Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.7 (plasmin)
 9,023 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

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing low-molecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium.
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PMID:Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature. 1614 2

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

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

Accumulation and deposition of beta amyloid (Abeta) play a critical role in the pathogenesis of Alzheimer's Disease (AD), and numerous approaches to control Abeta aggregation are being actively pursued. Brain Abeta levels are controlled by the action of several proteolytic enzymes such as neprilysin (NEP), insulin degrading enzyme (IDE) and plasmin. While up-regulation of these enzymes increased clearance of Abeta in transgenic mouse models of AD, these enzymes have other natural substrates and multiple cleavage sites in Abeta complicating their use for treating AD. Alternatively, immunotherapeutic approaches to clear Abeta are gaining interest. Active and passive immunization studies with Abeta can reduce plaque burden and memory loss, but clinical trials were stopped due to meningioencephalitis in some patients. Naturally occurring proteolytic antibodies have been shown to cleave Abeta, and their serum titers are increased in patients with AD reflecting a protective autoimmune response. These antibodies however cannot cross the blood brain barrier and depend entirely on peripheral clearance to clear Abeta. A potentially non-inflammatory approach to facilitate Abeta clearance and reduce toxicity is to promote hydrolysis of Abeta at its alpha-secretase site using affinity matured single chain antibody fragments (scFvs). Bispecific antibodies consisting of a proteolytic scFv and a targeting scFv can be engineered to selectively supplement and target extracellular alpha-secretase activity and to target toxic Abeta forms facilitating their degradation and clearance without generating an immune response. This strategy represents a suitable paradigm for treating other neurological diseases such as Parkinson's Disease, Lou Gehrig's Disease, and spongiform encephalopathies.
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PMID:Targeted hydrolysis of Beta-amyloid with engineered antibody fragment. 2008 8