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)

Mobilization of FFA in mice, triggered with an injection of thrombin, was followed within 24 h by a 2.5-fold increase in fibrinogen synthesis and a 30% increase in plasma fibrinogen concentration. In mouse liver slices, incubated in plasma, additions of palmitate or stearate in amounts similar to those expected in vivo in FFA mobilization stimulated fibrinogen synthesis 5.6- to 6.1-fold while unsaturated and short-chain FFA were less effective. Palmitate and linoleate also augmented albumin synthesis although not as stongly as fibrinogen synthesis. These observations raised the possibility that the greater effectiveness of saturated FFA in stimulating fibrinogen synthesis may reflect higher FFA/albumin ratios within hepatocytes in the presence of saturated FFA. Injection of exogenous defatted albumin into mice before thrombin injection prevented the FFA-associated rise in fibrinogen synthesis and plasma concentration. In incubated liver slices, defatted albumin abolished the FFA stimulation of fibrinogen synthesis when FFA/albumin ratios were maintained to the physiological range. These studies indicate that the FFA/ALBUMIN RATIO MAY PLAY A MAJOR ROLE IN THE REPLENISHMENT OF FIBRINOGEN AFTER PERIODS OF RAPID DEFIBRINOGENATION.
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PMID:Free fatty acids and albumin as mediators of thrombin-stimulated fibrinogen synthesis. 126 31

The 1,2-diacylglycerol (DAG) mass content was measured in thrombin-stimulated human platelets. Thrombin stimulates a biphasic accumulation of DAG, with an early phase reaching a peak at 10 s and a later phase reaching a peak at 2-3 min. The time course of first-phase DAG production corresponded well to that of Ins(1,4,5)P3 formation, which was rapid and transient. The second phase of DAG accumulation occurred after the level of Ins(1,4,5)P3 returned to nearly basal. Thrombin stimulated the decrease in PtdIns and phosphatidylcholine contents. The source of second-phase DAG was examined in platelets prelabelled with three radioactive fatty acids, i.e. arachidonic, palmitic and myristic. Thrombin stimulated the increase in radioactivity of DAG with decline of PtdIns in platelets labelled with [3H]arachidonic acid or [3H]palmitic acid, in which PtdIns was considerably labelled. In contrast, significant accumulation of [3H]DAG was not observed in [3H]myristic acid-labelled platelets, in which PtdIns was poorly labelled. In platelets prelabelled with [3H]inositol, an increase in InsP in response to thrombin was seen for more than 5 min. In contrast, upon stimulation, significant increases in [3H]phosphocholine and [3H]choline were not observed in [methyl-3H]choline-labelled platelets. Thrombin induced a small production of phosphatidylethanol, when ethanol was present during stimulation. However, the formation of DAG and phosphatidic acid was not significantly affected by ethanol. These results suggest that thrombin stimulates a biphasic accumulation of DAG, initially from PtdInsP2 and later from PtdIns in human platelets.
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PMID:Thrombin induces a biphasic 1,2-diacylglycerol production in human platelets. 190 64

Earlier studies showed that during the first 20 to 25 seconds of aggregation induced by thrombin (0.1 U/mL) or adenosine diphosphate (ADP) (2 microM) of rabbit or human platelets prelabeled with [3H]palmitic acid, labeled lipid became associated with the cytoskeleton (isolated after lysis with 1% Triton X-100, 5 mM EGTA [ethylene glycolbis-(beta-aminoethyl ether(N,N,N',N'-tetraacetic acid] in the presence of 0.5 mM leupeptin and 50 mM benzamidine). In comparison with labeled lipid in intact platelets, the labeled lipid that was associated with the cytoskeleton was enriched in phospholipids and ceramide. To determine whether these effects were specific for lipids labeled with palmitic acid, we studied rabbit platelets in which lipids had been labeled by incubation of the platelets with pairs of 14C- or 3H-labeled palmitic, stearic, arachidonic, and linoleic acids. Examination of the distribution of label among the lipid classes of intact platelets showed that phospholipids contained most of the label. Under the conditions of limited, thrombin-induced aggregation used, labeled lipids were not lost from the platelets and the distribution of label among the lipid classes was essentially unchanged. There were major differences in the incorporation of labeled lipids into the cytoskeleton. The greatest incorporation (2.1 to 2.8% of the label in the platelets) was observed with palmitic acid-labeled lipids; by direct comparison, only 44% as much of the label of stearic acid-labeled lipids, 21% as much of the label of linoleic acid-labeled lipids, and only 6% as much of the label of arachidonic acid-labeled lipids was incorporated into the cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Incorporation of lipids labeled with various fatty acids into the cytoskeleton of aggregating platelets. 194 90

The molecular species composition of rat platelet diacyl-glycerophosphocholine (GPC) was investigated by reverse-phase HPLC and by mass spectrometry. The two methods gave the same very high proportion of fully saturated phospholipids, the 16:0-16:0 and 16:0-18:0 species representing together about 40% of the overall molecular species. [14C]Palmitoyllyso-GPC was found to be acylated by resting platelets in equal amounts into 16:0-16:0 and into 16:0-20:4 species. The acylation rate of this lysophospholipid was increased by 3-fold and 14-fold when platelets were stimulated for 10 min with thrombin and the ionophore A23187, respectively. Essentially the same two molecular species were synthesized upon stimulation but with a higher preference for arachidonate than for palmitate. We investigated the mechanisms responsible for the incorporation of palmitate and arachidonate by examining the enzymatic acylation of [14C]palmitoyllyso-GPC by platelet homogenates. The percentage of the various molecular species formed when CoA, ATP, and Mg2+ were added excludes the CoA, ATP-dependent pathway as being involved in the acylation reactions previously observed. In the absence of ATP, CoA-independent transacylations appear to play a crucial role in the synthesis of the 16:0-20:4 species whereas the addition of CoA greatly favored dipalmitoyl-GPC synthesis. The involvement of CoA-dependent mechanisms in the synthesis of dipalmitoyl-GPC was demonstrated as follows: (i) the labeling in the sn-2 position of the dipalmitoyl-GPC synthesized in the presence of CoA was not modified when free unlabeled palmitic acid was added to the incubation medium and (ii) platelet homogenates were unable to esterify lysolecithin with added labeled palmitic acid in the presence of CoA only.
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PMID:Very high proportion of disaturated molecular species in rat platelet diacyl-glycerophosphocholine: involvement of CoA-dependent transacylation reactions. 211 66

The release of free fatty acids from rat platelets, triggered by thrombin stimulation, was monitored by high-performance liquid chromatography (HPLC) after precolumn derivatization with monodansylcadaverine (MDC). A rapid filtration procedure was devised for the precise determination of free fatty acids released from aggregated platelets, instead of the conventional method using a stop solution or enzyme reactions. The fatty acids thus collected were derivatized with MDC in the presence of diethyl phosphorocyanidate (DEPC). The simultaneous separation of MDC derivatives of fatty acids was achieved on a reversed-phase TSKgel ODS-80TM column within 60 min by linear gradient elution, using 0.2 M Tris-HCl buffer (pH 7.8)-methanol (50:50, v/v) and acetonitrile. The MDC derivatives were detected with excitation and emission wavelengths of 340 and 518 nm, respectively. The amounts of liberated fatty acids were in the range from 45.0 pmol for myristoleic acid (C14:1) to 395.0 pmol for palmitic acid (C16:0) per 1.9 x 10(7) platelets.
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PMID:Rapid determination by high-performance liquid chromatography of free fatty acids released from rat platelets after derivatization with monodansylcadaverine. 228 73

Previous experiments showed that during the early stages (20-30 seconds) of aggregation induced by adenosine diphosphate (ADP, 2 microM) or thrombin (0.1 U/mL) of rabbit or human platelets prelabeled with [3H]palmitic acid, labeled lipid became associated with the cytoskeleton isolated after lysis with 1% Triton X-100, 5 mM EGTA [ethylene glycol-bis-(beta-aminoethyl ether)]-N,N,N',N'-tetra-acetic acid. The association appeared to be related to the number of sites of contact and was independent of the release of granule contents. We have now investigated the nature of the labeled lipids by thin-layer and column chromatography and found differences between the distribution of the label in intact platelets (both stimulated and unstimulated) and the isolated cytoskeletons. In both species, and with either ADP or thrombin as aggregating agent, 70-85% of the label in both intact platelets and in the cytoskeletons was in phospholipids. The distribution of label among the phospholipids in the cytoskeletons was similar to that in intact platelets except that the percentage of label in phosphatidylcholine was significantly higher in the cytoskeletons of human platelets than in the intact platelets, and the percentage of label in phosphatidylserine/phosphatidylinositol was significantly lower in the cytoskeletons of rabbit platelets and thrombin-aggregated human platelets than in intact platelets. The cytoskeletons contained a lower percentage of label in triacylglycerol, diacylglycerol, and cholesterol ester than the intact platelets. Contrary to a report in the literature, we found no evidence for the incorporation of diacylglycerol and palmitic acid into the cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Palmitic acid-labeled lipids selectively incorporated into platelet cytoskeleton during aggregation. 239 15

The addition of arachidonic acid induced a rapid release of 45Ca2+ from human platelet membrane vesicles which accumulated 45Ca2+ in the presence of ATP. Docosahexaenoic acid, eicosapentaenoic acid, linolenic acid and linoleic acid were less active than arachidonic acid. In contrast, oleic acid, myristic acid and palmitic acid were without effect. The thromboxane A2 analogue induced no 45Ca2+ release. The cyclooxygenase/lipoxygenase inhibitor failed to suppress arachidonic acid-induced 45Ca2+ release at the concentration which inhibited the production of lipid peroxides. These data indicate that the activity of arachidonic acid may be due to fatty acid itself and not to its metabolites. The combination of arachidonic acid and inositol 1,4,5-trisphosphate (IP3) resulted in a greater 45Ca2+ release from platelet membrane vesicles than either compound alone. When the intracellular free Ca2+ concentration ([Ca2+]i) was measured using fura-2, the thrombin-induced [Ca2+]i increase was reduced in platelets which had been treated with a phospholipase A2 inhibitor, ONO-RS-082 (2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid). These results provide evidence that arachidonic acid alone may cause Ca2+ increase and also may induce an additional Ca2+ mobilization to IP3-induced Ca2+ release in human platelets.
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PMID:Evidence of Ca2+ mobilizing action of arachidonic acid in human platelets. 249 58

When washed human platelets were incubated with [3H]palmitic acid, radioactivity was incorporated into a major 38 kDa doublet and several minor proteins that were resolved on polyacrylamide gels. The radioactivity associated with the proteins remained after extractions with organic solvents, but it was lost after hydroxylamine treatment or mild alkali methanolysis. The products of these reactions were analyzed by thin-layer chromatography and HPLC. They were identified as palmitohydroxamate and methyl palmitate, respectively, indicating that the palmitic acid was covalently linked to the proteins via oxygenester or thioester bonds. In resting platelets, radioactivity was detected in the 38 kDa proteins 2 min after the addition of [3H]palmitic acid. A plateau was reached between 5 and 11 min, at which time radioactivity was also detected in a 23 kDa protein. Thrombin elicited faster and greater incorporation of label into both proteins. Phorbol 12-myristate 13-acetate (PMA) led to a similar, but slower increase of radioactivity in the 38 kDa proteins, while collagen and A23187 were less effective. Enhanced palmitoylation may be closely linked to platelet activation, as suggested by the following observations: (1) in thrombin- or PMA-activated platelets, the time-course of aggregation correlated with the time-course of enhanced palmitoylation of the 38 kDa proteins; (2) in platelets activated by various concentrations of thrombin with or without prostacyclin, aggregation was correlated with the enhanced incorporation of radioactivity into the 38 kDa proteins.
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PMID:Agonist-enhanced palmitoylation of platelet proteins. 265 44

We examined the level of 1,2-diacylglycerol and inositol phosphates in normal and EJ-H-ras-transformed BALB/3T3 fibroblasts by prelabelling the cells with [3H]glycerol, [3H]inositol, [14C]glucose, [14C]arachidonic acid, and [14C]palmitic acid. Steady-state level of inositol phosphates, however, was the same in control and transformed cells. Diacyglycerol labelling by [14C]arachidonic acid was the same in control and transformed cells. Insulin dramatically increased diacylglycerol labeling by [14C]glucose in normal cells, whereas it did not affect ras-transformed fibroblasts. Neurotransmitter-induced inositol lipid turnover was greatly enhanced in ras-transformed cells; conversely, platelet-derived growth factor and thrombin-stimulated normal cells to a greater extent than transformed fibroblasts. Taken together these results suggest that ras transformation may induce multifarious effects on signal transduction: it may cause de novo synthesis of diacylglycerol and subversion of neurotransmitter and growth factor receptor coupling to inositol lipid metabolism.
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PMID:Signal transduction in EJ-H-ras-transformed cells: de novo synthesis of diacylglycerol and subversion of agonist-stimulated inositol lipid metabolism. 266 28

The presence of covalently bound palmitic acid in fibrinogen receptors, glycoproteins (GP) IIb and IIIa, has been explored in human blood platelets. Membrane fractions were isolated from fresh blood platelets labeled with [9,10-3H]palmitic acid and then analyzed for radioactive proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with Coomassie Brilliant Blue, excised, and counted in a liquid scintillation counter. The results indicate that membrane proteins with electrophoretic mobility corresponding to glycoproteins IIb and IIIa incorporate [9,10-3H]palmitic acid. The palmitylated glycoproteins IIb and IIIa were immunoprecipitated by specific anti-GP IIb and GP IIIa antisera. It is interesting to note that the palmitylation of these glycoproteins occurred rapidly in platelets activated with 0.5 unit of thrombin or 30 microM ADP. At the concentration used (100 micrograms/ml), cycloheximide did not inhibit incorporation of [3H]palmitate into the glycoproteins showing that this process is not dependent upon protein synthesis. The acyl moiety was resistant to denaturating detergents, delipidation with organic solvents, and hydrolyzable with hydroxylamine. In the case of membrane protein with the electrophoretic mobility of GP IIb, the radioactive label was significantly decreased after reduction with 2-mercaptoethanol. Final identification of GP IIIa as an acylated product in human platelets incubated with [9,10-3H]palmitic acid was provided by two-dimensional polyacrylamide gel electrophoresis. In contrast to GP IIb alpha, GP IIIa isolated by this method showed the presence of attached radioactive palmitic acid residues. Analysis by high performance liquid chromatography after methanolysis of the [3H]palmitate-labeled glycoproteins confirmed the fatty acid nature of the label. Palmitylation is a newly identified post-translational modification of the fibrinogen receptor which may play an important role in its interaction with the membrane and/or its biological function.
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PMID:Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets. 274 34


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