154 results on '"Zwaal RF"'
Search Results
2. Long-term fatty acid modification of endothelial cells: implications for arachidonic acid distribution in phospholipid classes.
- Author
-
Vossen, RC, primary, Feijge, MA, additional, Heemskerk, JW, additional, van Dam-Mieras, MC, additional, Hornstra, G, additional, and Zwaal, RF, additional
- Published
- 1993
- Full Text
- View/download PDF
3. Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome
- Author
-
Bevers, EM, primary, Wiedmer, T, additional, Comfurius, P, additional, Shattil, SJ, additional, Weiss, HJ, additional, Zwaal, RF, additional, and Sims, PJ, additional
- Published
- 1992
- Full Text
- View/download PDF
4. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles
- Author
-
Tans, G, primary, Rosing, J, additional, Thomassen, MC, additional, Heeb, MJ, additional, Zwaal, RF, additional, and Griffin, JH, additional
- Published
- 1991
- Full Text
- View/download PDF
5. Enzymatic synthesis of phosphatidylserine on small scale by use of a one-phase system
- Author
-
Comfurius, P, primary, Bevers, EM, additional, and Zwaal, RF, additional
- Published
- 1990
- Full Text
- View/download PDF
6. The role of activated human platelets in prothrombin and factor X activation
- Author
-
Rosing, J, van Rijn, JL, Bevers, EM, van Dieijen, G, Comfurius, P, and Zwaal, RF
- Abstract
The effect of activated human platelets in intrinsic factor X activation was compared with their effect in prothrombin activation. Compared with unstimulated platelets, platelets triggered by the combined action of collagen plus thrombin showed a tenfold activity increase in prothrombin activation, and a 20-fold rate enhancement in factor X activation. Treatment of collagen plus thrombin-stimulated platelets with N.naja phospholipase A2 almost completely abolished their activity in prothrombin and factor X activation. Since no significant cell lysis occurs during phospholipase treatment, this indicates that platelet phospholipids, exposed at the membrane exterior, play an essential role in the interaction of platelets with the proteins of the prothrombin and factor X-activating complexes. The time course of generation of the procoagulant platelet surface was different when the amount of coagulation factors present in the assay systems was varied. At suboptimal concentrations of coagulation factors, maximum platelet activity was reached after a shorter time period than at saturating concentrations. When measured at suboptimal amounts of coagulation factors, the platelet activity in prothrombin and factor X activation is also more sensitive to phospholipase treatment. Experiments with synthetic phospholipid mixtures show that prothrombin and factor X activation are optimal at low mol% phosphatidylserine when high concentrations of factor Va and factor VIIIa are employed. The optimal mol% phosphatidylserine increases when the concentrations of nonenzymatic protein cofactors are lowered. These findings are discussed in relation to a model in which phosphatidylserine, exposed at the outer surface of activated platelets, plays an essential role in prothrombin and factor X activation. It is proposed that this phosphatidylserine is not homogeneously distributed in the platelet outer membrane, but that areas with different phosphatidylserine density participate in coagulation factor activation.
- Published
- 1985
- Full Text
- View/download PDF
7. Impaired factor X and prothrombin activation associated with decreased phospholipid exposure in platelets from a patient with a bleeding disorder
- Author
-
Rosing, J, Bevers, EM, Comfurius, P, Hemker, HC, van Dieijen, G, Weiss, HJ, and Zwaal, RF
- Abstract
Platelets from a platelet factor 3-deficient patient, which was first described by Weiss et al (Am J Med 67:206, 1979), were found to be equally impaired in their ability to promote factor X and prothrombin activation. Compared to normal platelets, the patient's platelets showed upon stimulation with thrombin plus collagen a much slower generation and a considerably lower level of platelet prothrombin- and factor X-converting activities. Treatment of stimulated platelets with phospholipases revealed a decreased exposure of negatively charged phospholipid at the outer surface of the patient's platelets, relative to control's. We suggest that the combined impairment of prothrombin- and factor X-converting activities in this patient is due to a defect in the mechanism by which phosphatidylserine becomes exposed at the outer surface of stimulated platelets.
- Published
- 1985
- Full Text
- View/download PDF
8. Platelet Phospholipid Asymmetry and its Significance in Hemostasis
- Author
-
Bevers Em and Zwaal Rf
- Subjects
chemistry.chemical_compound ,medicine.medical_specialty ,Endocrinology ,chemistry ,Hemostasis ,Internal medicine ,Phospholipid ,medicine ,Platelet - Published
- 1983
9. Direct inhibition of phospholipid scrambling activity in erythrocytes by potassium ions.
- Author
-
Wolfs JL, Comfurius P, Bekers O, Zwaal RF, Balasubramanian K, Schroit AJ, Lindhout T, and Bevers EM
- Subjects
- Calcium metabolism, Cell Shape, Erythrocyte Membrane drug effects, Erythrocytes cytology, Erythrocytes drug effects, Humans, Ionomycin pharmacology, Ionophores pharmacology, Ions metabolism, Sodium metabolism, Thromboplastin metabolism, Erythrocyte Membrane chemistry, Erythrocyte Membrane metabolism, Erythrocytes metabolism, Membrane Lipids metabolism, Phosphatidylserines metabolism, Phospholipid Transfer Proteins metabolism, Potassium metabolism
- Abstract
The exposure of phosphatidylserine (PS) at the cell surface plays a critical role in blood coagulation and serves as a macrophage recognition moiety for the engulfment of apoptotic cells. Previous observations have shown that a high extracellular [K(+)] and selective K(+) channel blockers inhibit PS exposure in platelets and erythrocytes. Here we show that the rate of PS exposure in erythrocytes decreases by approximately 50% when the intracellular [K(+)] increases from 0 to physiological concentrations. Using resealed erythrocyte membranes, we further show that lipid scrambling is inducible by raising the intracellular [Ca(2+)] and that K(+) ions have a direct inhibitory effect on this process. Lipid scrambling in resealed ghosts occurs in the absence of cell shrinkage and microvesicle formation, processes that are generally attributed to Ca(2+)-induced lipid scrambling in intact erythrocytes. Thus, opening of Ca(2+)-sensitive K(+) channels causes loss of intracellular K(+) that results in reduced intrinsic inhibitory effect of these ions on scramblase activity.
- Published
- 2009
- Full Text
- View/download PDF
10. Reversible inhibition of the platelet procoagulant response through manipulation of the Gardos channel.
- Author
-
Wolfs JL, Wielders SJ, Comfurius P, Lindhout T, Giddings JC, Zwaal RF, and Bevers EM
- Subjects
- Annexin A5 chemistry, Apamin pharmacology, Calcium metabolism, Cell Membrane metabolism, Charybdotoxin pharmacology, Clotrimazole pharmacology, Humans, Ionophores pharmacology, Peptides pharmacology, Phosphatidylserines chemistry, Potassium chemistry, Thromboplastin metabolism, Valinomycin pharmacology, Blood Coagulation Factors metabolism, Blood Platelets metabolism
- Abstract
The platelet procoagulant response requires a sustained elevation of the intracellular Ca2+ concentration, [Ca2+]i, causing exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. An increased [Ca2+]i also activates Ca2+-dependent K+ channels. Here, we investigated the contribution of the efflux of K+ ions on the platelet procoagulant response in collagen-thrombin-activated platelets using selective K+ channel blockers. The Gardos channel blockers clotrimazol, charybdotoxin, and quinine caused a similar decrease in prothrombinase activity as well as in the number of PS-exposing platelets detected by fluorescence-conjugated annexin A5. Apamin and iberiotoxin, inhibitors of other K+ channels, were without effect. Only clotrimazol showed a significant inhibition of the collagen-plus-thrombin-induced intracellular calcium response. Clotrimazol and charybdotoxin did not inhibit aggregation and release under the conditions used. Inhibition by Gardos channel blockers was reversed by valinomycin, a selective K+ ionophore. The impaired procoagulant response of platelets from a patient with Scott syndrome was partially restored by pretreatment with valinomycin, suggesting a possible defect of the Gardos channel in this syndrome. Collectively, these results provide evidence for the involvement of efflux of K+ ions through Ca2+-activated K+ channels in the procoagulant response of platelets, opening potential strategies for therapeutic interventions.
- Published
- 2006
- Full Text
- View/download PDF
11. Activated scramblase and inhibited aminophospholipid translocase cause phosphatidylserine exposure in a distinct platelet fraction.
- Author
-
Wolfs JL, Comfurius P, Rasmussen JT, Keuren JF, Lindhout T, Zwaal RF, and Bevers EM
- Subjects
- Annexin A5 metabolism, Antigens, Surface metabolism, Collagen pharmacology, Flow Cytometry, Fluorescein-5-isothiocyanate, Humans, Ionomycin pharmacology, Membrane Proteins agonists, Membrane Proteins antagonists & inhibitors, Milk Proteins metabolism, Phospholipid Transfer Proteins agonists, Phospholipid Transfer Proteins antagonists & inhibitors, Platelet Activation, Thrombin pharmacology, Thromboplastin metabolism, Blood Platelets metabolism, Membrane Proteins metabolism, Phosphatidylserines metabolism, Phospholipid Transfer Proteins metabolism
- Abstract
Platelet procoagulant activity is mainly determined by the extent of surface-exposed phosphatidylserine (PS), controlled by the activity of aminophospholipid translocase and phospholipid scramblase. Here, we studied both transport activities in single platelets upon stimulation with various agonists. Besides the formation of procoagulant microparticles, the results show that a distinct fraction of the platelets exposes PS when stimulated. The extent of PS exposure in these platelet fractions was similar to that in platelets challenged with Ca2+-ionophore, where all cells exhibit maximal attainable PS exposure. The size of the PS-exposing fraction depends on the agonist and is proportional to the platelet procoagulant activity. Scramblase activity was observed only in the PS-exposing platelet fraction, whereas translocase activity was exclusively detectable in the fraction that did not expose PS. We conclude that, irrespective of the agonist, procoagulant platelets exhibit maximal surface exposure of PS by switching on scramblase and inhibiting translocase activity.
- Published
- 2005
- Full Text
- View/download PDF
12. Surface exposure of phosphatidylserine in pathological cells.
- Author
-
Zwaal RF, Comfurius P, and Bevers EM
- Subjects
- Antiphospholipid Syndrome metabolism, Apoptosis physiology, Cell Membrane metabolism, Eukaryotic Cells pathology, Hematologic Diseases metabolism, Humans, Infections metabolism, Kidney Diseases metabolism, Membrane Transport Proteins metabolism, Membrane Transport Proteins physiology, Metabolic Diseases metabolism, Neoplasms metabolism, Phosphatidylserines physiology, Respiratory Tract Diseases metabolism, Eukaryotic Cells metabolism, Phosphatidylserines metabolism
- Abstract
The asymmetric phospholipid distribution in plasma membranes is normally maintained by energy-dependent lipid transporters that translocate different phospholipids from one monolayer to the other against their respective concentration gradients. When cells are activated, or enter apoptosis, lipid asymmetry can be perturbed by other lipid transporters (scramblases) that shuttle phospholipids non-specifically between the two monolayers. This exposes phosphatidylserine (PS) at the cells' outer surface. Since PS promotes blood coagulation, defective scramblase activity upon platelet stimulation causes a bleeding disorder (Scott syndrome). PS exposure also plays a pivotal role in the recognition and removal of apoptotic cells via a PS-recognizing receptor on phagocytic cells. Furthermore, expression of PS at the cell surface can occur in a wide variety of disorders. This review aims at highlighting how PS expression in different cells may complicate a variety of pathological conditions, including those that promote thromboembolic complications or produce aberrations in apoptotic cell removal.
- Published
- 2005
- Full Text
- View/download PDF
13. Anti-prothrombin IgG from patients with anti-phospholipid antibodies inhibits the inactivation of factor Va by activated protein C.
- Author
-
Galli M, Willems GM, Rosing J, Janssen RM, Govers-Riemslag JW, Comfurius P, Barbui T, Zwaal RF, and Bevers EM
- Subjects
- Case-Control Studies, Female, Humans, Lupus Coagulation Inhibitor immunology, Lupus Erythematosus, Systemic immunology, Male, Stroke immunology, Venous Thrombosis immunology, Antibodies, Antiphospholipid immunology, Antiphospholipid Syndrome immunology, Factor Va immunology, Immunoglobulin G blood, Protein C immunology, Prothrombin immunology
- Abstract
Interference of anti-phospholipid antibodies with the protein C pathway has been suggested to play a role in the development of thrombosis in the anti-phospholipid syndrome. We studied the effect of IgG preparations containing anti-prothrombin antibodies of 17 lupus anticoagulant-positive patients and 12 controls on the inactivation of factor Va (FVa) by activated protein C (APC) in a system with purified coagulation factors. Test IgG was incubated with human prothrombin, phospholipid vesicles and CaCl(2). Protein S, FVa and APC were added and the APC-dependent loss of FVa activity was monitored over time. The residual amount of FVa remaining after 10 min was 14 +/- 4% (mean +/- SD) when 1.5 mg/ml normal IgG was present and ranged between 17% and 82% with 1.5 mg/ml patient IgG. Twelve patients IgG gave values of residual FVa >22% (i.e. 2 SD above the mean of controls), indicating that APC-mediated inactivation of FVa was significantly inhibited. The inhibition was strictly dependent on the presence of prothrombin, proportional to the concentration of IgG and strongly diminished at a 20-fold higher phospholipid concentration. Most, although not all, IgG containing anti-prothrombin antibodies inhibit the APC-catalysed FVa inactivation, which may contribute to the increased risk of thrombosis in patients with the anti-phospholipid syndrome.
- Published
- 2005
- Full Text
- View/download PDF
14. Quantitative determination of the binding of beta2-glycoprotein I and prothrombin to phosphatidylserine-exposing blood platelets.
- Author
-
Bevers EM, Janssen MP, Comfurius P, Balasubramanian K, Schroit AJ, Zwaal RF, and Willems GM
- Subjects
- Binding Sites, Blood Platelets drug effects, Blood Platelets metabolism, Flow Cytometry methods, Humans, Ionomycin pharmacology, Osmolar Concentration, Protein Binding, beta 2-Glycoprotein I, Blood Platelets chemistry, Glycoproteins metabolism, Phosphatidylserines metabolism, Prothrombin metabolism
- Abstract
The plasma protein beta2GPI (beta2-glycoprotein I) has been proposed to mediate phagocytosis of apoptotic cells and to play a role in the antiphospholipid syndrome. This suggestion is based mainly on the presumption that beta2GPI has an appreciable interaction with PS (phosphatidylserine)-exposing cell membranes. However, quantitative data on the binding of beta2GPI to PS-exposing cells under physiologically relevant conditions are scarce and conflicting. Therefore we evaluated the binding of beta2GPI to PS-expressing blood platelets. Flow cytometry showed that binding of beta2GPI is negligible at physiological ionic strength, in contrast with significant binding occurring at low ionic strength. Binding parameters of beta2GPI and (for comparison) prothrombin were quantified by ellipsometric measurement of protein depletion from the supernatant following incubation with platelets. At low ionic strength (20 mM NaCl, no CaCl2), a dissociation constant (K(d)) of 0.2 microM was found for beta2GPI, with 7.4x10(5) binding sites per platelet. Under physiologically relevant conditions (120 mM NaCl and 3 mM CaCl2), binding of beta2GPI was not detectable (extrapolated K(d)>80 microM). Prothrombin binding (at 3 mM CaCl2) was much less affected by ionic strength: K(d) values of 0.5 and 1.4 muM were observed at 20 and 120 mM NaCl respectively. The low affinity and the presence of many lipid-binding proteins in plasma that can compete with the binding of beta2GPI suggest that only a small fraction (<5%) of the binding sites on PS-exposing blood cells are likely to be occupied by beta2GPI. These findings are discussed in relation to the alleged (patho-)physiological functions of beta2GPI.
- Published
- 2005
- Full Text
- View/download PDF
15. The effect of phospholipids on the formation of immune complexes between autoantibodies and beta2-glycoprotein I or prothrombin.
- Author
-
Bevers EM, Zwaal RF, and Willems GM
- Subjects
- Animals, Antibody Affinity, Antigen-Antibody Complex, Glycoproteins chemistry, Humans, Models, Immunological, beta 2-Glycoprotein I, Antibodies, Antiphospholipid immunology, Glycoproteins immunology, Lipid Bilayers immunology, Prothrombin immunology
- Abstract
In the last decennium, it became clear that antiphospholipid antibodies found in patients with antiphospholipid syndrome (APS) are in fact antibodies against lipid-bound plasma proteins. The most frequently occurring antigens are beta2-glycoprotein I and prothrombin, although several other lipid-bound plasma proteins have been reported as antigen for antiphospholipid antibodies. Both proteins bind to anionic phospholipids, mainly phosphatidylserine, which becomes exposed at the surface of activated platelets, apoptotic cells, or cell-derived microparticles. The binding of beta2-glycoprotein I and prothrombin to these cell surfaces or to artificial lipid vesicles with comparable amounts of anionic phospholipids is rather weak. Antiphospholipid antibodies from patients are predominantly of low affinity regarding their interaction with beta2-glycoprotein I or prothrombin in solution. In the presence of a suitable phospholipid surface, however, this interaction is strongly enhanced. There is now strong evidence that formation of bivalent, trimolecular immune complexes at the lipid membrane essentially contributes to the binding of these intrinsically low affinity patient antibodies. Depending on the affinity, the epitope specificity, and the polyclonality of a particular IgG preparation, multimeric structures of lipid-bound immune complexes may form a lattice with multiple interactions on the lipid (cell) surface. It is hypothesized that the functional activity, that is, the ability of antibodies to interfere with lipid-dependent reactions, not only depends on their affinity for the antigen, but also on their ability to form multiple interconnected bivalent trimolecular complexes at the lipid (or cell) surface. It is further proposed that the rate of desorption of immune complexes may present a better indicator for the functional properties of the antibodies than the amount of adsorbed immune complexes.
- Published
- 2004
- Full Text
- View/download PDF
16. Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids.
- Author
-
Zwaal RF, Comfurius P, and Bevers EM
- Subjects
- Humans, Membrane Lipids physiology, Phospholipids physiology, Syndrome, Blood Coagulation Disorders metabolism, Membrane Lipids metabolism, Phospholipids metabolism
- Abstract
Normal quescent cells maintain membrane lipid asymmetry by ATP-dependent membrane lipid transporters, which shuttle different phospholipids from one leaflet to the other against their respective concentration gradients. When cells are challenged, membrane lipid asymmetry can be perturbed resulting in exposure of phosphatidylserine [PS] at the outer cell surface. Translocation of PS from the inner to outer membrane leaflet of activated blood platelets and platelet-derived microvesicles provides a catalytic surface for interacting coagulation factors. This process is dramatically impaired in Scott syndrome, a rare congenital bleeding disorder, underscoring the indispensible role of PS in hemostasis. This also testifies to a defect of a protein-catalyzed scrambling of membrane phospholipids. The Scott phenotype is not restricted to platelets, but can be demonstrated in other blood cells as well. The functional aberrations observed in Scott syndrome have increased our understanding of transmembrane lipid movements, and may help to identify the molecular elements that promote the collapse of phospholipid asymmetry during cell activation and apoptosis.
- Published
- 2004
- Full Text
- View/download PDF
17. Influence of erythrocyte shape on the rate of Ca2+-induced scrambling of phosphatidylserine.
- Author
-
Wolfs JL, Comfurius P, Bevers EM, and Zwaal RF
- Subjects
- 4-Chloro-7-nitrobenzofurazan pharmacology, Calcium pharmacology, Carrier Proteins drug effects, Carrier Proteins metabolism, Cell Size drug effects, Cells, Cultured, Chlorpromazine pharmacology, Detergents pharmacology, Dibucaine pharmacology, Erythrocytes drug effects, Humans, Ionophores pharmacology, Lidocaine pharmacology, Membrane Proteins drug effects, Membrane Proteins metabolism, Phosphatidylcholines pharmacology, Secretory Vesicles metabolism, Thromboplastin drug effects, Thromboplastin metabolism, 4-Chloro-7-nitrobenzofurazan analogs & derivatives, Calcium metabolism, Erythrocytes cytology, Erythrocytes metabolism, Phosphatidylserines metabolism, Phospholipid Transfer Proteins
- Abstract
Membrane-perturbing agents that cause transformation of biconcave erythrocytes into echinocytes or stomatocytes were used to investigate the influence of erythrocyte shape on the rate of Ca(2+)-induced scrambling of phospholipids. Erythrocytes were treated with a variety of lipid-soluble compounds to induce these shape changes, followed by incubation with calcium and ionomycin to activate lipid scramblase. Prothrombinase activity of the cells was used to monitor the rate of surface exposure of phosphatidylserine, which is taken as a measure of scramblase activity. Echinocytes show an enhanced rate of scrambling, whereas stomatocytes show a reduced rate, relative to normocytes. This phenomenon appears to correlate with enhanced and diminished micro-exovesicle shedding from echinocytes and stomatocytes, respectively. It is concluded that the rate of calcium-induced phosphatidylserine exposure (rate of lipid scrambling) in erythrocytes depends for a considerable part on the cells' ability to form microvesicles.
- Published
- 2003
18. Kinetics of prothrombin-mediated binding of lupus anticoagulant antibodies to phosphatidylserine-containing phospholipid membranes: an ellipsometric study.
- Author
-
Willems GM, Janssen MP, Comfurius P, Galli M, Zwaal RF, and Bevers EM
- Subjects
- Biosensing Techniques, Humans, Immunoassay, Immunoglobulin G metabolism, Immunosorbents, Kinetics, Protein Binding immunology, Prothrombin immunology, Silicon, Antibodies, Antiphospholipid metabolism, Binding Sites, Antibody, Lipid Bilayers metabolism, Lupus Coagulation Inhibitor immunology, Phosphatidylserines metabolism, Prothrombin chemistry, Prothrombin physiology
- Abstract
Antiphospholipid antibodies interact with phospholipid membranes via lipid binding plasma proteins, mostly, prothrombin and beta(2)-glycoprotein I. Using ellipsometry, we characterized prothrombin-mediated binding of lupus anticoagulant (LA) positive IgG, isolated from patients with antiphospholipid syndrome, to phosphatidylserine (PS)-containing membranes. LA IgG did not bind to membranes in the absence of prothrombin, but addition of prothrombin resulted in high-affinity binding of prothrombin-LA IgG complexes; half-maximal binding was attained at IgG and prothrombin concentrations of 10 microg/mL and 4 nM, respectively. Adsorption to membranes containing 10-40 mol % PS revealed that membrane-bound rather than solution-phase prothrombin determines the adsorption kinetics. Depletion of prothrombin and LA IgG from the solution results in rapid desorption which is strongly inhibited by addition of prothrombin but not of LA IgG. Prothrombin-mediated adsorption of monovalent Fab1 fragments prepared from patient LA IgG was negligible, indicating that monovalent interaction between prothrombin and LA IgG is weak. The kinetics of adsorption and desorption indicate that divalent binding of LA IgG to prothrombin at the lipid membrane occurs.
- Published
- 2002
- Full Text
- View/download PDF
19. Anticoagulant and membrane-degrading effects of secretory (non-pancreatic) phospholipase A2 are inhibited in plasma.
- Author
-
Billy D, Speijer H, Zwaal RF, Hack EC, and Hermens WT
- Subjects
- Blood Platelets ultrastructure, Fibrin metabolism, Humans, Kinetics, Models, Biological, Phosphatidylserines metabolism, Phospholipases A antagonists & inhibitors, Phospholipases A metabolism, Phospholipases A2, Phospholipids metabolism, Platelet Activation drug effects, Thrombin antagonists & inhibitors, Thrombin biosynthesis, Blood Coagulation drug effects, Lipid Bilayers metabolism, Phospholipases A pharmacology, Plasma metabolism
- Abstract
Plasma concentrations of secretory (non-pancreatic) phospholipase A2 (sPLA2) may rise 1000-fold during inflammation, and this acute phase response has been related to anticoagulant effects. In the present study this hypothesis was further investigated. Prothrombinase activity was measured for model membranes mimicking the phospholipid composition of the outer membrane of resting and activated blood platelets. Using ellipsometry, membrane degradation by sPLA2 could be measured simultaneously with inhibition of thrombin production. The same technique was used to study clotting, by the sudden appearance of fibrin strands on the membrane. Results were compared with the effects of sPLA2 on the activation of washed platelets and platelets in plasma. In buffer solution, model membranes were degraded by (patho)physiological concentrations of sPLA2. Even when only partially degraded, membranes rapidly lost their prothrombinase activity, indicating preferential degradation of phosphatidylserine. Addition of diluted plasma interfered with membrane degradation, and also with inhibition of prothrombinase activity. In agreement with these observations, sPLA2 inhibited thrombin production and annexin V-binding of activated washed platelets, but had no effects on platelet activation or clotting in plasma. These findings indicate that the elevated plasma sPLA2 concentrations observed in inflammatory disease will not reduce hypercoagulability in such patients.
- Published
- 2002
20. Comparison between Ca2+-induced scrambling of various fluorescently labelled lipid analogues in red blood cells.
- Author
-
Dekkers DW, Comfurius P, Bevers EM, and Zwaal RF
- Subjects
- Erythrocytes drug effects, Humans, In Vitro Techniques, Kinetics, Lipid Bilayers, Membrane Lipids blood, Phospholipase D blood, Structure-Activity Relationship, 4-Chloro-7-nitrobenzofurazan analogs & derivatives, 4-Chloro-7-nitrobenzofurazan blood, Calcium pharmacology, Erythrocyte Membrane metabolism, Erythrocytes metabolism, Fluorescent Dyes pharmacokinetics, Glycerophospholipids blood, Ionomycin pharmacology
- Abstract
Treatment of red blood cells with calcium and ionomycin causes activation of the lipid scramblase, a putative membrane protein catalysing flip-flop of (phospho)lipids. Various fluorescent 1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl (C(6)-NBD) analogues were tested for transbilayer movement across the plasma membrane of red blood cells. Among these phospholipid analogues were phosphatidylgalactose, phosphatidylmaltose and phosphatidylmaltotriose, which were obtained from C(6)-NBD-phosphatidylcholine by phospholipase D-catalysed transphosphatidylation. The inward movement after the onset of scrambling was monitored by extraction of the non-internalized probe with BSA. We demonstrate that both the amino group and the size of the headgroup determine the kinetics of lipid scrambling, and that lipids with a ceramide backbone migrate much more slowly than glycerophospholipids with the same headgroup.
- Published
- 2002
- Full Text
- View/download PDF
21. Phospholipid scramblase activation pathways in lymphocytes.
- Author
-
Williamson P, Christie A, Kohlin T, Schlegel RA, Comfurius P, Harmsma M, Zwaal RF, and Bevers EM
- Subjects
- 4-Chloro-7-nitrobenzofurazan metabolism, Animals, Apoptosis genetics, B-Lymphocytes enzymology, B-Lymphocytes pathology, Blood Coagulation Disorders enzymology, Blood Coagulation Disorders genetics, Blood Coagulation Disorders pathology, Calcium metabolism, Carrier Proteins genetics, Cell Line, Transformed, Enzyme Activation genetics, Flow Cytometry, Humans, Hybridomas, Jurkat Cells, Lymphocytes cytology, Membrane Proteins genetics, Mice, Mutation, Phosphatidylcholines metabolism, Phosphatidylserines metabolism, Spectrometry, Fluorescence, Syndrome, 4-Chloro-7-nitrobenzofurazan analogs & derivatives, Carrier Proteins metabolism, Lymphocytes enzymology, Membrane Proteins metabolism, Phospholipid Transfer Proteins, Phospholipids metabolism
- Abstract
In erythrocytes and platelets, activation of a nonspecific lipid flipsite termed the scramblase allows rapid, bidirectional transbilayer movement of all types of phospholipids. When applied to lymphoid cells, scramblase assays reveal a similar activity, with scrambling rates intermediate between those seen in platelets and erythrocytes. Scrambling activity initiated in lymphoid cells by elevation of intracellular Ca(2+) proceeds after a lag not noted in platelets or erythrocytes. The rates of transbilayer movement of phosphatidylserine and phosphatidylcholine analogues are similar whether the scramblase is activated by elevated internal Ca(2+) or by apoptosis. Elevation of internal Ca(2+) levels in apoptotic cells does not result in an additive increase in the rate of lipid movement. In lymphoid cells from a patient with Scott syndrome, scramblase cannot be activated by Ca(2+), but is induced normally during apoptosis. These findings suggest that Ca(2+) and apoptosis operate through different pathways to activate the same scramblase.
- Published
- 2001
- Full Text
- View/download PDF
22. Multidrug resistance protein 1 regulates lipid asymmetry in erythrocyte membranes.
- Author
-
Dekkers DW, Comfurius P, van Gool RG, Bevers EM, and Zwaal RF
- Subjects
- 4-Chloro-7-nitrobenzofurazan pharmacology, Aminocaproic Acid pharmacology, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Erythrocytes ultrastructure, Fluorescent Dyes pharmacology, Humans, Hydrolysis, Indomethacin pharmacology, Lipid Bilayers metabolism, Microscopy, Electron, Multidrug Resistance-Associated Proteins, Phosphatidylcholines metabolism, Phospholipases A metabolism, Phospholipases A2, Phospholipids metabolism, Sphingomyelin Phosphodiesterase metabolism, Time Factors, Vasodilator Agents pharmacology, Verapamil pharmacology, 4-Chloro-7-nitrobenzofurazan analogs & derivatives, ATP-Binding Cassette Transporters metabolism, Aminocaproates, Cell Membrane metabolism, Erythrocytes metabolism
- Abstract
The role of multidrug resistance protein 1 (MRP1) in the maintenance of transbilayer lipid asymmetry in the erythrocyte membrane was investigated. The transbilayer distribution of endogenous phospholipids and [(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (NBD)-labelled lipid analogues was compared in the absence and the presence of inhibitors of MRP1. At equilibrium the transbilayer distribution of the NBD analogues (in the absence of MRP1 inhibitors) was very similar to that of the endogenous lipids. Inhibition of MRP1 by verapamil or indomethacin resulted in a shift in the amount of probe that was internalized: approx. 50% of NBD-labelled phosphatidylcholine (PtdCho) and 9% of NBD-sphingomyelin (NBD-Spm) were no longer extractable by BSA in cells treated with inhibitor, in comparison with 25% and 3% for control cells respectively. To verify whether inhibition of MRP1 also affected the distribution of the endogenous phospholipids, phospholipase A2 and sphingomyelinase were used to assess the amount of each of the various lipid classes present in the membrane outer leaflet. No shift in phospholipid distribution was observed after 5 h of incubation with verapamil or indomethacin. However, after 48 h of incubation with these inhibitors, significantly smaller amounts of PtdCho and Spm were present in the outer membrane leaflet. No appreciable change was observed in the distribution of phosphatidylethanolamine or phosphatidylserine. Decreased hydrolysis of PtdCho and Spm was not due to endovesicle formation, as revealed by electron microscopy. This is the first report to show that MRP1 has a role in the maintenance of the outwards orientation of endogenous choline-containing phospholipids in the erythrocyte membrane.
- Published
- 2000
23. No evidence for enhanced thrombin formation through displacement of annexin V by antiphospholipid antibodies.
- Author
-
Bevers EM, Janssen MP, Willems GM, and Zwaal RF
- Subjects
- Adsorption, Antiphospholipid Syndrome immunology, Autoimmune Diseases immunology, Binding, Competitive, Humans, Membrane Lipids metabolism, Models, Immunological, Protein Binding, Prothrombin Time, Thromboplastin metabolism, Annexin A5 metabolism, Antibodies, Antiphospholipid metabolism, Antiphospholipid Syndrome blood, Autoimmune Diseases blood, Thrombin biosynthesis
- Published
- 2000
24. Competition of annexin V and anticardiolipin antibodies for binding to phosphatidylserine containing membranes.
- Author
-
Willems GM, Janssen MP, Comfurius P, Galli M, Zwaal RF, and Bevers EM
- Subjects
- Annexin A5 chemistry, Antiphospholipid Syndrome metabolism, Binding, Competitive, Blood Platelets drug effects, Blood Platelets metabolism, Cell Membrane metabolism, Glycoproteins metabolism, Humans, Ionophores metabolism, Lipid Bilayers metabolism, Phosphatidylcholines metabolism, Phosphatidylserines metabolism, Time Factors, beta 2-Glycoprotein I, Annexin A5 metabolism, Antibodies metabolism, Cardiolipins immunology
- Abstract
Annexin V, an intracellular protein with a calcium-dependent high affinity for anionic phospholipid membranes, acts as an inhibitor of lipid-dependent reactions of the blood coagulation. Antiphospholipid antibodies found in the plasma of patients with antiphospholipid syndrome generally do not interact with phospholipid membranes directly, but recognize (plasma) proteins associated with lipid membranes, mostly prothrombin or beta(2)-glycoprotein I (beta(2)GPI). Previously, it has been proposed that antiphospholipid antibodies may cause thrombosis by displacing annexin V from procoagulant cell surfaces. We used ellipsometry to study the binding of annexin V and of complexes of beta(2)GPI with patient-derived IgG antibodies to beta(2)GPI, commonly referred to as anticardiolipin antibodies (ACA), to phospholipid bilayers composed of phosphatidylcholine (PC) and 20% phosphatidylserine (PS). More specifically, we investigated the competition of these proteins for the binding sites at these bilayers. We show that ACA-beta(2)GPI complexes, adsorbed to PSPC bilayers, are displaced for more than 70% by annexin V and that annexin V binding is unaffected by the presence of ACA-beta(2)GPI complexes. Conversely, annexin V preadsorbed to these bilayers completely prevents adsorption of ACA-beta(2)GPI complexes, and none of the preadsorbed annexin V is displaced by ACA-beta(2)GPI complexes. Using ellipsometry, we also studied the effect of ACA-beta(2)GPI complexes on the interaction of annexin V with the membranes of ionophore-activated blood platelets as a more physiological relevant model of cell membranes. The experiments with blood platelets confirm the high-affinity binding of annexin V to these membranes and unequivocally show that annexin V binding is unaffected by the presence of ACA-beta(2)GPI. In conclusion, our data unambiguously show that ACA-beta(2)GPI complexes are unable to displace annexin V from procoagulant membranes to any significant extent, whereas annexin V does displace the majority of preadsorbed ACA-beta(2)GPI complexes from these membranes.
- Published
- 2000
- Full Text
- View/download PDF
25. Lipid translocation across the plasma membrane of mammalian cells.
- Author
-
Bevers EM, Comfurius P, Dekkers DW, and Zwaal RF
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Animals, Apoptosis, Carrier Proteins metabolism, Cell Line, Humans, Membrane Proteins metabolism, Phosphatidylserines metabolism, Cell Membrane metabolism, Lipid Bilayers metabolism, Lipid Metabolism, Phospholipid Transfer Proteins
- Abstract
The plasma membrane, which forms the physical barrier between the intra- and extracellular milieu, plays a pivotal role in the communication of cells with their environment. Exchanging metabolites, transferring signals and providing a platform for the assembly of multi-protein complexes are a few of the major functions of the plasma membrane, each of which requires participation of specific membrane proteins and/or lipids. It is therefore not surprising that the two leaflets of the membrane bilayer each have their specific lipid composition. Although membrane lipid asymmetry has been known for many years, the mechanisms for maintaining or regulating the transbilayer lipid distribution are still not completely understood. Three major players have been presented over the past years: (1) an inward-directed pump specific for phosphatidylserine and phosphatidylethanolamine, known as aminophospholipid translocase; (2) an outward-directed pump referred to as 'floppase' with little selectivity for the polar headgroup of the phospholipid, but whose actual participation in transport of endogenous lipids has not been well established; and (3) a lipid scramblase, which facilitates bi-directional migration across the bilayer of all phospholipid classes, independent of the polar headgroup. Whereas a concerted action of aminophospholipid translocase and floppase could, in principle, account for the maintenance of lipid asymmetry in quiescent cells, activation of the scramblase and concomitant inhibition of the aminophospholipid translocase causes a collapse of lipid asymmetry, manifested by exposure of phosphatidylserine on the cell surface. In this article, each of these transporters will be discussed, and their physiological importance will be illustrated by the Scott syndrome, a bleeding disorder caused by impaired lipid scrambling. Finally, phosphatidylserine exposure during apoptosis will be briefly discussed in relation to inhibition of translocase and simultaneous activation of scramblase.
- Published
- 1999
- Full Text
- View/download PDF
26. Lipid-protein interactions in blood coagulation.
- Author
-
Zwaal RF, Comfurius P, and Bevers EM
- Subjects
- Animals, Binding Sites, Humans, Membrane Fluidity, Phospholipids chemistry, Thrombomodulin chemistry, Thromboplastin chemistry, Blood Coagulation, Blood Coagulation Factors chemistry, Membrane Lipids chemistry
- Abstract
It has long been appreciated that lipids, particularly anionic phospholipids, promote blood coagulation. The last two decades have seen an increasing insight into the kinetic and mechanistic aspects regarding the mode of action of phospholipids in blood coagulation. This essay attempts to review these developments with particular emphasis on the structure of lipid-binding domains of blood coagulation proteins, and the variable effect of phospholipid composition on the interaction with these proteins. Some examples are discussed of how lipid membranes direct the pathway of enzymatic conversions in blood coagulation complexes, also illustrating that the membrane lipid surface is more than an inert platform for the assembly of coagulation factors. Finally, the controlled exposure of procoagulant lipid on the surface of blood cells is shortly reviewed, and an example is discussed of how interference with lipid-protein interactions in blood coagulation may result in pathological phenomena.
- Published
- 1998
- Full Text
- View/download PDF
27. Transbilayer movement of NBD-labeled phospholipids in red blood cell membranes: outward-directed transport by the multidrug resistance protein 1 (MRP1).
- Author
-
Dekkers DW, Comfurius P, Schroit AJ, Bevers EM, and Zwaal RF
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, Animals, Biological Transport, Drug Resistance, Multiple genetics, Enzyme Activation, Erythrocyte Membrane enzymology, Humans, Lipid Bilayers blood, Mice, Mice, Knockout, Thromboplastin genetics, Thromboplastin metabolism, 4-Chloro-7-nitrobenzofurazan analogs & derivatives, 4-Chloro-7-nitrobenzofurazan blood, ATP Binding Cassette Transporter, Subfamily B, Member 1 blood, Erythrocyte Membrane metabolism, Lipid Bilayers metabolism, Phospholipids blood
- Abstract
The outward movement (flop) of fluorescently labeled analogues of phosphatidylserine (PS) and phosphatidylcholine (PC) in human and murine red blood cells (RBC) was examined. 1-Oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl (C6-NBD) analogues of PS and PC were incorporated in the inner leaflet of the plasma membrane through the action of aminophospholipid translocase or through equilibration upon prolonged incubation, respectively. After removal of noninternalized probe, externalization of C6-NBD-PS or C6-NBD-PC from the inner to outer leaflet was monitored by continuous incubation of the cells in the presence of bovine serum albumin. Flop rates for both probes in intact human RBC were virtually identical (t1/2 approximately 1.5 h), confirming earlier findings by Bitbol et al. [Bitbol, M., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6783-6787] and Connor et al. [Connor, J., et al. (1992) J. Biol. Chem. 267, 19412-19417]. Flop activity in resealed RBC ghosts could only be found upon coinclusion of both ATP and oxidized glutathione (GSSG). Furthermore, flop in intact cells was sensitive to verapamil (IC50 = 5-7 microM), vincristine (IC50 = 20 microM), and indomethacin (IC50 = 50 microM), suggesting the involvement of proteins conferring multidrug resistance (MDR). Experiments with RBC from knock-out mice for multidrug resistance P-glycoproteins (Mdr1a/1b-/- and Mdr2-/-) and multidrug resistance protein 1 (Mrp1-/-) revealed that Mrp1 is responsible for the observed flop of the fluorescent lipid analogues. We found no indications for outward transport of endogenous PS by any of these drug-transporting proteins as measured by a sensitive prothrombinase assay. Neither aminophospholipid translocase nor Ca2+-induced lipid scramblase activities were affected in RBC of these knock-out mice. We conclude that lipid floppase activity, as detected with lipid probes, reflects the activity of MRP1 recognizing the modified lipid analogues as xenobiotics to be expelled from the cell.
- Published
- 1998
- Full Text
- View/download PDF
28. Transmembrane phospholipid distribution in blood cells: control mechanisms and pathophysiological significance.
- Author
-
Bevers EM, Comfurius P, Dekkers DW, Harmsma M, and Zwaal RF
- Subjects
- Animals, Humans, Lipid Bilayers, Blood Cells metabolism, Membrane Lipids metabolism, Phospholipids blood
- Abstract
This review deals with current concepts on the regulation and function of phospholipid asymmetry in biological membranes. This ubiquitous phenomenon is characterized by a distinctly different lipid composition between the inner and outer leaflet of the membrane bilayer. Transbilayer asymmetry is controlled by different membrane proteins that function as lipid transporters, catalyzing uni- or bi-directional transbilayer movement of lipids. Under normal conditions, an ATP-dependent protein (aminophospholipid translocase) generates and maintains phospholipid asymmetry by promoting unidirectional transport of aminophospholipids from the outer- to the inner leaflet. The membrane lipid asymmetry may be compromised during cellular activation by a Ca2+-dependent transporter (lipid scramblase) that facilitates rapid bi-directional movement of all major phospholipid classes. A major consequence of this collapse of lipid asymmetry is the exposure of phosphatidylserine (PS) at the outer membrane surface. Surface exposure of PS has important physiological and pathological implications for blood coagulation, apoptosis, and cell-cell recognition.
- Published
- 1998
29. Impaired Ca2+-induced tyrosine phosphorylation and defective lipid scrambling in erythrocytes from a patient with Scott syndrome: a study using an inhibitor for scramblase that mimics the defect in Scott syndrome.
- Author
-
Dekkers DW, Comfurius P, Vuist WM, Billheimer JT, Dicker I, Weiss HJ, Zwaal RF, and Bevers EM
- Subjects
- Adenosine Triphosphate pharmacology, Blood Coagulation Disorders blood, Blood Platelets drug effects, Blood Platelets metabolism, Carrier Proteins blood, Carrier Proteins genetics, Cell Membrane drug effects, Cell Membrane metabolism, Enzyme Inhibitors pharmacology, Erythrocyte Membrane metabolism, Humans, Membrane Proteins blood, Membrane Proteins genetics, Methomyl pharmacology, Phosphatidylserines blood, Phosphatidylserines physiology, Phosphorylation drug effects, Staurosporine pharmacology, Syndrome, Thromboplastin metabolism, Blood Coagulation Disorders genetics, Calcium pharmacology, Carrier Proteins antagonists & inhibitors, Erythrocyte Membrane drug effects, Ionophores pharmacology, Membrane Lipids metabolism, Membrane Proteins antagonists & inhibitors, Methomyl analogs & derivatives, Phospholipid Transfer Proteins, Phospholipids metabolism, Protein Processing, Post-Translational drug effects, Protein-Tyrosine Kinases blood
- Abstract
Scott syndrome is an hereditary bleeding disorder characterized by a deficiency in platelet procoagulant activity. Unlike normal blood cells, Scott platelets, as well as erythrocytes and lymphocytes, are strongly impaired in their ability to scramble their membrane phospholipids when challenged with Ca2+. In normal cells this collapse of membrane asymmetry leads to surface exposure of phosphatidylserine. Here we report that Scott erythrocytes show an apparent defect in tyrosine phosphorylation on treatment with Ca2+-ionophore. Diminished tyrosine phosphorylation was also apparent in activated Scott platelets, but much less pronounced than observed in red blood cells. On the other hand, tyrosine phosphorylation profiles observed in Scott red blood cell ghosts after sealing in the presence of adenosine triphosphate (ATP) were indistinguishable from those obtained from normal ghosts. Several observations argue in favor of a mechanism in which tyrosine phosphorylation in red blood cells is facilitated by, rather than required for scrambling of membrane lipids. Staurosporin blocks tyrosine phosphorylation in normal red blood cells, but does not inhibit the lipid scrambling process. White ghosts from normal erythrocytes, resealed in the absence of ATP, exhibit Ca2+-induced lipid scrambling without tyrosine phosphorylation. A selective inhibitor of Ca2+-induced lipid scrambling also showed an apparent inhibition of tyrosine phosphorylation in ionophore-treated normal red blood cells, similar to that observed in Scott erythrocytes. While this inhibitor also suppressed Ca2+-induced lipid scrambling in ghosts that were sealed in the presence of ATP, it did not inhibit tyrosine kinase activity. We conclude that the apparent deficiency in tyrosine phosphorylation in Scott cells is an epiphenomenon, possibly associated with a defect in phospholipid scrambling, but not causal to this defect.
- Published
- 1998
30. Regulatory mechanisms of transmembrane phospholipid distributions and pathophysiological implications of transbilayer lipid scrambling.
- Author
-
Bevers EM, Comfurius P, Dekkers DW, Harmsma M, and Zwaal RF
- Subjects
- Animals, Anions, Antibodies, Antiphospholipid immunology, Antibodies, Antiphospholipid metabolism, Antiphospholipid Syndrome blood, Antiphospholipid Syndrome immunology, Apoptosis, Autoimmune Diseases blood, Autoimmune Diseases immunology, Blood Coagulation, Calcium Signaling, Carrier Proteins metabolism, Enzyme Activation, Humans, Lipid Bilayers, Mammals metabolism, Membrane Proteins metabolism, Mononuclear Phagocyte System physiology, Phosphatidylserines metabolism, Membrane Lipids metabolism, Phospholipid Transfer Proteins, Phospholipids metabolism
- Abstract
The various phospholipid classes that comprise mammalian cell membranes are distributed over both leaflets of the bilayer in a non-random fashion. While a specific and ATP-dependent transporter is responsible for rapid inward movement of aminophospholipids, its inhibition does not lead to spontaneous redistribution of lipids. Conditions of cellular activation which are accompanied with increased levels of intracellular Ca2+ may cause a collapse of lipid asymmetry by switching on an ATP-independently operating scramblase, which accelerates bidirectional movement of all phospholipid classes. The most prominent change in transmembrane lipid distribution is surface exposure of phosphatidylserine (PS), the more so since conditions which activate scramblase in most if not all cases lead to inhibition of aminophospholipid translocase activity, which will prevent PS from being pumped back to the inner leaflet of the membrane. Surface-exposed PS serves at least two important physiological functions: it promotes blood coagulation and offers a recognition signal for clearance by macrophages and other cells of the reticuloendothelial system. As such, PS exposure may form an important early event in the process of apoptosis to ensure rapid removal of these cells in order to avoid release of their inflammatory contents. Defective regulation of transbilayer lipid distribution may result in clinical manifestations such as in the Scott syndrome, a bleeding disorder caused by an impaired scramblase activity. Conversely, excessive PS exposure may lead to thrombosis or may explain formation of so-called antiphospholipid antibodies as occurring in patients with antiphospholipid syndrome.
- Published
- 1998
- Full Text
- View/download PDF
31. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells.
- Author
-
Zwaal RF and Schroit AJ
- Subjects
- Apoptosis, Blood Cells physiology, Calcium blood, Carrier Proteins blood, Complement Activation, Endothelium, Vascular physiopathology, Hemostasis physiology, Humans, Lipid Bilayers chemistry, Macrophages physiology, Membrane Proteins blood, Models, Biological, Phosphatidylethanolamines blood, Phosphatidylserines blood, Surface Properties, Thrombosis blood, Blood Cells chemistry, Membrane Lipids blood, Phospholipid Transfer Proteins, Phospholipids blood
- Published
- 1997
32. Role of divalency in the high-affinity binding of anticardiolipin antibody-beta 2-glycoprotein I complexes to lipid membranes.
- Author
-
Willems GM, Janssen MP, Pelsers MM, Comfurius P, Galli M, Zwaal RF, and Bevers EM
- Subjects
- Animals, Antibodies, Anticardiolipin pharmacology, Binding, Competitive, Blood Coagulation physiology, Calcium Chloride pharmacology, Cattle, Factor Xa Inhibitors, Humans, Immunoglobulin Fab Fragments pharmacology, Kinetics, Phosphatidylcholines metabolism, Phosphatidylserines metabolism, Phospholipids metabolism, Phospholipids pharmacology, Protein Binding, Sodium Chloride pharmacology, beta 2-Glycoprotein I, Antibodies, Anticardiolipin metabolism, Glycoproteins metabolism, Lipid Bilayers metabolism
- Abstract
beta 2-Glycoprotein I (beta 2GPI) is an essential cofactor for the binding to lipids of anticardiolipin antibodies (ACA), isolated from patients with anti-phospholipid syndrome. We used ellipsometry to study the binding of beta 2GPI and the beta 2GPI-mediated binding of ACA to planar membranes composed of phosphatidylcholine (PC) and 5-20 mol % phosphatidylserine (PS). No binding of beta 2GPI was observed to neutral (PC) membranes. Maximal binding of beta 2GPI was 3.2-3.6 pmol.cm-2. Affinity decreased strongly with decreasing PS content; increasing the NaCl and CaCl2 concentrations also led to a decrease in affinity. At physiologic conditions (10 mol % PS, 120 mM NaCl, and 3 mM CaCl2), a Kd of 14 microM was observed. Binding constants were insensitive to the chemical composition of the negatively charged phospholipid headgroup. ACA (1.25-10 micrograms.mL-1) caused a 30-40-fold enhancement of beta 2GPI binding to PS/PC membranes (20 mol % PS), resulting in the binding of about 2 pmol.cm-2 divalent ACA-(beta 2GPI)2 complexes at 100 nM beta 2GPI. In the absence of beta 2GPI, binding of ACA was negligible. Ad- and desorption kinetics of ACA-beta 2GPI complexes indicate that the initial monovalent association of ACA to membrane-bound beta 2GPI is rapidly followed by formation of divalent ACA-(beta 2GPI)2 complexes. Experiments with monovalent Fab1 fragments of ACA showed no appreciable effect on the beta 2GPI binding to lipid, substantiating the notion that divalent interactions are essential for the high-affinity binding of ACA-beta 2GPI. The anticoagulant effect of ACA is rationalized by the observation that binding of ACA-beta 2GPI complexes to the PSPC membrane severely restricts the adsorption of blood coagulation factor Xa.
- Published
- 1996
- Full Text
- View/download PDF
33. Regulatory mechanisms in maintenance and modulation of transmembrane lipid asymmetry: pathophysiological implications.
- Author
-
Bevers EM, Comfurius P, and Zwaal RF
- Subjects
- Antiphospholipid Syndrome complications, Humans, Lipid Bilayers analysis, Thrombosis etiology, Membrane Lipids physiology, Phosphatidylserines physiology
- Abstract
The two leaflets of the plasma membrane of eukaryotic cells differ in lipid composition: the outer leaflet comprises mainly neutral choline containing phospholipids, whereas the aminophospholipids reside almost exclusively in the cytoplasmic leaflet. The importance of transmembrane lipid asymmetry may be judged from the fact that the cell invests energy to maintain this situation for which at least two regulatory mechanisms are held responsible. A translocase, selective for aminophospholipids, acts as an ATP-dependent pump for rapid inward movement of phosphatidylserine (PS) and phosphatidylethanolamine; in addition, a non-selective, but also ATP-dependent pump causes outward movement of phospholipids, be it at a much lower rate compared to the inward transport by the aminophospholipid translocase. These two systems, acting in concert, are thought to be the main players in the maintenance of a dynamic equilibrium of the phospholipids over both membrane leaflets. Dissipation of membrane lipid asymmetry can be elicited in different cell types under a variety of conditions; in particular, platelets upon activation rapidly lose their normal plasma membrane lipid distribution, but also in other blood cells, lipid asymmetry can be lost, be it at a much lower rate and extent than in platelets. A putative protein, referred to as "scramblase' has been described, which requires the continuous presence of elevated intracellular Ca(2+)-levels, to allow a rapid, non-selective and bidirectional transbilayer movement of phospholipids. Although scrambling of lipids does not require ATP as such, preliminary studies suggest the possible involvement of one or more phosphorylated proteins. The most prominent consequence of the loss of phospholipid asymmetry is exposure of PS in the outer leaflet of the plasma membrane. Surface-exposed PS serves several important physiological functions: it promotes assembly of enzyme complexes of the coagulation cascade, it forms a signal for cell-cell recognition, which is important for cell scavenging processes. Surface-exposure of PS is an early phenomenon of apoptosis and appears to be involved in efficient removal of these cells. In addition, PS in the outer leaflet of cells is thought to play a role in cell fusion processes. It may be clear from the foregoing, that the amount of PS present at the cell surface needs to be tightly controlled, and that an impairment of this process leads to either excessive- or diminished exposition of PS which may have several pathophysiological consequences.
- Published
- 1996
- Full Text
- View/download PDF
34. Reconstitution of phospholipid scramblase activity from human blood platelets.
- Author
-
Comfurius P, Williamson P, Smeets EF, Schlegel RA, Bevers EM, and Zwaal RF
- Subjects
- 4-Chloro-7-nitrobenzofurazan, Calcium blood, Carrier Proteins isolation & purification, Chromatography, Ion Exchange, Cytosol metabolism, Detergents, Electrophoresis, Polyacrylamide Gel, Fluorescent Dyes, Humans, Kinetics, Membrane Proteins isolation & purification, Microscopy, Electron, Phospholipids metabolism, Spectrometry, Fluorescence, Blood Platelets metabolism, Carrier Proteins blood, Carrier Proteins chemistry, Lipid Bilayers metabolism, Membrane Proteins blood, Membrane Proteins chemistry, Phospholipid Transfer Proteins
- Abstract
Cellular activation, accompanied by elevation of cytoplasmic Ca2+ levels, can induce a progressive loss of plasma membrane phospholipid asymmetry, resulting from increased transbilayer movement (flip-flop) of phospholipids. While this process has been demonstrated in a variety of different cells, it is most active in blood platelets. In order to test whether this lipid scrambling process is mediated by a membrane protein, platelet membranes were solubilized in cholate and fractionated by anion exchange chromatography, and fractions were reconstituted into phospholipid vesicles by detergent dialysis in the presence of small amounts of fluorescent (NBD) phospholipids. Using dithionite reduction to monitor the transbilayer location of NBD phospholipids, it was shown that addition of Ca2+ and ionomycin to vesicles reconstituted with a particular fraction results in transbilayer movement of the fluorescent phospholipid analogs from the vesicle's inner to outer leaflet. Lipid vesicles reconstituted in the absence of membrane protein, or reconstituted with another platelet membrane protein fraction, were devoid of this activity. Heating the active fraction or incubating it with pronase or the SH reagent pyridyldithioethylamine markedly diminished the ability of the vesicles to translocate fluorescent phospholipid analogs across the bilayer in response to Ca2+ and ionophore. These results argue that a membrane protein (or proteins) from blood platelets is required to catalyze Ca2+-induced transbilayer movement of phospholipids, suggesting its (or their) involvement in the loss of lipid asymmetry that can occur during cellular activation.
- Published
- 1996
- Full Text
- View/download PDF
35. Critical micelle concentrations and stirring are rate limiting in the loss of lipid mass during membrane degradation by phospholipase A2.
- Author
-
Speijer H, Giesen PL, Zwaal RF, Hack CE, and Hermens WT
- Subjects
- Adsorption, Animals, Chromatography, Thin Layer, Drug Stability, Elapid Venoms, Elapidae, Kinetics, Micelles, Phosphatidylcholines chemistry, Phospholipases A2, Phospholipids chemistry, Protein Binding, Lipid Bilayers, Phosphatidylcholines metabolism, Phospholipases A metabolism, Phospholipids metabolism
- Abstract
In phospholipid membranes attacked by phospholipase A(2) (PLA(2)), accumulation of degradation products influences the binding affinity as well as the catalytic activity of PLA(2). Such accumulation in its turn depends on the rate of membrane degradation and the efflux of degradation products from the membrane, the latter being influenced by the stirring conditions in the system. This complicated process was investigated with a new ellipsometric technique for in situ measurement of membrane mass in a well-defined flow system. Planar phospholipid bilayers were formed on rotating silicon discs in buffer solution. After the addition of 0.05-100 ng/ml of PLA(2) (from Naja mocambique mocambique) to the buffer, mass desorption could be measured with a precision of 3-5 ng/cm(2), that is, about 1% of the surface mass of a single bilayer. Using radiolabeled phospholipids and thin-layer chromatography, it was verified that only the degradation products desorb from the membrane, which was confirmed by the desorption of mixtures of phospholipids, lysophospholipids, and fatty acids. The rotating disc allows the exact calculation of the mass transfer constant for transport-limited exchange of lipid between fluid and disc surface, as a function of rotation rate. By using the mass transfer constant, the critical micelle concentrations, and the mole fractions of products, desorption kinetics could be fully described. The amount of degraded phospholipid could be continuously monitored as the sum of the product mass still present in the membrane, as inferred from the desorption rate, and the mass already lost from the surface. It is concluded that ellipsometry is a suitable tool for studying the effects of PLA(2) on membranes.
- Published
- 1996
- Full Text
- View/download PDF
36. Contribution of different phospholipid classes to the prothrombin converting capacity of sonicated lipid vesicles.
- Author
-
Smeets EF, Comfurius P, Bevers EM, and Zwaal RF
- Subjects
- Cholesterol pharmacology, Humans, In Vitro Techniques, Phosphatidylserines pharmacology, Sonication, Sphingomyelins pharmacology, Thromboplastin analysis, Lipid Bilayers chemistry, Phospholipids chemistry, Platelet Activation drug effects, Prothrombin metabolism
- Abstract
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.
- Published
- 1996
- Full Text
- View/download PDF
37. Ultrastructural detection of surface exposed phosphatidylserine on activated blood platelets.
- Author
-
Stuart MC, Bevers EM, Comfurius P, Zwaal RF, Reutelingsperger CP, and Frederik PM
- Subjects
- Annexin A5 pharmacology, Blood Platelets ultrastructure, Humans, Ionophores, Microscopy, Electron, Platelet Activation drug effects, Blood Platelets physiology, Phosphatidylserines biosynthesis, Platelet Activation physiology
- Abstract
Phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane of cells (including blood platelets). Upon cell activation PS may become exposed to the outer surface of the cell. Cell membranes with surface exposed PS at the outside form a catalytic surface for coagulation reactions. When platelets are activated with ionophore or with thrombin in combination with thapsigargin, calcium induced scrambling of phospholipids takes place, resulting in PS exposure. Concomitant with PS exposition structural changes take place. On resting and activated platelets we combined the immunocytochemical detection of surface exposed PS with (ultra)structural information. Blood platelets were activated in the presence of annexin V, a protein which binds to PS in the presence of Ca2+. Annexin V was found to bind to lipid bilayers containing more than 5 mole % PS as estimated by binding of fluorescent-labelled annexin V to liposomes with varying PS concentrations. After vitrification, freeze-substitution and embedding of the platelets, annexin V was located on ultra thin sections, as detected by an anti-annexin V antibody and gold labelled protein A. Upon activation, the platelets show two different forms; irregular platelets with unchanged cytoplasm and round cells with apparently diluted cytoplasm. Activation with ionophore initially resulted in both forms, but after ten minutes only round platelets with diluted cytoplasm were observed. Both forms of these platelets as well as the microvesicles were found to be annexin V positive. However upon activation with thrombin in combination with thapsigargin, only the round cells with diluted cytoplasm and microvesicles were annexin V positive, whereas platelets with unchanged cytoplasm, even when microvesicles are present, are negative for annexin V.
- Published
- 1995
38. Continuous analysis of the mechanism of activated transbilayer lipid movement in platelets.
- Author
-
Williamson P, Bevers EM, Smeets EF, Comfurius P, Schlegel RA, and Zwaal RF
- Subjects
- Blood Platelets physiology, Calcium pharmacology, Disulfides pharmacology, Dithionite pharmacology, Egtazic Acid pharmacology, Humans, Ionomycin pharmacology, Kinetics, Oxidation-Reduction, Phosphatidylserines blood, Phosphatidylserines metabolism, Pyridines pharmacology, Terpenes pharmacology, Thapsigargin, Thrombin pharmacology, 4-Chloro-7-nitrobenzofurazan analogs & derivatives, Blood Platelets ultrastructure, Lipid Bilayers metabolism, Membrane Lipids blood, Platelet Activation drug effects
- Abstract
Dithionite reduction of fluorescent (NBD) phospholipids was used as the basis of a continuous assay of transbilayer lipid movement to the cell surface during platelet activation. This assay reveals that virtually all previously internalized phosphatidylserine passes through the external leaflet of the membrane within 90 s after activation with Ca2+ and ionophore or with thrombin and thapsigargin. We demonstrate that this lipid scrambling is reversible, bidirectional, and insensitive to the lipid headgroup. Prolonged activation gradually results in inactivation of the scramblase. The assay also reveals that activation of the scrambling activity is sensitive to the sulfhydryl reagent pyridyldithioethylamine, suggesting the involvement of a protein in the process of activated transbilayer lipid scrambling.
- Published
- 1995
- Full Text
- View/download PDF
39. Differential effects of endothelial cell fatty acid modification on the sensitivity of their membrane phospholipids to peroxidation.
- Author
-
Vossen RC, van Dam-Mieras MC, Hornstra G, and Zwaal RF
- Subjects
- Cells, Cultured, Copper pharmacology, Copper Sulfate, Fatty Acids metabolism, Fatty Acids, Monounsaturated metabolism, Fatty Acids, Monounsaturated pharmacology, Fatty Acids, Unsaturated metabolism, Fatty Acids, Unsaturated pharmacology, Humans, Hydrogen Peroxide pharmacology, Umbilical Veins, Endothelium, Vascular metabolism, Fatty Acids pharmacology, Lipid Peroxidation drug effects, Membrane Lipids metabolism, Phospholipids metabolism
- Abstract
In order to study the relationship between the fatty acid (FA) composition of human umbilical vein endothelial cells (HUVEC) and their susceptibility to oxidative stress, we modified their FA composition by long-term culturing in media supplemented with a saturated, monounsaturated, or polyunsaturated FA. Sensitivity of the cellular phospholipids to peroxidation was monitored by measuring conjugated diene formation and decrease of polyunsaturated FAs induced by CuSO4 and H2O2 in liposomes prepared from the respective phospholipid extracts. The extent of phospholipid peroxidation was found to increase with increasing content of polyunsaturated FAs. In addition, the sensitivity of individual polyunsaturated FAs to peroxidation was directly proportional to the number of double bonds present. However, no unequivocal relationship was observed between conjugated diene formation and the phospholipid unsaturation index (an indicator for the combined effect of number of double bonds and polyunsaturated FA contents of the membrane phospholipids). The results suggest that long-term FA modification of endothelial cells differentially alters the sensitivity of their membrane phospholipids to peroxidation: long-term modification with oleic acid may protect against lipid peroxidation, whereas linoleic acid may increase sensitivity to peroxidation. In contrast to what might be expected, long-term modification of endothelial cells with eicosapentaenoic or docosahexaenoic acid does not increase the sensitivity of phospholipids to peroxidation.
- Published
- 1995
- Full Text
- View/download PDF
40. Regulation of phospholipid asymmetry and induction of antiphospholipid antibodies.
- Author
-
Comfurius P, Bevers EM, Galli M, and Zwaal RF
- Subjects
- Antibodies, Antiphospholipid metabolism, Apoptosis, Humans, Phospholipids immunology, Phospholipids metabolism, Antibodies, Antiphospholipid immunology, Antiphospholipid Syndrome immunology, Lipid Bilayers chemistry, Phospholipids chemistry
- Published
- 1995
- Full Text
- View/download PDF
41. Calcium-induced transbilayer scrambling of fluorescent phospholipid analogs in platelets and erythrocytes.
- Author
-
Smeets EF, Comfurius P, Bevers EM, and Zwaal RF
- Subjects
- 4-Chloro-7-nitrobenzofurazan analogs & derivatives, 4-Chloro-7-nitrobenzofurazan metabolism, Biological Transport, Fluorescent Dyes metabolism, Humans, Phosphatidylcholines metabolism, Phosphatidylethanolamines metabolism, Phosphatidylserines metabolism, Platelet Activation, Blood Platelets metabolism, Calcium physiology, Erythrocyte Membrane metabolism, Lipid Bilayers metabolism, Phospholipids metabolism
- Abstract
The non-random distribution of phospholipids in the plasma membrane of human platelets and erythrocytes is at least partially maintained by the ATP-dependent aminophospholipid translocase, but can be disturbed by a calcium-induced scrambling of lipids. Using fluorescent NBD-phospholipid analogs, we demonstrate that in both cells the aminophospholipid translocase has a slightly higher preference for the naturally occurring L-isomer of the polar headgroup of phosphatidylserine as compared to the D-isomer. Calcium-induced outward movement of internalized phosphatidylserine probe, however, is not affected by the stereochemical configuration of the serine headgroup and is virtually identical to both the inward and outward movement of the phosphatidylcholine probe. The data also indicate that both in platelets and red blood cells the calcium-induced transbilayer movement is bidirectional and involves all major phospholipid classes, with reorientation rates of sphingomyelin being appreciably lower than that of the other phospholipid classes. While our results largely support earlier observations on red cells, they clearly differ from a recent study on platelets which suggested that calcium-induced scrambling is restricted to aminophospholipids and would not involve cholinephospholipids. The present results indicate that the same mechanism is responsible for calcium-induced lipid scrambling in red blood cells and platelets.
- Published
- 1994
- Full Text
- View/download PDF
42. Assembly of the prothrombinase complex on lipid vesicles depends on the stereochemical configuration of the polar headgroup of phosphatidylserine.
- Author
-
Comfurius P, Smeets EF, Willems GM, Bevers EM, and Zwaal RF
- Subjects
- Animals, Binding Sites, Cattle, Electrochemistry, Factor V chemistry, Factor X chemistry, In Vitro Techniques, Kinetics, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Phosphatidylserines chemistry, Phospholipids chemistry, Phospholipids metabolism, Protein Binding, Prothrombin metabolism, Stereoisomerism, Factor V metabolism, Factor X metabolism, Factor Xa, Phosphatidylserines metabolism
- Abstract
The conversion of prothrombin into thrombin is an imperative step in the sequence of reactions leading to the formation of a hemostatic plug. This reaction is catalyzed by the prothrombinase complex, composed of factors Xa and Va, which is assembled on a phospholipid surface through Ca-mediated interactions with the lipid polar headgroups. In this paper we describe experiments indicative for a major role of the stereochemical configuration of phosphatidylserine in the binding of the prothrombinase complex to a phospholipid surface. Using two stereoisomers of phosphatidylserine, i.e., L-alpha-glycerophosphoryl-L-serine (PLS) and L-alpha-glycerophosphoryl-D-serine (PDS), we demonstrate that membranes containing PLS are appreciably more favorable than membranes containing PDS in promoting assembly of the prothrombinase complex and catalysis of prothrombin conversion. Ellipsometric analysis of the binding of factor Va and factor Xa to a surface composed of phosphatidylcholine and 10 mol % of either PLS or PDS reveals that the apparent Kd for factor Va increases about 25-fold when substituting PDS for PLS. For factor Xa a 5-fold increase in Kd was observed on replacing PDS for PLS. When PLS is replaced by phosphatidyl-beta-lactate (PLac), a phospholipid resembling PS but lacking the amino group, a similar decrease in prothrombinase activity is found as observed with PDS, implicating the importance of both the amino group and the stereoconfiguration of the serine moiety for the assembly of the prothrombinase complex.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1994
- Full Text
- View/download PDF
43. Physiology of membrane lipid asymmetry.
- Author
-
Bevers EM, Smeets EF, Comfurius P, and Zwaal RF
- Subjects
- Animals, Antiphospholipid Syndrome etiology, Erythrocyte Membrane chemistry, Hemostasis, Humans, Phosphatidylserines physiology, Phospholipids physiology, Membrane Lipids physiology
- Published
- 1994
- Full Text
- View/download PDF
44. Prothrombin activation on dioleoylphosphatidylcholine membranes.
- Author
-
Govers-Riemslag JW, Janssen MP, Zwaal RF, and Rosing J
- Subjects
- 1-Carboxyglutamic Acid pharmacology, Animals, Calcium pharmacology, Cattle, Electrochemistry, Factor Xa metabolism, In Vitro Techniques, Kinetics, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Membranes, Artificial, Osmolar Concentration, Phosphatidylcholines chemistry, Phosphatidylglycerols chemistry, Phosphatidylglycerols metabolism, Phosphatidylserines chemistry, Phosphatidylserines metabolism, Phosphatidylcholines metabolism, Prothrombin metabolism
- Abstract
Factor-Xa-catalyzed prothrombin activation is greatly accelerated by negatively charged phospholipids plus calcium ions. In 1990, we reported that neutral phosphatidylcholine membranes also stimulated prothrombin activation [Gerads, I., Govers-Riemslag, J.W.P., Tans, G., Zwaal, R. F. A. & Rosing, J. (1990) Biochemistry 29, 7967-7974]. In the present study, we have performed a detailed analysis of the prothrombin-converting activity of phosphatidylcholine membranes. Stimulation of prothrombin activation by phosphatidylcholine vesicles was particularly observed (a) with phosphatidylcholine molecules that contained unsaturated hydrocarbon side chains, (b) in the presence of factor Va, (c) at low ionic strength and (d) when Ca2+ were present in the reaction medium. It is unlikely that the prothrombinase activity of phosphatidylcholine preparations was due to contaminating anionic phospholipids. This is concluded from the fact that thin-layer chromatographic analysis showed that dioleoylphosphatidylcholine [(Ole)2GroPCho] contained less than 0.1 mol/100 mol anionic phospholipid, and that incorporation of such amounts of anionic lipids in (Ole)2-GroPCho membranes hardly increased their prothrombin-converting activity. At low ionic strength and in the presence of factor Va and Ca2+ (Ole)2GroPCho membranes accelerated prothrombin activation about 100-fold. At ionic strength (I) 0.06, prothrombin activation on 100 microM (Ole)2-GroPCho was characterized by a Km for prothrombin of 2 microM, a Vmax of 3020 IIa min-1.Xa-1 and a Kd for factor XaVa complex formation at the membrane surface of 7.5 nM. Prothrombin activation on (Ole)2GroPCho membranes was drastically reduced when the ionic strength was increased. The inhibition at high ionic strength could be explained by an effect on the Kd for XaVa complex formation which increased from 7.5 nM at I = 0.06 to 100 nM at I = 0.22. Prothrombin activation on (Ole)2GroPCho required Ca2+ and was dependent on the presence of gamma-carboxyglutamic acid domains in prothrombin and factor Xa. This indicates that similar interactions may account for the assembly of prothrombinase complexes on phosphatidylcholine and an anionic lipid-containing membranes.
- Published
- 1994
- Full Text
- View/download PDF
45. Prothrombinase complex as a tool to assess changes in membrane phospholipid asymmetry.
- Author
-
Comfurius P, Bevers E, and Zwaal RF
- Subjects
- Animals, Biological Assay methods, Cattle, Chromatography, Gel, Chromatography, Ion Exchange, Factor Va isolation & purification, Liposomes, Molecular Conformation, Factor V chemistry, Factor X chemistry, Factor Xa, Membrane Lipids chemistry, Phospholipids chemistry
- Published
- 1994
- Full Text
- View/download PDF
46. Thapsigargin amplifies the platelet procoagulant response caused by thrombin.
- Author
-
Smeets EF, Heemskerk JW, Comfurius P, Bevers EM, and Zwaal RF
- Subjects
- Drug Synergism, Humans, Thapsigargin, Blood Coagulation drug effects, Blood Platelets drug effects, Calcium-Transporting ATPases antagonists & inhibitors, Terpenes pharmacology, Thrombin pharmacology
- Abstract
The platelet procoagulant response involves an increase in surface-exposed phosphatidylserine, which allows binding and assembly of enzyme complexes of the coagulation pathway resulting in acceleration of the clotting process. This response essentially requires the presence of extracellular Ca2+, and varies in extent with the type of agonist used. In the present paper we demonstrate that the moderate procoagulant response of human platelets caused by thrombin is strongly amplified by the presence of thapsigargin, an inhibitor of the microsomal Ca(2+)-ATPase. Thapsigargin, like thrombin, has only a weak effect on procoagulant activity. The large increase in procoagulant activity observed with the combined action of these two agonists is associated with increased shedding of microvesicles from the platelet plasma membrane as well as with inhibition of transport of a fluorescent-labeled analog of phosphatidylserine from the outer to the inner leaflet of the plasma membrane by the aminophospholipid translocase. The latter two observations support current concepts regarding the mechanism of development of procoagulant activity. Although the synergistic effect of thapsigargin on thrombin-induced procoagulant activity is at least in part due to the high levels of intracellular [Ca2+] evoked by these agonists, the data clearly indicate that a rise of the intracellular [Ca2+] is insufficient to completely explain this response. The present findings suggest that additional factors control expression of procoagulant activity upon stimulation of platelets by thrombin.
- Published
- 1993
47. Continuous monitoring of lipid peroxidation by measuring conjugated diene formation in an aqueous liposome suspension.
- Author
-
Vossen RC, van Dam-Mieras MC, Hornstra G, and Zwaal RF
- Subjects
- Chromatography, Gas, Chromatography, Thin Layer, Fatty Acids analysis, In Vitro Techniques, Spectrum Analysis, Lipid Metabolism, Lipid Peroxidation physiology, Liposomes metabolism
- Abstract
A method is described for the direct and continuous monitoring of lipid peroxidation in an aqueous suspension of sonicated liposomes. By means of ultraviolet difference spectroscopy using tandem cuvettes, the formation of conjugated dienes during liposome peroxidation can be followed. Using this technique, the effect of the fatty acid composition of liposomes on lipid peroxidation can be studied. The results show that both the extent and the time scale of lipid peroxidation are influenced by the fatty acid composition of the phospholipid liposomes. This was confirmed also by other methods, such as measurement of the formation of lipid hydroperoxides and measurement of the decrease in polyunsaturated fatty acids. The advantage of the method described is the direct and continuous monitoring of phospholipid peroxidation in an aqueous environment, without subsampling and extraction of peroxidation products into organic solvents. Using this experimental approach based on difference spectra the contributions from changes in liposome, CuSO4 and H2O2 concentrations are canceled, thus improving sensitivity. The method can be employed for measuring the susceptibility to peroxidation of membrane phospholipids from fatty acid modified endothelial cells.
- Published
- 1993
- Full Text
- View/download PDF
48. Mechanism and function of changes in membrane-phospholipid asymmetry in platelets and erythrocytes.
- Author
-
Zwaal RF, Comfurius P, and Bevers EM
- Subjects
- Blood Coagulation Disorders blood, Humans, Syndrome, Blood Platelets chemistry, Erythrocyte Membrane chemistry, Membrane Lipids chemistry, Phospholipids chemistry
- Published
- 1993
- Full Text
- View/download PDF
49. Effect of antiphospholipid antibodies on procoagulant activity of activated platelets and platelet-derived microvesicles.
- Author
-
Galli M, Bevers EM, Comfurius P, Barbui T, and Zwaal RF
- Subjects
- Adult, Aged, Apolipoproteins, Female, Glycoproteins, Humans, Male, Middle Aged, Platelet Activation, Prothrombin, Time Factors, beta 2-Glycoprotein I, Antibodies, Anticardiolipin immunology, Blood Platelets immunology, Factor VIII immunology, Lupus Coagulation Inhibitor immunology
- Abstract
We have recently described the in vitro mechanism of action of anticardiolipin (aCL) and lupus anticoagulant (LA) antibodies in patients with the antiphospholipid syndrome. LA antibodies inhibit coagulation reactions in plasma because they appear to recognize the complex of lipid-bound (human) prothrombin, whereas aCL antibodies require beta 2-glycoprotein I (beta 2-GPI) for binding to anionic phospholipids. aCL antibodies can be divided into two subgroups, according to their behaviour in lipid-dependent coagulation reactions: aCL-type A enhances the anti-coagulant effect of beta 2-GPI, whereas aCL-type B does not. In the present study we investigated the effect of purified aCL-type A and B and of LA antibodies on the procoagulant activity of both Ca-ionophore activated platelets and platelet-derived microvesicles, using an assay system with highly purified bovine coagulation factors Xa, Va, and prothrombin from human and bovine origin. In the absence of beta 2-GPI neither type of aCL was able to inhibit the prothrombinase activity of platelets or microvesicles. However, a strong and dose-dependent inhibition of the prothrombinase activity of both platelets and platelet-derived microvesicles was observed within a few minutes, when aCL-type A antibodies were added in combination with beta 2-GPI. This inhibitory effect was dependent also on the concentration of beta 2-GPI. Conversely, no inhibitory effect of aCL-type B antibodies on platelet- (or microvesicle) prothrombinase activity in the presence of beta 2-GPI could be observed. LA antibodies were able to inhibit in a dose-dependent way the procoagulant activity of activated platelets and platelet-derived microvesicles. With two LA preparations this inhibition was only apparent when human prothrombin was used as substrate, while a third preparation exhibited its inhibitory effect both in the presence of human and bovine prothrombin. The data indicate that, in the presence of their respective cofactors beta 2-GPI and prothrombin, aCL and LA antibodies interact with the membrane of activated platelets and platelet-derived microvesicles in a very similar way as previously observed for their interaction with anionic phospholipid surfaces.
- Published
- 1993
- Full Text
- View/download PDF
50. Antiphospholipid antibodies: involvement of protein cofactors.
- Author
-
Galli M, Barbui T, Zwaal RF, Comfurius P, and Bevers EM
- Subjects
- Animals, Antibodies, Antiphospholipid metabolism, Blood Coagulation drug effects, Blood Platelets metabolism, Glycoproteins metabolism, Glycoproteins pharmacology, Humans, Immunoglobulin G immunology, Immunoglobulin G metabolism, Lupus Coagulation Inhibitor immunology, Lupus Coagulation Inhibitor metabolism, Phospholipids metabolism, Protein Binding, Species Specificity, beta 2-Glycoprotein I, Antibodies, Antiphospholipid immunology, Antigen-Antibody Reactions, Glycoproteins immunology, Phospholipids immunology
- Published
- 1993
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.