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)

KRDS (Lys-Arg-Asp-Ser), a tetrapeptide from human lactotransferrin, was tested for its effects in vitro on dog platelet function and in vivo on femoral arterial thrombus formation in dogs. KRDS inhibited ADP (8 microM)-induced platelet aggregation (IC50: 350 microM) and arachidonic acid (2 mM)-induced thromboxane B2 generation (IC50: 175 microM). In addition, the thrombin (0.2 U/ml)-induced serotonin release was inhibited by KRDS (IC50: 525 microM) and the expression of alpha-granule membrane protein (GMP-140) was also inhibited (IC50: 350 microM). The results show that KRDS is an inhibitor for platelet aggregation and secretion to which the inhibition is more potent. Meanwhile, in the experiment of arterial thrombosis in dogs, KRDS (5 microM/kg) and 125I-SZ-51 (a monoclonal antibody against GMP-140) were injected before operation and immediately after the thrombus formation, respectively. In the KRDS group, the weight of removed thrombi was reduced to 50% of that in controls and the radioactivity per mg of labeled thrombi to 33.3% while in blood the radioactivity increased 2 times that in controls at the 4th hour after the injection of 125I-SZ-51. The radioactivity ratio between removed thrombi and blood was only 16% of that in controls. These results indicate that KRDS can inhibit thrombus formation in vivo and is a promising antithrombotic agent.
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PMID:Inhibition effects of KRDS, a peptide derived from lactotransferrin, on platelet function and arterial thrombus formation in dogs. 138 97

In the A alpha-chain gene coding for an abnormal fibrinogen (fibrinogen Marburg) we identified a single base substitution (A-->T) that changes the codon A alpha 461 AAA (Lys) to TAA (Stop). The propositus was found to be homozygous for the mutation, whereas the father and five siblings were heterozygous, and three other siblings contained only the normal sequence. The stop codon at position 461 results in the deletion of the carboxyl-terminal segment A alpha 461-610. Purified fibrinogen Marburg contained an A alpha-chain with a relative molecular weight of approximately 47,000. The FpA release by thrombin was not affected by this deletion, whereas the fibrin polymerization was strongly decreased. The binding of endothelial cells to immobilized fibrinogen Marburg was almost completely abolished compared with normal fibrinogen. Fibrinogen Marburg contained a substantial amount of albumin linked to the fibrinogen molecule by disulfide bonds, and these fibrinogen-albumin complexes were also present in plasma. The plasma fibrinogen concentration of the propositus was measured by three different methods: a functional method (< 0.25 mg/mL), an immunologic method using polyclonal antibodies (0.6 mg/mL), and an immunologic method based on two monoclonal antibodies specific for the amino-terminus and carboxyl-terminus of the A alpha-chain (< 0.05 mg/mL). Using the two immunologic methods, it appeared that only 10% to 15% of the plasma fibrinogen of the heterozygous siblings was abnormal.
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PMID:Fibrinogen Marburg: a homozygous case of dysfibrinogenemia, lacking amino acids A alpha 461-610 (Lys 461 AAA-->stop TAA). 139 54

Poly-L-lysine with molecular masses of 3.3-290 kDa increased the amidolytic activities of leukocyte elastase and cathepsin G at low concentration, but had little effect on the activities of pancreatic elastase, alpha-chymotrypsin, plasmin and thrombin. Highly purified cathepsin G was obtained from column of EAH Sepharose 4B or Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose (affinity chromatography) by elution with poly-L-lysine solution (0.4 mg/ml, molecular weight (MW.) 290000 or 2.2 mg/ml, MW. 3300). Leukocyte elastase, adsorbed to Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose, was not eluted with poly-L-lysine solution. The amino acid composition of purified cathepsin G has been determined.
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PMID:Application of poly-L-lysine to purification of leukocyte cathepsin G by affinity chromatography. 139 85

Radioactivity from I125-labeled human platelets was measured to estimate the extent of binding of platelet surface proteins to immobilized thrombin. 1-3% of the radioactivity was bound with 10-20% of this amount apparently irreversibly bound to the thrombin matrix. Site-specific chemical modification of thrombin with pyridoxal-5'-phosphate, N-bromosuccinimide or tetranitromethane resulted in a variable reduction of the amount of radiolabel bound. When thrombin modified with H-D-PheProArg-chloromethyl ketone (PPACK) was coupled to the matrix, there was no difference in the binding of platelet membrane proteins when compared to a control thrombin preparation while thrombin modified with tosyl-Lys-chloromethyl ketone (TLCK) coupled to the matrix did not bind radiolabel any more effectively than albumin which served as the control. However, when thrombin was modified with PPACK after coupling to the agarose matrix, ability to bind radiolabel was lost. Thrombin bound to platelets remained catalytically active when assayed with a peptide nitroanilide substrate. These results suggest tight binding between thrombin and platelets that is not only not dependent on active site integrity but leaves the bound thrombin catalytically competent.
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PMID:Affinity chromatography of platelets on immobilized thrombin: retention of catalytic activity by platelet-bound thrombin. 141 20

Platelet suspensions, that secreted about 50% of their dense granule contents upon stimulation with alpha-thrombin, showed a dose-dependent increase in secretion after 30 min preincubation with 0.5-3.0 g low density lipoprotein (LDL) protein/1. A 1-5 min preincubation had no effect. The enhancement by LDL only occurred at about 20% secretion or more, indicating that a minimal degree of activation was required for LDL to become effective. Lysine-modified LDL was equally effective as native LDL. The effect of LDL on secretion was accompanied by enhanced thromboxane B2 formation caused by stimulation of the liberation of arachidonate from phosphatidylcholine and/or phosphatidylinositol. However, when thromboxane formation was inhibited or the prostaglandin H2-thromboxane A2-receptor was blocked, LDL remained a potent stimulator of the secretion response. Thus, LDL enhances platelet secretion by a thromboxane A2-dependent and a thromboxane A2-independent mechanism via an effect that is independent of specific binding sites on the platelet.
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PMID:Enhancement of platelet functions by low density lipoproteins. 142 Mar 43

Recombinant hirudin variants have been designed which inhibit alpha-thrombin by the hirudin mechanism and which in addition exhibit disintegrin activity. These proteins, called "hirudisins," have been engineered by replacing the Ser-Asp-Gly-Glu sequence at the tip of hirudin's finger-like structure (residues 32-35) by Arg-Gly-Asp-Ser (RGDS) to yield hirudisin and Lys-Gly-Asp-Ser (KGDS) to obtain hirudisin-1. Comparison of thrombin inhibition activities showed that hirudisin is 2-fold more potent (K(i) = 160 +/- 70 fM) than hirudisin-1 (K(i) = 370 +/- 44 fM) and recombinant (r)-hirudin (K(i) = 270 +/- 50 fM). alpha-Thrombin-stimulated platelet aggregation was effectively inhibited by r-hirudin, hirudisin, and hirudisin-1 with IC50 of 5.7 to 6.8 nM. Unlike r-hirudin, hirudisin inhibits ADP-induced platelet aggregation (IC50 = 65 microM) 3- to 5-fold stronger than the linear GRGDS- and RGDS-peptide. Direct interaction of hirudisin with purified glycoprotein IIb-IIIa demonstrated that antiplatelet aggregation activity is due to the integrin-directed RGD motif. Disintegrin activity of hirudisin relative to that of reduced and carboxymethylated hirudisin suggests that the conformational strain favors binding to integrins. On the basis of these results, hirudisins appear to be interesting molecules for the design of potential antithrombotic agents with antithrombin as well as antiplatelet aggregation activities.
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PMID:Hirudisins. Hirudin-derived thrombin inhibitors with disintegrin activity. 144 73

Some, if not all, of the cellular actions of alpha-thrombin are now believed to be mediated by proteolytic cleavage of the cell surface thrombin receptor to yield a tethered ligand that initiates signal transduction via the receptor. We have investigated the actions of alpha-thrombin on the regulation of cytosolic free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) in human osteoblast-like Saos-2 cells. After acidification with nigericin, thrombin induced an acute increase of [Ca2+]i and a rise in pHi. The action of thrombin on pHi was dependent on activation of the Na(+)-H+ antiporter. Thrombin elicited parallel concentration-dependent increases in both [Ca2+]i and pHi, and the rise in [Ca2+]i was a prerequisite for the increase in pHi. Preincubation of thrombin with the active site proteolytic inhibitor, BOC-D-Phe-L-Pro-D,L-Lys-CF3, prevented the alkalinization response to thrombin but had little or no effect on the thrombin-induced rise in [Ca2+]i. Hirudin, a natural inhibitor of thrombin, acts by tight binding to several discrete regions on the thrombin molecule. Preincubation of thrombin with hirudin completely blocked the rise in both [Ca2+]i and pHi. These results demonstrate that the thrombin-induced rise in [Ca2+]i alone is not sufficient to cause alkalinization in Saos-2 cells. More importantly, our findings reveal that not all of the cellular actions of thrombin can be explained by proteolytic cleavage of the thrombin receptor and suggest that different domains on the thrombin molecule may be required for eliciting signals that raise [Ca2+]i and pHi in Saos-2 cells.
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PMID:Regulation of pHi in Saos-2 cells by thrombin: roles of proteolytic activity and cytosolic calcium transients. 147 67

Trypsin and trypsin-like enzymes cleave C-terminal bonds of the basic amino acids Arg and Lys. Inhibitors of these enzymes have been found not only among Arg and Lys derivatives but also with structurally related benzamidines. Especially cyclic amides of 4-amidinophenylalanine were found to be inhibitors of thrombin. The most potent selective thrombin inhibitor of these type is N alpha-(beta-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidine. From the X-ray crystal structures of thrombin and trypsin-inhibitor complexes the thrombin complexes formed with inhibitors derived from amidinophenylalanine have been modeled. These models allow valuable predictions to design inhibitors of improved selection and binding properties. Most recently, also the X-ray crystal structures of complexes of inhibitors with bovine thrombin have been solved.
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PMID:Interactions of thrombin with benzamidine-based inhibitors. 151 80

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.
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PMID:The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution. 151 14

A metalloprotease from the rattlesnake Crotalus atrox venom was isolated and purified from multiple-step chromatographies including anion-exchange chromatography, gel permeation and reversed-phase HPLC. The fraction was shown to be homogeneous as judged by SDS-gel electrophoresis. It also showed a high proteolytic activity against alpha- and beta-chains of fibrinogen molecules. Further characterization of the purified fraction with fibrinogenase activity indicated that it is a single-chain protease with a molecular mass of about 24 kDa and an acidic isoelectric point. It is relatively heat stable up to about 65 degrees C, inhibited by EDTA, beta-mercaptoethanol, but not by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitor and aprotinin. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to some metalloproteases characterized before from the closely related rattlesnake venoms. N-Terminal sequence analysis of the enzyme corroborated some similarity between this enzyme and the reported sequences of these enzymes characterized from the Crotalidae snake family. This study indicated the presence of a novel fibrinogenase (termed Catroxase) with N-terminal sequence different from the metalloprotease with hemorrhagic activity isolated from the same Western diamondback rattlesnake.
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PMID:Characterization of a protease with alpha- and beta-fibrinogenase activity from the Western diamondback rattlesnake, Crotalus atrox. 152 Mar 24


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