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)

A method providing a biochemical index for the evaluation of promoters or inhibitors of angiogenesis in the chick chorioallantoic membrane (CAM) is here described and validated. This method is based on the determination of collagenous protein synthesis which takes place during new vessel formation. Validation was done by comparing collagenous protein synthesis to morphological methods of determining vascular density either by counting the number of vessels intersecting three concentric rings or by computer-assisted image analysis. Five compounds which promote or inhibit angiogenesis in the CAM were used for this purpose. The protein kinase C activator 4-beta-phorbol 12-myristate 13-acetate and alpha-thrombin increased collagenous protein synthesis and these results correlated with those obtained by using the two morphological methods mentioned above. Similarly, the inhibitors of angiogenesis, Ro318220, tricyclodecan-9-yl xanthate (D609), and 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide (GPA1734), reduced collagenous protein synthesis and vascular density (determined by image analysis or by counting the number of vessels intersecting three concentric rings) to a comparable degree. These results indicate that collagenous protein synthesis can be used as a reliable, reproducible, and unbiased index of angiogenesis in the chick chorioallantoic membrane.
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PMID:Validation of collagenous protein synthesis as an index for angiogenesis with the use of morphological methods. 853 1

We investigated the anticoagulating and heparin-neutralizing properties of protamine and polybrene (hexadimethrine bromide), using the endogenous thrombin potential (ETP) as the parameter to access plasma coagulability. The hypocoagulability induced by high doses of heparin (3 IU/ml) could be reversed by addition of protamine to a very limited extent only. Polybrene on the other hand did neutralize heparin at the equivalent concentration and a two-fold excess did not influence the ETP parameters. In vivo neutralization of high-dose heparin with protamine should therefore be reconsidered. Our experiments suggest polybrene to be superior over protamine with respect to neutralization of high doses of heparin.
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PMID:Influence of heparin, protamine and polybrene on the time integral of thrombin generation (endogenous thrombin potential). 869 72

We have compared the effects of thrombin and of the 14-amino acid peptide agonist (TRAP-14) of the thrombin protease activated receptor (PAR) on cholinergic neurons in pure cultures of rat septal neurons and in co-cultures of septal neurons and glial cells. In pure septal cultures, low concentrations of thrombin (up to 10 nM) did not affect choline acetyltransferase (ChAT) activity, a marker of cholinergic neurons, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, an index of cell viability. However, 100 nM thrombin decreased ChAT activity and MTT reduction by 44 and 17%, respectively. In co-cultures, a low concentration of thrombin (1 nM) increased ChAT activity (+75%), whereas a high concentration (100 nM) decreased it (-83%). At this high concentration, thrombin was neurotoxic, as indicated by a large decrease in MTT reduction (-80%). Thrombin effects on ChAT activity were mimicked by TRAP-14 both in pure septal cultures (no effect at 0.1 microM and -63% at 100 microM) and in co-cultures (+25% at 0.1 microM and -28% at 100 microM). In contrast, this peptide did not affect MTT reduction. These dual effects of thrombin and TRAP-14 on ChAT activity in co-cultures, were also observed on pure cultures of septal cells supplied with NGF. The activation and inhibition by TRAP-14 of the expression of ChAT activity in septal neuron/glial cell cultures were inhibited by a 9-amino acid peptide antagonist of thrombin PAR. Thus, the effects of thrombin on cholinergic neurons seem to be mainly mediated by thrombin PAR and glial cells seem to play a major role in these thrombin actions.
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PMID:Dual effects of thrombin and a 14-amino acid peptide agonist of the thrombin receptor on septal cholinergic neurons. 872 Aug 72

Spreading of human umbilical vein endothelial cells (ECs) on fibrin requires thrombin cleavage of fibrinopeptide B (FPB) and subsequent exposure of the new beta 15-42 N-terminus. To further understand the interactions between ECs and fibrin beta 15-42 sequences, binding of fibrin(ogen) to EC monolayers was measured with polyclonal anti-fibrinogen (FBG) in parallel with monoclonal anti-FBG (18C6, beta 1-21; J88B, gamma 63-78) and anti-fibrin (T2G1, beta 15-21) antibodies in an indirect enzyme-linked immunosorbent assay. To accomplish this, large, soluble fragments of fibrin were prepared by cyanogen bromide (CNBr) cleavage (fibrin-CNBr); CNBr-cleaved FBG (FBG-CNBr) served as the control ligand. N-terminal fibrin-CNBr bound to EC monolayers and cells in suspension in a dose-dependent and saturable manner. By contrast, FBG-CNBr bound only 50% as well to EC monolayers, with no significant binding of intact FBG, C-terminal FBG plasmic fragment D, or N-terminal plasmic fragment E, which lacks beta 1-53. ECs bound the peptide beta 15-42-bovine serum albumin (BSA) conjugate but neither a scrambled beta 15-42 peptide conjugate nor conjugates of beta 24-42, beta 18-27, or beta 18-31. Binding of fibrin-CNBr was inhibited 54% by the beta 15-42-BSA conjugate and 17% by the B beta 1-42-BSA conjugate but not by free beta 15-42 peptide or RGDS-cell binding peptide. Binding of fibrin-CNBr was inhibited > 95% by heparin in a concentration-dependent manner; the same concentrations of heparin inhibited binding of beta 15-42-BSA by > 75% but not the dose-dependent binding of fibronection to ECs. These data suggest that in their native conformation, FBG B beta 15-42 sequences are unavailable for binding to ECs and that thrombin-induced exposure of beta 15-42 is required for binding by a heparin-dependent, RGD-independent mechanism at the new N-terminus of fibrin.
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PMID:Heparin-binding domain of fibrin mediates its binding to endothelial cells. 897 60

It has been previously reported that Alzheimer's amyloid beta protein (A beta) induces reactive astrocytosis in culture. In the present study, we found that A beta potently inhibits cellular redox activity of cultured astrocytes, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. The following comparative studies revealed several differences between these two actions of A beta on astrocytes. First, A beta-induced reactive morphological change was suppressed by the presence of serum or thrombin, and A beta inhibition of cellular redox activity was observed in either the presence or the absence of serum. Second, micromolar concentrations (10 microM or more) were required for A beta to induce reactive astrocytosis, whereas nanomolar concentrations (0.1-100 nM) were sufficient to inhibit cellular redox activity. Third, the effect of micromolar A beta was virtually irreversible, but nanomolar A beta-induced inhibition of cellular redox activity was reversed by washing out A beta. Furthermore, as it has been reported that A beta neurotoxicity is mediated by reactive oxygen species, we also examined if similar mechanisms are involved in astrocytic response to A beta. However, neither A beta-induced morphological change nor inhibition of redox activity was blocked by antioxidants, suggesting that these effects are not caused by oxidative stress.
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PMID:Nanomolar amyloid beta protein-induced inhibition of cellular redox activity in cultured astrocytes. 910 14

Crude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources. LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercaptoethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lysophosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet's response to several agonists.
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PMID:Mechanism of inhibitory action on platelet activation of a phospholipase A2 isolated from Lachesis muta (Bushmaster) snake venom. 940 22

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A2 activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O(-)2) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O(-)2 release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase A2 inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A2 inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.
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PMID:Thrombin stimulated reactive oxygen species production in cultured human endothelial cells. 993 Jun 45

Ovomucoid, a major allergen in hen's egg white, consists of three tandem domains. The third domain (DIII) cDNA was sublconed into pGEMT-vector and the resultant plasmid (pGEMDIII) was inserted into a pGEM-4T-2 glutathione-S-transferase (GST) fusion vector. The GST-DIII fusion protein was expressed in Escherichia coli. The 56-residue fragment corresponding to DIII (Leu131-Cys186) was liberated using cyanogen bromide to cleave off the GST that had been hydrolized with thrombin, which left an additional peptide at the terminus of the recombinant protein. Measurement of circular dichroism spectra indicated that the recombinant third domain (DIII*) had a structure that was slightly less compact than that of the native form. Immunoblot analysis showed that the human IgE binding activity of DIII* was identical to that of native DIII, while its activity was significantly increased to IgE antibodies from egg-allergic patients when tested with an enzyme-linked immunosorbent assay. These results indicate that recombinant DIII* has similar sequential epitopes, but may have more predominant conformational epitopes than native analogues. This might have important implications in egg-allergic reactions.
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PMID:IgE binding properties of the recombinant ovomucoid third domain expressed in Escherichia coli. 1135 43

Single-chain urokinase-type plasminogen activator (scu-PA) can be cleaved by thrombin into a virtually inactive form called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), a process accelerated by thrombomodulin, which contains six epidermal growth factor (EGF)-like domains. In this study, we identified the EGF-like domains of thrombomodulin required for the acceleration of the inactivation of scu-PA by thrombin using various forms of thrombomodulin (TM). scu-PA was treated with thrombin in the absence and presence of full-length rabbit TM (containing EGF1-6), recombinant TM comprising all of the extracellular domains including EGF1-6 (TMLEO) and recombinant TM comprising EGF4-6 plus the interconnecting region between EGF3 and EGF4 (TMEi4-6), and the tcu-PA/T generated was quantitated in each case. Rabbit TM accelerated the inactivation of scu-PA approximately 35-fold, while both recombinant forms accelerated it only threefold due to the absence of a critical chondroitin sulfate moiety. Subsequently, TME5-6 was prepared by cyanogen bromide digestion of TMEi4-6. TME5-6 bound to thrombin but did not accelerate the activation of protein C. In contrast, the inactivation of scu-PA by thrombin was accelerated to the same extent as that induced by TMLEO and TMEi4-6. This study demonstrates that, in addition to the chondroitin sulfate moiety, only EGF-like domains 5 and 6 are essential for the acceleration of the inactivation of scu-PA by thrombin. This differs from the domains that are critical for activation of protein C (EGF-like domains i4-6) and thrombin activatable fibrinolysis inhibitor (EGF-like domains 3-6).
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PMID:Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator. 1168 79

The amino acid sequence of a thrombin like enzyme, named elegaxobin, isolated from the venom of Trimeresurus elegans (Sakishima-habu) was determined by Edman sequencing of the peptides, derived from digests with cyanogen bromide, hydroxylamine, achromobacter protease I, trypsin, V8 protease, and chymotrypsin. Elegaxobin showed conservation of the catalytic amino acid residues (His, Asp, and Ser) of trypsin like serine protease in the amino acid sequence. The carboxy-terminal amino acid, Leu, was determined using carboxypeptidase Y. Elegaxobin consisted of 233 amino acids and had a calculated molecular weight of 25,439. Elegaxobin was 53, 59, 26 and 40% homologous in sequence to ancrod, flavoxobin, bovine thrombin and trypsin, respectively.
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PMID:Amino acid sequence of a thrombin like enzyme, elegaxobin, from the venom of Trimeresurus elegans (Sakishima-habu). 1207 50


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