmon resonance) analyses, radioactive dot-blot analyses and heterologous expression on the surface of Streptococcus gordonii confirmed the plasminogen-binding capability of SCM. The binding domain was located within the N-terminus of SCM, which specifically bound to the C-terminal part of plasminogen (mini-plasminogen) comprising kringle domain 5 and the catalytic domain. In the presence of urokinase, SCM mediated plasminogen activation on the bacterial surface that was inhibited by serine protease inhibitors and lysine amino acid analogues. Surface-bound plasmin effectively degraded purified fibrinogen as well as fibrin clots, resulting in the dissolution of fibrin thrombi. Electron microscopic illustration and time-lapse imaging demonstrated bacterial transmigration through fibrinous thrombi. The present study has led, for the first time, to the identification of SCM as a novel receptor for (mini)-plasminogen mediating the fibrinolytic activity of S. canis.     

Structural basis for sequential cleavage of fibrinopeptides upon fibrin assembly

Nonsubstrate interaction of thrombin with fibrinogen promotes sequential cleavage of fibrinopeptides A and B (fpA and fpB, respectively) from the latter, resulting in its conversion into fibrin. The recently established crystal structure of human thrombin in complex with the central part of human fibrin clarified the mechanism of this interaction. Here, we reveal new details of the structure and present the results of molecular modeling of the fpA- and fpB-containing portions of the Aalpha and Bbeta chains, not identified in the complex, in both fibrinogen and protofibrils. The analysis of the results reveals that in fibrinogen the fpA-containing portions are in a more favorable position to bind in the active site cleft of bound thrombin. Surface plasmon resonance experiments establish that the fpB-containing portions interact with the fibrin-derived dimeric D-D fragment, suggesting that in protofibrils they bind to the newly formed DD regions bringing fpB into the vicinity of bound thrombin. These findings provide a coherent rationale for the preferential removal of fpA from fibrinogen at the first stage of fibrin assembly and the accelerated cleavage of fpB from protofibrils and/or fibrils at the second stage.     

STUDIES ON BLOOD COAGULATION : I. THE R?LE OF PROTHROMBIN AND OF PLATELETS IN THE FORMATION OF THROMBIN

1. A method is described for the preparation and titration of prothrombin and thrombin. 2. Confirming the views of Morawitz, Howell (1916–17, 1925), and Bordet, thrombin cannot be regarded as an artificial by-product of coagulation (Wooldridge, Nolf (both quoted from Morawitz)). Calcium, a platelet factor, and a plasma factor (prothrombin) interact to form thrombin, and this then acts upon fibrinogen to form fibrin. The amount and rate of thrombin formation in the first reaction are independent of the presence or absence of fibrinogen. After a variable latent period, thrombin suddenly appears in large quantities, coincident with or immediately preceding the deposition of fibrin if fibrinogen is present. 3. The amount of thrombin formed in a mixture of prothrombin, Ca and platelets is independent of the platelet or Ca concentration, and depends primarily upon the amount of prothrombin used. The platelets (or cephalin) enormously accelerate the transformation of prothrombin to thrombin, and this acceleration seems to be their physiological rôle in the coagulation process. 4. Contrary to previous reports, platelets have not been demonstrated to contain significant quantities of prothrombin. 5. The available data do not allow any definite decision as to whether the platelet factor actually combines with prothrombin to form thrombin, or merely catalyzes the transformation. The very slow formation of thrombin in the complete absence of platelets may be due to dissolved traces of platelet material released during the physical manipulation of the plasma (centrifuging, Berkefeld filtration). 6. There was no evidence for a species-specific activity of platelets in the transformation of prothrombin to thrombin.     

Modification of fibrinogen by homocysteine thiolactone increases resistance to fibrinolysis: a potential mechanism of the thrombotic tendency in hyperhomocysteinemia

We have previously shown functional differences in fibrinogen from hyperhomocysteinemic rabbits compared to that in control rabbits. This acquired dysfibrinogenemia is characterized by fibrin clots that are composed of abnormally thin, tightly packed fibers with increased resistance to fibrinolysis. Homocysteine thiolactone is a metabolite of homocysteine (Hcys) that can react with primary amines. Recent evidence suggests that Hcys thiolactone-lysine adducts form in vivo. We now demonstrate that the reaction of Hcys thiolactone with purified fibrinogen in vitro produces fibrinogen (Hcys fibrinogen) with functional properties that are strikingly similar to those we have observed in homocysteinemic rabbits. Fibrinogen purified from homocysteinemic rabbits and Hcys fibrinogen are similar in that (1) they both form clots composed of thinner, more tightly packed fibers than their respective control rabbit and human fibrinogens; (2) the clot structure could be made to be more like the control fibrinogens by increased calcium; and (3) they both form clots that are more resistant to fibrinolysis than those formed by the control fibrinogens. Further characterization of human fibrinogens showed that Hcys fibrin had similar plasminogen binding to that of the control and an increased capacity for binding tPA. However, tPA activation of plasminogen on Hcys fibrin was slower than that of the control. Mass spectrometric analysis of Hcys fibrinogen revealed twelve lysines that were homocysteinylated. Several of these are close to tPA and plasminogen binding sites. Lysines are major binding sites for fibrinolytic enzymes and are also sites of plasmin cleavage. Thus, modification of lysines in fibrinogen could plausibly lead to impaired fibrinolysis. We hypothesize that the modification of lysine by Hcys thiolactone might occur in vivo, lead to abnormal resistance of clots to lysis, and thereby contribute to the prothrombotic state associated with homocysteinemia.     

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.     

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)     

华广虻溶纤活性蛋白(TAFP)的性质和对大鼠血液流变学的影响

华广虻溶纤活性蛋白 (TAFP)经血纤蛋白平板法和试管凝块法测定表明 ,TAFP只具有纤溶酶作用 ,不具有激活纤溶酶原的作用 .TAFP的最适 p H为 7.5,且在 p H为 6.0时最稳定 .蛋白水解酶抑制剂对 TAFP的抑制作用显示 :STI>antipain>SBBI>antitrypsin>TLCK>leupeptin>bacteracin>PMSF>TPCK,金属蛋白酶抑制剂 1 ,1 0 - phenanthroline对 TAFP没有抑制作用 .TAFP能显著的延长大鼠出血时间、抑制血小板聚集性 ;显著降低血浆中血纤蛋白原含量、全血粘度、血浆粘度、红细胞压积 ;减慢血沉速度     

Activation and Proteolytic Activity of the Treponema pallidum Metalloprotease,Pallilysin

Treponema pallidum is a highly invasive pathogen that undergoes rapid dissemination to establish widespread infection. Previous investigations identified the T. pallidum adhesin, pallilysin, as an HEXXH-containing metalloprotease that undergoes autocatalytic cleavage and degrades laminin and fibrinogen. In the current study we characterized pallilysin''s active site, activation requirements, cellular location, and fibrin clot degradation capacity through both in vitro assays and heterologous treponemal expression and degradation studies. Site-directed mutagenesis showed the pallilysin HEXXH motif comprises at least part of the active site, as introduction of three independent mutations (AEXXH [H¹⁹⁸A], HAXXH [E¹⁹⁹A], and HEXXA [H²⁰²A]) abolished pallilysin-mediated fibrinogenolysis but did not adversely affect host component binding. Attainment of full pallilysin proteolytic activity was dependent upon autocatalytic cleavage of an N-terminal pro-domain, a process which could not occur in the HEXXH mutants. Pallilysin was shown to possess a thrombin cleavage site within its N-terminal pro-domain, and in vitro studies confirmed cleavage of pallilysin with thrombin generates a truncated pallilysin fragment that has enhanced proteolytic activity, suggesting pallilysin can also exploit the host coagulation process to facilitate protease activation. Opsonophagocytosis assays performed with viable T. pallidum demonstrated pallilysin is a target of opsonic antibodies, consistent with a host component-interacting, surface-exposed cellular location. Wild-type pallilysin, but not the HEXXA mutant, degraded fibrin clots, and similarly heterologous expression of pallilysin in the non-invasive spirochete Treponema phagedenis facilitated fibrin clot degradation. Collectively these results identify pallilysin as a surface-exposed metalloprotease within T. pallidum that possesses an HEXXH active site motif and requires autocatalytic or host-mediated cleavage of a pro-domain to attain full host component-directed proteolytic activity. Furthermore, our finding that expression of pallilysin confers upon T. phagedenis the capacity to degrade fibrin clots suggests this capability may contribute to the dissemination potential of T. pallidum.