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)

We have utilized HPLC to develop optimal conditions for assaying the transformation of arachidonic acid in thrombin-treated human platelets. In the presence of increasing amounts of albumin, the total amount of radioactivity released from thrombin-treated platelets pre-labeled with 3H-arachidonic acid is first enhanced and then inhibited. Maximal release, reflecting primarily enhanced amounts of free labeled arachidonic acid, occurs at a final albumin concentration of 0.5 mg/ml. Calcium promoted the release of all radiolabeled metabolites, but it specifically enhanced HETE formation and release. Magnesium was without effect. Cyclo-oxygenase derived products constituted the bulk of released label at short time intervals, but after ten minutes exposure to thrombin in the presence of albumin (0.5 mg/ml) and 3 mM calcium, radioactivity in the released products was equally distributed among cyclo-oxygenase derived products (TXB2 + PGD2 + HHT), HETE and free arachidonic acid.
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PMID:The use of high pressure liquid chromatography (HPLC) for the separation of radiolabeled arachidonic acid and its metabolites produced by thrombin-treated human platelets. II. Establishment of optimal assay conditions. 11 87

This study investigates the effect of platelet/neutrophil interactions on eicosanoid production. Human platelets and polymorphonuclear leukocytes (PMNs) were stimulated alone and in combination, with calcium ionophore A23187 and the resulting eicosanoids 12S-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), 12S-heptadecatrienoic acid (HHT), 5S,12R-dihydroxy-(6Z,8E,10E,14Z)-eicosatetraenoi c acid (LTB4) and 5S-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) were measured by HPLC. The addition of PMNs to platelet suspensions caused a 104% increase in 12-HETE, a product of 12-lipoxygenase activity, but had only a modest effect on the cyclooxygenase product HHT (increase of 18%). By using PMNs labelled with [14C]arachidonic acid it was shown that the increases in these platelet eicosanoids could be accounted for by translocation of released arachidonic acid from PMNs to platelets and its subsequent metabolism. The observation that 12-lipoxygenase was about five times more efficient than cyclooxygenase at utilising exogenous arachidonic acid during the platelet/PMN interactions was confirmed in experiments in which platelets were stimulated with A23187 in the presence of [14C]arachidonic acid. Stimulations of platelets with thrombin in the presence of PMNs resulted in a decrease in 12-HETE and HHT levels of 40% and 26%, respectively. The presence of platelets caused a small increase in neutrophil LTB4 output but resulted in a decrease in 5-HETE production of 43% during stimulation with A23187. This study demonstrates complex biochemical interactions between platelets and PMNs during eicosanoid production and provides evidence of a mechanism to explain the large enhancement in 12-HETE production.
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PMID:Enhancement of platelet 12-HETE production in the presence of polymorphonuclear leukocytes during calcium ionophore stimulation. 173 56

Stimulation of platelets induces a rapid release of arachidonate from specific phospholipids and subsequent remodeling of arachidonate-containing phospholipids. This process is accompanied by transformation of released arachidonate by cyclooxygenase and lipoxygenase enzymes. We addressed the question of whether the cyclooxygenase and the lipoxygenase products originated from the same arachidonate-containing phospholipids. [14C]Arachidonate prelabeled platelets were stimulated by thrombin or by ionophore A 23187. We monitored the cyclooxygenase pathway by following 12-hydroxy-5,8,10-heptadecatrienoic acid [12(S)-HHT] formation and the lipoxygenase pathway by following 12-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE] formation and compared specific activities. The data showed that the same pool of released arachidonate can be utilized by either cyclooxygenase or by lipoxygenase. Indeed, the specific activity of both products was identical when both enzymes were acting. Since cyclooxygenase was rapidly deactivated while lipoxygenase continued to be active, the specific activity of 12(S)-HETE became lower than the specific activity of 12(S)-HHT when large amounts of 12(S)-HETE were synthesized. Based on comparison of specific activity between phospholipids and oxygenated products, the pools of arachidonate-containing phospholipids involved in the synthesis of oxygenated products are dependent on the amount of arachidonate released.
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PMID:A unique pool of free arachidonate serves as substrate for both cyclooxygenase and lipoxygenase in platelets. 181 90

Since crotalase has thrombin-like and kallikrein-like functional and structural properties, we compared the actions of crotalase, thrombin and plasma kallikrein on 13 tripeptide nitroanilide substrates. Initial rates of hydrolysis were determined at 27 degrees C, pH 8.3, and used to construct Lineweaver-Burk plots from which Km and Vmax were determined. The ratio of kcat/Km was taken as a measure of enzymatic specificity. Crotalase yielded kcat/Km values for the following nitroanilide substrates in descending order of magnitude: H-D-NLeu-CHA-Arg, H-D-Pro-HHT-Arg, Tos-Gly-Pro-Arg, H-D-PhGly-Phe-Arg, Cbo-Glu(BuO)-Gly-Arg, H-D-But-CHA-Lys, H-D-CHG-But-Arg, H-D-NLeu-HHT-Lys, H-D-HHT-Ala-Arg, Bz-Pro-Phe-Arg, Tos-Gly-Pro-Lys, MeS-Leu-Gly-Arg, MeO-CO-CHG-Gly-Arg. This pattern of specificity correlated only roughly with those of thrombin and kallikrein.
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PMID:Action of crotalase, an enzyme with thrombin-like and kallikrein-like specificities, on tripeptide nitroanilide derivatives. 293 64

Although HHT accounts for approximately one third of the arachidonic acid (AA) metabolites produced by stimulated platelets, no well defined function has been attributed to this product. We report that HHT stimulates prostacyclin production by endothelial cells, and have identified the mechanism for this effect. In human umbilical venous endothelial cells, HHT (0.5 and 1 microM) stimulated prostacyclin (RIA for 6KPGF1 alpha) by 32 +/- 22% (1SD) and 42 +/- 38% (P less than 0.05 and less than 0.01). Similar changes were observed when the effect of HHT on exogenous [1-14C] AA metabolism in fetal bovine aortic endothelial cells (FBAECs) was studied. Kinetic analyses revealed that HHT affected vascular cyclooxygenase. HHT (1 microM) increased Vmax in test microsomes (706 +/- 21 pmol/mg/min, mean +/- 1SE) when compared to controls (529 +/- 20; P less than 0.02). No concomitant effect on Km was observed. A further effect of HHT on AA release from endothelial cell membrane phospholipids was noted. Prelabeling experiments revealed that HHT (1 microM) increased the ionophore stimulated release of AA from FBAECs (20952 +/- 555 cpm/well control mean +/- 1SE vs 25848 +/- 557 for paired HHT treated cells; P less than 0.05). The effect of HHT on platelet AA metabolism was next studied. Preincubation of washed platelets with HHT (1 microM) did not enhance thrombin or arachidonic acid induced platelet TXB2 formation. In platelets prelabelled with [1-14C]AA, HHT (1 microM) had no effect on AA release post thrombin stimulation. Conversion to cyclooxygenase metabolites was also not enhanced. HHT stimulates vascular prostacyclin without a concomitant effect on platelet AA metabolism. HHT may thus be an important local modulator of platelet plug formation.
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PMID:The platelet cyclooxygenase metabolite 12-L-hydroxy-5, 8, 10-hepta-decatrienoic acid (HHT) may modulate primary hemostasis by stimulating prostacyclin production. 312 19

A randomized, placebo-controlled double-blind trial was conducted on 20 adults to assess the effect of vitamin E (800 IU/d 727 mg/d for 5 wk) on platelet function, arachidonic acid metabolism, and prostacyclin generation. Platelet aggregation was measured in response to collagen, arachidonic acid, and adenosine diphosphate. Thromboxane B2 was assayed in serum and in the supernatant plasma after platelet aggregation. Platelets were labeled with [3H]arachidonic acid to assess production and release of cyclooxygenase products (MDA, TXB2, and HHT), a lipoxygenase product (12-HETE), and arachidonic acid in response to stimulation by thrombin or collagen. Prostacyclin was measured in plasma and in blood collected from bleeding-time incisions by a sensitive HPLC-RIA procedure. Despite marked increases in plasma and erythrocyte vitamin E levels in the vitamin E group, there were no significant differences between the vitamin E and placebo groups in any of the variables measured.
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PMID:Vitamin E supplementation effect on human platelet function, arachidonic acid metabolism, and plasma prostacyclin levels. 312

Kinetic parameters [the Michaelis-Menten (Km), catalytic (kcat), and specificity (kcat/Km) constants] were determined for human alpha- and gamma-thrombins with the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-p-nitroanilide), Chromozym-TH (Tos-Gly-Pro-Arg-p-nitroanilide), and Spectrozyme-TH (H-D-HHT-Ala-Arg-p-nitroanilide) under physiologically relevant conditions (0.15 M NaCl buffered with 10 mM HEPES and 10 mM Tris-HCL, pH 7.4 at 37 degrees C). No major differences were found between alpha-thrombin with high fibrinogen clotting activities (greater than 3,500 killo clotting units/g) and gamma-thrombin with essentially no clotting activities (less than 10 kCU/g), although the Km values and in most cases kcat values for alpha-thrombin were slightly lower than for the gamma-thrombin. At 37 degrees C, relative to 23 degrees C, Km values increased 2-fold for S-2238, approximately 1.5-fold for Chromozym-TH, and remained essentially the same for Spectrozyme-TH (e.g., reciprocal substrate binding), whereas the kcat values increased for all 3 substrates (e.g., enzyme turnover). This caused kcat/Km values to decrease slightly for S-2238, remain the same for Chromozym-TH, and increase for Spectrozyme-TH (e.g., enzyme efficiency). Since spontaneous hydrolysis was not limiting at 37 degrees C, assays employing these substrates may be satisfactorily performed under physiologically relevant conditions. Under these conditions, kcat/Km ratios for the 3 substrates are similar to that for the A alpha cleavage in fibrinogen by alpha-thrombin.
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PMID:Human alpha- and gamma-thrombin specificity with tripeptide p-nitroanalide substrates under physiologically relevant conditions. 361 13

Thrombin, histamine and ionophore A23187 stimulated human endothelial cells to release arachidonic acid and synthesize prostaglandins. To compare the activation of arachidonic acid release by these three stimuli in endothelial cells, we examined the intracellular lipid metabolism by prelabeling the cells with [14C]stearic acid and [3H]arachidonic acid. Thrombin stimulated the loss of 3H and 14C label from intracellular phospholipids. At the same time [3H]arachidonic acid and prostaglandins were released into the incubation medium. Thin layer chromatography analysis indicated that prostacyclin is the major metabolite formed followed by PGF2 alpha, PGE2, HHT and PGD2. In addition, several intracellular lipid metabolites were accumulated. These include: phosphatidic acid and 1,2-diacylglycerol detected by increase of both 14C and 3H radioactivity; lysophosphatidylinositol, lysophosphatidylethanolamine, and to a smaller extent lysophosphatidylcholine and lysophosphatidylserine detected by increase of 14C radioactivity. Like thrombin, both histamine and ionophore A23187 also stimulated release of arachidonic acid and synthesis of prostaglandins. Despite the different nature of the agonists, the type and the relative amount of prostaglandins synthesized in response to histamine and A23187 were similar to that stimulated by thrombin. The relative extents of hydrolysis of phospholipids and the accumulation of phosphatidic acid, 1,2-diacylglycerol and lysophospholipids are similar to that of 3H radioactivity and prostacyclin released into the medium and follow the order: ionophore A23187 greater than thrombin greater than histamine. These results suggest that in human endothelial cells, histamine, thrombin and ionophore A23187 directly or indirectly activated both phospholipase C and phospholipase A2 and these activations most likely involve mobilization of Ca2+.
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PMID:Prostacyclin synthesis and deacylation of phospholipids in human endothelial cells: comparison of thrombin, histamine and ionophore A23187. 392 46

Incubation of cultured human umbilical vein endothelial cells with [1-14C]-arachidonic acid, followed by RP-HPLC analysis, resulted in the appearance of two principal radioactive products besides 6-keto-PGF1 alpha. The first peak was HHT, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by GC/MS and was shown to be the lipoxygenase product 15-HETE. Stimulation of endothelial cells with thrombin enhanced 15-HETE synthesis from arachidonate. Subsequent experiments showed that 5-HETE and 12-HETE were also synthesized by endothelial cells, but no evidence of leukotriene synthesis was found. Incubation of the 15-HETE precursor 15-HPETE with endothelial cells resulted in the formation of four distinct ultraviolet light-absorbing peaks. Ultraviolet and GC/MS analysis showed these peaks to be 8,15-diHETEs that differed only in their hydroxyl configuration and cis-trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14,15-LTA4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggest that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-LTA4.
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PMID:Evidence for 15-HETE synthesis by human umbilical vein endothelial cells. 392 91

Flunarizine, a calcium (Ca2+)-entry blocker, selective for vascular tissues, inhibits in a concn-dependent way the contraction of isolated rat caudal artery preparations induced by mediators derived from thrombin-stimulated rat platelets. This inhibition is slow in onset and is of prolonged duration. Specific measurements and pharmacological analysis show 5-hydroxytryptamine (5HT) and thromboxane A2 (TXA2) to be the main mediators involved. Comparison with exogenously added agonists shows amplification between 5HT and TXA2 at the level of the vascular smooth muscle cells. Combined treatment with ketanserin, a selective 5HT2 receptor antagonist, and suprofen, a fatty acid cyclo-oxygenase inhibitor, shows that flunarizine inhibits the 5HT-induced as well as the prostaglandin-induced components of the contraction. The compound does not affect the release of 5HT from the platelet and does not interfere with the biosynthesis of TXA2 from endogenous platelet arachidonic acid; it reduces the amounts of TXB2 and HHT and increases the production of HETE from exogenous [14C]arachidonic acid by washed rat platelets.
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PMID:Platelet-mediated vascular contractions. Inhibition by flunarizine, a calcium-entry blocker. 640 80


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