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)

Proteolytic digestion of the human T lymphoblastoid cell line (Molt-4) and of peripheral blood lymphocytes by trypsin, chymotrypsin, and pronase results in a progressive, time-and dose-dependent diminution of T lymphocyte-sheep red bloock cell (SRBC) rosette formation, whereas thrombin, plasmin, collagenase, DNAse, and phospholipase have not effect. Complete abrogation of SRBC binding is achieved when lymphocytes (1 x 108/ml) are incubated with either trypsin or chymotrypsin at 10 mug/ml for 30 min, and greater than 50% abrogation is observed between 3 to 10 min. Preincubation of SRBC with the 10 min and 20 min lymphocyte digest supernatants inhibited their subsequent binding by normal T lymphocytes by as much as 64%. Thirty-minute digests were less inhibitory. Equivalent digests from several human B lumphoblastoid cell lines and from a non-rosetting clone of Molt-4 cells were not inhibitory. Polyacrylamide gel electrophoresis followed by elution of serial gel slices revealed four distinct inhibitory bands (I-IV) in the 20-min digest supernatant whereas only bands I-III and band IV were present in the 10-min and 30-min digest supernatants, respectively, suggesting progressive proteolysis of a distinct receptor. These experiments indicate that the binding of SRBC by human T lymphocytes represents a receptor-ligand interaction rather than a nonspecific electrical charge phe nomenon and that the receptor is a discrete molecular species which can be isolated from the surface of T but not B lymphocytes by limited enzymatic proteolysis.
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PMID:Recovery of soluble sheep erythrocyte receptor from the T lymphocyte surface by proteolytic cleavage. 30 Mar 98

Factor V was isolated from human citrate plasma by very mild purification steps. Cryoprecipitation, fractionation with polyethylene glycol 6000, gel filtration of AcA 44 and adsorption of haptoglobin to immobilized hemoglobin were applied successively, resulting in factor V preparations with a specific activity of 14.5 unit/mg. The yield was 28 percent. A molecular weight of 296 000 was determined by gel filtration and the apparent sedimentation constant found by ultracentrifugation in a sucrose gradient was 7.8 S. Parallel experiments with citrate plasma resulted in the same molecular weight and sedimentation constant. Polyacrylamide gel electrophoresis of factor V in the presence or absence of sodium dodecyl sulfate showed a single protein band. Incubation with human thrombin resulted in an 8-fold activation of the purified factor V.
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PMID:Isolation and partial characterization of human factor V. 45 68

In the presence of a procoagulant fraction (Echis carinatus procoagulant) isolated from the venom of the saw-scaled viper Echis carinatus sochureki, purified human prothrombin (P1) is completely converted to thrombin. The first step is the removal of an NH2-terminal peptide (F1) representing approximately one-third of the prothrombin molecule. The remaining peptide (P2) is then cleaved by the action of E.c. procoagulant to yield a two-chain, disulfide-bridged protein (P'2) which has the same molecular weight as P2. P'2 has enzymic (thrombin) activity, as evidence by incorporation of radiolabeled diisopropylphosphate into its heavy chain (TB), hydrolysis of p-toluenesulfonylarginine methyl ester, and clotting of fibrinogen. Relative to thrombin, its esterolytic activity greatly exceeds its clot-promoting activity. Examination of the polypeptide chains obtained by reducing P'2 has shown that its larger chain (TB) is indistinguishable from the heavy chain of thrombin. Its other chain (F2TA) consists of the light chain (TA) of thrombin bound by peptide linkage to the protion of the prothrombin molecule which had been adjacent to F1. Removal of this portion (F2) is catalyzed by thrombin (and, evidently, by P'2), but not by the E.c. procoagulant. When F2 is removed from P'2, the remaining two-chian protein is indistinguishable from thrombin by any of the criteria applied--molecular weight, subunit chain composition, or enzymic activity. Polyacrylamide gel electrophoresis was carried out in sodium dodecyl sulfate before and after disulfide reduction of samples generated in the presence and in the absence of diisopropylphosphorofluoridate, which inhibits thrombin but not the E.c. procoagulant. Such experiments showed that thrombin (and probably P'2), as well as E.c. procoagulant, catalyzes the release of F1. Furthermore, thrombin brings about the cleavage of F1 to yield a two-chain, disulfidebridged protein (F'1). These observations, particularly those made in the course of characterizine P'2, have led to the conclusion that cleavage of the peptide bond connecting the TA and TB portions of the prothrombin molecule (or its derivatives) produces a serine active center and, hence, a molecule possessing thrombin activity. This cleavage is catalyzed by the E.c. procoagulant but not by thrombon itself.
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PMID:Activation of human prothrombin by a procoagulant fraction from the venom of Echis carinatus. Identification of a high molecular weight intermediate with thrombin activity. 115 98

Fibrinogen-based adhesive, derived from pooled human plasma, has been used in Europe with great success in otologic surgery, but has not been approved for use in the U.S. because of the risk of transmitting hepatitis. Autologous fibrinogen, derived by polyethylene glycol precipitation from the blood of an individual patient would avoid this risk, and has been shown to be relatively safe to the ear in animal studies. A study of the biochemical composition of this autologous fibrinogen concentrate derived from 15 human volunteer donors was performed. The mean starting plasma fibrinogen was 2.12 mg/ml (range 1.59-3.22 mg/ml). When 10% polyethylene glycol was used to precipitate the fibrinogen, the concentrate contained, on the average, 31.8 mg of fibrinogen/ml. The percent yield averaged 54.9%, and the protein in the final product was 91.9% fibrinogen. Increasing the polyethylene glycol concentration in the precipitation process to as high as 15% resulted in an increased yield as high as 91%, but the protein in the final product was only 42.5% fibrinogen. Polyacrylamide gel electrophoresis confirmed that the predominant protein in the 10% polyethylene glycol precipitate was fibrinogen. These data suggest that highly concentrated fibrinogen can be derived with relative ease from single donor human plasma, and that the product is relatively pure. When combined with thrombin and calcium chloride, this concentrate should provide an adhesive that avoids the risks associated with fibrinogen adhesive derived from pooled blood.
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PMID:Biochemical characterization of autologous fibrinogen adhesive. 244 81

Heparin cofactor II (HCII) is a highly specific serine proteinase inhibitor, which complexes covalently with thrombin in a reaction catalyzed by heparin and other polyanions. The molecular basis for the thrombin specificity may be explained by the identification here of a segment of HCII including residues 54-75 that binds to thrombin. A synthetic peptide, HCII(54-75), based on this segment of HCII, Gly-Glu-Glu-Asp-Asp-Asp-Tyr-Leu-Asp-Leu-Glu- Lys-Ile-Phe-Ala-Glu-Asp-Asp-Asp-Tyr-Ile-Asp inhibited thrombin's cleavage of fibrinogen. Clotting activity of thrombin was inhibited 50% at a concentration of 28 microM. Polyacrylamide gel electrophoresis showed that HCII(54-75) inhibited thrombin's cleavage of both the A alpha and B beta polypeptides in fibrinogen. However, the peptide did not block thrombin's active site, as hydrolysis of chromogenic substrates was not inhibited. HCII(54-75) probably binds to the same site on thrombin as do carboxyl-terminal residues of hirudins, thrombin inhibitors of leeches. HCII(54-75) inhibited binding of thrombin to a synthetic peptide corresponding to residues 54-66 of hirudin PA, but the hirudin peptide was about 30-fold more potent in binding and clotting assays. Both synthetic peptides, as a result of their polyanionic character, might be expected to stimulate the reaction of HCII with thrombin. However, the hirudin-related peptide inhibited this reaction, suggesting that it blocked a site on thrombin required for interaction with HCII. HCII(54-75) had a net stimulatory effect on the thrombin-HCII reaction as a consequence of its lower affinity for thrombin and greater negative charge relative to the hirudin-related peptide. These studies suggest that residues 54-75 of HCII interact with a noncatalytic binding site on thrombin and that this interaction contributes to efficient inhibition of thrombin by HCII.
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PMID:Antithrombin activity of a peptide corresponding to residues 54-75 of heparin cofactor II. 276 54

Coagulation Factor V (Mr = 330,000), upon cleavage by thrombin, produces Factor Va, which is composed of two subunits with Mr values of 94,000 and 74,000, along with two activation fragments possessing no known function. Studies were undertaken to assess the ability of the transamidase Factor XIIIa to covalently incorporate the lysine analogs [3H]putrescine and dansylcadaverine into the thrombin-cleaved (activated) and unactivated forms of human and bovine Factor V. The incorporation of either probe into thrombin-activated Factor V proceeded at an initial rate approximately twice that for unactivated Factor V. The extent of the incorporation of [3H]putrescine or dansylcadaverine into activated or unactivated human Factor V was identical; 4 mol of either probe per mol of Factor V. In the case of bovine Factor V, however, while 4 mol of probe were bound per mol of the unactivated pro-cofactor, 5 mol of either lysine analog were covalently linked to 1 mol of thrombin-cleaved Factor V. Polyacrylamide gel fluorography, immunoaffinity chromatography, and immunoprecipitation identified the largest activation fragment of human Factor V (Mr = 150,000) and bovine Factor V (Mr = 120,000) to contain the sites of incorporation of the covalently bound probes. High molecular weight, apparently covalent polymers of Factor V were produced by the action of Factor XIIIa on activated and unactivated human or bovine Factor V. The absence of either probe in the reaction mixtures did not appear to allow an enhancement of protein polymerization.
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PMID:Factor V is a substrate for the transamidase factor XIIIa. 287 32

We perfused the endothelia of isolated human corneas mounted in the specular microscope with BSS Plus containing 1,000-U/ml or 100-U/ml dilutions of two commercially available topical thrombin preparations. Corneas perfused with thrombin at 1,000 U/ml showed intracellular and intercellular vacuole formation and altered junctional complexes. As listed on the package inserts, the thrombin preparations contained preservatives and other additives that present a significant osmotic load in 1,000-U/ml preparations. Corneas perfused with 100-U/ml thrombin solutions showed a significant attenuation in their deswelling rate but no ultrastructural alterations. One available thrombin preparation when diluted to 100 U/ml had a glycine concentration associated with previous retinal electroretinography changes. Polyacrylamide gel electrophoresis of one manufacturer's thrombin solution showed multiple high and low molecular weight constituents. Analysis of particulate contamination showed one 100-U/ml thrombin preparation to have a large quantity of particulates. Although thrombin may be useful when applied topically as an aid in surgical hemostasis, its use intraocularly presents substantial concern regarding the preparation's purity, additives, contaminants, and adverse effects on ocular tissues.
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PMID:Intracameral thrombin and the corneal endothelium. 317 58

Lymphocyte egress from the vascular compartment into the lymph node (LN) parenchyma occurs at the postcapillary venules, termed high endothelial venules (HEVs). Lymphocyte adhesion and migration through the HEVs is a receptor-mediated, energy-dependent, process. The aim of this study was to investigate the role of MHC Class II antigen expression on lymphocyte-HEV interaction in normal (CBA) and autoimmune (MRL/l) mice. Using the HEV binding assay, lymphocyte adhesion to LN sections pretreated with monoclonal antibody (MAb; 10-2.16) was decreased compared to diluent (mean of the differences +/- standard deviation; xd +/- SD: 0.749 +/- 0.22, P less than 0.0075)- and myeloma immunoglobulin-pretreated controls (xd = 0.462 +/- 0.13, P less than 0.005). Similar inhibition of binding was found in MRL/l LN sections pretreated with MAb 10-2.16. Binding inhibition was concentration dependent, but total inhibition was never achieved. Several other anti-Ia MAb's were used, but failed to inhibit lymphocyte attachment. Lymphocyte binding to control sections treated with MAb's against MHC Class I antigen, plasminogen activator (PAM-3), anti-thrombin III (AT-IIIm), and MECA-325 antigen was not significantly different from diluent controls. LN cell suspensions pretreated with MAb 10-2.16 bound normally to LN sections. By contrast, MAb to lymphocyte homing receptor (MEL-14) inhibited lymphocyte adhesion. The role of Class II antigens in lymphocyte-HEV interactions is discussed.
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PMID:Anti-Ia monoclonal antibody (10-2.16) inhibits lymphocyte-high endothelial venule (HEV) interaction. 318 Feb 28

Prothrombin isolated from duck sodium citrate plasma was activated in a system containing duck factor Xa and calcium ions. Polyacrylamide gel electrophoresis showed that intermediates and the final product, thrombin, of Mr in the range 21 500-52 000 were present in the incubation mixture Serine and isoleucine were found to be the N-terminal amino acids of the intermediate form 1 and thrombin, respectively.
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PMID:Activation of duck prothrombin by factor Xa and thrombin. 383 1

It was demonstrated that partial reduction of disulfide bonds in thrombin by dithiothreitol in the absence of denaturating agents leads to a decrease of enzymatic activity with respect to fibrinogen coagulation and tosylarginine methyl ester hydrolysis. Polyacrylamide gel electrophoresis and determination of the number of SH-groups liberated in the course of reduction suggest that the observed inactivation is primarily due to the disruption of the S-S-bridge between the A- and B-chains of thrombin.
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PMID:[Effect of partial reduction of disulfide bonds on the enzymatic activity of thrombin]. 407 91


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