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)

Platelets from individuals with familial hypercholesterolemia show increased sensitivity to the aggregating atents, epinephrine and ADP. Since the mechanism of this abnormal sensitivity is unknown, we examined, in vitro, the influence of the plasma lipid environment on the function of platelets. The composition of plasma lipids was altered by the addition of sonicated cholesterol-dipalmitoyl lecithin liposomes which were "cholesterol normal" (cholesterol-phospholipid mole ratio [C/P] equals 1.0, "cholesterol rich" (C/P eauals 2.2), or "cholesterol poor" (C/P equals 0). Cholesterol-normal liposomes had no influence on platelet lipids or platelet function. In contrast, after incubation for 5 h at 37 degrees C with cholesterol-rich liposomes, normal platelets acquired 39.2% excess cholesterol with no change in phospholipids or protein. The percent increase in platelet membrane cholesterol was three-fold that of the granule fraction. The acquisition of cholesterol by platelets was associated with a 35-fold increase in sensitivity to epinephrine-induced aggregation (P less than 0.001) and 15-fold increase to ADP aggregation (P less than 0.001), as determined both by aggregometry and by [13C]serotonin release. Response to thrombin or collagen was unchanged. Platelets incubated with cholesterol-poor liposomes underwent a selective loss of 21.4% cholesterol and this was associated with an 18-fold reduction in their sensitivity to epinephrine. These studies demonstrate that the cholesterol content of platelets is dependent on the lipid composition of the milier. Cholesterol acquired by platelets may exert its effect on platelet function by a modification of the platelet membrane.
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PMID:Platelet hypersensitivity induced by cholesterol incorporation. 111 69

The effect of cholesterol enrichment on vascular smooth muscle cell (VSMC) calcium homeostasis was studied by evaluating calcium uptake, efflux, and intracellular content in cultured VSMC derived from the rat pulmonary artery. Incubation of VSMC with liposomes consisting of free cholesterol (FC) and phospholipid (2:1 molar ratio, 1 mg FC/ml medium) for 24 h resulted in a 69 +/- 19% increase (P less than 0.01; n = 10) in FC which was associated with a 73 +/- 11% increase (P less than 0.005; n = 10) in intracellular calcium content as assessed by isotopic equilibrium with 45Ca2+ and a 65 +/- 11% increase (P less than 0.024; n = 3) as assessed by atomic absorption spectroscopy. Cholesterol enrichment caused a marked increase in the unidirectional calcium uptake rate from 0.026 +/- 0.03 to 0.158 +/- 0.022 nmol calcium/s per mg protein (P less than 0.01; n = 3), but had no effect on calcium efflux. Nifedipine (1 microM) reduced (P less than 0.05; n = 6) the effect of cholesterol enrichment on unidirectional calcium uptake by 78 +/- 16%; and verapamil (10 microM), diltiazem (1 microM), and nifedipine (1 microM) each significantly inhibited the effect of cholesterol enrichment on intracellular calcium accumulation. Exposure of cholesterol-enriched VSMC to cholesterol-poor liposomes for 24 h returned both FC and calcium contents to control levels. Serum- and serotonin-stimulated calcium uptakes were potentiated 3.7- and 1.7-fold, respectively, in cholesterol-enriched VSMC, whereas endothelin, vasopressin, and thrombin-stimulated calcium uptakes were not affected. We conclude that VSMC FC content plays a role in regulating cellular calcium homeostasis, both under basal conditions and in response to selected agonists.
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PMID:Cholesterol enrichment increases basal and agonist-stimulated calcium influx in rat vascular smooth muscle cells. 175 51

Male Fisher rats were fed chow diets for two weeks after which they were divided into seven groups of ten rats each and fed 20% Canola, 20% menhaden, 20% partially hydrogenated soy oil (PHSO) or chow only, with or without 500 mg/Kg dietary vitamin E in chow containing 2% cholesterol for six weeks. Triglycerides were lower in the menhaden group and were essentially the same in the E supplemented groups as in their unsupplemented cohorts. Plasma cholesterol was higher in the Canola, and lower in the menhaden, groups, compared to the PHSO group. Cholesterol was the same in the E supplemented groups as in their unsupplemented cohorts. Plasma thiobarbituric acid reactant substances (TBARS) were higher in the menhaden group, compared to the chow group. Vitamin E supplementation lowered TBARS in the menhaden and PHSO groups, compared to the unsupplemented cohorts. Collagen induced platelet aggregation was lower in both Canola and menhaden groups, compared to the PHSO group. Vitamin E supplementation lowered collagen induced platelet aggregation only in the PHSO group. Thrombin induced platelet aggregation was lower in the Canola group, compared to the PHSO group. Vitamin E supplementation did not affect thrombin induced platelet aggregation compared to unsupplemented cohorts. Plasma vitamin E levels were lowest in the menhaden supplemented group compared to all other groups not receiving E, suggesting a greater requirement for E in this group. Finally, vitamin E supplementation raised the plasma E levels in all groups except the menhaden group when compared to unsupplemented cohorts.
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PMID:Effect of dietary menhaden, Canola and partially hydrogenated soy oil supplemented with vitamin E upon plasma lipids and platelet aggregation. 202 42

A ninhydrin positive compound (L2) now identified as 2-amino-5-(N-ethylcarboxyamido)-pentanoic acid, from unprocessed tea leaves was a potent inhibitor of thrombin-stimulated thromboxane formation in rabbit whole blood (Ali and Afzal; Prostaglandins, Leukotrienes and Medicine, 27: 9, 1987). In the present study, processed and unprocessed tea leaf extracts were given to rats to consume for a period of eight weeks. Cholesterol and thromboxane levels were measured in the serum obtained from clotting the blood at 37 degrees C. A significant reduction in thromboxane levels was observed in rats taking unprocessed tea extract. This reduction was equally distributed in adult as well as in juvenile rats. However no appreciable changes in the levels of thromboxane were noticed in the serum of rats taking processed tea extracts. This might be due to the presence of a labile component which is destroyed during the processing of green tea leaves. A decreased level of cholesterol was observed in rats consuming unprocessed tea extract. This decrease could be linked to the decrease in thromboxane levels as observed. Processed tea refers to commercially available tea of different brands while unprocessed tea refers to dried green tea leaves.
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PMID:A potent thromboxane formation inhibitor in green tea leaves. 227 65

To investigate the mechanism of enhanced responsiveness of cholesterol-enriched human platelets, we compared stimulation by surface-membrane-receptor (thrombin) and post-receptor (AlF4-) G-protein-directed pathways. Platelets were labelled with [32P]Pi and [methyl-3H] choline chloride, incubated with sonicated lipid dispersions of various ratios of cholesterol and phospholipid, and loaded with the fluorescent Ca2+ indicator fura-2. We report the following. (1) Cholesterol enrichment enhances cytosolic Ca2+ accumulation and phospholipase A activation in response to both receptor-directed and post-receptor-directed agonists. No enhancement by cholesterol of phospholipase A activity at fixed Ca2+ concentrations is observed in lysed platelets, implying that no perturbation by cholesterol of phospholipase A/substrate interaction occurs in our preparations. (2) In both normal and cholesterol-enriched platelets, Ca2+ mobilization is promoted by a factor(s) apart from InsP3 that appear(s) to be modulated by cholesterol. A disproportionate increase in cytosolic Ca2+ relative to [32P]InsP3 is observed with increasing doses of thrombin in normal, and to a larger extent in cholesterol-enriched, platelets. When AlF4- is the agonist, there is no cholesterol-associated enhancement in [32P]InsP3 to account for the heightened Ca2+ rise seen with cholesterol enrichment. (3) Enhanced phospholipase A activation is not necessarily proportional to cytosolic Ca2+ increase. The magnitude of the increase in phospholipase A activity for a given rise in cytosolic Ca2+ is greater in cholesterol-enriched platelets that are stimulated by AlF4- than in those stimulated by thrombin. We conclude that increased membrane microviscosity associated with cholesterol enrichment may promote G-protein/phospholipase A interaction as well as the Ca2(+)-release mechanism, without significantly altering G-protein/phospholipase C interaction.
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PMID:Stimulated cholesterol-enriched platelets display increased cytosolic Ca2+ and phospholipase A activity independent of changes in inositol trisphosphates and agonist/receptor binding. 230 12

Dextran sulfate and polybrene were immobilized on polypropylene tubes with plastic adhesive. Serum albumin was also immobilized on the tube by heat-denaturation. In the those tubes thrombin was protected from inactivation in the presence of 0.1 M NaCl remarkably. Cholesterol coated on the tube also protected the enzyme from inactivation. The results indicate that amounts of thrombin protected by these four coating materials corresponded to the amount of thrombin inactivated on surface of the tube. At least half of the thrombin was also inactivated within 20 sec at 37 degrees C in the presence of quartz sand, supporting a significant contribution of a solid surface to temperature-dependent inactivation of enzyme in dilute solution.
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PMID:Possible contribution of solid surface for inactivation of thrombin. 245 60

Cholesterol-loaded human monocyte-derived macrophages increase their production of apolipoprotein E (apoE). Although cholesterol loading is often achieved with modified plasma lipoproteins, macrophages can be loaded also by coculture with platelets. Therefore, the relationship between platelet-mediated cholesteryl ester accumulation and apoE secretion was examined. Macrophages were isolated by adherence and cultured for 6 days in serum-free medium. Secreted apoE was measured with a sensitive solid-phase radioimmunoassay. Maximum apoE secretion by the adherent macrophages from 5 x 10(6) peripheral blood mononuclear cells was obtained with 3 x 10(8) platelets and was ten-fold greater than control cells cultured in the absence of platelets. Platelet-mediated apoE secretion was consistently greater than that obtained by culture with either native or acetylated low density lipoproteins. Whereas the 1000 g supernatants of unstimulated platelets were poor inducers of apoE secretion, substances rich in cholesterol that were shed from thrombin-stimulated platelets and recovered in the 1000 g supernatants were almost as active as intact platelets. In all studies, platelet-induced secretion of apoE paralleled the capacity of platelets to induce macrophage cholesterol accumulation, indicating that macrophage apoE secretion was readily influenced by macrophage cholesterol metabolism.
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PMID:Platelet-enhanced apolipoprotein E production by human macrophages: a possible role in atherosclerosis. 341 Dec 46

Platelets are a source of lipid that facilitates the coagulation process and that potentially accumulates in cells within atherosclerotic lesions. With this in mind, we have examined the release of lipid-containing particles from activated rat and human platelets. When washed platelets were activated with thrombin and incubated, cholesterol and phospholipid were continuously released for a period up to 2 hours. The amount of cholesterol released was approximately 20% of the platelet cholesterol content. Cholesterol release was also stimulated by strong platelet agonists such as collagen and the calcium ionophore A23187 but not by the weak agonist ADP. The release of cholesterol did not simply result from lysis of platelets since lactate dehydrogenase and cholesterol release could be dissociated. Colchicine substantially inhibited release of cholesterol but did not substantially inhibit release of lactate dehydrogenase. Cholesterol-phospholipid particles were isolated from platelet-release supernatants by centrifugation, microfiltration, and gel filtration chromatography. The particles, which eluted in the void volume, were composed of 57% protein, 32% phospholipids, 8% cholesterol, and 3% triglyceride. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of particle-associated protein showed a major protein component of Mr 42,000, presumably actin. The density of the particles in a sucrose density gradient was 1.16 g/ml. The cholesterol to phospholipid molar ratio of isolated particles from rat and human platelets was about 0.6, a value typical of plasma membranes, whereas the cholesterol to phospholipid molar ratio in particles released from platelets of rats fed a high-cholesterol diet increased to 1.0. Electron microscopic analysis of the particles showed them to be spherical or irregular in shape with sizes ranging from 50 to 550 nm and with projections that extended from their surfaces. We suggest that these cholesterol-phospholipid particles released from platelets may represent a mechanism by which the membrane becomes dispersed following platelet activation.
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PMID:Biochemical characterization of isolated cholesterol-phospholipid particles continuously released from rat and human platelets after activation. 341 35

Exchange and net mass efflux of cholesterol were investigated in [3H]cholesterol-labeled or cholesteryl ester-loaded murine peritoneal macrophages, respectively. Macrophages were subjected to mild proteolysis prior to measurements of mass efflux or exchange to assess whether plasma membrane proteins participated in either process. Cholesterol exchange and mass efflux were inhibited up to 70% following trypsinization. The inhibitory effect was reversible as cells regained normal efflux and exchange 6-8 hr following treatment. Incubation of trypsinized cells with cycloheximide prevented recovery, indicating that protein synthesis was necessary for restoration of normal cholesterol efflux. Studies with peptide and nonpeptide inhibitors of proteolysis suggested that active catalytic activity of trypsin was necessary for the inhibitory effect to be expressed. The degree of inhibition for both cholesterol exchange and mass efflux was dependent in a quantitatively similar manner on the time of incubation and the concentration of trypsin, suggesting that the mechanism of cholesterol exchange and mass efflux were similar at the level of the plasma membrane. Two other serine-proteases, thrombin and elastase, were also capable of inhibiting cholesterol removal in a similar manner. No cell death was observed by altered morphology, detachment, changes in DNA or protein content, or trypan blue exclusion even under the most severe proteolytic conditions. These studies suggest that protease-sensitive plasma membrane proteins play a role in cholesterol efflux in macrophages.
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PMID:Exchange and mass efflux of cholesterol in macrophages. Evidence for a common mechanism and a role for plasma membrane proteins. 363 4

Modifications in the membrane lipid organization of human platelets activated with different agents (adenosine 5'-diphosphate, thrombin, collagen type I, and monosaccharides such as fucose, mannose, and galactose) were analyzed in vitro by using three lipid markers. Cholesterol was detected upon interaction with filipin, the anionic phospholipids were reacted with polymyxin B, and alterations in the degree of lipid packing were evaluated with the lipophilic fluorescent probe merocyanine 540, which reportedly inserts into bilayer domains whose lipids are more disordered. Filipin-sterol complexes and polymyxin B-anionic phospholipid complexes form characteristic membrane deformations which were examined in freeze-fracture preparations, whereas the merocyanine 540 binding to platelet membrane was recorded by fluorescent microscopy. In contrast to the resting cells, thrombin-stimulated platelets displayed an uneven distribution of filipin-sterol complexes which occurred in much higher density on the cell body than on pseudopods: on the latter, apparently cholesterol-free domains were very common. Unlike the non-stimulated cells, the platelets aggregated with the various agents employed showed characteristic polymixin B-anionic phospholipid complexes deformations of plasmalemma suggesting the appearance in uneven concentration of anionic phospholipids in the outer membrane leaflet. Incubation with merocyanine 540 did not result in staining of resting platelets when these were maintained in plasma, but a slight fluorescence was observed when platelets were kept in Tyrode buffer. However, platelets stimulated with thrombin, collagen type I, and monosaccharides bound very heavily the fluorescent dye; platelets aggregated with adenosine-5'-diphosphate bound only small amounts of merocyanine 540. The results showed that, during activation by different agents, modifications in lipid membrane organization include alterations in cholesterol and anionic phospholipid distribution, transbilayer movement of anionic phospholipids accompanied by more disordered membrane.
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PMID:Changes in the organization of membrane lipids during human platelet activation. Study by fluorescent and freeze-fracture cytochemistry. 394 48


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