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)

Normal and mutant forms of human antithrombin-III (AT-III) were synthesized in a cell-free system in order to identify putative functional domains required for heparin binding and complex-formation with alpha-thrombin. Heparin-Sepharose chromatography resulted in the elution of approx. 70% of cell-free-derived normal AT-III-(1-432)-polypeptide as a peak between 0.2 M- and 0.7 M-NaCl. The cell-free-derived normal AT-III also reacted with alpha-thrombin. Approx. 15% of this AT-III formed covalent complexes with alpha-thrombin in 2 min. Unfractionated heparin accelerated the rate of formation of such complexes. Two truncated forms of AT-III (amino acid residues 219-432 and 251-432), containing only the putative thrombin-binding domain, were synthesized independently in this cell-free system. These truncated AT-III polypeptides did not bind heparin and were unable to form stable covalent complexes with alpha-thrombin. However, both of these AT-III polypeptides were cleaved by alpha-thrombin, presumably at the reactive centre Arg-393-Ser-394. The formation of the disulphide bond between Cys-247 and Cys-430 in AT-III-(219-432)-polypeptide had no effect on the results obtained. Mutations in full-length AT-III at Cys-430 had no effect on the ability of AT-III to bind heparin. There was, however, a slight decrease in the formation of stable inhibitory complexes with alpha-thrombin. A cell-free-derived AT-III mutant, devoid of amino acid residues 41-49, which comprise heparin-binding region 1 of AT-III, had slightly decreased heparin binding compared with cell-free-derived normal AT-III-(1-432)-polypeptide. This mutant AT-III polypeptide was unable, however, to form a stable complex with alpha-thrombin. We conclude therefore that the N-terminal domain of AT-III is essential for both heparin binding and complex-formation with alpha-thrombin, but not for the cleavage of AT-III at its reactive centre by alpha-thrombin.
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PMID:The N-terminal domain of antithrombin-III is essential for heparin binding and complex-formation with, but not cleavage by, alpha-thrombin. 154 50

To evaluate the effect of thrombin on the dynamics of thrombolysis, we infused rabbits with heparin or hirudin alone or in conjunction with tissue-type plasminogen activator (t-PA) and monitored the kinetics of fibrinolysis and changes in ex vivo platelet aggregation responses over time. Both heparin and hirudin enhanced total fibrinolysis in an ex vivo arteriovenous shunt preparation: 82 +/- 2% and 79 +/- 2%, respectively, compared with 51 +/- 8% for t-PA alone (P less than 0.05) and 50 +/- 4% for t-PA plus aspirin (p less than 0.05). Heparin coadministered with t-PA significantly reduced the half-time for clot lysis compared with t-PA alone (p less than 0.05), whereas hirudin coadministered with t-PA significantly reduced the half-time for clot lysis compared with that for t-PA alone, t-PA plus aspirin, and t-PA plus heparin (5.5 +/- 0.6 versus 12.1 +/- 2.0 versus 12.6 +/- 2.2 versus 10.0 +/- 0.8 minutes, respectively; p less than 0.05). Both heparin and hirudin prevented the increase in ADP-induced platelet aggregation normally seen with t-PA alone (p less than 0.01 by t test; p less than 0.05 by two-way analysis of variance). These data demonstrate that selective, antithrombin III-independent thrombin inhibitors can enhance the efficacy of thrombolysis by modulating the dynamics of the process and preventing platelet activation associated with plasminogen activator therapy.
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PMID:Effect of thrombin inhibition on the dynamics of thrombolysis and on platelet function during thrombolytic therapy. 155 Dec 6

The interaction of heparin with glia-derived nexin (GDN) has been characterized and compared to that observed between heparin and antithrombin III (ATIII). Heparin was fractionated according to its affinity for immobilized GDN, and the ability of various fractions to accelerate the inhibition rate of thrombin by either GDN or ATIII was examined. Fractions with different affinities for GDN accelerated the thrombin-GDN reaction to a similar extent; heparin with a high affinity for immobilized GDN stimulated the reaction only about 30% more than the fraction that did not bind to immobilized GDN. Slightly greater differences were observed for the effect of these fractions on the thrombin-ATIII reaction; heparin that did not bind to the GDN affinity column was about 60% more effective than heparin with a high affinity for GDN in accelerating the inhibition of thrombin by ATIII. The CNBr fragment of GDN between residues 63 and 144 was able to reduce the heparin-accelerated rate of inhibition of thrombin by GDN indicating that this region of GDN was able to bind the heparin molecules responsible for the acceleration. Shorter synthetic peptides within this sequence did not significantly reduce the rate, suggesting that the heparin-binding activity of fragment 63-144 depends on a specific conformation of the polypeptide chain. Fragment 63-144 was less effective in decreasing the heparin-accelerated rate of inhibition of thrombin by ATIII. The results are discussed in terms of the heparin species that are responsible for the acceleration of the GDN- and ATIII-thrombin reactions and the heparin-binding sites of GDN and ATIII.
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PMID:Characterization of the heparin-binding site of glia-derived nexin/protease nexin-1. 155 34

Although the benefits of coronary thrombolysis are well established, the optimal therapeutic strategy for ensuring rapid and sustained coronary artery patency remains controversial. The available data suggest that the success of coronary thrombolysis depends not only on the induction of clot lysis, but also on the extent to which procoagulant activity that promotes recurrent thrombosis is inhibited. Procoagulant activity increases almost immediately in patients treated with fibrinolytic agents, and persistent increases in thrombin activity have been associated with recurrent coronary thrombosis. Heparin administered intravenously appears to markedly attenuate the thrombin activity associated with thrombolysis and, in patients treated with tissue plasminogen activator (t-PA), prevents early recurrent coronary thrombosis. The results of clinical trials of coronary thrombolysis indicate that conjunctive treatment of patients with heparin improves survival compared with treatment with fibrinolytic agents alone. Although recent clinical trials in which patients were treated with streptokinase suggested that 12,500 units of heparin administered subcutaneously twice daily decreases mortality, this dosage regimen does not induce therapeutic levels of anticoagulation within the first 24 h in most patients. The failure to achieve early therapeutic anticoagulation may account for the lack of mortality benefit in trials in which patients given t-PA were treated with conjunctive subcutaneous heparin therapy. Thus, the available experimental and clinical data suggest that intravenous heparin should be given to patients treated with fibrinolytic agents for acute myocardial infarction.
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PMID:Role of heparin in coronary thrombolysis. 155 78

The antithrombotic activity of the tripeptide thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766), was compared to heparin in a model of canine coronary artery thrombosis. Thrombogenesis was initiated by electrolytic injury of the intimal surface of the left circumflex coronary artery. Drug administration was started 15 min before initiation of intimal injury. Clotting times and ex vivo platelet aggregation were determined on citrated blood samples. Gingival template bleeding times were determined. Clotting times (thrombin time; activated partial thromboplastin time, APTT; prothrombin time, PT) increased in a dose-dependent manner with both anticoagulants. The two anticoagulants selectively inhibited thrombin-induced platelet aggregation. GYKI-14766 and heparin were found to delay thrombosis significantly when compared to vehicle-treated animals; minimum effective antithrombotic doses were 0.25 mg/kg/h and 80 U/kg + 30 U/kg/h, respectively. GYKI-14766 (0.25 mg/kg/h) had no effect on template bleeding time, APTT or PT. Heparin (80 U/kg + 30 U/kg/h), however, was associated with a 2.5- to 3.0-min increase in template bleeding time, a 1.8-fold and 1.7-fold increase in APTT and PT, respectively. Antithrombotic efficacy was achieved at doses of GYKI-14766 that did not affect APTT, PT or template bleeding time, whereas antithrombotic efficacy observed with heparin was associated with significant increases in APTT, PT and template bleeding time. These data demonstrate that the tripeptide thrombin inhibitor, GYKI-14766, could potentially prove to be a safer and more effective antithrombotic agent than heparin.
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PMID:Pharmacological assessment of the antithrombotic activity of the peptide thrombin inhibitor, D-methyl-phenylalanyl-prolyl-arginal (GYKI-14766), in a canine model of coronary artery thrombosis. 157 72

The antithrombotic properties of bolus i.v. injections of heparin, of recombinant hirudin (r-hirudin) or of the synthetic competitive thrombin inhibitor Argatroban were investigated in a quantitative hamster femoral vein platelet-rich mural thrombosis model. Heparin at a dose of 100 U/kg prolonged the activated partial thromboplastin time from 26 +/- 15 to 177 +/- 45 sec (P = .001), but did not significantly inhibit platelet-rich thrombus formation (7 +/- 44% inhibition, P = NS vs. placebo). However, 400 U/kg of heparin produced total inhibition of thrombus formation (101 +/- 14+, P less than .06 vs. control). R-hirudin and argatroban inhibited thrombus formation in a dose-dependent manner: 50% inhibition was obtained with 1.4 mg/kg for r-hirudin and with 2.0 mg/kg for Argatroban. A linear correlation was observed between the percentage of inhibition of thrombus formation vs. Activated partial thromboplastin time (r = 0.57, P = .003 for r-hirudin and r = 0.66, P = .002 for Argatroban). These results suggest that thrombin plays a pivotal role in platelet-rich mural thrombus formation, that this small animal model may be useful for investigation of the pharmacodynamics of synthetic thrombin inhibitors and that platelet-rich thrombus formation is inhibited effectively by heparin, r-hirudin and Argatroban. However, r-hirudin and Argatroban cause less profound changes in the coagulant function at doses that inhibit platelet-rich thrombus formation than heparin.
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PMID:Comparative antithrombotic effects of heparin, recombinant hirudin and argatroban in a hamster femoral vein platelet-rich mural thrombosis model. 160 94

The venous antithrombotic effects of a novel chemical entity, LF 1351, were investigated in rats following single oral administration, in comparison with i.v. administered heparin. LF 1351 demonstrated a dose-related antithrombotic effect in three models of venous thrombosis. The compound was approximately equipotent in two models involving complete stasis of the vena cava and administration of factor Xa or porcine serum, giving respective ED50 values of 48.7 mg/kg and 36.7 mg/kg. LF 1351 was less effective in a model involving partial stasis in the presence of an endothelial lesion. In this case, the antithrombotic effect did not exceed 60-65%, the ED50 being 150 mg/kg. Heparin (50-300 micrograms/kg; 7.5-45.0 U/kg) was effective in all three models. At the approximate ED80 value against factor Xa-induced thrombosis (75 mg/kg) the antithrombotic effect of LF 1351 persisted for 6 h. The antithrombotic effect of LF 1351 (300 mg/kg) occurred without significant changes in APTT or thrombin time.
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PMID:The venous antithrombotic effect of LF 1351 in the rat following oral administration. 161 73

Although heparin is currently used in concomitance with thrombolytic agents to improve their efficacy, its effect on fibrinolysis is controversial. We have evaluated the sensitivity to t-PA-induced lysis of clots prepared from plasma preincubated in vitro with therapeutic concentrations of heparin. The extent of t-PA-induced lysis was significantly increased by preincubation of plasma with 0.5 and 1.0 U/ml heparin. The concentration of t-PA required to give similar lysis rates were reduced by up to five times after adding 1.0 U/ml heparin to plasma prior to clot formation. Heparin added to the t-PA-containing medium after clot formation did not exert any significant effect. The effect of heparin was not mediated by the inhibition of thrombin as preincubation of plasma with hirudin did not modify clot sensitivity to t-PA. We also found that heparin significantly modified fibrin assembly and clot structure as assessed by a turbidimetric assay. Pre-incubation of fibrinogen with heparin caused an increase in the speed of fibrin fibre polymerization and in the turbidity of the final fibrin gel; changes known to be associated with the formation of thicker fibrin fibres. Thus the effect of heparin on clot sensitivity to lysis appears to be due to an increased permeability of these clots to fibrinolytic components. This may contribute to the antithrombotic activity and to the haemorrhagic risk of heparin. These findings could be particularly important for clinical thrombolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fibrin clots obtained from plasma containing heparin show a higher sensitivity to t-PA-induced lysis. 164 5

Thrombotic or thromboembolic occlusion of a cerebral artery is the most common pathophysiologic mechanism of acute ischemic stroke. An antithrombotic agent would therefore appear to be an ideal medication for treatment of this condition. Heparin is an effective anticoagulant, but it has poor bioavailability and effects on thrombin and platelets that predispose it to life-threatening complications such as hemorrhage and thrombocytopenia. Low-molecular-weight (LMW) heparins have better bioavailability, a higher anti-Xa:anti-IIa ratio, and less effect on platelets than heparin; yet their heterogeneity has hampered their proper investigation in clinical trials and it has not yet been proven that they exhibit less tendency toward hemorrhage and thrombocytopenia than conventional heparin. The LMW heparinoid, Org 10172, is superior to standard heparin in terms of its bioavailability, anti-Xa:anti-IIa ratio, and lack of effect on platelets. It is less likely than heparin and many LMW heparins to induce thrombocytopenia. Like the various heparins, Org 10172 exhibits dose-dependent hemorrhagic tendencies, yet preliminary studies have found doses that are safe for use in patients with acute ischemic stroke. These studies also suggest that Org 10172 may improve outcome and lessen mortality in this population. A prospective, randomized, double-blind, controlled trial is needed to establish the potential efficacy of Org 10172 in patients who suffer acute or progressing ischemic stroke.
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PMID:Low-molecular-weight heparins and heparinoids and their use in acute or progressing ischemic stroke. 170 55

Heparin interacts with platelets to impair collagen-induced aggregation and adhesion to collagen. Low molecular weight heparin and heparinoids have little or no inhibitory activity in these platelet-collagen interactions. Thrombin-induced aggregation of platelets is inhibited by any of the glycosaminoglycans that will block the action of thrombin on fibrinogen. However, heparin is a much more potent inhibitor than low molecular weight heparins or heparinoids at equigravimetric concentrations in these reactions. The inhibition of ristocetin or asialo-von Willebrand factor aggregation of platelets is partially blocked by high dose heparin but not by low molecular weight heparin or heparinoids. The heparin-induced IgG antibody, produced to a heparin-platelet complex, aggregates platelets strongly in the presence of heparin, less strongly in the presence of low molecular weight heparins and pentosan polysulfate and not at all with dermatan sulfate or the pentasaccharide. Heparan sulfate does interact with platelets and this antibody, which is of interest because of the heparan sulfate on endothelial cells. Clinical information to date suggests a low incidence of heparin antibodies in patients receiving only low molecular weight heparin of the depolymerized type. Whether long-term clinical use of low molecular weight heparins, heparan sulfate, and dermatan sulfate will give rise to specific antibodies that would cause a similar problem as heparin remains to be seen.
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PMID:Interaction of heparinoids with platelets: comparison with heparin and low molecular weight heparins. 171 12


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