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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Heparin is often used as an adjunct to thrombolytic therapy in order to prevent reocclusion of the patent vessels in patients with thrombotic disease. Controversy exists as to whether heparin is required for effective clot lysis with tissue-type plasminogen activator, while in vitro data and small scale clinical trials have suggested an enhancement of pro-urokinase efficacy by heparin. The present study was conducted to determine whether heparin pre-treatment is required to produce optimal clot lysis and blood flow restoration in response to recombinant pro-urokinase (r-proUK). In four groups of dogs, blood clots labelled with 125Iodine were formed in the femoral artery and were monitored continuously for loss of counts as an indicator of clot lysis. Femoral artery blood flow was measured simultaneously. Group 1 received vehicle (n = 5), while group 2 was given vehicle + heparin (n = 6; 500 U bolus + 350 U/h). This dose of heparin increased the activated partial thromboplastin time (APTT) by at least 1.5 times the control level for the 4 h observation period. Group 3 received r-proUK alone at a dose of 100,000 U/kg (50% given as a 1-min bolus injection, 50% as a 30 min infusion) (n = 8), while group 4 was treated with the same dose of r-proUK in the presence of heparin as described (n = 8).2
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PMID:Recombinant pro-urokinase requires heparin for optimal clot lysis and restoration of blood flow in a canine femoral artery thrombosis model. 849 50

The sole administration of urokinase causes no initial prolongation of activated partial thromboplastin time (A-PTT), but thereafter produces serious progressive prolongation of A-PTT; it also causes a progressive, severe decrease in fibrinogen levels and alpha 2-plasmin inhibitor activity by depletion. The antithrombogenicity of urokinase is not caused by prevention of blood coagulation system activation by antithrombin effect, but by secondary fibrinolysis by plasmin. Consequently, the administration of urokinase as a sole anticoagulant results in activation of coagulation and fibrinolysis, and, as a result, induces disseminated intravascular coagulation. Therefore, it is concluded that administration of urokinase is an inadequate anticoagulation therapy unless it is combined with other antithrombin agents.
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PMID:Danger of urokinase as an anticoagulant with left ventricular assist devices. 857 15

Procoagulant activity, thrombin and fibrinolytic system activation have been demonstrated in the first 24-48 h after acute myocardial infarction treated with thrombolytic therapy. Little is known about what happens in the subsequent days, during which the incidence of ischaemic recurrence is high. In 21 patients treated with streptokinase and in 20 patients treated with urokinase we evaluated, with multiple plasma determinations, D-dimer and fibrinogen plasma levels in the first week after myocardial infarction. From the 2nd hour after the beginning of thrombolysis to the 4th day, all patients received intravenous heparin in doses sufficient to raise the partial thromboplastin time to twice its normal level; subcutaneous calcium heparin (12,000 U/day) was subsequently substituted for the intravenous route. Coronary angiography was performed 7 days after infarction. From the basal values 2.22 +/- 1.44 nmol.1(-1) in the streptokinase group and 3.28 +/- 3.05 nmol.1(-1) in the urokinase group, D-dimer rose consistently in the 1st hour after thrombolysis 269.4 +/- 206.7 nmol.1(-1) and 44.5 +/- 35.5 nmol.1(-1) in the streptokinase and urokinase groups, respectively; P < 0.001. After the peak value, which in both groups was reached after 5 h, D-dimer slowly decreased during the study period. It reverted to normal values only in 10/21 patients in the streptokinase group; in the urokinase group normalization was attained in 14/20 patients between the 3rd and 6th days. After withdrawal of i.v. heparin in patients of both groups with TIMI 0 or 1 grade of coronary patency, D-dimer rose to levels four to seven times greater than normal; in patients of both groups with TIMI 2 or 3 grade coronary flow, D-dimer showed a monophasic pattern of progressive normalization (P < 0.05 and P < 0.01 at the 6th and 7th days, respectively, for differences between TIMI 0-1 and TIMI 2-3 groups). After myocardial infarction, thrombolysis is followed by active and persistent fibrin degradation more marked and lasting after streptokinase than after urokinase. When occurring sooner, it is a consequence of plasmin activation induced by thrombolytic agents; later it seems to be related to intracoronary substrate, as suggested by the relationship of plasma elevation of D-dimer with the presence of occluded or suboccluded infarction-related vessels.
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PMID:Late activation of the fibrinolytic system in myocardial infarction treated with thrombolytic therapy. Influence of the coronary anatomical substrate. 873 76

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R155-S156. Cleavage at R271-T272 generated fragment 1.2 and prethrombin 2 whilst cleavage at R284-T285 yielded truncated prothrombin 2 (prethrombin 2'). However, cleavage at R320-I321 which, during prothrombin activation produces two-chain alpha-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1-3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R271-S272 and by alpha-thrombin at R155-S156 and R284-T285. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30-40 kDa.
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PMID:Prothrombin cleavage by human vascular smooth muscle cells: a potential alternative pathway to the coagulation cascade. 874 20

Three new tripeptide arginal thrombin inhibitors were shown to have potent anticoagulant and antithrombotic activity: D-MePhg-Pro-Arg-H (LY287045), D-1-Tiq-Pro-Arg-H (LY294291), and D-MePhe-Pro-Arg-H (Efegatran). Efegatran and the related arginals differ mechanistically from old and from new anticoagulant agents. As illustrated with x-ray diffraction analysis of crystals of the LY294291 complex with human thrombin, the family of arginals binds thrombin with the P3, P2, and P1 residues interacting with the putative S3, S2, and S1 fibrinogen-binding sites. A hemi-acetal bond at Ser 195 was shown to contribute to the tight-binding reversible competitive thrombin inhibition properties observed with the arginal family. Tight-binding Kass values from thrombin inhibition studies correlated with thrombin clottin inhibition potency. The thrombin time (TT) assay was prolonged twofold with 33 nM Efegatran, which demonstrated an apparent Kass value of 0.8 x 10(8) L/mol (for comparison, 17 nM hirudin was required to prolong the TT assay two-fold). There are empirical anticoagulant selectivity differences between Efegatran and hirudin, manifested by large activated partial thromboplastin time (aPTT)/TT effect ratios (30 to 55) found with the arginals, as compared to the small aPTT/TT effect ratio (2 to 3) found with hirudin. The underlying anticoagulant mechanism differences between the arginals and hirudin appear to be confined to the aPTT pathway and, therefore, might involve different effects toward thrombin feedback activation of factor VIII. The arginals did not substantially inhibit other coagulation factor serine proteases. Antithrombotic effects of Efegatran and the arginal family occur at low infusion doses in dogs and appear to correlate with effects on TT without requiring perturbation of the aPTT. Selectivity properties regarding the fibrinolytic enzymes were shown to be important for successful use of the arginals in vivo as adjunctive agents during tissue plasminogen activator (t-PA) thrombolysis. The data suggest that LY287045, LY294291, and Efegatran should be expected to be useful as antithrombotic adjuncts to thrombolytic therapy with t-PA, urokinase, or streptokinase and should be expected to spare endogenous fibrinolysis. Efegatran has been evaluated in phase I clinical studies and is currently under clinical investigation in phase II protocols as a new cardiovascular anticoagulant.
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PMID:A family of arginal thrombin inhibitors related to efegatran. 880 15

Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol. Chem., in press). We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas and leukocytes, a fungal trypsin and three subtilisins. Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k(ass)) of 4.5 X 10(3)-1.3 x 10(5) M(-1) s(-1) at 22 degrees C. Only factor Xa turned a significant fraction of BSZx over as substrate. Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass)=1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected. The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction with subtilisin Carlsberg and Novo was rapid but most BSZx was cleaved as a substrate. Identification of a monoclonal antibody specific for native BSZx indicate that complex formation and loop cleavage result in similar conformational changes.
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PMID:Inhibition of coagulation factors by recombinant barley serpin BSZx. 884 56

Coagulation is initiated by the binding of factor VIIa to tissue factor, with resultant limited factor IX and X activation and thrombin production. Owing to the feedback inhibition of the factor VIIa/tissue factor complex by tissue factor pathway inhibitor (TFPI), additional factor X activation and thrombin generation must proceed through a pathway involving factors VIII, IX, and XI. Experiments designed to elucidate the requirement for amplified factor Xa and thrombin generation in normal hemostasis show that the resistance of plasma clots to tissue plasminogen activator (tPA)- and urokinase-induced fibrinolysis is related to the extent of thrombin generation. Inhibition of fibrinolysis is mediated in part by plasma carboxypeptidase-U ([CPU] carboxypeptidase-R, procarboxypeptidase-B, thrombin-activatable fibrinolysis inhibitor), a proenzyme that is proteolytically activated by thrombin in a process enhanced dramatically by the cofactor thrombomodulin. A clot induced in factor IX-deficient plasma with limited amounts of tissue factor in the presence of urokinase (100 U/mL) lyses prematurely, and this defect is corrected by supplementation of the deficient plasma with factor IX (5 micrograms/mL) or thrombomodulin (20 ng/mL). These additions enhance the rate and extent of CPU activation: in the case of factor IX, presumably by permitting amplified generation of factor Xa and thrombin, and in the case of thrombomodulin, presumably by increasing the degree of CPU activation produced by the low levels of thrombin generated in the absence of factor IX. Pretreatment of the factor IX-deficient plasma with specific anti-CPU antibodies prevents the increased resistance to fibrinolysis produced by addition of factor IX and thrombomodulin. Likewise, when coagulation is induced by thrombin (2 U/mL) in the presence of tPA (60 U/mL), clots formed from plasmas deficient in factors VIII, IX, X, or XI lyse prematurely unless the missing factor is replaced or thrombomodulin (20 ng/mL) is added.
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PMID:Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma. 891 45

The kinetic parameters were determined for the hydrolysis of a peptide based on the activation site of the thrombin receptor (residues 38-60) by thrombin and 12 other proteases. The kcat and Km values for the cleavage of this peptide (TR39-40) by thrombin were 107 s-1 and 1.3 microM; the kcat/Km of TR39-40 is among the highest observed for thrombin. A model is presented that reconciles the parameters for cleavage of the peptide with the concentration dependence of cellular responses to thrombin. Cleavage of TR39-40 was not specific for thrombin. The pancreatic proteases trypsin and chymotrypsin hydrolysed TR39-40 efficiently (kcat/Km > 10(6) M-1.s-1). Whereas trypsin cleaved TR39-40 at the thrombin activation site (Arg41-Ser42), chymotrypsin hydrolysed the peptide after Phe43. This chymotryptic cleavage would result in inactivation of the receptor. The efficient cleavage of TR39-40 by chymotrypsin (kcat/Km approximately 10(6) M-1.s-1) was predominantly due to a low Km value (2.8 microM). The proteases factor Xa, plasmin, plasma kallikrein, activated protein C and granzyme A also hydrolysed TR39-40 at the Arg41-Ser43 bond, but exhibited kcat/Km values that were at least 10(3)-fold lower than that observed with thrombin. Both tissue and urokinase plasminogen activators as well as granzyme B and neutrophil elastase were unable to cleave TR39-60 at appreciable rates. However, neutrophil cathepsin G hydrolysed the receptor peptide after Phe55. Like the chymotryptic cleavage, this cleavage would lead to inactivation of the receptor, but the cathepsin G reaction was markedly less efficient; the kcat/K(m) value was almost four orders of magnitude lower than that for thrombin. In addition to the above cleavage sites, a secondary site for thrombin and other arginine-specific proteases was identified at Arg46, but the cleavage at this site only occurred at very low rates and is unlikely to be significant in vivo.
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PMID:Cleavage of the thrombin receptor: identification of potential activators and inactivators. 894 6

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach.
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PMID:Interactions of mast cell tryptase with thrombin receptors and PAR-2. 902 Jan 12

Plasminogen has been immobilized onto a segmented polyurethane containing amino groups, using glutaraldehyde as coupling agent. It was also aspecifically adsorbed, for sake of comparison, onto polyurethane films containing different functional groups and, in particular, epsilon-amino caproic acid and lysine residues. The differently immobilized plasminogen has been converted to plasmin by activation with urokinase, and the percentage of active plasmin for the various polymer films was determined using a tripeptide (S-2251) as a synthetic substrate. The biological behaviour of the differently treated polymer films has been evaluated in vitro by measurements of partial thromboplastin time (PTT) and platelet adhesion.
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PMID:Preparation and evaluation of polyurethane surfaces containing immobilized plasminogen. 904 Oct 39


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