thromboplastin and 1-2-dioleoylphosphatidylserine

The activation of coagulation factor X by tissue factor (TF) and coagulation factor VIIa (VIIa) on a phospholipid surface is thought to be the key step in the initiation of blood coagulation. In this reaction, the product, fXa, is transiently and reversibly bound to the TF-VIIa enzyme complex. This in effect leads to a probabilistic inhibition of subsequent fX activations; a new fX substrate molecule cannot be activated until the old fXa molecule leaves. In this study, we demonstrate that benzamidine and soybean trypsin inhibitor-conjugated Sepharose beads, which bind fXa and sequester it away from the reaction, serve to enhance fX activation by the TF-VIIa complex. Thus, removal of fXa from the reactive zone, by either flow, fXa sequestration, or binding to distant lipid surfaces, can serve to enhance the levels of TF-VIIa activity. Using resonance energy transfer, we found the dissociation constants of fX and fXa for 100 nm diameter phospholipid vesicles to be on the order of 30-60 nM, consistent with previous measurements employing planar lipid surfaces. On the basis of the measurements of binding of fXa to phospholipid surfaces, we demonstrate that the rates of fX activation by the TF-VIIa complex under a variety of experimental conditions depend inversely on the amount of product (fXa) bound to the TF-phospholipid surface. These data support an inhibitory role for the reaction product, fXa, and indicate that models previously employed in understanding this initial coagulation reaction must now be re-evaluated to account for both the product occupancy of the phospholipid surface and the binding of the product to the enzyme. Moreover, the inhibitory properties of fXa can be described on the basis of the estimated surface density of fXa molecules on the TF-phospholipid surface.

Topics: Benzamidines; Down-Regulation; Factor VIIa; Factor Xa; Humans; Microspheres; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Protein Binding; Substrate Specificity; Thromboplastin; Time Factors; Trypsin Inhibitor, Kunitz Soybean; Up-Regulation

Although the phospholipid requirement for tissue factor (TF) activity has been well-established, the mechanism by which the surface regulates enzymatic activity remains unclear. We added phospholipid vesicles to already relipidated TF (30/70 PS/PC) and found that added lipid can both enhance and inhibit the rate of factor X (F.X) activation. Using active-site-inhibited F.Xa we demonstrate that F.Xa is a more potent inhibitor of TF/VIIa at lower lipid concentrations, and that this inhibition is attributable to high surface occupancy by F.Xa near the enzyme. We also find that exactly twice as many F.Xa molecules are bound to a lipid surface at saturation as F.X, and that a dimer model of F.Xa binding to the lipid can account for the experimentally observed, preferential binding of F.Xa (compared to F.X) to phospholipid surfaces. We manipulated the amount of phospholipid available to each TF molecule by controlling vesicle size and the number of TF molecules per vesicle and found that, as the 2D radius of phospholipid available to each TF molecule was increased, the observed k(cat) increased hyperbolically toward a maximum or "true k(cat)". At a 2D lipid radius of approximately 37 nm, the observed k(cat) was 50% of the "true k(cat)". Thus, phospholipid surface serves as a conduit for F.X presentation and F.Xa removal, and the rate at which F.Xa leaves the vicinity of the enzyme, either by lateral diffusion or desorption from the surface, regulates the rate of F.X activation. We argue that these findings require reevaluation of existing models of coagulation.

Topics: Binding, Competitive; Catalysis; Dimerization; Enzyme Activation; Factor VIIa; Factor X; Factor Xa; Factor Xa Inhibitors; Humans; Kinetics; Lipid Bilayers; Models, Chemical; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Protein Binding; Substrate Specificity; Thromboplastin; Titrimetry

This is a study of the morphology and transbilayer lipid distribution of human erythrocytes treated with chlorpromazine (CPZ) over extended time courses. At 0 degree C, treatment of dilauroylphosphatidyl[1-14C]choline-labeled erythrocytes with 120 microM CPZ produced an immediate stomatocytic transformation (t1/2 < 5 min) with no concurrent change in transbilayer distribution of radiolabeled lipid, as determined by bovine serum albumin extractability. At 37 degrees C, CPZ treatment of cells produced two sequential morphological effects: immediate stomatocytosis (t1/2 < 1 min) with no concurrent change in radiolabel transbilayer distribution, followed by gradual increase in stomatocytic extent over several hours, with concurrent redistribution of radiolabeled lipid to the inner monolayer. Cells pretreated with vanadate at 37 degrees C exhibited a triphasic morphological response: CPZ produced immediate stomatocytosis, followed by a transient reversion to echinocytes lasting about 2 h, before returning to stomatocytic morphologies over the next several hours. The echinocytic reversion was accompanied by exposure of phosphatidylserine on the cell surface, as indicated by increased activation of exogenous prothrombinase. These findings suggest that while CPZ induces transbilayer lipid redistribution over extended time periods (which may mediate the complex morphological transformations observed), the early stomatocytic response elicited by addition of CPZ is not due to lipid reorganization.

Topics: Antipsychotic Agents; Cell Size; Chlorpromazine; Erythrocytes; Humans; Membrane Lipids; Phosphatidylcholines; Phosphatidylserines; Serum Albumin, Bovine; Thromboplastin; Vanadates

The influence of different neutral phospholipids and cholesterol on the procoagulant properties of sonicated vesicles containing phosphatidylserine was studied, using the prothrombinase assay. When incorporated into membranes composed of phosphatidylcholine and phosphatidylserine, a stimulating effect of phosphatidylethanolamine and an inhibiting effect of sphingomyelin was observed. Cholesterol slightly increased the activities of all vesicles tested. In lipid vesicles with a composition mimicking that of the outer leaflet of the plasma membrane of the activated platelet, the inhibitory effect of sphingomyelin was overruled by an overall stimulatory effect of phosphatidylethanolamine, suggesting an accessory role for phosphatidylethanolamine in the procoagulant properties of activated platelets.

Topics: Cholesterol; Humans; In Vitro Techniques; Lipid Bilayers; Phosphatidylserines; Phospholipids; Platelet Activation; Prothrombin; Sonication; Sphingomyelins; Thromboplastin

The kinetics of inhibition of prothrombinase during prothrombin conversion by antithrombin and antithrombin-heparin complexes was studied in a tubular flow reactor. Prothrombinase was assembled at a macroscopic phospholipid membrane, composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine, deposited on the inner wall of a glass capillary, by perfusion with a factor Xa-factor Va mixture. Measurement of thrombin production allowed estimation of the amount of prothrombinase present at the capillary wall. Perfusion with a mixture of prothrombin and antithrombin or antithrombin-heparin complexes caused a progressive decline of the prothrombinase activity. The rate of inactivation steeply decreased with increasing prothrombin concentrations, indicating competitive inhibition. Analysis of competitive inhibition data requires estimation of the time-dependent substrate concentration, Co, near the prothrombin converting surface using earlier developed transport theory [Billy, D., et al. (1995) J. Biol. Chem. 270, 1029-1034]. It appears that the inhibition rate is proportional to the fraction of enzyme, Km/(Km+Co), not occupied by substrate. The value of Km of prothrombinase estimated from the dependence of the inhibition rate on the prothrombin concentration (Km = 2-3 nM) is in excellent agreement with the value estimated from the substrate conversion rate (Km = 3 nM). Therefore inhibition of prothrombinase by antithrombin and antithrombin-heparin complexes is fully competitive with the substrate: prothrombin. Our results show that prothrombinase assembled on macroscopic lipid surfaces by virtue of its low Km value is protected for inhibition due to highly effective competition of prothrombin with antithrombin for the active site of factor Xa.

Topics: Animals; Antithrombins; Binding, Competitive; Capillary Action; Cattle; Factor Va; Humans; Kinetics; Mathematics; Models, Theoretical; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Prothrombin; Thromboplastin; Time Factors

The activation of prothrombin is catalyzed by prothrombinase, a complex of factor Xa and factor Va assembled on a negatively charged phospholipid membrane. We used a tubular flow reactor to identify the relative contributions of factor Va, prothrombin, and the negatively charged phosphatidylserine to the assembly of prothrombinase. Perfusion of phospholipid-coated capillaries with a mixture of factor Xa, factor Va, and prothrombin resulted in a steady-state rate of thrombin production that increased with (i) the phosphatidylserine content of the phospholipid bilayer, (ii) the factor Va concentration, and, most interestingly, (iii) the prothrombin concentration of the perfusion solution. Incorporation of 20 mol % phosphoatidylethanolamine, a phospholipid with poor ability to promote prothrombinase activity, into a 5 mol % phosphatidylserine membrane also increased the steady-state rate of thrombin production. Direct measurements of the amount of prothrombinase in the flow reactor demonstrated that increased catalytic activities were the result of an increased steady-state amount of membrane-associated prothrombinase. Thus, similar turnover numbers of prothrombin activation (3100 min-1) were calculated, irrespective of the phosphatidylserine content of the membrane. We established for membranes with low phosphatidylserine content (< 10 mol%) a linear relationship between the prothrombinase activity and the arithematical product of the factor Va concentration in the perfusion solution and the prothrombin concentration near the catalytic surface. Our results indicate that, in addition to factor Va, prothrombin also is essential to the assembly of prothrombinase at macroscopic surfaces with low phosphatidylserine content. The data further suggest that the prothrombin concentration near the surface, controlled by the prothrombinase activity and mass transfer, is an important regulator of the prothrombinase surface density.

Topics: Factor Va; Factor Xa; Humans; In Vitro Techniques; Kinetics; Membrane Lipids; Perfusion; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Prothrombin; Surface Properties; Thrombin; Thromboplastin

The response of coagulation tests to heparin can be expressed as the coagulation time of plasma containing heparin divided by the coagulation time of the same plasma without heparin (CT ratio). The purpose of the present study was to assess the influence of liposomes on these response to heparin of four coagulation tests: the kaolin-induced coagulation time, the tissue factor-induced coagulation time, the factor Xa-induced coagulation time, and the thrombin-induced coagulation time. Liposomes were prepared from dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and dioleoylphosphatidylserine (DOPS). High concentrations of DOPS/DOPE/DOPC (20:40:40) liposomes enhanced the CT ratio of the four coagulation tests, more than DOPS/DOPC (20:80) or DOPE/DOPC (40:60) or a mixture of DOPS/DOPC and DOPE/DOPC liposomes. These experiments demonstrate that there is synergism between DOPS and DOPE in promoting heparin's anticoagulant effect if both phospholipids are incorporated into the same liposome surface.

Topics: Blood Coagulation; Blood Coagulation Tests; Drug Synergism; Heparin; Humans; Kaolin; Liposomes; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Thromboplastin

Planar-supported phospholipid bilayers formed by the adsorption of vesicles are increasingly used in the investigation of lipid-dependent reactions. We have studied the way in which these bilayers are formed with phospholipid vesicles containing the transmembrane protein Tissue Factor (TF). TF complexed with the serine protease, factor VIIa, is the primary initiator of blood coagulation by way of activation of the zymogen factor X. TF has been shown to orient randomly on the inner and outer leaflets of vesicles. We used proteolytic digestion to produce vesicles in which the extracellular domain of TF is located on the inner leaflet. These vesicles show no cofactor activity for factor VIIa as a result of the inability of the extracellular domain of TF to bind VIIa. After freeze/thawing, 50% of the cofactor activity was regained, indicating reorientation of the sequestered, inner leaflet TF. Adsorption of these vesicles to the inner surface of glass microcapillaries results in a continuous phospholipid bilayer. The microcapillaries were perfused with a solution of factors VIIa and X, and the effluent was monitored for factor Xa production, a sensitive measure of the activity of the TF-VIIa complex. For coatings produced with the digested vesicles, minimal TF-VIIa activity was observed, showing that the supported bilayer preserves the orientation of the leaflets in the vesicles, i.e., the outer leaflet of the vesicles forms the outer leaflet of the supported bilayer.

Topics: Adsorption; Biophysical Phenomena; Biophysics; Factor VIIa; Factor X; Humans; In Vitro Techniques; Lipid Bilayers; Membrane Proteins; Models, Chemical; Molecular Probes; Phosphatidylcholines; Phosphatidylserines; Phospholipids; Thromboplastin

Blood coagulation is initiated on cells which present a macroscopic surface to the flowing blood stream. We have used a continuous flow enzyme reactor to model this system and to investigate the effects of shear rate and mass transport on the activation of factor X by the complex of the transmembrane protein, tissue factor, and the serine protease, factor VIIa. This initial step of blood coagulation was found to be half-maximal at very low enzyme densities (0.03-0.06%) on the wall of the capillaries. In agreement with hydrodynamic theory, the apparent Km in the flow reactor was correlated with the cube root of the wall shear rate. These data indicate that at high tissue factor densities (> 0.6%) the activation of 150 nM factor X is controlled by the flux of X toward the surface, which is controlled by wall shear rate and substrate concentration. The appearance of the product, Xa, in the effluent was delayed to 8-12 min, which was caused by high-affinity binding of Xa to the phospholipid. This delay was considerably shortened by embedding tissue factor into PC or by coating the PS/PC surface with the phospholipid binding protein, annexin V. At low tissue factor densities, annexin V inhibited X activation by 45%, while no inhibition was observed at high densities. We demonstrate that when the reaction is limited by substrate flux, addition of further enzyme does not increase reaction rates. This contrasts with classical three-dimensional catalysis in which the initial velocity is ordinarily linear with the enzyme concentration.

Topics: Amino Acid Sequence; Annexin A5; Biological Transport; Catalysis; Enzymes, Immobilized; Factor VIIa; Factor X; Humans; Kinetics; Molecular Sequence Data; Phosphatidylcholines; Phosphatidylserines; Rheology; Thromboplastin