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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles. 139 Jul 58

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.
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PMID:Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation. 173 85

Human prothrombin and prothrombin fragment 1 were demonstrated to bind to Phenyl-TSK columns in the presence of 5.0 mM calcium ions but not in the presence of either magnesium ions or manganese ions. The calcium-dependent interaction of prothrombin fragment 1 is markedly reduced upon oxidation of approximately one mole of tryptophan per mole of protein. The ability of prothrombin fragment 1 to inhibit prothrombin activation by factor Xa in the presence of calcium ions and phospholipid is also markedly reduced by reaction with N-bromosuccinimide. These results provide the first demonstration of a calcium-specific site in prothrombin outside of the "GLA domain".
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PMID:A hydrophobic site in human prothrombin present in a calcium-stabilized conformer. 319 39

We have examined initial assembly of the extrinsic pathway of blood coagulation on cell surfaces with radiolabeled human factor VIIa and a human fetal lung cell line possessing abundant functional tissue factor activity. Binding of factor VIIa to these cells was observed and was time- and temperature-dependent. Binding of factor VIIa was quantitatively equivalent at 37 and 6 degrees C, although the kinetics of binding differed. The radiolabeled ligand bound by the cell was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel analysis from the factor VIIa offered. Factor VIIa binding was influenced by calcium ions. The binding appears to involve at least two classes of calcium-dependent binding sites. Optimal binding occurred at 2 mM calcium for both classes of sites, and there was inhibition of binding to the high affinity sites at higher calcium. Association of factor VIIa was specific, saturable, had a Kd of 123 +/- 37 pm, and factor VIIa interacted with about 100,000 binding sites per cell. Once established, specific binding was rapidly reversible. Direct cellular binding of human factor X also was observed and was calcium, time- and temperature-dependent. Factor X binding was specific and saturable with half-maximal binding at 87.6 +/- 27.4 nM to 6.03 +/- 1.03 X 10(6) sites per cell. Specific high affinity binding of factor VIIa correlated with generation of factor Xa. A direct linear relationship was observed at low factor VIIa binding; however, at higher bound factor VIIa, the relationship was nonstoichiometric, i.e. less factor Xa was formed per mole of factor VIIa. Expression of specific binding sites for factors VIIa and X provides further substantiation for the molecular assembly hypothesized to initiate the extrinsic coagulation protease cascade on cells.
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PMID:Initiation of the extrinsic pathway of coagulation. Association of factor VIIa with a cell line expressing tissue factor. 349 35

In this paper we describe the effects of the activation peptides prothrombin fragment 1 and fragment 1.2 on factor Xa-catalyzed prothrombin activation. Prothrombin activation in free solution by either factor Xa or factor Xa together with factor Va is unaffected by the activation fragments. When negatively charged phospholipids are present we observed considerable inhibition of prothrombin activation by both fragment 1 and fragment 1.2. For the activation of 0.25 microM prothrombin by factor Xa in the presence of 50 microM phospholipid (phosphatidylserine/phosphatidylcholine, 25/75; mol/mol) and 5 mM CaCl2 50% inhibition was obtained at 0.28 microM fragment 1 or fragment 1.2. Much higher fragment concentrations were required for 50% inhibition of a prothrombinase complex consisting of factor Xa, factor Va, Ca2+ and phospholipid. This shows that factor Va protects prothrombin activation against inhibition by its own activation peptides. Less inhibition by activation fragments was also observed at higher phospholipid and prothrombin concentrations or when the mole fraction phosphatidylserine in the phospholipid vesicles was decreased. The effects of fragment 1 and fragment 1.2 on prothrombin activation were identical throughout all experiments, indicating that the inhibition is due to the gamma-carboxyglutamic acid containing region of the activation peptides. Our observations suggest that the activation fragments inhibit prothrombin activation by competing with prothrombin and factor Xa for binding sites at the phospholipid surface. In such a model factor Va will protect against the inhibition since it is known to promote the assembly of the prothrombinase complex through interactions with factor Xa and prothrombin that are independent of the gla-residues. The kinetic properties of fragment inhibition also suggest that in vivo prothrombin activation will not be affected by the generation of activation peptides.
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PMID:The effects of bovine prothrombin fragment 1 and fragment 1.2 on prothrombin activation. 392 88

The interaction between factor Xa and factor Va was investigated both in solution and in the presence of phospholipid vesicles with varying contents of phosphatidylserine. The binding parameters were inferred from the kinetics of prothrombin activation. Factor Xa and factor Va form in solution an equimolar complex with a dissociation constant of 3.3 X 10(-9) M. Phospholipid vesicles promote the formation of the factor Xa-Va complex. The Kd of complex formation is dependent on both the phospholipid concentration and the composition of the phospholipid vesicle. For the interaction between factor Xa and factor Va in the presence of phospholipid vesicles containing 40 mol % dioleoylphosphatidylserine (DOPS) and 60 mol % dioleoylphosphatidylcholine (DOPC), the Kd increases linearly with increasing phospholipid concentration. In the presence of 10 microM phospholipid (DOPS/DOPC, 40/60 mol/mol) Kd = 3 X 10(-11) M. When the mole percentage of DOPS in the phospholipid vesicles is lowered from 20 to 5 mol %, there is a gradual increase of the Kd. In the presence of 10 microM phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC Kd = 2.8 X 10(-10) M. The Kd measured in the presence of phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC is independent of the phospholipid concentration. Two models are discussed that can quantitatively explain the effect of phospholipid vesicles on the complex formation between factor Xa and factor Va. Studies on the effect of the polypeptides with Mr 80 000 and Mr 94000 of which factor Va is composed on the Kd of the factor Xa-Va complex suggest that factor Xa binding to factor Va requires a Ca2+-mediated interaction between the two polypeptides.
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PMID:Factor Va-factor Xa interaction. Effects of phospholipid vesicles of varying composition. 717 70

Elevation of cytoplasmic Ca2+ levels in human erythrocytes induces a progressive loss of membrane phospholipid asymmetry, a process that is impaired in erythrocytes from a patient with Scott syndrome. We show here that porcine erythrocytes are similarly incapable of Ca2+-induced redistribution of membrane phospholipids. Because a complex of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ has been proposed as the mediator of enhanced transbilayer movement of lipids (J Biol Chem 269:6347,1994), these cell systems offer a unique opportunity for testing this mechanism. Analysis of both total PIP2 content and the metabolic-resistant pool of PIP2 that remains after incubation with Ca2+ ionophore showed no appreciable differences between normal and Scott erythrocytes. Moreover, porcine erythrocytes were found to have slightly higher levels of both total and metabolic-resistant PIP2 in comparison with normal human erythrocytes. Although loading of normal erythrocytes with exogenously added PIP2 gave rise to a Ca2+-induced increase in prothrombinase activity and apparent transbilayer movement of nitrobenzoxadiazolyl (NBD)-phospholipids, these PIP2-loaded cells were also found to undergo progressive Ca2+-dependent cell lysis, which seriously hampers interpretation of these data. Moreover, loading Scott cells with PIP2 did not abolish their impaired lipid scrambling, even in the presence of a Ca2+-ionophore. Finally, artificial lipid vesicles containing no PIP2 or 1 mole percent of PIP2 were indistinguishable with respect to transbilayer movement of NBD-phosphatidylcholine in the presence of Ca2+. Our findings suggest that Ca2+-induced redistribution of membrane phospholipids cannot simply be attributed to the steady-state concentration of PIP2, and imply that such lipid movement is regulated by other cellular processes.
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PMID:The complex of phosphatidylinositol 4,5-bisphosphate and calcium ions is not responsible for Ca2+-induced loss of phospholipid asymmetry in the human erythrocyte: a study in Scott syndrome, a disorder of calcium-induced phospholipid scrambling. 765 25

In studies on the binding of proteins to small unilamellar phospholipid vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solution. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a vesicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentrations of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obtain well-defined initial adsorption rates. Therefore, an off-null technique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation factor Xa was measured in SUV suspensions prepared from a 20% dioleoylphosphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) phospholipid mixture. For prothrombin, a dissociation constant Kd = 140 +/- 27 nM (mean +/- S.E.) and maximal surface concentration gamma max = (8.9 +/- 0.8) x 10(-3) mole of protein per mole of lipid, were obtained. For factor Xa, these values were Kd = 49.6 +/- 6.3 nM and gamma max = (23.0 +/- 1.4) x 10(-3) mole of protein per mole of lipid. These binding parameters are similar to those obtained earlier for planar bilayers. Apparently, the binding of factor Xa and prothrombin is not dependent on surface curvature.
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PMID:Monitoring of unbound protein in vesicle suspensions with off-null ellipsometry. 846 22

The anti-factor Xa activity of the synthetic pentasaccharide SR 90107A/ORG 31540 was assayed by a chromogenic method at pH 8.4 and pH 7.35, comparatively to the 4th International Heparin Standard (IHS) or to the Ist International Low Molecular Weight Heparin Standard (LMWHS). At pH 8.4, SR 90107A/ORG 31540 was found to have a specific anti-factor Xa activity of 639 +/- 14 and 659 +/- 19 IU/mg (mean +/- sem, n = 6) when assayed in comparison with the 4th ISH and the Ist LMWHS respectively. At pH 7.35, the corresponding figures were 864 +/- 6 and 1160 +/- 51 IU/mg (mean +/- sem, n = 6) respectively. The dissociation constants of the ATIII-pentasaccharide complex formed by SR 90107A/ORG 31540 and by two close analogues: SR 80327A and SR 80027A in the presence of purified human ATIII were found to be 41 +/- 8, 96 +/- 1 and 3 +/- 1.4 nM (mean +/- sem, n = 3) respectively. For the three compounds, the pseudo-first order molar catalytic constants for factor Xa inactivation by the ATIII-pentasaccharide complex were shown to be statistically comparable, in the range of 7-8 x 10(7) min-1 per mole. It is concluded that the differences in specific anti-factor Xa activities between SR 90107A/ORG 31540 and its synthetic chemical analogues can be attributed to variations of the dissociation constants whereas the catalytic constants for factor Xa inactivation remain unchanged.
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PMID:Determination of the anti-factor Xa activity of the synthetic pentasaccharide SR 90107A/ORG 31540 and of two structural analogues. 898 27

Blood coagulation factor V (FV) circulates in the blood in two forms designated FV1 and FV2. In model systems containing purified proteins FV1 appears to be more thrombogenic than FV2. Recently, we reported that in plasma from carriers of the R2 haplotype, a polymorphism which encodes several amino acid changes in FV and which is associated with an increased risk of thrombosis, the FV1/FV2 ratio is shifted in favor of the more thrombogenic form FV1. Here we describe in detail the assay that enables quantification of the plasma levels of FV1 and FV2. FV present in highly diluted plasma samples was activated with thrombin and the FVa generated was subsequently quantified in two prothrombinase-based assay systems. In the first assay, which is performed at saturating amounts of FXa and phospholipid vesicles with a high mole fraction phosphatidylserine, FVa1 and FVa2 express the same cofactor activity in prothrombin activation. Hence, this assay quantifies the total FV level (FV1 + FV2) present in plasma. In the second assay, which is performed at suboptimal amounts of FXa and phospholipid vesicles with a low mole fraction phosphatidylserine, FVa2 has approximately an 8-fold higher cofactor activity than FVa1. Therefore, the response in this assay depends on the relative amounts of FV1 and FV2 in the plasma sample. Calibration curves made with samples containing known concentrations of purified FVa1 and FVa2 subsequently allowed calculation of the amounts of FV1 and FV2 present in plasma.
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PMID:An assay to quantify the two plasma isoforms of factor V. 1115 15


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