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 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

Previous studies have reported that polyunsaturated fatty acids (PUFAs) of nutritional interest may influence arachidonic acid (20:4n-6) metabolism in both platelets and endothelium, when tested separately. In the present study, platelets (PL) and cultured endothelial cells (EC) were first pre-enriched with eight different PUFAs for a two hour incubation in the presence of free fatty acid albumin pre-coated with each acid. EC, PL or both cell populations in combination, were then stimulated by thrombin (0.1 U/ml) for five minutes. Prostanoids were extracted, purified by thin-layer chromatography, and TxB2, 6-keto-PGF1 alpha and PGE2 were quantitated by radioimmunoassays. Prostanoids or dihomoprostanoids formed from cyclooxygenase substrates other than 20:4n-6 were measured by gas chromatography-negative chemical ionisation mass-spectrometry (GC-MS). When co-incubated with EC, PL produced less TxB2 (-15 and -85% in the absence and presence of thrombin, respectively). In contrast, 6-keto-PGF1 alpha increased by 189 (basal conditions) and 358% (thrombin stimulation) when PL were added to EC, in agreement with PGH2 transfers from PL to EC. PGE2, produced by both cell populations, reached amounts which roughly represent the sum of those measured in PL and EC alone, except when cells were pre-enriched with linoleic (18:2n-6) and the n-3 family fatty acids (18:3-, 20:5- and 22:6n-3). 6-keto-PGF1 alpha was markedly inhibited by adrenic acid (22:4n-6), while this acid was converted into dihomo-6-keto-PGF1 alpha, the stable metabolite of dihomoprostacyclin. 22:4n-6 also inhibited TxB2 formation and was converted into dihomo-TxA2.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Modulation of prostanoid formation by various polyunsaturated fatty acids during platelet-endothelial cell interactions. 211 Jun 76

Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC or HUVEC with 100 nM PMA for 5-10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5-fold. Pretreatment with 100 nM PMA for 5 min also caused a 2-fold enhancement of thrombin-stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2 alpha, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin-stimulated (1 U/ml) PAF production was enhanced 2.6-fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H-7 and staurosporine ablated the enhancing effect of PMA on thrombin-stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.
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PMID:Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester. 211 37

The effects of newly discovered vasoconstrictor peptide endothelin was studied on human, rabbit and canine platelet function. Endothelin (0.01 nM-1 microM) did not promote platelet aggregation. In human platelets, endothelin (0.1 microM) did not significantly affect aggregation responses to ADP, collagen, epinephrine, arachidonic acid, PGH2 or thrombin. Endothelin did not promote the mobilization of intracellular calcium in Fura2 loaded human platelets. In rabbit and canine platelets endothelin produced significant potentiation of platelet aggregation mediated by low concentrations of ADP. Aggregation responses to higher concentration of ADP (5 microM) were unaffected by endothelin. These data reveal that under certain circumstances endothelin may potentiate rabbit and canine platelet aggregation responses to ADP, however endothelin does not produce direct effects on human platelet function.
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PMID:Endothelin and platelet function. 211 86

Number and affinity of thromboxane A2/PGH2 receptors on gel-filtered human platelets was determined using the receptor antagonist 3H-SQ 29548 as a tracer compound. Preincubation of platelet rich plasma (PRP) with U 46619 reduced receptor number to 31% of control platelets without affecting the affinity (2 x 10(-9) mol/l). Pretreated platelets lost their reactivity to stimulation by U 46619 but not to thrombin within 3 h. It is concluded that preincubation of platelets leads to a homologeous platelet desensitisation and down regulation of thromboxane receptors.
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PMID:Desensitisation of human platelets against stimulation by thromboxane mimics--reduction in receptor capacity and loss of functional response. 247 Mar 62

Pathways of arachidonic acid metabolism were identified in freshly prepared and in cultured bovine corneal endothelial cells. The principal pathway of arachidonic acid metabolism in the bovine corneal endothelial cells appears to be the cyclooxygenase pathway with the resultant synthesis of PGI2, PGF2 alpha and PGE2. At least two of these products, PGI2 and PGF2 alpha, are formed by the enzymatic conversion of the substrate, PGH2. Measurements of endogenous prostaglandin production by radioimmunoassay demonstrated that PGE2 was the major arachidonic acid metabolite released, with smaller amounts of PGF2 alpha and the stable hydrolysis product of PGI2, 6-keto PGF1 alpha. The release of all three prostanoids was significantly increased by the addition of the calcium ionophore (A23187), human thrombin, bradykinin and histamine. Basal and stimulated release of prostaglandins by the corneal endothelium may contribute to the regulation of intraocular pressure and also in the modulation of the corneal response to injury.
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PMID:Arachidonic acid metabolism by cultured bovine corneal endothelial cells. 249 58

We examined the effects of various cytokines on alpha-thrombin-stimulated prostaglandin (PG) I2 production, von Willebrand factor (vWF) secretion, and platelet-activating factor (PAF) synthesis in cultured human umbilical vein endothelial cells (HUVEC). A 24-h pretreatment with IL-1 beta doubled the low level of constitutive PGI2 production. In contrast, alpha-thrombin increased PGI2 production fivefold in untreated HUVEC. The most striking increase in PGI2 production was observed in IL-1 beta-treated HUVEC that were subsequently stimulated with thrombin. PGI2 production was two to three times greater than in untreated, thrombin-stimulated HUVEC and nearly eightfold greater than in IL-1 beta-treated but unstimulated HUVEC. Enhanced thrombin-stimulated PGI2 production was also observed in HUVEC pretreated with the related cytokines IL-1 alpha, TNF, or lymphotoxin. This cytokine effect was selective for PGI2 production because none of these cytokines altered either constitutive or thrombin-stimulated vWF secretion or PAF biosynthesis. IL-1 beta enhancement of thrombin-stimulated PGI2 production was concentration and time dependent and required protein synthesis. IL-1 beta pretreatment also enhanced PGI2 production in response to another agonist, histamine, and to exogenously added substrates, arachidonic acid or PGH2. Our results indicate that activation by IL-1 and related cytokines selectively primes endothelial cells for enhanced PGI2 production, but not vWF secretion or PAF synthesis, in response to thrombin and histamine. The evidence suggests that this effect is mediated through specific induction of biosynthetic enzymes for PGI2.
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PMID:IL-1 and related cytokines enhance thrombin-stimulated PGI2 production in cultured endothelial cells without affecting thrombin-stimulated von Willebrand factor secretion or platelet-activating factor biosynthesis. 249 85

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

Vascular endothelial cells (EC) play an active role in the synthesis and assembly of components of the fibrinolytic system and the generation of the major fibrinolytic protease plasmin. However, the reciprocal effects of plasmin on EC function have not been previously examined. We have studied the actions of plasmin on the production of prostacyclin (PGI2) by cultured human umbilical vein (HUVEC) and bovine aortic (BAEC) endothelial cells. Plasmin causes little or no direct stimulation of PGI2 formation by EC. Preincubation of EC with plasmin, however, produces a time- and concentration-dependent inhibition of ionophore A23187-, thrombin-, and histamine-induced PGI2 synthesis; a smaller inhibitory effect on arachidonate- and PGH2-induced PGI2 synthesis is found. Incubation of HUVEC or BAEC with a physiologic concentration of plasminogen (180 micrograms/mL) and recombinant tissue plasminogen activator (tPA) generates tPA dose-dependent plasmin activity that exceeds that generated in the absence of EC. In the presence of plasminogen, tPA also causes a tPA dose-dependent inhibition of thrombin- and ionophore A23187-stimulated PGI2 production. PGI2 inhibitory plasmin activity is generated within the concentration range of tPA achieved in plasma during pharmacologic therapy with tPA. These findings suggest that vascular endothelial cells not only regulate activation of the fibrinolytic system but may also be targets of plasmin action on PGI2 synthesis in the modulation of hemostasis and thrombosis.
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PMID:Inhibition of vascular endothelial cell prostacyclin synthesis by plasmin. 252 69

The inhibition of human platelet aggregation produced by PGF2 alpha is not specific for thromboxane A2 mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF2 alpha (8 microM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH2). In contrast the thromboxane receptor antagonist EP 045 (up to 20 microM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC50 = 0.5 microM), but not PGF2 alpha (28 microM), displaces the specific binding of [3H] 9,11-epoxymethano PGH2 to washed human platelets. PGF2 alpha produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF2 alpha is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF2 alpha. We suggest that the very weak effect of PGF2 alpha on cyclic AMP production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.
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PMID:Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2 alpha. 298 22


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