Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the effects of fibrinogen degradation product (FDP) fragment D on endothelial monolayer integrity and the mechanisms of fragment D-induced endothelial cell detachment from the substratum. Incubation of bovine pulmonary artery endothelial cells (BPAEC) with fragment D caused concentration- and time-dependent cell detachment from the substratum. The optimal response occurred at fragment D concentrations of 2 microM and required an incubation time of 24 h. BPAEC challenged with fragment D increased the concentration and activity of urokinase-type plasminogen activator (uPA) in the conditioned medium within 2 to 4 h of incubation. Fragment D also induced the release of tissue-type plasminogen activator, but to a lesser extent than uPA. Fragment D concurrently increased plasminogen activator (PA) activity in a concentration-dependent manner. Increased PA activity was followed by augmentation of cell-associated plasmin activity and subsequent increase in the degradation of 125I-fibrinogen and 125I-vitronectin precoated in the subendothelial matrix. Pretreatment of BPAEC with anti-uPA antibody, and inhibitors of uPA (dansyl-GGACK) and plasmin (aprotinin) prevented approximately 60% of the fragment D-induced endothelial cell detachment. We conclude that FDP fragment D increases secretion of endothelial PAs and enhances the generation of plasmin, thereby contributing to proteolysis of extracellular matrix and endothelial cell detachment. Fragment D may be a critical mediator linking activation of fibrinolysis to vascular endothelial injury in inflammatory disorders.
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PMID:Fibrinogen degradation product fragment D induces endothelial cell detachment by activation of cell-mediated fibrinolysis. 128 36

The vampire bat salivary plasminogen activator (BatPA) is virtually inactive toward Glu-plasminogen in the absence of a fibrin-like cofactor, unlike human tissue-type plasminogen activator (tPA) (the kcat/Km values were 4 and 470 M-1 s-1, respectively). In the presence of fibrin II, tPA and BatPA activated Glu-plasminogen with comparable catalytic efficiencies (158,000 and 174,000 M-1 s-1, respectively). BatPA's cofactor requirement was partially satisfied by polymeric fibrin I (54,000 M-1 s-1), but monomeric fibrin I was virtually ineffective (970 M-1 s-1). By comparison, a variety of monomeric and polymeric fibrin-like species markedly enhanced tPA-mediated activation of Glu-plasminogen. Fragment X polymer was 2-fold better but 9-fold worse as cofactor for tPA and BatPA, respectively, relative to fibrin II. Fibrinogen, devoid of plasminogen, was a 10-fold better cofactor for tPA than fibrinogen rigorously depleted of plasminogen, Factor XIII, and fibronectin; the enhanced stimulatory effect of the less-purified fibrinogen was apparently due to the presence of Factor XIII. By contrast, the two fibrinogen preparations were equally poor cofactors of BatPA-mediated activation of Glu-plasminogen. BatPA possessed only 23 and 4% of the catalytic efficiencies of tPA and two-chain tPA, respectively, in hydrolyzing the chromogenic substrate Spectrozyme tPA. However in the presence of fibrin II, BatPA and tPA exhibited similar kcat/Km values for the hydrolysis of Spectrozyme tPA. Our data revealed that BatPA, unlike tPA, displayed a strict and fastidious requirement for polymeric fibrin I or II. Consequently, BatPA may preferentially promote plasmin generation during a narrow temporal window of fibrin formation and dissolution.
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PMID:Vampire bat salivary plasminogen activator exhibits a strict and fastidious requirement for polymeric fibrin as its cofactor, unlike human tissue-type plasminogen activator. A kinetic analysis. 138 41

To explore whether fibrin fragments have binding affinity for the tissue-type plasminogen activator (t-PA) molecule, the interactions were studied of (DD)E complex and fragments DD, E1, and E3 with one-chain and two-chain t-PA. For this purpose, a solid-phase binding assay was developed using microtiter plates with nitrocellulose filters. It was found that (DD)E complex and fragments DD and E3 retained the t-PA binding function of the parent fibrin molecule, thus demonstrating that t-PA binds to both the D and E domains of fibrin. Unexpectedly, fragment E1 did not bind t-PA. Fibrin fragments had different binding properties for one-chain and two-chain t-PA. (DD)E complex had the highest and fragment E3 the lowest affinity for one-chain t-PA, both binding curves being consistent with one class of binding sites. However, binding of the fragments with two-chain t-PA was distinguished by more than one class of binding sites, with fragment E3 having the highest affinity for this form of the activator. epsilon-Aminocaproic acid, even at 50 mmol/L concentration, had only minimal effect on binding of (DD)E complex or fragment DD to either one-chain or two-chain t-PA. The potentiating effect of fibrin fragments on plasminogen activation by t-PA was measured by a chromogenic substrate assay. Fragment DD was the most effective stimulator of plasminogen activation by t-PA. In conclusion, (DD)E complex and fragment DD retained most of the regulatory functions of fibrin, which included t-PA binding and t-PA-mediated acceleration of plasminogen activation to plasmin.
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PMID:Binding of fibrin fragments to one-chain and two-chain tissue-type plasminogen activator. 157 44

Prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complexes (TAT), as well as other coagulation and fibrinolysis parameters, were studied in a series of 13 patients affected by thrombotic thrombocytopenic purpura (TTP) or hemolytic-uremic syndrome (HUS). Fragment F1 + 2 was found to be increased in all patients at diagnosis (patients' range, 1.21-19.03 nmol/l; normal limits, 0.28-1.08 nmol/l), and remained also higher than normal after treatment with plasma exchange (patients' range, 1.5-4.01 nmol/l). Even though the analysis of fibrinolysis markers did not show a definite state of hypo or hyperfibrinolysis in the systemic circulation, enhanced circulating D-dimer levels (0.53-12.6 micrograms/ml, normal levels of 0.03-0.29 micrograms/ml) indicated that a certain grade of fibrin lysis was present at previously formed thrombi. Plasma PAI-1 activities either on admission (9.2-38.2 U/ml) and after plasma exchange therapy (2.6-38.6 U/ml) showed a behavior irrespective of t-PA:Ag changes, and post-plasmapheresis values remained high only in patients with fatal neurological outcome. Nevertheless, no correlations between clinical and laboratory data could be established useful for the TTP/HUS prognosis. We conclude that increased thrombin generation occurring in damaged areas is appropriately inhibited by antithrombin III in the systemic circulation, avoiding consumption coagulopathy to develop in uncomplicated patients. In addition, fibrinolysis data suggest that elevated PAI-1 may decisively favor the development of microvascular thrombi.
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PMID:Thrombin generation and fibrinolysis in the thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome. 151 82

An enzyme-linked immunosorbent assay for fragment D-dimer was developed with the use of two monoclonal antibodies directed against specific non-overlapping antigenic determinants, present in fragment D-dimer of crosslinked fibrin but not in fragment D of non crosslinked fibrin or of fibrinogen. The lower limit of sensitivity of the assay when applied to human plasma, is 25 ng/ml. Concentration of fragment D-dimer in plasma from healthy individuals was 177 +/- 83 ng/ml (mean +/- SD). In plasma of 11 out of 12 patients with phlebographically confirmed acute deep vein thrombosis, fragment D-dimer levels were significantly increased. Fragment D-dimer was not increased in 9 out of 10 patients with recurrent idiopathic deep vein thrombosis during clinically silent episodes. Total t-PA antigen and free t-PA antigen concentrations were measured using previously developed ELISAs. Nine of the 12 patients with acute deep vein thrombosis showed a significant increase of total t-PA antigen (from 8.6 +/- 6.9 ng/ml to 21 +/- 16 ng/ml) after venous occlusion but in 3 of these free t-PA remained undetectable. Five of the 10 patients with recurrent deep vein thrombosis responded to venous occlusion with a significant increase of total t-PA antigen (from 6.7 +/- 3.2 ng/ml to 14 +/- 7.9 ng/ml) but, in all patients, free t-PA antigen remained undetectable. It is concluded that the combined assays of total and free t-PA antigen and of fragment D-dimer may be useful for the evaluation of the dynamics of the fibrinolytic system in physiological and pathological conditions.
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PMID:Fibrinolytic response and fibrin fragment D-dimer levels in patients with deep vein thrombosis. 312 14

Lipoprotein lipases from human, bovine or guinea-pig milk were purified, judged for domain relationships by characterization of sites sensitive to proteases, and structurally compared. The subunit of human lipoprotein lipase migrated slightly slower than those of bovine or guinea-pig lipoprotein lipases on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Bovine lipoprotein lipase is known to be a dimer of two non-covalently linked subunits of equal size, and the lipases from all three sources now yielded homogeneous N-terminal amino acid sequences (followed for 15-27 residues). The results indicate that the two subunits are identical. Bovine lipoprotein lipase had two additional N-terminal residues, Asp-Arg, compared to the human and guinea-pig enzymes, and the next two positions revealed residue differences, but further on homologies were extensive between all three enzymes as far as presently traced. Exposure of bovine lipoprotein lipase to trypsin led to production of three fragments (T1, T2a, and T2b), suggesting cleavage at exposed segments delineating domain borders. Time studies gave no evidence for precursor-product relationships between the fragments, and prolonged digestion did not lead to further cleavage. Fragments T2a and T2b had the same N-terminal sequence as intact lipase. Fragment T1 revealed a new sequence, and represents the C-terminal half of the molecule. Plasmin caused a similar cleavage as trypsin, whereas thrombin, factor Xa, and tissue plasminogen activator did not cleave the enzyme. Chymotrypsin cleaved off a relatively small fragment from the C-terminal of the molecule, after which exposure to trypsin still resulted in cleavage at the same sites as in intact lipase. Tryptic cleavage of guinea-pig lipoprotein lipase yielded two fragments. One had a similar size as bovine fragment T2b; the other had a similar size as bovine fragment T1 and an N-terminal sequence homologous with that of T1. Thus, trypsin recognizes the same unique site in guinea-pig lipoprotein lipase as in the bovine enzyme. This confirms the conclusion that this segment is the border between two domains in the subunit. The binding site for heparin was retained after both tryptic and chymotryptic cleavages and was identified as localized in the C-terminal part of the molecule.
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PMID:Lipoprotein lipases from cow, guinea-pig and man. Structural characterization and identification of protease-sensitive internal regions. 353 11

Plasminogen activators initiate the fibrinolytic process by converting plasminogen to plasmin. Though plasminogen activators are effective in the treatment of thrombotic disorders, bleeding complications are associated with their use. The development of plasminogen activators with greater fibrin specificity was expected to reduce the incidence of bleeding complications; however, this has not occurred. In our rabbit model (a) bleeding from standardized ear incisions induced by tissue-type plasminogen activator (t-PA) is attenuated when fibrinogenolysis is reduced by the coadministration of alpha 2-antiplasmin and (b) when used in doses that produce equivalent thrombolysis, vampire bat plasminogen activator (b-PA), an agent that is more fibrin specific than t-PA, causes less bleeding than t-PA. In addition, we have found that the (DD)E complex formed as a result of degradation of crosslinked fibrin is a potent stimulator of t-PA-induced plasminogen activation but has no effect on b-PA. Fragment X, a high-molecular-weight clottable fibrinogen degradation product, accumulates after treatment with t-PA but not with t-PA given with alpha 2-antiplasmin or with b-PA. These findings suggest that there is a link between plasminogen activator-induced fibrinogenolysis and bleeding, and that the composition of fibrin within hemostatic plugs may influence susceptibility to lysis. Whether these results mean that fibrin-specific plasminogen activators like b-PA will have a better risk-to-benefit profile in humans requires rigorous testing in well-designed clinical trials. However, at the very least, our findings suggest that the development of plasminogen activators that are more fibrin specific than t-PA is a worth-while exercise.
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PMID:Limited fibrin specificity of tissue-type plasminogen activator and its potential link to bleeding. 877 Aug 37

Bleeding, the most serious complication of thrombolytic therapy with tissue-type plasminogen activator (t-PA), is thought to result from lysis of fibrin in hemostatic plugs and from the systemic lytic state caused by unopposed plasmin. One mechanism by which systemic plasmin can impair hemostasis is by partially degrading fibrinogen to fragment X, a product that retains clottability but forms clots with reduced tensile strength that stimulate plasminogen activation by t-PA more than fibrin clots. The purpose of this study was to elucidate potential mechanisms by which fragment X accelerates t-PA-mediated fibrinolysis. In the presence of t-PA, clots containing fragment X were degraded faster than fibrin clots and exhibited higher rates of plasminogen activation. Although treatment with carboxypeptidase B, an enzyme that reduces plasminogen binding to fibrin, prolonged the lysis times of fragment X and fibrin clots, clots containing fragment X still were degraded more rapidly. Furthermore, plasmin or trypsin also degraded clots containing fragment X more rapidly than fibrin clots, suggesting that this effect is largely independent of plasminogen activation. Fragment X-derived degradation products were not preferentially released by plasmin from clots composed of equal concentrations of fibrinogen and fragment X, indicating that fragment X does not constitute a preferential site for proteolysis. These data suggest that structural changes resulting from incorporation of fragment X into clots promote their lysis. Thus, attenuation of thrombolytic therapy-induced fragment X formation may reduce the risk of bleeding.
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PMID:Incorporation of fragment X into fibrin clots renders them more susceptible to lysis by plasmin. 1656