Fibrinolytic Protease Research Articles

A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): Purification and characterization

A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH2-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5′RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-β-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577×10−3mol/L and 4.09×10−3μmol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed Aα and Bβ chains followed by γ-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent.

Antithrombotic effects of a newly purified fibrinolytic protease from Urechis unicinctus

The prevalence of thromboembolic disease, one of the top 3 leading causes of mortality worldwide, is being reported continually. More effective and safer antithrombotic drugs may overcome the underlying problems in antithrombotic therapy. In the present work, antithrombotic effects of UFEIII, a newly purified fibrinolytic protease from Urechis unicinctus were evaluated. UFEIII was purified from the marine invertebrate, Urechis unicinctus, using anion exchange and gel filtration chromatography. Molecular weight, fibrinolytic activity and fibrinogenolysis pattern of UFEIII were determined. Furthermore, antithrombotic effects of UFEIII in vivo were investigated through electrical induced carotid arterial thrombosis in rats, FeCl3 induced carotid arterial thrombus model in rabbits and stasis induced vena caval thrombus model in rats. SDS-PAGE of the purified enzyme showed a single polypeptide chain with molecular weight of 20.8kDa. In fibrin plate assays, UFEIII could not only directly degrade fibrin but also activate plasminogen. The fibrinogenolysis pattern of UFEIII was Aα-chains>Bβ-chains>γ-chain. Moreover, UFEIII could effectively prolong the time to occlusion in electrical induced carotid arterial thrombosis. Besides, both in rabbits and rats, the administration of UFEIII not only prolonged the activated partial thromboplastin time (APTT) and thrombin time (TT) also decreased the fibrinogen (FIB) content. Further, the thrombus lysis was observed after administration of UFEIII both in rabbits and rats. UFEIII can possibly be a new potential source of fibrinolytic agent.

Integrated Process Production and Extraction of the Fibrinolytic Protease from Bacillus sp. UFPEDA 485

Fibrinolytic proteases are enzymes that degrade fibrin; these enzymes are a promising alternative for thrombolytic therapy, and microorganisms produce them. The aim of this study was to evaluate the optimum conditions for the integrated production and purification of fibrinolytic protease from Bacillus sp. UFPEDA 485. Extractive fermentation was carried out in a culture medium containing soybean flour and by adding polyethylene glycol (PEG) and Na2SO4 according to a 2(3) experimental design. In all assays, the enzyme preferentially partitioned to the bottom phase (K < 1), with an optimum activity of 835 U ml(-1) in the bottom phase (salt-rich phase). The best conditions for extractive fermentation were obtained with 18 % PEG 8000 and 13 % Na2SO4. Characterization showed that it is a metalloprotease, as a strong inhibition-residual activity of 3.13 %-occurred in the presence of ethylenediaminetetraacetic acid. It was also observed that enzymatic activity was stimulated in the presence of ions: CaCl2 (440 %), MgCl2 (440 %), FeSO4 (268 %), and KCl (268 %). The obtained results indicate that the use of a low-cost substrate and the integration of fermentation with an aqueous two-phase system extraction may be an interesting alternative for the production of fibrinolytic protease.

Enhanced production of fibrinolytic protease from Bacillus cereus NS-2 using cotton seed cake as nitrogen source

Abstract Cardiovascular complications due to thrombosis have become one of the major causes of mortality. High cost and fatally undesired side effects associated with the available thrombolytic agents motivated the researchers to investigate potentially better agents for therapeutic applications. In the current study, production of an efficacious fibrinolytic protease from a bacterial isolate Bacillus cereus NS-2 was optimized by employing low-cost agricultural residues as substrates (wheat bran and cotton cake at 1%, w/v, each). Wheat bran supported fibrinolytic protease production (148 U/ml) that was comparable with control (145.5 U/ml on glucose). Cotton cake as nitrogen source enhanced fibrinolytic protease production substantially (71%) as compared to control (315 U/ml vs 184 U/ml). Fibrinolytic protease was partially purified (2.35-fold) by ammonium sulfate precipitation and diethylaminoethyl-sepharose chromatography with the yield of 58.27%. Maximum activity of partially purified fibrinolytic protease was observed at 40 °C and at pH 9. Fibrinolytic protease activity was increased immensely by Fe 2+ (76.6%) and moderately by Ca 2+ , Mn 2+ , Zn 2+ , Cu 2+ and Mg 2+ (29–44%), however, Pb 2+ and Hg 2+ strongly inhibited the protease. B. cereus NS-2 protease showed reasonable stability in presence of Triton X-100 and Tween 20 (relative activity 87 and 80%, respectively) but poor stability in presence SDS (relative activity 29%). Retention of considerable activity (46%) in presence of EDTA reflects that requirement of divalent ions is not absolute for catalysis by B. cereus NS-2 fibrinolytic protease.

Herinase: A Novel Bi-functional Fibrinolytic Protease from the Monkey Head Mushroom, Hericium erinaceum

Herinase, a new bi-functional fibrinolytic metalloprotease, was purified from a medicinal and edible mushroom Hericium erinaceum. The enzyme was monomeric with a molecular mass of 51 kDa. Analysis of fibrin zymography showed an active band with a similar molecular mass. The N-terminal sequence of herinase VPSSFRTTITDAQLRG was highly distinguished from known fibrinolytic enzymes. Moreover, the enzyme activity was strongly inhibited by EDTA and EGTA, indicating that herinase is a metalloprotease. Herinase exhibited high specificity for the substrate t-PA followed by plasmin. The K(m) and V(max) values for H-D-Ile-Pro-Arg-PNA were found to be 4.7 mg and 26.7 U/ml respectively. Similarly, fibrin plate assays revealed that it was able to degrade fibrin clot directly and also able to activate plasminogen. Herinase provoked a rapid degradation of fibrin and fibrinogen α chains and slower degradation of γ chains. It had no activity on the β chains of fibrin and fibrinogen. This result suggests that herinase could possibly contain higher amount of α-fibrinogenase. The activity of herinase was stimulated by metal ions such as Ca(2+), Mg(2+), and Mn(2+), but inhibited by Cu(2+), Fe(2+), and Zn(2+). Herinase exhibited maximum activity at 30 °C and pH 7.0. These results demonstrate that herinase could be a novel fibrinolytic enzyme.

Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Three distinct alkaline serine proteases (named CTSP-1, -2, and -3) were purified from the polychaete Cirriformia tentaculata and characterized in terms of their enzymatic properties and kinetics. The estimated molecular masses of CTSP-1, -2, and -3 enzymes were found to be 28.8, 30.9, and 28.4kDa, respectively. The enzymes were active at the temperature range of 50–60°C under pH 8.5–9.0 and completely inactivated by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, but not by 1,10-phenanthroline and bestatin, suggesting that they are all typical serine proteases and not metalloproteases or cysteine proteases. CTSP-1 and -2 cleaved arginine, whereas CTSP-3 digested tyrosine residue at the carboxyl sides in their peptide substrates. A typical hepta-sequence (I-X-X-G-X-X-A) conserved in serine proteases from annelid species was found in N-termini of all CTSPs. CTSP-2 was the most active enzyme among the proteases purified as shown by kinetic values. The enzymes cleaved all chains of fibrinogen within 20min and also hydrolyzed actively fibrin polymer as well as cross-linked fibrin. In addition, the enzymes could actively digest the fibrin clot in blood plasma milieu. Taken together, the results obtained demonstrate that CTSP enzymes have a potential of becoming therapeutic agents for thrombus dissolution.

Deficiency of urokinase plasminogen activator suppresses epithelial sodium channels in tracheal epithelial cells

Epithelial sodium channels (ENaC) govern transepithelial salt and fluid transport in tracheal, bronchi, and lungs. Fibrinolytic proteases play a crucial role in airway and lung injury and repair. We hypothesized that urokinase‐like plasminogen activator (uPA) regulates respiratory ENaC activity in primary mouse tracheal epithelial cells. The short‐circuit currents in confluent monolayer cells were compared between wild type and uPA null mice. Knockout of uPA led to depressed ENaC activity in both intact and permeabilized cells significantly. A significant decrease in Na+/K+‐ATPase activity was observed as well. Application of uPA and plasmin partially restored ENaC activity and increased cleavage of alpha ENaC proteins in confluent monolayers. ERK1/2 but not Akt phosphorylation was augmented in uPA−/− cells and lungs. Taken together, uPA may regulate ENaC activity via multifaceted mechanisms, including proteolytic cleavage of α ENaC proteins, reduction in vectorial Na+ driving force, and upregulation of ERK1/2 phosphorylation.Supported by NIH HL87017 and HL95435

Comparative proteomic analysis of the sun- and freeze-dried earthworm Eisenia fetida with differentially thrombolytic activities

The dried earthworm is a traditional thrombolytic medicine in East Asia. Its thrombolytic mechanism has been extensively studied. However, the effects of drying process on thrombolysis were rarely investigated. Herein, we compared the thrombolytic activity of earthworm Eisenia fetida processed by sun-drying to that by freeze-drying. Fibrin plate and blood clot lysis assays showed that freeze-dried earthworms gave dramatically higher fibrinolytic and thrombolytic activities than the sun-dried earthworms. To address the thrombolytic difference, comparative proteomic analysis was carried out using fibrin zymography and two-dimensional gel electrophoresis (2-DE). The freeze- and sun-dried earthworms generated remarkably different 2-DE protein spot patterns. A total of 126 differential protein spots were detected, 83 of them were identified by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry and database searching with 13 quantitative changes and 70 qualitative changes. Five of these differential proteins were identified as fibrinolytic proteases (lumbrokinases), responsible for dissolving fibrin, the main protein component of thrombus. The total abundance of these fibrinolytic proteases in the freeze-dried earthworms was significantly higher, consistent with the results of fibrin zymography. Therefore, the higher concentration of fibrinolytic enzymes along with their broad substrate specificity explained the stronger fibrinolytic and thrombolytic activities of the freeze-dried earthworms. This study suggests that freeze-drying represents an improved processing method for earthworm as the thrombolytic therapy in the future. Thrombosis has become one of the biggest concerns all over the world. The dried earthworms have been intensively used as thrombolytic agents. Its thrombotic mechanism has been studied by the modern pharmacological researches. However, the drying procedure of the earthworm and its effects on the thrombolysis were rarely investigated. The present study compared the thrombolytic effects of the freeze-dried and the normal dried earthworm E. fetida. To better understand the underlying mechanisms for differential thrombolytic effects, the fibrin zymography and the two-dimensional gel electrophoresis (2-DE) were employed to identify sets of differential proteins. Therefore, this study provides not only the comparative proteomic analysis but also molecular mechanism underlying the differential thrombolytic effects.

Annexin A2 system in human biology: cell surface and beyond.

Annexin A2 (A2) is a multicompartmental, multifunctional protein that orchestrates a growing spectrum of biologic processes. At the endothelial cell surface, A2 and S100A10 (p11) form a heterotetramer, which accelerates tissue plasminogen activator-dependent activation of the fibrinolytic protease, plasmin. In antiphospholipid syndrome, anti-A2 antibodies are associated with clinical thrombosis, whereas overexpression of A2 in acute promyelocytic leukemia promotes hyperfibrinolytic bleeding. A2 is upregulated in hypoxia, and mice deficient in A2 are resistant to oxygen-induced retinal neovascularization, suggesting a role for A2 in human retinal vascular proliferation. In solid malignancies, the (A2•p11)(2) tetramer may promote cancer cell invasion, whereas in multiple myeloma A2 enables malignant plasmacyte growth and predicts prognosis. In the central nervous system, the p11 enables membrane insertion of serotonin receptors that govern mood. In the peripheral nervous system, p11 directs sodium channels to the plasma membrane, enabling pain perception. In cerebral cortex neurons, A2 stabilizes the microtubule-associated tau protein, which, when mutated, is associated with frontotemporal dementia. In inflammatory dendritic cells, A2 maintains late endosomal/lysosomal membrane integrity, thus modulating inflammasome activation and cytokine secretion in a model of aseptic arthritis. Together, these findings suggest an emerging, multifaceted role for A2 in human health and disease.

Thrombolytic, anticoagulant and antiplatelet activities of codiase, a bi-functional fibrinolytic enzyme from Codium fragile

Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. In this study, codiase, a new bi-functional fibrinolytic serine protease having thrombolytic, anticoagulant, and antiplatelet activities was purified from marine green alga, Codium fragile. The molecular weight of the enzyme was estimated to be 48.9 kDa by SDS-PAGE, and mass spectrometry. Fibrin zymography analysis showed an active band with similar molecular weight. The N-terminal sequence was found to be APKASTDQTLPL, which is different from that of other known fibrinolytic enzymes. Codiase displayed maximum activity at 30 °C and pH 6.0, and the activity was inhibited by Zn(2+) and Fe(2+). Moreover, the enzyme activity was strongly inhibited by serine protease inhibitor such as PMSF. Codiase exhibited high specificity for the substrate S-2288, and the Km and Vmax values for this substrate were found to be 0.24 mM and 79 U/ml respectively. Fibrin plate assays revealed that it was able to hydrolyze fibrin clot either directly or by activation of plasminogen. Codiase effectively hydrolyzed fibrin and fibrinogen, preferentially degrading α- and Aα chains, followed by γ-γ, and γ-chains. However, it provoked slower degradation of Bβ and β-chains. The structural change of fibrin clot and fibrinogen by codiase was also detected by FTIR-ATR spectroscopy analysis. In vitro and in vivo studies revealed that codiase reduces thrombosis in concentration-dependent manner. Codiase was found to prolong activated partial thromboplastin time (APTT), and prothrombin time (PT). PFA-100 studies showed that codiase prolonged the closure time (CT) of citrated whole human blood. These favorable antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to mice and NIH-3T3 cells, make it a potential agent for thrombolytic therapy.

Molecular cloning and functional expression of a fibrinolytic protease gene from the polychaeta, Periserrula leucophryna

Full-length cDNA encoding a fibrinolytic protease (PLFP) from the cDNA library of the polychaete, Periserrula leucophryna was cloned, sequenced and expressed in Escherichia coli. The entire cDNA of the PLFP clone was 921 bp (CDS: 41-837), including a coding nucleotide sequence of 798 bp, a 5′-untranslaed region of 40 bp, and a 3′-noncoding region of 83 bp. The ORF encoded a 265-amino acid polypeptide precursor consisting of a 36-residue signal sequence and a 229-residue mature polypeptide. The sequence alignment results of PLFP revealed sequence similarity with several fibrinolytic enzymes. Sequence analysis revealed a conserved catalytic triad of His78, Asp126 and Ser219 residues, suggesting that PLFP is a serine protease. Mature PLFP had an apparent molecular weight of approximately 25 kDa and was produced in inclusion bodies when expressed in E. coli. Substrate specificity results that recombinant PLFP was active towards Arg-X or Lys-X and did not hydrolyze substrates with nonpolar amino acids at the P1 site. Recombinant PLFP was strongly inhibited by typical serine protease inhibitors, further indicating that PLFP is a member of the serine protease family. PLFP was able to dissolve artificial plasminogenfree fibrin, and its fibrinolytic behavior was similar to that of plasmin. Taken these results together, PLFP is a new member of the fibrinolytic enzyme family with selective specificity on fibrin, and the availability of PLFP offers an attractive alternative approach for thrombolysis therapy because rPLFP is believed to have advantages over currently used plasminogen activators, that is, lower price and lower side effect.

Elastase and Tryptase Govern TNFα-Mediated Production of Active Chemerin by Adipocytes

Chemerin is a leukocyte chemoattractant and adipokine with important immune and metabolic roles. Chemerin, secreted in an inactive form prochemerin, undergoes C-terminal proteolytic cleavage to generate active chemerin, a ligand for the chemokine-like receptor-1 (CMKLR1). We previously identified that adipocytes secrete and activate chemerin. Following treatment with the obesity-associated inflammatory mediator TNFα, unknown adipocyte mechanisms are altered resulting in an increased ratio of active to total chemerin production. Based on these findings we hypothesized adipocytes produce proteases capable of modifying chemerin and its ability to activate CMKRL1. 3T3-L1 adipocytes expressed mRNA of immunocyte and fibrinolytic proteases known to activate chemerin in vitro. Following treatment with a general protease inhibitor cocktail (PIC), the TNFα-stimulated increase in apparent active chemerin concentration in adipocyte media was amplified 10-fold, as measured by CMKLR1 activation. When the components of the PIC were investigated individually, aprotinin, a serine protease inhibitor, blocked 90% of the TNFα-associated increase in active chemerin. The serine proteases, elastase and tryptase were elevated in adipocyte media following treatment with TNFα and their targeted neutralization recapitulated the aprotinin-mediated effects. In contrast, bestatin, an aminopeptidase inhibitor, further elevated the TNFα-associated increase in active chemerin. Our results support that adipocytes regulate chemerin by serine protease-mediated activation pathways and aminopeptidase deactivation pathways. Following TNFα treatment, increased elastase and tryptase modify the balance between activation and deactivation, elevating active chemerin concentration in adipocyte media and subsequent CMKLR1 activation.

Extraction of fibrinolytic proteases from Streptomyces sp. DPUA1576 using PEG-phosphate aqueous two-phase systems

Extraction of fibrinolytic protease from Streptomyces sp. DPUA1576 fermentation broth was performed using polyethylene glycol (PEG) – phosphate salts aqueous two phase system (ATPS). The influence of pH, PEG molar mass, PEG and phosphate concentrations on fibrinolytic protease partition coefficient (K) were evaluated by 24 full factorial design. Fibrinolytic protease partitioned preferentially to the (PEG)-rich phase with the highest partition coefficient (K=37.00) obtained in the system 20% (w/w) PEG 1500 (g/mol)-14% (w/w) phosphate at pH 8.0. The system that allowed for the highest extraction consisted of 15% (w/w) PEG 3350-12% (w/w) phosphate, at pH 7.0. In these conditions, a purification factor of 1.51 was obtained for a partition coefficient of 6.41 with the fibrinolytic activity (FA) retained in the top phase. Obtained results confirm the interest of considering ATPS as an alternative extraction method for fibrinolytic protease from Streptomyces sp. DPUA1576.

Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.

Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa

AbstractEP217609 is a new dual-action parenteral anticoagulant that combines an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (α-NAPAP analog) in a single molecule together with a biotin tag to allow avidin neutralization. EP217609 exhibits an unprecedented pharmacologic profile in showing high bioavailability, long plasma half-life, and potent antithrombotic activity in animals without the complications of thrombin rebound. Here we report the exceptional specificity and selectivity profile of EP217609. EP217609 inhibited thrombin with rapid kinetics (kon > 107M−1s−1), a high affinity (KI = 30-40pM), and more than 1000-fold selectivity over other coagulation and fibrinolytic protease targets, comparing favorably with the best direct thrombin inhibitors known. EP217609 bound antithrombin with high affinity (KD = 30nM) and activated the serpin to rapidly (kass ∼ 106M−1s−1) and selectively (> 20-fold) inhibit factor Xa. The dual inhibitor moieties of EP217609 acted largely independently with only modest linkage effects of ligand occupancy of one inhibitor moiety on the potency of the other (∼ 5-fold). In contrast, avidin binding effectively neutralized the potency of both inhibitor moieties (20- to 100-fold). These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone.

The X-ray Crystal Structure of Full-Length Human Plasminogen

Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.

Bafibrinase: A non-toxic, non-hemorrhagic, direct-acting fibrinolytic serine protease from Bacillus sp. strain AS-S20-I exhibits in vivo anticoagulant activity and thrombolytic potency

A non-toxic, direct-acting fibrinolytic serine protease (Bafibrinase) demonstrating thrombolytic and anticoagulant properties was purified from Bacillus sp. strain AS-S20-I. Bafibrinase was monomeric, with a molecular mass of 32.3 kDa. The peptide mass fingerprinting of Bafibrinase revealed only 8.3% sequence coverage, suggesting it was a novel fibrinolytic enzyme. However, two of the tryptic digested de novo peptide sequences of Bafibrinase demonstrated good similarity with endopeptidases possessing serine in their catalytic triad. Further, catalytic activity of Bafibrinase was inhibited by serine protease inhibitor reinforcing this is a subtilisin-like serine protease. The apparent Km and Vmax values of Bafibrinase towards fibrin were determined as 0.24 μM and 2.8 μmol/min, respectively. It showed a Km value of 0.139 mM towards a chromogenic substrate for plasmin (d-Val-Leu-Lys-p-Nitroanilide dihydrochloride) and optimum activity at physiological conditions (37 °C and pH 7.4). Based on the cleavage pattern of fibrin and fibrinogen, Bafibrinase may be classified as an α,β-fibrinogenase. Bafibrinase could not degrade collagen and was non-cytotoxic to HT29 cells or mammalian erythrocytes. Further, Bafibrinase at a dose of 2 mg/kg was devoid of toxicity as well as hemorrhagic activity on BALB/c mouse model, supporting its suitability for the development of a better and safer thrombolytic drug. Bafibrinase was also superior to human plasmin in degrading in vitro thrombus. The in vivo anticoagulant nature of Bafibrinase is being explored for the treatment and prevention of thrombosis and other cardiovascular diseases.

Characterization of Fibrinolytic Proteases from Gloydius Blomhoffii Siniticus Venom

Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods : The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1) The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2) The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3) Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II) chloride inhibited it. 4) The fibrinolytic protease cleaved preferentially A-chain and slowly B-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions : The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

Herpesviruses enhance fibrin clot lysis

The incorporation of virus- and host-derived procoagulant factors initiates clotting directly on the surface of herpesviruses, which is an explanation for their correlation to vascular disease. The virus exploits the resulting thrombin to enhance infection by modulating the host cell through protease activated receptor (PAR) 1 signalling. Prior reports demonstrated that at least one herpesvirus expresses surface annexin A2 (A2), a cofactor for tissue plasminogen activator (tPA)-dependent activation of plasminogen to plasmin. Since plasmin is both a fibrinolytic protease and PAR agonist, we investigated whether herpesviruses enhance fibrinolysis and the effect of plasmin on cell infection. Herpes simplex virus types 1 (HSV1) and 2, and cytomegalovirus (CMV) purified from various cell lines each accelerated the proteolytic activation of plasminogen to plasmin by tPA. Ligand blots identified A2 as one of several plasminogen binding partners associated with the virus when compared to an A2-deficient virus. This was confirmed with inhibitory A2-antibodies. However, A2 was not required for virus-enhanced plasmin generation. HSV1, HSV2 and CMV accelerated tPA-dependent fibrin clot lysis by up to 2.8-fold. Modest plasmin generation and fibrinolysis was detected independent of exogenous tPA, which was inhibited by plasminogen activator inhibitor type-1 and ε-aminocaproic acid; however, the molecular basis remains speculative. Up to a ~6-fold enhancement of infection was provided by plasmin-mediated cell infection. Inhibitory antibodies revealed that plasmin increased HSV1 infection through a mechanism involving PAR2. Thus, virus-enhanced fibrinolysis may help explain the paradox of the highly procoagulant in vitro herpesvirus surface eliciting only relatively weak independent vascular disease risk.

A two-dimensional electrophoresis reference map of the earthworm &amp;lt;i&amp;gt;Eisenia fetida&amp;lt;/i&amp;gt;

The molecular mechanisms underlying innate immunity in the earthworm E. fetida remain unclear. For the recognition of innate immunity in the earthworm E. fetida, a detailed knowledge of this proteome is a prerequisite. The absence of a high-resolution E. fetida proteome map prompted us to determine E. fetida protein spots that can be visualised on 2-D protein gels. In this study, we present a preliminary description of the whole earthworm E. fetida proteome. A highly detailed two-dimensional gel electrophoresis (2-DE) map of the E. fetida proteome was established and approximately 1500 protein spots were detected from the earthworm sample when applying a 500 μg protein 2-DE in the pH range 3.0 - 10.0. We present a 2-DE proteome map of E. fetida, identifying 76 different proteins by matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry (MALDI-TOF/ TOF-MS) analysis. These identified proteins, including heat shock protein 90 (Hsp90), chaperonine protein HSP60, caspase-8, fibrinolytic protease 0, gelsolin-like protein, lombricine kinase, coelomic cytolytic factor1 (CCF 1), manganous superoxide dismutase (MnSOD), triosephosphate isomerase, extracellular globin-4, lysenin, and intermediate filament protein, glyceraldehyde-3- phosphate dehydrogenase, et al., are involved in several processes, including transcripttion, translation, the tricarboxylic acid cycle, the cellular amino acid metabolic process, protein amino acid phosphorylation, glycolysis, and the glucose metabolic process. These 2-DE data will enhance future comparisons of immunity, toxicology, biotic processes and other challenges, thereby allowing for further study of the molecular mechanisms in response to environmental stressors, and it will be useful to investigate environmental proteomics in invertebrate earthworms.