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 Authors, thanks to experimental works, have established that piribedil at high concentrations inhibits ADP and thrombin aggregation effect on the rabbit PRP.
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PMID:[Platelet aggregation and piribedil]. 722 47

Synthesis of prostaglandin endoperoxides was evaluated in paired maternal and cord blood samples. Platelets from mothers and neonates aggregated normally in response to arachidonic acid (AA). Cyclooxygenase activity was evaluated by monitoring the incorporation of radioactivity into prostaglandin endoperoxide metabolites after incubation with 1-14C-AA. Thin layer radiochromatograms of methylated incubation products revealed three main peaks corresponding to 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and 8-(1-hydroxy-3-oxoproply)-9,12-L-dihydroxy-5,10-heptadecadienoic acid (TXB2). Maternal and neonatal platelets incorporated similar amounts of radioactivity into HHT and TXB2. Radioimmunoassay for TXB2 in thrombin-clotted PRP revealed no significant differences between maternal and neonatal platelets. Since these metabolites are derived from cyclic endoperoxides formed by the action of cyclooxygenase on AA, we conclude that prostaglandin endoperoxide synthesis is fully developed in neonatal platelets. Mutual correction of collagen-induced platelet aggregation and ADP release was observed when equal volumes of neonatal and aspirin-treated adult platelet-rich plasma were mixed. Therefore, since neonatal platelets contain normal amounts of storage pool nucleotides, we also conclude that the defective secondary aggregation and release seen in neonatal platelets is caused by a failure in the release of AA from membrane phospholipids upon stimulation with collagen or epinephrine.
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PMID:Neonatal platelet function: a membrane-related phenomenon? 725 Jul 85

Evaluation of platelet physiology, biochemistry, ultrastructure, and function in a young woman without history of hemorrhagic problems revealed that her platelets were deficient in cyclooxygenase activity. Her citrate platelet-rich plasma (C-PRP) responded monophasically when stirred with aggregating agents in the same manner as aspirin-treated normal C-PRP, but could be irreversible aggregated by high concentrations of thrombin, collagen, and ADP. Her platelets did not aggregate when stirred with AA at concentrations as high as 2 mM. Ultrastructure and levels of serotonin and adenine nucleotides wer normal. Amounts of 14C-AA released after stirring with thrombin were similar to normal cells. However, evaluation of prostaglandin synthesis after stirring with 14C-AA revealed no evidence of endoperoxide or thromboxane production, although products of the lipoxygenase pathway were produced in normal amounts. Aggregation in response to AA was completely corrected after mixing with 10% normal C-PRP. However, equal volumes of her C-PRP and normal aspirin-treated C-PRP did not respond to AA, whereas 10% normal platelets combined with aspirin-treated cells corrected aggregation to AA. Since epinephrine pretreatment corrects the response of dog platelets that are not aggregated by AA, we evaluated the influence of epinephrine on her platelets. Preexposure to 5 micro M epinephrine, a concentration that gave only primary waves of aggregation, resulted in normalization of her response to AA, even though correction was not associated with the generation of endoperoxides or thromboxanes. The results may explain why patients with platelet cyclooxygenase deficiency have mild or absent bleeding symptoms.
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PMID:Epinephrine potentiation of arachidonate-induced aggregation of cyclooxygenase-deficient platelets. 733 91

The interactions of alpha-thrombin with platelets are critical in haemostasis and arterial thrombosis. This study established methods for characterizing the binding of alpha-thrombin to platelets and some of its consequences in platelet-rich plasma. The binding of alpha-thrombin to platelets and the subsequent platelet activation were quantified by flow cytometry, using affinity purified polyclonal antibodies to human alpha-thrombin and a monoclonal antibody to GMP-140, respectively. Dose-dependent binding of alpha-thrombin to platelets and their activation occurred in parallel, both reaching the maxima for each enzyme concentration within 10s after > or = 1.0 nM alpha-thrombin was added to recalcified PRP containing 1 microM recombinant tick anticoagulant peptide. The tick anticoagulant peptide abrogated prothrombin activation in the platelet-rich plasma. alpha-Thrombin binding to platelets, and their activation, were abrogated by a monoclonal antibody to the hirudin tail-like domain of the seven transmembrane thrombin receptor on platelets. Therefore this receptor represents an important site for alpha-thrombin binding to platelets suspended in plasma. D-Phe-Pro-ArgCH2-alpha-thrombin only bound to platelets when its concentration was > or = 100 nM, and it did so without inhibiting platelet activation by alpha-thrombin. Whereas concentrations of hirudin equimolar to those of alpha-thrombin failed to abrogate alpha-thrombin-mediated activation of platelets, a 10-fold molar excesses of hirudin over alpha-thrombin abrogated alpha-thrombin binding to platelets. The demonstration that > or = 1.0 nM alpha-thrombin can bind to platelets and initiate their activation raises the possibility that the levels of thrombin generated in venous and arterial thrombosis contribute to platelet activation in vivo.
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PMID:Thrombin binding to platelets and their activation in plasma. 752 34

Two high molecular mass proteins, flavocetin-A and flavocetin-B, were purified from Trimeresurus flavoviridis venom. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the apparent molecular mass of flavocetin-A and -B were 149 and 139 kDa, respectively, under nonreducing conditions. On reduction, flavocetin-A showed two distinct subunits (17 and 14 kDa), and flavocetin-B three distinct subunits (17, 15 and 14 kDa). At 1 microgram/ml, flavocetin-A and -B (flavocetins) inhibited the von Willebrand factor (vWF)-dependent aggregation of fixed human platelets. However, flavocetins (10 micrograms/ml) had no effect on ADP- and collagen-induced platelet aggregation in PRP. Flavocetins (3 micrograms/ml) also inhibited shear-induced platelet aggregation at high shear stress. Furthermore, flavocetin-A completely inhibited the aggregation of and ATP release from washed platelets stimulated with a low concentration of thrombin. Flavocetin-A specifically bound to platelet with high affinity (Kd = 0.35 +/- 0.13 nM) at 21,500 +/- 1760 binding sites per platelet. The N-terminal amino acid sequences of the subunits of flavocetin-A show a high degree of homology with those of echicetin, botrocetin, alboaggregin-B and factor IX/factor X-binding protein. These results suggest that flavocetins may be a useful tool for further investigation of the GPIb-vWF interaction.
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PMID:Flavocetin-A and -B, two high molecular mass glycoprotein Ib binding proteins with high affinity purified from Trimeresurus flavoviridis venom, inhibit platelet aggregation at high shear stress. 759 52

The effect of NEM pretreatment on hydrogen peroxide accumulation and aggregation in non stimulated and activated human platelets was studied. It was shown that low concentrations of NEM potentiate hydrogen peroxide formation induced specifically by thrombin. The cooperative effect of NEM with thrombin is dose-dependent and reaches the maximum at 5 microM NEM. Thrombin is particularly effective at low concentrations (0.02-0.1 U). Extracellular Ca2+ significantly increases the NEM effect. Moreover NEM inhibits aggregation both in PRP and in washed platelets, exhibiting the greatest activity in washed platelets stimulated by thrombin.
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PMID:N-ethylmaleimide inhibits platelet aggregation and potentiates hydrogen peroxide formation induced by thrombin. 784 14

The binding energetics of eight synthetic peptides capable of interfering with thrombin function have been studied by steady-state measurements and clotting assays. The synthetic peptides are bifunctional inhibitors consisting of three domains: (i) a fragment of the C-terminus of recombinant hirudin, hir55-65, which binds to the fibrinogen-recognition site of thrombin; (ii) a small active site inhibitor, Ac-(DF)PRP, binding to the catalytic pocket of the enzyme, and (iii) a linker spanning these two portions with variable length and chemical composition. All these synthetic peptides are competitive inhibitors of fibrinogen. On the other hand, a linker of at least 13 carbon atoms is required for full competitive inhibition of the hydrolysis by thrombin of small synthetic substrates, which only bind to the catalytic pocket of the enzyme. The best inhibitory effect is observed with a linker of 13 carbon atoms, with a value of KI in the nanomolar range. Studies conducted as a function of temperature, in the range 15-40 degrees C, have revealed the enthalpic and entropic components of inhibitor binding to thrombin. Chemical compensation is observed for all synthetic peptides that bridge-bind to the fibrinogen-recognition site and the catalytic pocket of the enzyme thereby inhibiting in a competitive fashion either fibrinogen binding or the hydrolysis of small synthetic substrates. The extrathermodynamic relationship between delta H and delta G also includes the enthalpy and free energy of binding for the natural substrate fibrinogen and the potent natural inhibitor hirudin, measured under identical solution conditions. Preferential binding of hirudin over fibrinogen is an entropy-driven process.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Chemical compensation in macromolecular bridge-binding to thrombin. 845 59

N alpha-Acetyl[D-Phe45,Arg47]hirudin45-65 (P53) is a bivalent thrombin inhibitor (Ki = 5.6 nM) that consists of an active site inhibitor segment, [N alpha-acetyl-(dF)PRP]; a fibrinogen recognition exo site inhibitor segment, hirudin55-65 (DFEEIPEEYLQ-OH); and a linker, hirudin49-54 (QSHNDG), connecting these inhibitor segments (DiMaio et al., 1990). The structure-function relationships of the linker were studied using a combination of various omega-amino acids, which modified the length of the linker as well as the number and the locations of peptide bonds. Linkers with 14-18 atoms (counting only the atoms contributing to the length of the linker) showed a competitive inhibition with Ki = 1.7-3.4 nM. The potency of the inhibitors with 12-13-atom linkers was sensitive to the chemical structure of the linker. The high-potency inhibitors showed a competitive inhibition, while the low-potency inhibitors showed a hyperbolic inhibition. Among them, an inhibitor with a 13-atom linker showed the highest potency (Ki = 0.51 nM, an 11-fold improvement from that of P53 above), indicating that this is an optimal linker length. Since linkers with 6-10 atoms failed to bridge the active site and exo site inhibitor segments, a minimum of 11 atoms was required to bridge them, even though the potency of the inhibitor with an 11-atom linker was weak (Ki = 26 nM). Molecular dynamics simulation of the inhibitors with 13-atom linkers suggested that some linkers serve as a functional domain with the amide bond of the linker interacting with thrombin through hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Design of a linker for trivalent thrombin inhibitors: interaction of the main chain of the linker with thrombin. 846 3

The aim of the present study was to evaluate platelet responsiveness in rats following E.coli endotoxin administration. Injection of E.coli to rats caused a reduction in ADP-induced pulmonary 111In labelled platelet accumulation four hours later. Similarly, when platelet aggregation was evaluated on PRP obtained from rats four hours after endotoxin administration, we found that platelet response to both ADP and collagen was significantly reduced. When platelets obtained from endotoxemic rats were suspended in normal plasma, the aggregating response to ADP and collagen was not different from that obtained with control platelets. Similarly, platelets from control rats suspended in plasma from endotoxemic rats showed hyporesponsiveness to ADP and collagen. There was no difference in the aggregatory response to collagen or to thrombin of washed platelet suspension (WPS) obtained from endotoxemic and normal rats. In conclusion, by using an in vivo minimally invasive technique and an ex vivo platelet aggregation test we demonstrate that during endotoxemia platelet are functionally unaltered and the platelet hyporesponsiveness is only observed in presence of plasma.
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PMID:A study on rat platelet responsiveness following intravenous endotoxin administration. 900 Jan 23

Vipera lebetina fibrinogenase (VlF) was shown to render fibrinogen incoagulable and to solubilize fibrin. The fibrinogenolytic activity of this enzyme was found to be 33 mg fibrinogen/min/mg protein. The study of the specificity of this enzyme revealed that it has no effect on purified factor X, prothrombin and protein C and on the specific chromogenic substrates of their active form. Plasminogen was not activated by VlF but slightly degraded. We have also compared the effect of VlF and plasmin on fibrinogen and shown that these two enzymes have a different sites of cleavage. This enzyme inhibited human platelet aggregation on PRP initiated by ADP and collagen but was without effect on the aggregation of washed rabbit platelets using thrombin as agonist. Administration of VlF in rat did not show any necrosis or hemorrhage in treated rats organ's. We therefore, examined the thrombolytic activity of VlF in a rat model of venous thrombosis. Thrombus was produced in the posterior vena cava by injection of human fibrinogen and thrombin. Injection of 5 mg/Kg body weight showed an evident flow restoration after one hour and measurement of the fibrinogen level a decrease of about 30% after 3 hrs. VlF's action is not dependent on plasminogen activators and may act synergistically with them, thereby providing an intriguing potential clinical application for dissolution of blood clots.
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PMID:Further characterization and thrombolytic activity in a rat model of a fibrinogenase from Vipera lebetina venom. 917 44


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