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)

The interaction of alpha-thrombin with the hirudin (HV1) fragment N alpha-acetyl desulfo hirudin45-65 (P51) was investigated. Kinetic analysis revealed that P51 inhibits the proteolysis of a tripeptidyl substrate with Ki = 0.72 +/- 0.13 and 0.11 +/- 0.03 microM for bovine and human alpha-thrombins, respectively. The inhibition was partially competitive, affecting substrate binding to the enzyme-inhibitor complex by a factor alpha = 2 (bovine) and alpha = 4 (human) characteristic of hyperbolic inhibitors. P51 also inhibited thrombin-induced fibrin clot formation with IC50 values of 0.94 +/- 0.20 and 0.058 +/- 0.006 microM for bovine and human alpha-thrombins, respectively. The enhanced antithrombin activity for human thrombin could be attributed to species variations in the putative auxiliary "anion" exosite since N alpha-acetyl desulfo hirudin55-65 displayed the same rank order of potency shift in a clotting assay without inhibiting the amidolytic activity of either enzyme. From these observations, a potent thrombin inhibitor was designed having modified residues corresponding to the P1 and P3 recognition sites. N alpha-Acetyl[D-Phe45, Arg47] hirudin45-65 (P53) emerged as a pure competitive inhibitor with a Ki = 2.8 +/- 0.9 nM and IC50 = 4.0 +/- 0.8 nM (human alpha-thrombin) and is designated as a "bifunctional" inhibitor. Its enhanced potency could be explained by a cooperative intramolecular interaction between the COOH-terminal domain of the inhibitor and the auxiliary exosite of thrombin on the one hand, and the modified NH2-terminal residues with the catalytic site on the other.
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PMID:Bifunctional thrombin inhibitors based on the sequence of hirudin45-65. 225 23

Hirudin, a thrombin inhibitor of the leech, was expressed in BHK cells; the alpha 1-antitrypsin signal peptide was used to direct secretion into the culture medium. The recombinant hirudin so produced inhibited thrombin and was shown by labelling experiments with [35S]sulphate to have been posttranslationally modified.
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PMID:Sulphation of hirudin in BHK cells. 226 97

The effect of heparin and of the synthetic competitive thrombin inhibitor (2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfon yl)-L-arginyl]-2-piperidinecarboxylic acid monohydrate (argatroban) on platelet-rich arterial thrombosis was studied in a rabbit model, consisting of a 4-6-mm everted ("inside-out") femoral arterial segment. Intravenous injection of heparin (200 units/kg) failed to prevent occlusion within 60 minutes in all 10 rabbits, whereas intravenous argatroban infusion at a rate of 100 or 200 micrograms/kg/min for 60 minutes, which prolonged the thrombin time more than fourfold, prevented thrombosis in nine of 13 rabbits (p = 0.002 vs. i.v. heparin). Intra-arterial infusion of 200 units/kg heparin over 60 minutes prevented occlusion in six of nine rabbits (p = 0.003 vs. i.v. heparin), whereas intra-arterial argatroban at a rate of 100 micrograms/kg/min for 60 minutes prevented thrombosis in all 10 rabbits (p = 0.00001 vs. i.v. heparin). Patency of femoral arterial segments was maintained after the end of the intra-arterial heparin and intravenous or intra-arterial argatroban infusion for up to 3 hours despite normalization of the thrombin time and partial thromboplastin time. Pathologic examination of the graft revealed that the inverted adventitial surface was covered by layers of platelets without platelet aggregation or fibrin deposition. These findings indicate that thrombin plays an important role in platelet-rich arterial thrombosis, and that the thrombogenic stimulus is rapidly attenuated by short-term infusion of the synthetic thrombin inhibitor. Selective thrombin inhibition can constitute an alternative approach to the prevention of arterial occlusion after angioplasty or thrombolytic therapy in patients with unstable coronary syndromes.
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PMID:Prevention of platelet-rich arterial thrombosis by selective thrombin inhibition. 229 28

Protease nexin-1 (PN-1) is a protein proteinase inhibitor recently shown to be identical with the glial-derived neurite-promoting factor or glial-derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN-1:thrombin complexes might mediate the neurite outgrowth activity of PN-1. However, the present studies showed that such complexes, unlike free PN-1, did not promote neurite outgrowth. The neurite outgrowth activity of PN-1 was only detected in the presence of thrombin or serum (which contains thrombin). PN-1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum-free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN-1. The neurite-promoting activity of PN-1 was inhibited by an anti-PN-1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN-1 corresponded to their inhibition of thrombin. Together, these observations show that PN-1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.
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PMID:Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition. 229 18

Human leuserpin-2 (hLS2) cDNA variants generated by site-directed mutagenesis were expressed in a transient COS cell system. Functional analysis of the mutants revealed two regions in the NH2-terminal half of hLS2 which are essential for glycosaminoglycan-enhanced thrombin inhibition by hLS2. One of these regions, which encompasses a dimeric structure enriched in basic amino acids, is required for both glycosaminoglycan binding and glycosaminoglycan-mediated acceleration of thrombin inhibition. Deletion of another dimeric region, which spans a sequence with a high negative charge density, resulted in a strong reduction in the glycosaminoglycan-enhanced activity of hLS2. This polyanionic region displays structural and functional similarities to the COOH-terminal end of hirudin, another potent thrombin inhibitor, indicating that both inhibitors may have a common binding site on thrombin. Based on our observations we propose a model for the activation of hLS2 by glycosaminoglycans. The key feature of this model is the suggestion that the glycosaminoglycan-enhanced reaction between hLS2 and thrombin is mediated by at least two regions of contact, involving both the reactive center region and the acidic domain of hLS2. Binding of glycosaminoglycans to hLS2 is suggested to result first in the release of the acidic region from intramolecular interactions. Then, amino acid sequences in thrombin are proposed to interact with the acidic dimer of hLS2.
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PMID:On the activation of human leuserpin-2, a thrombin inhibitor, by glycosaminoglycans. 231 89

The specific thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (FPRCH2Cl, PPACK) has been used as an anticoagulant with human blood to permit the preparation of platelet-rich plasma in which the physiological concentration of Ca2+ is maintained. When we attempted to use FPRCH2Cl (40 microM) as an anticoagulant for rabbit blood, clotting was prevented for at least 6 h at room temperature, but nearly all the platelets agglutinated. Thromboxane B2 was not formed and indomethacin and prostaglandin E1 were not inhibitory. The presence of citrate delayed this agglutination, but only EDTA (not EGTA) prevented it, indicating a dependence on Mg2+. Addition of FPRCH2Cl to suspensions of rabbit platelets in Tyrode-albumin solution had no effect on responses to ADP or collagen, but completely blocked all responses to human or rabbit thrombin. Thus plasma is required for the agglutination. Intravenous injection of FPRCH2Cl (5 mg/3 kg rabbit) reduced the platelet count to less than 50% by 15 to 25 min. Thus FPRCH2Cl cannot be used as an anticoagulant for rabbit blood when platelets are to be examined, and has a profound effect on the platelet count in vivo in rabbits. These effects should be considered when FPRCH2Cl is used as an antithrombotic agent for in vivo experiments with rabbits.
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PMID:Agglutination of rabbit platelets in plasma by the thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone. 236 29

Human factor VIII:C has been purified over 300 000-fold from cryoprecipitate by polyelectrolyte purification followed by affinity chromatography on Sepharose linked to antibody to factor VIIIR:Ag (monoclonal or polyclonal) and Sepharose linked to monoclonal antibody to factor VIII:C. The purified material has been analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies. PAGE shows predominant bands at 360K (unreduced), 210K, and 90K and an 80K/79K doublet; Western blotting showed all the monoclonal antibodies used bound the 360K form. In a small-scale purification, plasma from blood taken directly into thrombin inhibitor Kabi S-2581 was applied directly to the monoclonal anti-factor VIII:C column. Western blot analysis of this material showed the 360K band on reduction. The purified factor VIII:C could be activated 13-fold by human thrombin. Gel analysis of the activated material showed intensification followed by fading of the band at 90K and generation of bands at 70K/69K, 55K, and 40K. Western blotting shows that the 70K/69K doublet derives from the 80K/79K moiety and the 40K peptide derives from the 90K and is presumed to contain the active site. From these studies an epitope map of the factor VIII:C molecule has been constructed.
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PMID:Purification of human factor VIII:C and its characterization by Western blotting using monoclonal antibodies. 241 85

We have recently reported that exogenous thrombin produced a dose- and endothelium-dependent coronary vasodilation in both intact open-chested dogs and in isolated dog coronary artery preparations. To determine whether the observed vasodilatory effect may be related to thrombin proteolytic enzymatic activity, effects of other proteases, such as trypsin, chymotrypsin, and pepsin, on the mechanical responses of isolated dog coronary arteries were studied. Among the four proteases evaluated, only thrombin (0.01-0.1 U/ml) and trypsin (0.03-0.67 U/ml) consistently produced a potent dose- and endothelium-dependent relaxation, that was reproducible with repeated testings. Addition of chymotrypsin (0.01-1.0 U/ml) produced only a minimal effect and was not reproducible, while addition of pepsin, as much as 10 U/ml, did not produce any effect. The specific soybean trypsin inhibitor and aprotinin, but not heparin and hirudin, competitively shifted the trypsin dose-response to the right, whereas heparin, hirudin, and antithrombin III proved to be more effective than trypsin inhibitors in inhibiting the thrombin-induced vasodilation. In all cases, the thrombin- and trypsin-induced vasodilation were equally sensitive to inhibition by the specific synthetic thrombin inhibitor, PPACK (D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone, 1-30 nM). PPACK, however, had no effect on the other endothelium-dependent coronary vasodilators, such as acetylcholine and adenosine triphosphate, in our isolated dog coronary artery preparations. Biochemical determinations of the amidolytic activity of thrombin, using Tosylglycyl-L-prolyl-L-arginine-p-nitroanilide as a chromogen, also indicated a similar PPACK and heparin-antithrombin III dose-dependent inhibition of the thrombin enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of thrombin-induced endothelium-dependent coronary vasodilation in dogs: role of its proteolytic enzymatic activity. 241 89

The content of free calcium ions in stored blood can be maintained by stabilizing it with thrombin inhibitors. With regard to a long-term stabilization of blood or blood plasma, satisfactory results can only be achieved with the competitive synthetic thrombin inhibitor 4-amidinophenylpyruvic acid by adding slight amounts of heparin and by storing it in a cool room. By using hirudin (greater than 50 ATE/ml of blood) the stabilization of blood enables a long-lasting anticoagulative effect to be achieved, such as it is required for the purpose of reprocessing blood to plasma fractions.
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PMID:[Stabilization of donor blood with thrombin inhibitors]. 243 79

Three p-amidinophenyl esters have been synthesized and characterized as irreversible inhibitors of the vitamin-K dependent proteinases; factors IXa, Xa and thrombin (Turner et al. [4]).+ In the present report we describe the in vitro and in vivo effects of these agents on standard coagulation tests in vitro and in blood from animals treated with the compounds. At a concentration of 500 microM, the three esters increased the activated partial thromboplastin time (PTT) of pooled human plasma 3 to 5-fold. The prothrombin time increased 1.4 to 3.7-fold under similar conditions. The p-amidinophenyl ester of cinnamic acid (CINN) showed the most pronounced effect on both assays. This ester also is the best inhibitor of human factors IXa and Xa, while the p-amidinophenyl ester of benzoic acid (BENZ) is a slightly better alpha-thrombin inhibitor (4). The effect of these esters on the thrombin clotting time correlated with in vitro kinetic measurements of alpha-thrombin inhibition rates. Both BENZ and CINN increased the assay endpoint more than 6-fold. The three esters also were studied using mouse plasma. A comparable effect on the PTT was noted. Intravenous administration of 300 microliter of 1 mM CINN as a single bolus in mice caused a 2.3-fold increase in the PTT which remained 1.2-fold normal 2 h later. The BENZ and alpha-methyl-cinnamic acid (MECINN) esters were somewhat less effective as predicted from their in vitro effect on the PTT. This investigation and previous studies indicate that these compounds demonstrate low toxicity at therapeutic levels. It is concluded that the p-amidinophenyl esters may be useful in antithrombotic therapy.
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PMID:Evaluation of p-amidinophenyl esters as potential antithrombotic agents. 243 36


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