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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphatidylserine (PS) is asymmetrically distributed in mammalian cell membranes, being preferentially localized in the inner leaflet. Some studies have suggested that a disturbance in the normal asymmetric distribution of PS--e.g., PS exposure in the outer leaflet of the cell membrane, which can occur upon platelet activation as well as in certain pathologic red cells--serves as a potent procoagulant surface and as a signal for triggering their recognition by macrophages. These studies suggest that the regulation of PS distribution in cell membranes may be critical in controlling coagulation and in determining the survival of pathologic cells in the circulation. In this paper we describe a sensitive technique, based on PS-dependent prothrombinase complex activity, for assessing the amount of PS on the external leaflet of intact viable cells. Our results indicate that tumorigenic, undifferentiated murine erythroleukemic cells express 7- to 8-fold more PS in their outer leaflet than do their differentiated, nontumorigenic counterparts. Increased expression of PS in the tumorigenic cells directly correlated with their ability to be recognized and bound by macrophages.
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PMID:Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation. 271 15

Phosphatidylserine (PS) in the plasma membrane of nonactivated human platelets is almost entirely located on the cytoplasmic side. Stimulation of platelets with the Ca2+ ionophore A23187 or combined action of collagen plus thrombin results in a rapid loss of the asymmetric distribution of PS. Also, treatment with the sulfhydryl-reactive compounds diamide and pyridyldithioethylamine (PDA) causes exposure of PS at the platelet outer surface. PS exposure is sensitively measured as the catalytic potential of platelets to enhance the rate of thrombin formation by the enzyme complex factor Xa-factor Va, since this reaction is essentially dependent on the presence of a PS-containing lipid surface. In this paper we demonstrate that endogenous PS, previously exposed at the outer surface during cell activation or sulfhydryl oxidation, can be translocated back to the cytoplasmic leaflet of the membrane by addition of dithiothreitol (DTT) but not by nonpermeable reducing agents like reduced glutathione. Treatment of platelets with trypsin or chymotrypsin, prior to addition of DTT, inhibits the inward transport of exposed PS. Moreover, severe depletion of metabolic ATP, as obtained by platelet stimulation with A23187 in the presence of metabolic inhibitors, though not inhibiting PS exposure at the outer surface, blocks the translocation of endogenous PS to the internal leaflet of the plasma membrane. These results strongly indicate the involvement of a membrane protein in the inward transport of endogenous PS. Recently, an aminophospholipid-specific translocase in the platelet membrane was postulated on the basis of the inward transport of exogenously added PS (analogues) [Sune, A., Bette-Bobillo, P., Bienvenue, A., Fellmann, P., & Devaux, P.F. (1987) Biochemistry 26, 2972-2978].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelets is reversed by restoration of aminophospholipid translocase activity. 273 Aug 70

Erythrocytes and spectrin-free DMPC-induced vesicles released from the cells were incubated for 3 weeks at 6 degrees C under conditions of metabolic ATP-depletion. Phosphatidylserine (PS) asymmetry was monitored during this period by use of the prothrombinase assay. Prothrombinase activities measured at the beginning of the incubation period indicated that approximately 0.06% of PS was located at the outer layer of the red cell membrane, whereas in DMPC-induced vesicles approximately 1.5% the PS was exposed on the outside. After completion of the incubation period PS exposure on the outside of red cells and vesicles was increased by no more than 5-fold. On the other hand, with vesicles prepared with a significantly increased (4-fold) ATP-content to sustain translocase activity, the incubation process resulted in a surprisingly high (20-fold) increase of PS exposure. With vanadate, an inhibitor of the aminophospholipid translocase, included in the incubation medium, the redistribution of PS was even more pronounced. These observations indicate that PS asymmetry in spectrin-free vesicles can not be directly correlated to either ATP content or translocase activity and suggest that besides the aminophospholipid translocase and the membrane skeleton, other mechanisms must be involved in maintaining phospholipid asymmetry.
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PMID:Phospholipid asymmetry in red blood cells and spectrin-free vesicles during prolonged storage. 865 96

Phosphatidylserine was exposed on the surface of human umbilical endothelial cells (ECV304) a few minutes after adding thrombin in vitro, as monitored by prothrombinase assays with and without annexin V. Jurkat T cells adhered to the thrombin-treated cells. The adhesion was inhibited by annexin V, indicating that it was mediated by exposed phosphatidylserine on the endothelial cells.
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PMID:Phosphatidylserine-mediated adhesion of T-cells to endothelial cells. 871 56

Prothrombin activation to thrombin is a key control reaction in blood coagulation. During the process, prothrombin is sequentially cleaved at two peptide bonds (Arg323-Ile and Arg274-Thr) by factor X(a) to generate meizothrombin and then thrombin. Phosphatidylserine (PS)-containing membranes from platelets are believed to facilitate this two-step process. Using fluorescence energy transfer (FRET), we determined the distances of closest approach between a specifically located C-terminal fluorescein of a double mutant bovine prothrombin (P(S528A, G581C)-FM) or meizothrombin (M(S528A, G581C)-FM) and phosphatidylethanolamine-N-rhodamine B (PE-Rh; 0-8.7 mol %) contained in membranes composed of PS (25 mol %) and phosphatidylcholine (66.3-75 mol %). Plots of the energy transfer efficiency as a function of membrane concentration, at six PE-Rh surface densities, were analyzed globally to obtain dissociation constants and binding stoichiometries as global parameters and saturating energy transfer efficiencies characteristic of each surface density. From the global analysis, the dissociation constants were estimated to be 0.32 +/- 0.10 and 0.28 +/- 0.12 microM with stoichiometries of 42 +/- 12 and 44 +/- 9 lipid/protein for prothrombin and meizothrombin, respectively. The distance of closest approach was obtained from the dependence of the saturating energy transfer efficiency on the acceptor (PE-Rh) surface density. With the assumptions of kappa2 = 2/3 and n = 1.4, the distances were 94 +/- 3 A for prothrombin and 114 +/- 2 A for meizothrombin. Since both prothrombin and meizothrombin behave in solution as oblate ellipsoids of revolution with a long axis of 120 A, our FRET measurements suggest that binding to PS-containing membranes induced tighter folding of the prothrombin molecule but not of the meizothrombin intermediate. This observation is consistent with our hypothesis that membrane binding plays an essential role in the sequential alignment of the bond Arg323-Ile in prothrombin and Arg274-Thr in meizothrombin with the active site of the membrane-bound prothrombinase in the two-step thrombin-generating process.
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PMID:Fluorescence resonance energy transfer study of shape changes in membrane-bound bovine prothrombin and meizothrombin. 910 82

The phospholipid content of different activated partial thromboplastin time (APTT) reagents was determined and compared to heparin sensitivity. The seven reagents included were those most widely used amongst participants of the U.K. National External Quality Assessment Scheme (NEQAS) at the time of study. Heparin sensitivity was assessed using the APTT ratios obtained by more than 300 NEQAS participants on five plasmas prepared from patients receiving unfractionated heparin. The concentrations of three neutral lipids and six phospholipids present in the seven APTT reagents were determined by high-performance thin-layer chromatography (HPTLC) and densitometry. Both the concentrations and the relative percentages of individual phospholipid components varied markedly between reagents. The total phospholipid concentration included a 12-fold range from 16 to 205 microgram/ml. Phosphatidylserine (PS) was completely lacking from one reagent prepared from vegetable material and ranged from 3 to 22 microgram/ml in the other six reagents containing extracts from animal tissue. The concentration of phosphatidylcholine ranged from 3 to 109 microgram/ml. There was no demonstrable relationship between the concentration of any individual lipid components and heparin sensitivity. However, the relative percentage phospholipid composition was important since a lower % of PS or phosphatidylinositol (PI) correlated with increasing heparin sensitivity.
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PMID:Lipid composition of seven APTT reagents in relation to heparin sensitivity. 1046 76

Phosphatidylserine (PhtdSer) is an anionic aminophospholipid necessary for the development of optimal tissue factor (TF) activity at the cell surface. This study investigates the implication of a restricted lipid environment with respect to PhtdSer availability on TF expression and activity. K562 cells, showing a reduced ability to externalize PhtdSer, were transfected with human TF cDNA. PhtdSer exposure and TF activity were examined in transfected cells and compared to monocytic THP-1 cells expressing constitutive and inducible TF or megakaryocytic HEL cells showing a high PhtdSer externalization potency. TF expression was evidenced by flow cytometry and its activity measured using functional assays. PhtdSer exposure was monitored by enzymatic prothrombinase assay. One clone (DC9) expressed a stable amount of TF antigen without global modification of its membrane status. Despite a noticeable TF expression level, clone DC9 presented only a weak TF activity even after ionophore stimulation. The apparent Km, relative to factor X (FX) activation by TF-factor VIIa (FVIIa) complex, was 335 nM versus 70 nM for THP-1 cells. The velocity of the reaction was found 3-fold slower in DC9 than THP-1 cells. Ionophore treatment resulting in slightly enhanced amounts of available PhtdSer abolished this difference. The DC9 clone appears suitable for further investigations on the biology of TF expressed at the surface of cells where the contribution of PhtdSer is significantly attenuated. Such cells should enable further assessment of the role of TF as a receptor coupled to intracellular signaling pathways and its fate during apoptotic cell death.
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PMID:The influence exerted by a restricted phospholipid microenvironment on the expression of tissue factor activity at the cell plasma membrane surface. 1073 87

We have previously demonstrated that the loss of glutathione (GSH) and GSH-peroxidase (GSH-PX) in banked red blood cells (RBCs) is accompanied by oxidative modifications of lipids, proteins and loss of membrane integrity. The objective of this study was to determine whether artificial increases in antioxidant (GSH) or antioxidant enzyme (catalase) content could protect membrane damage in the banked RBCs following an oxidant challenge. RBCs stored at 1-6 degrees C for 0, 42 and 84 days in a conventional additive solution (Adsol) were subjected to oxidative stress using ferric/ascorbic acid (Fe/ASC) before and after enriching them with GSH or catalase using a hypotonic lysis-isoosmotic resealing procedure. This lysis-resealing procedure in the presence of GSH/catalase raised intracellular GSH and catalase concentrations 4-6 fold, yet produced only a small reduction in mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentrations (MCHC). Indicators of oxidative stress and membrane integrity were measured, including acetylcholinesterase (AChE) activity, GSH concentration, phosphatidylserine (PS) externalization (prothrombin-converting activity) and transmembrane lipid movements (14C-lyso phosphatidylcholine flip-flop and PS transport). GSH-enrichment protected AChE activity in fresh (0 day) and stored (42 and 84 days) RBCs from Fe/ASC oxidation by 10, 23 and 26%, respectively, compared with not-enriched controls. Following oxidative stress, the rate of transbilayer lipid flip-flop did not increase in fresh cells, but increased 9.3% in 42-day stored cells. Phosphatidylserine exposure, as measured by prothrombinase activity, increased 2.4-fold in fresh and 5.2-fold in 42-day stored cells exposed to Fe/ASC. Previous studies have shown that 42-day storage causes a moderate decrease in PS transport (approximately 50%), whereas transport rates declined by up to 75% in stored RBCs when challenged with Fe/ASC. GSH-enrichment prevented the increase in passive lipid flip-flop and the increase in prothrombinase activity, but offered no protection against oxidative damage of PS transport. In contrast to these effects, catalase-enrichment failed to protect GSH levels and AChE activity upon oxidative stress. Membrane protein thiol oxidation was assessed by labeling reactive protein thiols with 5-acetalamidofluorescein followed by immunoblotting with antifluorescein antibodies. Significant oxidation of membrane proteins was confirmed by a greater loss of thiols in stored RBCs than in fresh RBCs. These results demonstrate that it may be possible to prevent storage-mediated loss of AChE, increased lipid flip-flop, and increased PS exposure, by maintaining or increasing GSH levels of banked RBCs.
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PMID:Glutathione loading prevents free radical injury in red blood cells after storage. 1120 85

Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.
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PMID:Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin. 1504 12

Phosphatidylserine (PS) plays a crucial role, in the conversion of prothrombin into thrombin by the protease, factor Xa. Physiologically, the conversion occurs in the prothrombinase complex. The question of how water-soluble proteins that normally circulate in plasma bind remains to be unambiguously determined. We previously found that the amphitropic proteins (prothrombin, factors V and Va) penetrate into phospholipid layers. AC polarography has allowed the detection for the first time of insertion of factor Xa into condensed monolayers containing phosphatidylserine (PS) and phosphatidylcholine (PC) either 100% PS or 25% PS in the presence of Ca2+. This observation demonstrates that part of factor Xa can cross the phospholipid polar headgroup/hydrocarbon chain interface. In parallel experiments, radioactive surface measurements permitted measuring binding of tritium-labeled factor Xa onto a PS monolayer and calculate an association constant, 6x10(6) M(-1). Penetration of factor Xa into PS-containing vesicles was investigated also using photoactivable 5-[125I]iodonaphthalene-1-azide, which binds selectively to the lipid embedded domains of the protein. These experiments suggest that Factor Xa penetrates preferentially by its heavy chain, an alternative mode of binding to the commonly accepted binding via its Gla domain. Interaction of factor Xa with PS vesicles also changes its apparent K(m) for S 2222.
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PMID:Interaction of an amphitropic protein (factor Xa) with membrane models in a complex system. 1596 78


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