Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase. 148 77

Glycoprotein VI is a type I membrane protein identified as a key platelet receptor for collagen. In vitro binding of the GPVI receptor with collagen leads to activation and ultimately to aggregation of platelets. In vivo, GPVI-collagen interactions could cause formation of occlusive thrombi within vessels with damaged endothelial barriers. GPVI antagonists are therefore important therapeutics in patients suffering from collagen-mediated ischemic disorders such as myocardial infarction or stroke. Polyclonal antibodies to GPVI prepared from one patient serum have previously been described. However, only their monovalent Fab fragments, incapable of receptor crosslinking, were found to protect platelets from collagen-mediated aggregation. Here we describe GPVI-neutralizing human antibodies derived from a combinatorial phage display library of single-chain antibodies. By selecting phage on GPVI-expressing U937 cells, we isolated five specific antibodies - A4, A9, A10, C3 and C9. Of the set A10 and C3 specifically blocked GPVI binding to collagen-rich adventitial layers in aorta sections. The higher affinity antibody A10 inhibited binding of snake-venom convulxin to GPVI. It also specifically protected human platelets from collagen-induced aggregation in vitro. A10-bound platelets could still be activated by ADP or thrombin suggesting that this human scFv may represent an original anti-platelet agent for the treatment of collagen-mediated thrombotic diseases.
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PMID:Anti GPVI human antibodies neutralizing collagen-induced platelet aggregation isolated from a combinatorial phage display library. 1245 70

A recombinant prodrug, single-chain urokinase-type plasminogen activator (scuPA) fused to an anti-PECAM-1 antibody single-chain variable fragment (anti-PECAM scFv/scuPA) targets endothelium and augments thrombolysis in the pulmonary vasculature.(1) To avoid premature activation and inactivation and to limit systemic toxicity, we replaced the native plasmin activation site in scFv/low-molecular-weight (lmw)-scuPA with a thrombin activation site, generating anti-PECAM scFv/uPA-T that (1) is latent and activated by thrombin instead of plasmin; (2) binds to PECAM-1; (3) does not consume plasma fibrinogen; (4) accumulates in mouse lungs after intravenous injection; and (5) resists PA inhibitor PAI-1 until activated by thrombin. In mouse models of pulmonary thrombosis caused by thromboplastin and ischemia-reperfusion (I/R), scFv/uPA-T provided more potent thromboprophylaxis and greater lung protection than plasmin-sensitive scFv/uPA. Endothelium-targeted thromboprophylaxis triggered by a prothrombotic enzyme illustrates a novel approach to time- and site-specific regulation of proteolytic reactions that can be modulated for therapeutic benefit.
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PMID:Prophylactic thrombolysis by thrombin-activated latent prourokinase targeted to PECAM-1 in the pulmonary vasculature. 1804 68

Coagulation factor VIII (FVIII) is a ligand for two members of the low-density lipoprotein receptor family, low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein receptor, which cooperate in regulating clearance of FVIII from the circulation. This study was aimed to explore the mechanism of interaction of FVIII with very low density lipoprotein receptor (VLDLR), another member of the family, and map receptor-binding sites. Binding of plasma-derived FVIII and its fragments to recombinant soluble ectodomain of VLDLR (sVLDLR) was studied in solid-phase and surface plasmon resonance assays. Full-length FVIII and its light chain bound to sVLDLR with similar affinities (KD = 114 +/- 14 and 95 +/- 11 nmol/l, respectively); in contrast, exposure of high-affinity VLDLR-binding site within the heavy chain (KD = 30 +/- 2 nmol/l) required proteolytic cleavage by thrombin. The VLDLR-binding sites within heavy and light chains were mapped to the A2 domain residues 484-509 and the A3-C1 fragment, based on the inhibitory effects of anti-A2 monoclonal antibody 413 and anti-A3-C1 antibody fragment scFv KM33, respectively, previously shown to inhibit FVIII/LRP interaction. Soluble ligand-binding fragment of VLDLR inhibited activation of factor X by the intrinsic Xase in purified system. In cell culture, a higher Xase activity was associated with wild-type human embryonic kidney cells compared with transfected cells that express VLDLR on the cell surface. We conclude that the binding sites for VLDLR and LRP within FVIII overlap and the A2 site becomes exposed upon physiological activation of FVIII. A functional role of FVIII/VLDLR interaction may be related to regulation of intrinsic Xase activity.
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PMID:The binding sites for the very low density lipoprotein receptor and low-density lipoprotein receptor-related protein are shared within coagulation factor VIII. 1827 39

Thrombin activatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis and is considered as an attractive drug target. We generated two different antibody fragments, an antigen-binding fragment (Fab) and a single-chain variable fragment (scFv), derived from three distinct monoclonal antibodies (MAs) that inhibit the activation of TAFI by the thrombin/thrombomodulin complex (T/TM) and plasmin (MA-T1C10 and MA-T94H3) or by T/TM alone (MA-T12D11). The Fabs were obtained by papain digestion of the purified MAs, whereas the scFvs were cloned and subsequently expressed in bacteria. All antibody fragments revealed similar or slightly decreased affinities compared to those of the respective MAs, except scFv-T94H3. In the presence of a 16-fold molar excess of all antibody fragments, activation of TAFI by T/TM was completely blocked. Furthermore, Fab and scFv-derivatives from MA-T1C10 and MA-T94H3 were capable of interfering with the plasmin-mediated activation of TAFI. Addition of 850 nM of MA, Fab or scFv to an in-vitro clot lysis assay caused a significant reduction of clot lysis time (except for scFv-T94H3) and this effect was comparable to that of potato tuber carboxypeptidase inhibitor, a well-known TAFIa inhibitor. Dose-response experiments with the antibody (derivatives) in clot lysis and chromogenic assay revealed that the inhibitory capacity of the Fabs was comparable to that of the MAs, whereas the scFvs had a more reduced potency. In conclusion, these highly specific TAFI inhibitors are interesting tools to further evaluate the concept of TAFI inhibition in various in-vitro and in-vivo models.
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PMID:Comparative study of inhibitory antibody derivatives towards thrombin activatable fibrinolysis inhibitor. 1957 70

A novel protein equalizer was developed with single chain variable fragment (scFv) library displaying M13 phage covalently bonded on monolithic cryogel. Due to the great number and various kinds of displayed scFv fragments, as well as strong and specific binding capacity between scFv fragments and proteins, a new protein equalizer technology is preferable in the pretreatment of complex protein samples. After the sample dissolved in phosphate buffer solution (PBS), it was repeatedly loaded onto the equalizer for five times, the bound proteins were in sequence eluted by 2 mol/L NaCl and 50 mmol/L Gly-HC1 (pH 2.5) solution, followed by digestion with thrombin. All proteins or peptides collected from each fraction were further analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (RPLC-ESI-MS/MS) with a serially coupled long microcolumn. Compared with the untreated samples, the identified protein number was increased from 142 to 396. Furthermore, from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis results, it was found that the protein concentration difference was reduced obviously in the eluant of direct sample loading, and most high abundance proteins were identified in the eluant of NaCl. All these results demonstrate that the novel protein equalizer with scFv display M13 phage library immobilized on cyrogel could effectively reduce the dynamic range of proteins in complex samples, enabling the identification of more low abundance proteins.
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PMID:[A novel protein equalizer based on single chain variable fragment display M13 phage library for nephropathy patient urine study]. 1980 25

Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.
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PMID:Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation. 2041 May 3

Excessive expression of CTGF (connective tissue growth factor)/CCN2 has been observed in many fibrotic diseases. The inhibition of the CTGF/CCN2 by antibody has been shown to be clinically useful for the management of fibrosis. A phage display humanized single-chain Fv antibody library was screened using CTGF/C (CTGF/CCN2 C-terminal domain) as the target. A phage ELISA was performed after four rounds of biopanning, and ten positive clones were further evaluated by ELISA and were chosen for DNA sequencing. The DNA encoding scFv (single-chain variable fragment) containing a full-length variable domain fragment of heavy chain and light chain of human immunoglobulin was inserted into pET-32(a)+ vector, and the fusion protein (TrxA-scFv) containing a thrombin cleavage site was expressed mainly in soluble form. The scFv was obtained by purified fusion protein digested with thrombin and then separated from the fusion partner TrxA by gel-filtration chromatography. An immunological assay showed that the purified scFv reacted with CTGF/CCN2 in a concentration-dependent manner. The result of the cell migration assay demonstrated that the scFv at 100 ng/ml could effectively inhibit the migration of HUVEC (human umbilical-vein endothelial cells) caused by CTGF/C. The number of migratory cells was significantly decreased as compared with the negative control (1062+/-92 versus 3269+/-288, P<0.001) and the inhibition rate was 90.5%.
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PMID:Bioscreening of phage display antibody library and expression of a humanized single-chain variable fragment antibody against human connective tissue growth factor (CTGF/CCN2). 2049 54

Delayed cerebral ischemia (DCI) is a significant cause of morbidity and mortality for patients surviving the rupture of an intracranial aneurysm. Despite an association between vasospasm and DCI, thrombosis and thromboembolism may also contribute to DCI. In this study we investigate the time course of intravascular microclot formation after experimental subarachnoid hemorrhage (SAH) and assess the effects of the following two drugs on microclot burden: mutant thrombin-activated urokinase-type plasminogen activator (scFv/uPA-T), which is bound to red blood cells for use as a thromboprophylactic agent, and clazosentan, an endothelin antagonist. In the first study, adult male C57BL/6 mice were sacrificed at 24 (n=5), 48 (n=6), 72 (n=8), and 96 (n=3) hours after SAH induced by filament perforation of the anterior cerebral artery. Sham animals (n=5) underwent filament insertion without puncture. In the second study, animals received scFv/uPA-T (n=5) 3 hours after hemorrhage, clazosentan (n=5) by bolus and subcutaneous pump after SAH just prior to skin closure, or a combination of scFv/uPA-T and clazosentan (n=4). Control (n=6) and sham (n=5) animals received saline alone. All animals were sacrificed at 48 hours and underwent intra-cardiac perfusion with 4% paraformaldehyde. The brains were then extracted and sliced coronally on a cryostat and processed for immunohistochemistry. An antibody recognizing thrombin-anti-thrombin complexes was used to detect microclots on coronal slices. Microclot burden was calculated for each animal and compared among groups. Following SAH, positive anti-thrombin staining was detected bilaterally in the following brain regions, in order of decreasing frequency: cortex; hippocampus; hypothalamus; basal ganglia. Few microclots were found in the shams. Microclot burden peaked at 48 hours and then decreased gradually. Animals receiving scFv/uPA-T and scFv/uPA-T+clazosentan had a lower microclot burden than controls, whereas animals receiving clazosentan alone had a higher microclot burden (p<0.005). The overall mortality rate in the time course study was 40%; mortality was highest among control animals in the second study. Intravascular microclots form in a delayed fashion after experimental SAH. Microclots may be safely reduced using a novel form of thromboprophylaxis provided by RBC-targeted scFv/uPA-T and represent a potential target for therapeutic intervention in the treatment of DCI.
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PMID:Microthrombosis after experimental subarachnoid hemorrhage: time course and effect of red blood cell-bound thrombin-activated pro-urokinase and clazosentan. 2207 56

Recombinant single-chain variable fragment (scFv) antibodies have wide applications in the areas of biotechnology and medicine. However, there is currently no universal expression-purification system for generating different soluble scFvs. In this study, A15 and E34, two genes coding scFvs against human IL-17A, were fused with N-terminal signal peptide sequences pelB or STII, or with highly hydrophilic tags Trx, NusA, or MBP, respectively. These constructs were expressed in Escherichia coli. We found that the scFvs fused with either NusA or MBP showed a higher solubility than fused with signal peptides or Trx. The scFvs were aggregated when the NusA or MBP was removed by thrombin. Interestingly, we observed a reduction of precipitation when the fusion proteins were expressed in Origami B(DE3)pLysS cells but not in BL21(DE3)pLysS. Because cleaving the tags resulted in the aggregation of scFvs, several solubility-enhancing additives were added in the digestion buffer and only L-arginine (Arg) or Tween20 promoted the solubility. After an affinity chromatography, the scFvs were separated from the tags with the purity up to 90%. The final yield of scFvs from the scFv-MBP system was approximately 8.9 mg/L of culture medium and 1.5 mg/g of wet weight cells, which was 1.6-fold higher than the yield from the scFv-NusA system. The obtained scFvs exhibited normal binding affinities and activities after endotoxin removal. In conclusion, we describe a strategy combining the fusion tags, the Escherichia coli with oxidizing bacterial cytoplasm, and the solubility-enhancing additives for expressing and purifying the soluble and functional scFvs.
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PMID:A combined strategy improves the solubility of aggregation-prone single-chain variable fragment antibodies. 2238 83


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