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)

Imidazole and compound L8027 (selective inhibitors of thromboxane synthase) produced parallel inhibition of malonaldehyde and thromboxane B2 secretion induced by collagen or thrombin in gel-filtered suspensions of human platelets. Comparing the effects of these inhibitors and aspirin on secretion of granule constituents indicated that platelet degranulation depends mainly on thromboxane production; prostaglandin endoperoxides contributed little.
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PMID:Malonaldehyde formation in intact platelets is catalysed by thromboxane synthase. 74 61

Platelets participate in formation of thrombin through secretion of coagulation factors and by providing a catalytic surface on which prothrombinase complex is assembled. We studied the effects of four antiplatelet drugs on thrombin formation in healthy volunteers. Thrombin generation was monitored both in vitro--in recalcified plasma--and ex vivo--in blood emerging from a standardized skin microvasculature injury, which also served to determine bleeding time. A mathematical model has been developed to describe the latter reaction. It is based on estimation of the rate of increase in fibrinopeptide A (FPA), a specific marker of thrombin activity, in blood emerging from skin incisions. Two hours after the ingestion of 500 mg of aspirin, thrombin formation became significantly impaired both in vitro and ex vivo. In contrast, 2 hours after the oral administration of placebo, indomethacin 50 mg, or OKY-046 (a thromboxane synthase inhibitor) 400 mg, thrombinogenesis remained unaltered. Ticlopidine, studied either 3 hours after 500 mg oral administration, or after 5 days of intake at a daily dose of 500 mg, had no effect on thrombin generation. Thus, aspirin, contrary to other antiplatelet drugs, depresses thrombin formation in clotting blood, a phenomenon that might be of clinical relevance. It is suggested that aspirin exerts this effect by acetylating prothrombin and/or macromolecules of platelet membrane.
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PMID:Antiplatelet drugs and generation of thrombin in clotting blood. 139 58

A technically simple model of arterial thrombosis in the rat, induced by a crush injury to the dorsal aorta is described. The mechanical injury to the artery caused deep medial injury and the formation of a platelet-rich thrombus with associated fibrin formation which was assessed both radiometrically and morphometrically. No significant inclusion of erythrocytes was noted in the thrombus. Administration of the platelet inhibitors aspirin, BM 13505 (a thromboxane receptor antagonist) or CGS 12970 (a thromboxane synthase inhibitor) reduced the extent of platelet deposition on the injured vessel, but no decrease in fibrin(ogen) was observed. In contrast, infusion of prostacyclin resulted in reductions in both these components of the thrombus. In studies involving inhibition of thrombin activity, the direct thrombin inhibitor CGP 39393 (recombinant desulphatohirudin) inhibited both the platelet and fibrin(ogen) deposition. The indirect thrombin inhibitors were less effective; unfractionated heparin and low-molecular-weight heparin inhibited both platelet and fibrin(ogen) deposition but only at doses which rendered the blood uncoagulable, as evaluated by the activated partial thromboplastin time. Dermatan sulphate only inhibited platelet deposition. The results suggest that thrombin plays a key role in the initiation of thrombus formation in this experimental model. The agonist prostaglandins (PGG2, PGH2, and TXA2) would appear to have a supporting role in the platelet deposition onto the thrombotic surface but do not have a role to play with respect to fibrin(ogen) deposition.
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PMID:A non-occlusive model of arterial thrombus formation in the rat and its modification by inhibitors of platelet function, or thrombin activity. 160 87

Human erythroleukemia cells transformed arachidonic acid and prostaglandin endoperoxide H2 into thromboxane A2. Stimulation of these cells with A23187 or thrombin, however, produced no thromboxane. Similarly, cells labeled with [3H]-arachidonic acid released no detectable label upon stimulation. Data suggest that human erythroleukemia cells contain the enzymatic capacity for thromboxane formation from exogenous precursors, but lack the endogenous mechanisms for arachidonate release. The presence of thromboxane synthase messenger RNA was verified using the polymerase chain reaction. Amplification and sequence analysis of a 528 bp cDNA demonstrated virtually 100% identity to a published thromboxane synthase cDNA fragment.
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PMID:Thromboxane A2 synthesis in human erythroleukemia cells. 193 Feb 41

The covalent modification of proteins by metabolites of arachidonic acid (AA) was investigated in human platelets. Following incubation of washed human platelets with radiolabeled AA, ethanol precipitation of the proteins, and lipid extraction by organic solvents, a small fraction of the radioactivity added (0.3%) was tightly bound to the protein pellet. A dozen labeled protein bands were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Exhaustive hydrolysis of platelet proteins by proteases released an amphipathic radiolabeled material which had a chromatographic behavior similar to that of a known peptidolipid, leukotriene C4. These findings suggest a covalent nature for the observed binding. This binding was specific for AA since palmitate, myristate, or linoleate did not bind to a significant extent. It involved products of both cyclooxygenase and lipoxygenase pathways: it was indeed inhibited to a greater extent by eicosatetraynoic acid than by indomethacin. The protein-associated radioactivity was increased by the thromboxane synthase inhibitor dazoxiben. Indomethacin completely abolished this increase in binding, which could not be reproduced by exogenous prostaglandin (PG) E2, F2 alpha, or D2, and might thus involve PGG2 and/or PGH2. Diamide, an agent known to inhibit the reduction of 12-hydroperoxyeicosatetraenoic acid in platelets, produced an increase of the covalent binding, which was abolished by eicosatetraynoic acid but not by indomethacin: this suggests that the lipoxygenase product bound was 12-hydroperoxyeicosatetraenoic acid or a by-product. Dazoxiben and diamide produced distinct patterns of protein labeling after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One labeled band had a Mr of 70,000 as the PGH synthase monomer. Addition of AA at 17 microM enhanced the labeling of this band, while 100 microM was inhibitory. Labeling of this band was also induced by thrombin in prelabeled platelets. Two monoclonal antibodies against PGH synthase caused immune precipitation of a 70-kDa labeled protein in homogenates of [3H]AA-labeled platelets. PGH synthase, purified from ram seminal vesicles, was covalently modified after incubation with [3H]AA: this labeling was almost completely abolished by indomethacin. As much as 40% of platelet PGH synthase was covalently modified after incubation with 17 microM AA. It can be concluded that in intact platelets PGH synthase is covalently modified by an eicosanoid following incubation with exogenous AA or after AA mobilization from phospholipids by thrombin.
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PMID:Covalent binding of arachidonic acid metabolites to human platelet proteins. Identification of prostaglandin H synthase as one of the modified substrates. 210 68

Hydrogen peroxide (H2O2) and methyl mercury induced the liberation of arachidonate and its metabolites from human washed platelets. [14C]Eicosanoids were extracted from the supernatants of [14C] arachidonate-prelabelled platelets and analysed by thin layer chromatography and radioscanning. Thromboxane B2 (TXB2), 12(S)-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) were found as stable metabolites, together with unreacted arachidonate. In the presence of dazoxiben, a shift in eicosanoid metabolism was observed towards prostaglandin E2 (PGE2), prostaglandin D2 (PGD2) and prostaglandin F2 alpha (PGF2 alpha), while in the presence of indomethacin there was a shift towards 12-HETE and unmetabolized arachidonate. The concentration pattern of those metabolites resembled that found with the physiological agonist, thrombin. H2O2 and methyl mercury also induced platelet shape change, aggregation and secretion. The EC50 values for the induction of shape change and aggregation were 27 and 850 mumol/l for H2O2 and 0.33 and 2.7 mumol/l for methyl mercury, respectively. The [3H]serotonin release required higher stimulus concentrations and amounted to 45% with 2 mumol/l H2O2 and to 16% with 3 mumol/l methyl mercury. These effects on platelet function were absent in platelets exposed to acetylsalicylic acid and prevented by indomethacin, the prostaglandin H2 (PGH2)/thromboxane A2 (TXA2) receptor antagonist, daltroban, and the functional antagonist, iloprost. In contrast, none of these drugs suppressed the formation of [14C]eicosanoids, indicating that the platelet activation by H2O2 and methyl mercury essentially requires previous PGH2/TXA2 formation. As expected, the thromboxane synthase inhibitor, dazoxiben, did not prevent, but instead potentiated the activation by H2O2 and methyl mercury through accumulated PGH2.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hydrogen peroxide and methyl mercury are primary stimuli of eicosanoid release in human platelets. 248 9

Human platelets were prelabelled with [14C]arachidonate and stimulated with thrombin or methyl mercury. [14C]PGH2 and the more stable of the other [14C]eicosanoids formed were rapidly extracted with organic solvent cooled to -30 degrees C and analyzed by radio-TLC. TXB2 and PGH2 were also quantified by radioimmunoassay, the latter as its index metabolite PGE2. PGH2 reached its peak concentration of 12 nmol/l after 20-30 s when it amounted to approximately 2/3 of the TXB2 concentration. In the presence of the thromboxane synthase inhibitor dazoxiben, PGH2 peaked after 60 s and afterwards declined in favour of PGE2, PGD2 and PGF2 alpha. Thirty seconds after stimulation with thrombin 1 IU/ml or methyl mercury 20 mumol/l, PGH2 amounted to 35 or 28% of the cyclooxygenase products in the absence and to 66 or 63% in the presence of dazoxiben, respectively. The platelet-activating potency of PGH2 was evaluated with purified PGH2 in platelets pretreated with acetylsalicylic acid. The EC50 values of PGH2 were 0.69 and 19 nmol/l for shape change and aggregation, respectively. U 46619 produced the same effects at 4.1 and 23 nmol/l. PGH2-induced [3H]serotonin release did not exceed 25%, whereas U 46619 was able to induce approximately 50% [3H]serotonin release. Dazoxiben enhanced the aggregation induced by PGH2. Human serum albumin inhibited the aggregating effect of PGH2, suppressed the enhancing effect of dazoxiben and shifted the metabolism of PGH2 to the inhibitory PGD2. The TXA2/PGH2 receptor antagonist daltroban suppressed the agonistic effects of endogenous or added PGH2, demonstrating that the TXA2/PGH2 receptor was its site of action.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transient concentrations and agonist potency of PGH2 in platelet activation by endogenous arachidonate. 251 34

The arachidonic acid metabolites produced by human peripheral blood monocytes were studied to determine which metabolites could have a role in thrombogenesis. Monocytes were found to be free of platelets by scanning electron microscopy and by measurement of 12-HETE. Human peripheral blood monocytes produce thromboxane as their major metabolite. Thromboxane levels reached a plateau at 12-16 hours of culture. Monocytes produced relatively little prostaglandin E2 or F2. In contrast to our control platelet preparation, neither A23187 (1-10 microM) nor exogenous arachidonic acid (0-40 microM) caused an increase in monocyte thromboxane B2. On the other hand, lipopolysaccharide (20 micrograms per ml), collagen (2.5 mg per 10(7) cells), and thrombin (5-10 units per ml) caused a two- to fivefold increase in monocyte thromboxane B2 in most donors but had no effect on prostaglandin F1 alpha levels. Blockage of thromboxane synthase by 1-benzylimidazole abolished thromboxane B2 production but did not increase prostaglandin F1 alpha. Finally, aspirin-treated platelets from a volunteer donor, which were refractory to 30 microM arachidonate, could be aggregated by isolated blood monocytes. Our data indicate that monocytes are capable of producing thromboxane in large amounts. The regulation of this increase, however, appears to be quite different from platelets. We postulate that monocytes may have a role in hemostasis by virtue of their ability to adhere at sites of vascular injury and release thromboxane, which may enhance platelet aggregation and thrombus formation.
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PMID:Arachidonic acid metabolites produced by platelet-depleted human blood monocytes: a possible role in thrombogenesis. 274 10

It has been proposed that thromboxane synthase inhibition (TXSI) may be a useful form of anti-thrombotic therapy and that this is due, in part, to redirection of PGH2 metabolism in favour of PGI2, a potent vasodilator and anti-platelet agent. While redirection has been observed ex vivo there are conflicting reports of its occurrence in vivo. We now describe the characterisation of an acute intravenous challenge model using thrombin, collagen, arachidonic acid (AA) and PGH2 for the study of PGH2 metabolism. Following challenge, plasma concentrations of TXB2, 6-oxo-PGF1 alpha, alleged metabolites of PGI2 (PGI2m) and PGE2 were measured by radioimmunoassay (RIA). Thrombin and collagen challenge resulted in a dose-related increase in plasma TXB2 while AA and PGH2, in addition, elevated 6-oxo-PGF1 alpha and PGI2m. Injection of PGH2 elevated 6-oxo-PGF1 alpha, PGI2m, TXB2 and PGE2 levels. Experimental conditions were defined such that challenge with thrombin (40 NIH units kg-1), collagen (100 micrograms kg-1), AA (1 mg kg-1) and PGH2 (5 micrograms kg-1) and measurement of eicosanoids 0.5 min following challenge were optimal for detection of redirection of PGH2 metabolism in vivo. The identity of immunoreactive TXB2 and 6-oxo-PGF1 alpha was further supported by experiments in which the extracted immunoreactive eicosanoids co-eluted with authentic [3H]standards when subject to reverse phase high performance liquid chromatography (RPHPLC). Evidence is also presented that the levels of plasma eicosanoids measured in this model reflect in vivo biosynthesis.
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PMID:Thromboxane synthase inhibition: implications for prostaglandin endoperoxide metabolism. I. Characterisation of an acute intravenous challenge model to measure prostaglandin endoperoxide metabolism. 308 69

The thrombolytic efficacy of recombinant tissue-type plasminogen activator (tPA) in the presence and absence of a thromboxane synthase inhibitor was studied in a model of femoral artery thrombosis in the anesthetized rabbit. The thrombus was formed by injection of thrombin and whole blood into an isolated segment of the femoral artery. After 30 min of stable thrombotic occlusion of the femoral artery, sodium heparin (300 U/kg, i.v.) was administered and tPA was infused locally to the site of the thrombus for 30 min at 0.01, 0.10 or 1.0 microgram/kg/min. In other experiments, CGS 13080, a selective thromboxane synthase inhibitor, was administered at a dose of 2 mg/kg i.v., 5 min before tPA was infused and at the end of the 30 min tPA infusion. Pretreatment with CGS 13080 resulted in a shorter time to tPA-induced reperfusion, greater incidence of reperfusion and increased the magnitude of femoral artery blood flow achieved after effective thrombolysis. Furthermore, pretreatment with CGS 13080 resulted in a greater than 10-fold enhancement in the effective dose of tPA. These data indicate that thromboxane synthase inhibition may be beneficial as an adjunct to thrombolytic therapy with tPA.
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PMID:Effect of thromboxane synthase inhibition on the thrombolytic action of tissue-type plasminogen activator in a rabbit model of peripheral arterial thrombosis. 314 20


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