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)

Hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(53-64) (HV1)] was designed to bind by its first four and last 12 residues to the alpha-thrombin catalytic site and anion-binding exosite for fibrin(ogen) recognition respectively, with a [Gly]4 bridge and an Arg-Pro bond at the scissional position. Human alpha-, gamma- and zeta-thrombins, as well as bovine trypsin, readily hydrolyse Spectrozyme-TH (D-hexahydrotyrosyl-Ala-Arg p-nitroanilide) at pH 7.4 and approx. 23 degrees C. Both alpha- and zeta-thrombins, which have high fibrinogen-clotting activities (greater than 3000 kunits/g), were inhibited with this substrate by hirulog-1 [Ki = 2.56 +/- 0.35 nM (n = 3) and 1.84 +/- 0.15 nM (n = 3) respectively] and slowly cleaved the inhibitor [k = 0.326 +/- 0.082 min-1 (n = 12) and 0.362 +/- 0.056 min-1 (n = 18) respectively], whereas gamma-thrombin, which has essentially no clotting activity (approx. 4 kunits/g), and trypsin were not inhibited with greater than 1000-fold molar excess of hirulog-1. Similar inhibition parameters were also obtained for hirulog-1 incubated with alpha-thrombin or zeta-thrombin at approx. 23 degrees C and by measuring thrombin activity with fibrinogen in the clotting assay at 37 degrees C. Cleavage of the Arg-3-Pro-4 bond in hirulog-1 by either alpha- or zeta-thrombin was shown by identical cleavage products of either thrombin on h.p.l.c. and by sequence analysis of the alpha-thrombin products. These data demonstrate that hirulog-1 is a specific inhibitor of thrombin forms with high fibrinogen-procoagulant activities and that its Arg-3-Pro-4 bond is slowly cleaved by these thrombin forms.
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PMID:Thrombin-specific inhibition by and slow cleavage of hirulog-1. 144 27

The isomorphous structures of the hirugen (N-acetylhirudin 53'-64' with sulfato-Tyr63') and hirulog 1 (D-Phe-Pro-Arg-Pro-(Gly)4 desulfato-Tyr63'-hirugen) complexes of human alpha-thrombin have been determined and refined at 2.2 A resolution to crystallographic R-factors of 0.167 and 0.163, respectively. The binding of hirugen to thrombin is similar to that of the binding of the C-terminal dodecapeptide of hirudin, including that of the terminal 3(10) helical turn. The sulfato Tyr63', which, as a result of sulfation, increases the binding affinity by an order of magnitude, is involved in an extended hydrogen bonding network utilizing all three sulfato oxygen atoms. The hirugen-thrombin complex is the first thrombin structure determined to have an unobstructed active site; this site is practically identical in positioning of catalytic residues and in its hydrogen bonding pattern with that of other serine proteinases. Hirulog 1, which is a poor thrombin substrate, is cleaved at the Arg3'-Pro4' bond in the crystal structure. The Arg3' of hirulog 1 occupies the specificity site, the D-Phe-Pro-Arg tripeptide is positioned like that of D-Phe-Pro-Arg chloromethylketone in the active site and the Pro4'(Gly)4 spacer to hirugen is disordered in the structure, as is the 3(10) turn of hirugen. The latter must be related to the simultaneous absence both of sulfation and of the last residue of hirudin (Gln65'). In addition, the autolysis loop of thrombin (Lys145-Gly150) is disordered in both structures. Changes in circular dichroism upon hirugen binding are therefore most likely the result of the flexibility associated with this loop.
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PMID:Structure of the hirugen and hirulog 1 complexes of alpha-thrombin. 194 57

A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of alpha-thrombin. These peptides, called "hirulogs", consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 A in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ([D-Phe)-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile- Pro-Glu-Tyr-Leu] inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with Ki = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir53-64, which binds to the thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with Ki = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. In addition, the pentapeptide (D-Phe)-Pro-Arg-Pro-Gly, which comprises the catalytic-site inhibitor moiety of hirulog-1, was determined to have a Ki for thrombin inhibition greater than 2 microM. Hirulog-1, but not S-Hir53-64, was found to inhibit the incorporation of [14C]diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Design and characterization of hirulogs: a novel class of bivalent peptide inhibitors of thrombin. 222 63

Hirulog is a thrombin catalytic site inhibitor which exhibits specificity for the anionic binding exosite of alpha thrombin. Here, we have evaluated the effect of Hirulog (1, 5 and 10 mg/kg, 30 min pretreatment) in a rat model of endotoxemia. Intravenous injection of lipopolysaccharide from E. coli (25 mg/kg; serotype 0127:B8) caused decreases in blood pressure which were significantly reduced (about 60%) in animals pretreated with Hirulog. Rat survival to endotoxin was significantly increased in Hirulog pretreated group (5 and 10 mg/kg) up to 24 hours. Hirulog at the dose of 10 mg/kg inhibited both endotoxin-induced leukopenia at 30 and 60 minute points and thrombocytopenia at 30 minute point but not at 90 and 120 minute points. Fibrinogen levels were significantly reduced after 2 hours following endotoxin administration. Pretreatment with Hirulog (5-10 mg/kg i.v.) 30 min prior to administration of endotoxin prevented changes in fibrinogen plasma levels. These results demonstrate that Hirulog-induced inhibition of thrombin is effective in reducing toxic and lethal effects of endotoxin.
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PMID:Hirulog effect in rat endotoxin shock. 747 25

111Indium-labelled platelets were continuously monitored in the cranial vasculature of anaesthetised rabbits and thrombin inhibitors and anti-coagulants were tested on the sustained platelet accumulation induced by intracarotid injection of thrombin (90 U/kg). Pretreatment, commencing 30 min prior to thrombin, with a 1-h intracarotid infusion of D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK; 0.25-1.0 micrograms/kg per min), unfractionated heparin (Multiparin; 5-20 U/kg bolus + 0.75-3.0 U/kg per min infusion) or low molecular weight heparin (Fragmin; 2.4-9.6 U/kg per min) produced dose-related reductions in platelet accumulation. Continuous infusion of acetyl-D-phenylalanyl-prolyl-boroarginine (DuP-714 ester; 30 micrograms/kg per min) for 30 min induced marked accumulation of platelets in the pulmonary circulation in the absence of thrombin. Bolus intracarotid injection, 1 min before thrombin, of Hirulog (0.05-0.2 mg/kg), PPACK (10-30 micrograms/kg), Multiparin (25-100 U/kg), Fragmin (150 U/kg) or DuP-714 ester (15-30 micrograms/kg) caused significant reductions in platelet accumulation. When injected 1 min after thrombin, Hirulog (1 mg/kg), PPACK (100 micrograms/kg), Fragmin (150 U/kg) and DuP-714 ester (30 micrograms/kg) had no significant effect and Multiparin (100 U/kg) increased platelet accumulation. The results demonstrate that pretreatment with a range of thrombin inactivators, acting via different mechanisms, can inhibit thrombin-induced cerebral thromboembolism in the rabbit.
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PMID:Thrombin inhibitors and anti-coagulants on thrombin-induced embolisation in rabbit cranial vasculature. 785 81

Hirulog (D-FPRPGGGGDGDFEEIPEEYL) is a bivalent inhibitor of thrombin consisting of a moiety (D-FPRP) that binds to the active-site cleft and a hirudin-like C-terminal region (DGDFEEIPEEYL) that binds to the positively charged surface groove of thrombin known as the anion-binding exosite. The formation of the thrombin-Hirulog complex was studied using steady-state and rapid kinetics at 37 degrees C. The inhibition constant for Hirulog was found to be 1.9 nM. Hirulog was slowly degraded by thrombin with a kcat value of 0.01 s-1. The formation of the complex resulted in an enhancement of 44% in the intrinsic fluorescence of thrombin. The kinetics of the increase in thrombin fluorescence were described by a double-exponential decay. The dependence of the rate constant for the fast phase on the concentration of Hirulog could be described by the Michaelis-Menten equation with Km and kmax values of 0.75 +/- 0.12 microM and 325 +/- 17 s-1. The data were consistent with a mechanism in which the C-terminal region of Hirulog binds to the anion-binding exosite with a dissociation constant of 0.75 microM in the first step, followed by two intramolecular steps with rate constants of about 300 and 30 s-1. A C-terminal fragment of hirudin was found to compete in the first step confirming that this process corresponded to the binding of the hirudin-like C-terminus of Hirulog to the anion-binding exosite.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic mechanism for the interaction of Hirulog with thrombin. 799 8

The anticoagulant effect of recombinant hirudin (rHir) and Hirulog has been monitored in patients with the activated partial thromboplastin time. Accurate monitoring with this test cannot be achieved if plasmas contain heparin, lupus anticoagulants, low concentrations of fibrinogen or other factors, or elevated fibrinogen-fibrin degradation products (FDP). We have therefore developed a simple, rapid, sensitive clot-based method, the quantitative thrombin time (QTT), to measure levels of rHir and Hirulog in patient plasma (or whole blood). The QTT is performed by mixing a 1:10 dilution of patient plasma (50 microliters) with human fibrinogen (50 microliters, 128 mg/dl) at 37 degrees C; the clotting time is initiated by adding human thrombin (50 microliters, 5-7.5 U/ml). The concentration of Hirulog or rHir in plasma can be determined by comparing the QTT in patient plasma with a standard curve that is generated by adding different concentrations of anticoagulant to pooled normal plasma. Studies with whole blood using the same procedure yield similar results. In the absence of Hirulog or rHir, the baseline QTT is the same in normal and abnormal plasmas (fibrinogen < 150 mg/dl and FDP as high as 1024 micrograms/ml, elevated FDP alone, lupus anticoagulant, or heparin < 0.9 U/ml). When known concentrations of either rHir or Hirulog are added to abnormal plasmas, the mean observed concentrations as determined by the QTT deviate from the expected values by less than 10% (range 0-19%). The data indicate that the QTT is a simple, rapid, and accurate test for the determination of levels of rHir and Hirulog in plasma or whole blood.
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PMID:A quantitative thrombin time for determining levels of hirudin and Hirulog. 811 87

Recent angiographic studies after thrombolytic therapy have shown that the rates of effective coronary reperfusion (TIMI grade 3) is still quite low with currently available thrombolytic regimens. Approaches to improving thrombolytic efficacy include the development of mutants of t-PA, recombinant staphylokinase and conjugations of thrombolytic agents to monoclonal antibodies targeted to components of the thrombus. The development of more potent conjugative antithrombolysis therapy includes newer antiplatelet agents and selective thrombin inhibitors such as hirudin and Hirulog. Clinical trials of these new approaches are now underway and hold hope for improvement of reperfusion efficacy in coronary thrombolysis.
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PMID:New thrombolytic strategies. 814 22

A brief outline is presented of a proposed trial of Hirulog versus heparin after thrombolytic therapy with streptokinase (SK). The lower patency rates achieved with SK compared with tissue plasminogen activator (t-PA) suggest that a potent thrombin specific agent may be more important for SK than for t-PA therapy. This study will involve more than 400 patients with two dose levels of Hirulog. Endpoints will be angiographic patency, and ST segment monitoring showing evidence of coronary reperfusion.
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PMID:Hirudin and Hirulog. 814 24

Prosthetic vascular grafts become coated with a layer of fibrin that contributes to graft thrombosis and occlusion. We compared the effect of antithrombin III-independent inhibitors of thrombin with heparin for their ability to prevent fibrin accretion onto a model of a vascular graft formed in vitro by coating polyethylene tubing with thrombin bound to a layer of polymerized fibrin. Equivalent antithrombin concentrations of heparin, D-Phe-Pro-Arg CH2Cl (PPACK), recombinant hirudin (r-hirudin), and Hirulog-1 were added to barium chloride-absorbed plasma containing radiolabelled fibrinogen. Whereas, PPACK and r-hirudin persistently inhibited fibrin accretion, the inhibition by heparin was transient. Hirulog-1 had no effect on early fibrin accretion and was actually associated with enhanced accretion at 30 min (control 11.7 +/- 2.0 micrograms fibrin/cm2; Hirulog-1, 18.4 +/- 3.5 micrograms fibrin/cm2, p < 0.001). Both Hirulog-1 and r-hirudin displaced radiolabelled thrombin from the fibrin surface. Whereas hirudin-thrombin complexes are stable, Hirulog-1 produces only transient inhibition of the displaced thrombin thereby accounting for the enhanced fibrin accretion with this anticoagulant. These studies show that the antithrombin III-independent inhibitors, r-hirudin and PPACK, are more effective inhibitors of fibrin accretion onto fibrin-coated polyethylene than heparin or Hirulog-1. In addition, they emphasize the importance of determining the ability of anticoagulants to displace thrombin from fibrin and to form stable thrombin-inhibitor complexes; lack of stability of thrombin-inhibitor complexes must be countered by levels of anticoagulant that are adequate to maintain its effectiveness.
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PMID:The effect of antithrombin III-independent thrombin inhibitors and heparin on fibrin accretion onto fibrin-coated polyethylene. 845 25


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