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)

Trypsin, thrombin, and ionophore A23187 activate phospholipid breakdown of platelets that have been labeled with [(14)C]arachidonate, releasing their cyclooxygenase and lipoxygenase products. Intact platelets can also very effectively directly degrade low concentrations of exogenous, free [(14)C]arachidonate. Pretreatment of platelets with trypsin, thrombin, or ionophore A23187 for a minimum time of 30 sec leads to complete inactivation of cyclooxygenase activity, as demonstrated by subsequent exposure to [(14)C]arachidonate. Lipoxygenase activity is lost after 5 min. The thrombin-induced inactivation of cyclooxygenase and lipoxygenase is prevented by cyclic AMP (which inhibits the stimulated activity of phospholipase A(2)), although cyclic AMP does not affect the degradation of exogenous [(14)C]arachidonate. Exposure of platelets labeled with [(14)C]arachidonate to unlabeled arachidonate under conditions that lead to use of the latter also results in a similarly rapid inhibition of cyclooxygenase activity, as determined by subsequent challenge with thrombin. Under these conditions lipoxygenase activity is much less markedly inactivated. The arachidonate-induced inhibition of cyclooxygenase activity is not prevented by cyclic AMP. Trypsin does not induce platelet aggregation, and platelets whose cyclooxygenase activity has been inactivated are intact insofar as they are still able to undergo aggregation. These studies demonstrate that operation in intact platelets of the cyclooxygenase pathway, through use of endogenous or exogenous substrate, leads to a very rapid, irreversible inactivation of this enzyme. The lipoxygenase pathway is also progressively impaired, but much less rapidly than the cyclooxygenase enzyme and much less markedly on use of exogenous compared to endogenous substrate. The possible consequences of these physiological processes of spontaneous inactivation are considered.
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PMID:Rapid inactivation of cyclooxygenase activity after stimulation of intact platelets. 21 91

Platelets enzymatically convert prostaglandin H(3) (PGH(3)) into thromboxane A(3). Both PGH(2) and thromboxane A(2) aggregate human platelet-rich plasma. In contrast, PGH(3) and thromboxane A(3) do not. PGH(3) and thromboxane A(3) increase platelet cyclic AMP in platelet-rich plasma and thereby: (i) inhibit aggregation by other agonists, (ii) block the ADP-induced release reaction, and (iii) suppress platelet phospholipase-A(2) activity or events leading to its activation. PGI(3) (Delta(17)-prostacyclin; synthesized from PGH(3) by blood vessel enzyme) and PGI(2) (prostacyclin) exert similar effects. Both compounds are potent coronary relaxants that also inhibit aggregation in human platelet-rich plasma and increase platelet adenylate cyclase activity. Radioactive eicosapentaenoate and arachidonate are readily and comparably acylated into platelet phospholipids. In addition, stimulation of prelabeled platelets with thrombin releases comparable amounts of eicosapentaenoate and arachidonate, respectively. Although eicosapentaenoic acid is a relatively poor substrate for platelet cyclooxygenase, it appears to have a high binding affinity and thereby inhibits arachidonic acid conversion by platelet cyclooxygenase and lipoxygenase. It is therefore possible that the triene prostaglandins are potential antithrombotic agents because their precursor fatty acids, as well as their transformation products, PGH(3), thromboxane A(3), and PGI(3), are capable of interfering with aggregation of platelets in platelet-rich plasma.
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PMID:Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological properties. 21 23

Thrombin rapidly induces the formation of labeled phosphatidic acid from platelets prelabeled with [17C]arachidonate or 32PO34- and specifically decreases by 50--75% the content of phosphatidylinositol. Ionophore A23187 also stimulates phosphatidate labeling, but less effectively than thrombin. This effect on phosphatidic acid is blocked by increasing the levels of cyclic AMP by preincubation with dibutyryl cyclic AMP, cyclic AMP-phosphodiesterase inhibitors or prostacyclin. Indomethacin and eicosatetraynoic acid do not alter the production of phosphatidate, indicating independence from cyclooxygenase or lipoxygenase products. Increased turnover of [14C]- or [32P]phosphatidate occurs within 2--5 s after platelet activation by thrombin and is observed before endogenous, 14C-labeled arachidonate can be detected. The rate of phosphatidate formation parallels the induced rate of serotonin release. Release of [3H]serotonin is not affected by eicosatetraynoic acid. Phosphatidate production reflects the generation of diacylglycerol by C-type phospholipase degradation of phosphatidylinositol. Diacylglycerol and phosphatidic acid may participate in the membrane modification related to the early changes in platelet shape, release reactions or aggregation which occur on stimulation.
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PMID:Stimulation of phosphatidic acid production in platelets precedes the formation of arachidonate and parallels the release of serotonin. 37 88

Aspirin treatment of cultured endothelial cells from the umbilical vein increased the adherence of 51Cr-platelets when thrombin was present. If the cyclooxygenase activity of endothelium was inhibited by aspirin, as it is in the platelet, reduction of endogenous prostacyclin (PGI2) production could have been responsible. By correlating thrombin-induced adherence of platelets to endothelial monolayers with PGI2 release (as measured by radioimmunoassay for 6-keto-prostaglandin FI1 alpha [6-keto-PGF1 alpha]), we have demonstrated an inverse relationship between platelet adherence and PGI2 levels. Untreated endothelial monolayers exposed to thrombin and platelets resulted in 4% platelet adherence and 107 nM 6-keto-PGF1 alpha. With 0.1 mM aspirin treatment, which is known to block platelet cyclooxygenase, adherence was 5% and 6-keto-PGF1 alpha decreased to 45 nM. Increasing the aspirin concentration to 1 mM resulted in 44% adherence and less than 3 nM 6-keto-PGF1 alpha. When 25 nM exogenous PGI2 was added to 1 mM aspirin-treated endothelium, adherence returned to 5%. The increase in thrombin-induced platelet adherence to 1 mM aspirin-treated monolayers was reversed 2 h after removal of the aspirin solution. 6-Keto-PGF1 alpha returned to 37% of the untreated monolayer value. Recovery from the aspirin effect did not occur when cycloheximide, an inhibitor of protein synthesis, was present during the 2-h period.
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PMID:Inhibition of prostacyclin by treatment of endothelium with aspirin. Correlation with platelet adherence. 37 48

Human platelets were separated into 2 density populations by repeated centrifugations of platelet-rich plasma at increasing gravitational force. The heaviest platelet fraction was rich in larger platelets. The lightest platelet fraction was rich in smaller platelets. In both fractions and in the platelet button, lipid peroxidation (malonaldehyde-MDA-production after addition of thrombin) was measured at basal condition, on the 1st, 3rd, 5th, 7th and 9th day after aspirin ingestion. At basal conditions and after ingestion of aspirin, MDA production was higher in the heavy-large platelets than in light-small ones, but a parallel increase of MDA production was observed in the light and in the heavy population and in the platelet button. The data are not compatible with the hypothesis that platelet density and size are age-related. Aspirin inhibits platelet lipid peroxidation by permanently acetylating their cyclooxygenase and if the heaviest platelets were the young ones, lipid peroxidation should reappear sooner in them.
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PMID:Platelet heterogeneity. Relationship between buoyant density, size, lipid peroxidation and platelet age. 50 68

All agents capable of triggering the platelet release reaction also stimulate prostaglandin biosynthesis in these cells. Information concerning the endoperoxides, thromboxanes, and more stable metabolites generated by the action of cyclooxygenase and lipoxygenase on arachidonic acid has accumulated rapidly, but little is known about the preliminary steps in the cleavage and preparation of arachidonic acid for insertion into the enzymatic pathways of prostaglandin synthesis. Studies in this laboratory have shown that the combination of nitroblue tetrazolium (NBT) and vitamin E which prevents oxygenation of arachidonic acid to a free radical also blocks platelet prostaglandin biosynthesis. The present study has evaluated the influence of NBT, vitamin E, and the combination of NBT and vitamin E on the fine structure and biochemistry of platelets during incubation, and the effects of these compounds on the aggregation and secretion of platelets stimulated by collagen, thrombin, epinephrine, and ADP. Results of the study demonstrate that NBT and vitamin E, rather than injuring platelets, appear to protect them during incubation. Together NBT and vitamin E blocked aggregation by epinephrine, collagen, and thrombin, but permitted a small first wave stimulated by ADP. Both ADP and thrombin induced shape change, pseudopod formation, and limited degrees of internal contraction in vitamin E-NBT-treated platelets, whereas epinephrine and collagen failed to significantly alter discoid form. This pattern of response to aggregating agents was identical to reactions observed in platelets pretreated with aspirin and indomethacin, both potent inhibitors of platelet prostaglandin synthesis. In addition, NBT-vitamin E virtually blocked the first wave of aggregation which is not affected by aspirin and indomethacin. The findings support the concept that conversion of arachidonic acid to an activated state is an important step in prostaglandin synthesis and that electron transfer or oxidation-reduction reactions are intimately involved in the development of platelet stickiness.
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PMID:Effects of nitroblue tetrazolium and vitamin E on platelet ultrastructure, aggregation, and secretion. 87 76

We wished to determine whether the metabolism of arachidonic acid, through lipoxygenase and cytochrome P-450 pathways, is involved in production of endothelium-derived relaxing factor(s) (EDRFs) in canine femoral veins. Veins were removed from anesthetized dogs and cut into rings. Endothelium was deliberately removed from some rings. In separate sets of experiments, rings were incubated with either AA861 (10(-5) M) or TMK777 (10(-6) M), inhibitors of 5-lipoxygenase, nordihydroguaiaretic acid (NDGA 3 x 10(-6) M), an inhibitor of lipoxygenase or proadifen (SKF 525A, 10(-6) M), an inhibitor of cytochrome P-450. In addition, some rings were incubated with a combination of indomethacin (10(-5) M) and NG-monomethyl-L-arginine (L-NMMA 10(-4) M) or, where appropriate, a solvent control. Concentration-response curves were obtained for acetylcholine, adenosine diphosphate, thrombin, A23187, and nitric oxide in rings contracted with a submaximal concentration of prostaglandin F2 alpha. AA861 and TMK777 did not alter endothelium-dependent relaxations to the agonists, whether with or without indomethacin and L-NMMA. However, indomethacin plus L-NMMA reduced endothelium-dependent relaxations to thrombin. These results suggest that metabolism of arachidonic acid, through lipoxygenase and cytochrome P-450 pathways, does not produce an EDRF in veins. However, thrombin receptor-activated relaxations are mediated in part by products of the cyclooxygenase pathway and nitric oxide.
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PMID:Role of lipoxygenase and cytochrome P-450 in production of endothelium-derived relaxing factors in canine femoral veins. 127 84

We examined the effect of human recombinant granulocyte-colony stimulating factor (G-CSF) on the release of immunoreactive endothelin-1 (ET-1) from cultured bovine vascular endothelial cells. G-CSF dose dependently (10(-8)-10(-6) M) increased the release of immunoreactive ET-1 as a function of time under a serum-free condition. Coaddition of G-CSF and thrombin induced an additive effect on immunoreactive ET-1 release. Neither Ca2+ channel antagonist nor cyclooxygenase inhibitor affected immunoreactive ET-1 release stimulated by G-CSF. These results suggest that G-CSF, in addition to its effect on granulocyte progenitors, has a direct effect on vascular endothelium to induce the release of immunoreactive ET-1.
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PMID:Granulocyte-colony stimulating factor stimulates immunoreactive endothelin-1 release from cultured bovine endothelial cells. 128 2

Eicosanoid biosynthesis was examined with a human megakaryocytic cell line (Dami). Megakaryocytes incubated with [1-14C]arachidonic acid and either ionophore A23187 or thrombin generated both thromboxane and 12-hydroxyheptadecatrienoic acid (HHTrE). Exposure to phorbol myristate acetate (PMA) for 1 through 9 days induced differentiation and revealed an increase in the conversion of [1-14C]arachidonate to cyclooxygenase- and lipoxygenase (LO)-derived products. The LO-derived product was identified as 12S-HETE by its physical characteristics including GC/MS and chiral column SP-HPLC. PMA-treated Dami cells did not generate 5-HETE, leukotrienes or lipoxins from exogenous arachidonic acid while they did convert leukotriene A4 (LTA4) to lipoxin A4, lipoxin B4 and their respective all-trans isomers. In addition, COS-M6 cells transfected with a human 12-lipoxygenase cDNA and incubated with either arachidonic acid or LTA4 generated 12-HETE and lipoxins, respectively. The lipoxin profile generated by transfected COS-M6 cells incubated with LTA4 was similar to that generated by the PMA-treated Dami cells. Results indicate that human megakaryocytes can transform arachidonate and LTA4 to bioactive eicosanoids and that the 12-lipoxygenase appears upon further differentiation of these cells. In addition, they indicate that the 12-LO of human megakaryocytes and the 12-LO expressed by transfected COS cells can generate both lipoxins A4 and B4. Together they suggest that the human 12-LO can serve as a model of LX-synthetase activity with LTA4.
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PMID:Lipoxin generation by human megakaryocyte-induced 12-lipoxygenase. 131 55

The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Platelets do not adhere to blood-borne microfilariae, and thrombo-occlusive phenomena are not observed in patients with circulating microfilariae. We studied the ability of microfilariae to inhibit human platelet aggregation in vitro. Brugia malayi microfilariae incubated with human platelets caused dose-dependent inhibition of agonist-induced platelet aggregation, thromboxane generation, and serotonin release. As few as one microfilaria per 10(4) platelets completely inhibited aggregation of platelets induced by thrombin, collagen, arachidonic acid, or ionophore A23187. Microfilariae also inhibited aggregation of platelets in platelet-rich plasma stimulated by ADP, compound U46619, or platelet-activating factor. The inhibition required intimate proximity but not direct contact between parasites and platelets, and was mediated by parasite-derived soluble factors of low (less than 1,000 Mr) molecular weight that were labile in aqueous media and caused an elevation of platelet cAMP. Prior treatment of microfilariae with pharmacologic inhibitors of cyclooxygenase decreased both parasite release of prostacyclin and PGE2 and microfilarial inhibition of platelet aggregation. These results indicate that microfilariae inhibit platelet aggregation, via mechanisms that may include the elaboration of anti-aggregatory eicosanoids.
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PMID:Intravascular filarial parasites inhibit platelet aggregation. Role of parasite-derived prostanoids. 131 45


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