RESUMEN

Immune cells and platelets maintain plasma membrane phospholipid asymmetry. Upon activation, this asymmetry is disrupted by phospholipid scrambling (PS), which is a major step during activation of immune cells, hemostasis and apoptosis. Anoctamin 6 (Ano6; TMEM16F) causes chloride (Cl(-)) and cation currents and is required for Ca(2+)-dependent PS. It is defective in blood cells from patients with Scott syndrome, a rare bleeding disorder. We examined if Cl(-) currents and PS are related, whether both processes are Ca(2+) dependent, and whether Ca(2+)-independent scrambling during intrinsic and extrinsic apoptosis is controlled by Ano6. Ca(2+) increase by ionomycin activated Ano6 Cl(-) currents and PS in normal lymphocytes, but not in B-lymphocytes from two different patients with Scott syndrome. Fas ligand (FasL) did not increase intracellular Ca(2+), but activated Cl(-) currents in normal but not in Scott lymphocytes. Whole-cell currents were inhibited by Cl(-) channel blockers and by siRNA knockdown of Ano6. In contrast, intrinsic mitochondrial apoptosis by ABT-737 did not induce Cl(-) currents in lymphocytes. PS was not inhibited by blockers of Ano6 or removal of Cl(-) ions. Remarkably, Ca(2+)-independent scrambling due to extrinsic (FasL) or intrinsic (ABT-737) apoptosis was unchanged in Scott cells. We conclude that: (i) Ano6 Cl(-) currents are activated by increase in cytosolic Ca(2+), or Ca(2+) independent by stimulation of Fas receptors; (ii) Ca(2+)-dependent PS induced by Ano6 does not require Cl(-) currents; (iii) Ca(2+)-independent PS does not require Ano6; (iv) Ano6 is necessary for Ca(2+)-dependent PS, but not by increasing intracellular Ca(2+).

Asunto(s)

Calcio/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Anoctaminas , Apoptosis/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/fisiología , Compuestos de Bifenilo/farmacología , Trastornos de la Coagulación Sanguínea/fisiopatología , Ionóforos de Calcio/farmacología , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/metabolismo , Proteína Ligando Fas/farmacología , Células HEK293 , Humanos , Transporte Iónico/efectos de los fármacos , Ionomicina/farmacología , Células Jurkat , Nitrofenoles/farmacología , Técnicas de Placa-Clamp , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Proteínas de Transferencia de Fosfolípidos/genética , Piperazinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Sulfonamidas/farmacología

RESUMEN

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.

Asunto(s)

Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Lípidos de la Membrana/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Potasio/metabolismo , Calcio/metabolismo , Forma de la Célula , Membrana Eritrocítica/efectos de los fármacos , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Humanos , Ionomicina/farmacología , Ionóforos/farmacología , Iones/metabolismo , Sodio/metabolismo , Tromboplastina/metabolismo

RESUMEN

Microparticles (MPs), shed during the storage of platelets, support blood coagulation and could be helpful in restoring the haemostatic system in thrombocytopenic patients. The mechanisms by which MPs support haemostasis under flow conditions were investigated. Fluorescent-labelled MPs were perfused at shear rates of 100 and 1000/s over surfaces coated with collagen, fibrinogen, von Willebrand factor (VWF) or surface-adherent platelets. Adhesion was monitored in real-time by fluorescence microscopy. In addition, thrombin-antithrombin (TAT) complex formation was measured in flowing thrombocytopenic blood. MPs attained the capacity to firmly adhere to collagen, VWF, fibrinogen and surface-adherent platelets at high and low shear rate. Antibodies against glycoprotein Ibalpha and alpha(IIb)beta(3) were used to demonstrate the specificities of these interactions. The addition of MPs to thrombocytopenic blood did not affect platelet adhesion. TAT complex formation was increased in the presence of MPs in capillaries coated with fibrinogen, but not on collagen fibres. We confirmed that MPs adhere to a damaged vascular bed in vivo after infusion in denuded arteries in a mouse model. MPs have platelet-like adhering properties and accelerate thrombin generation. These properties strongly support the notion that MPs can be beneficial in maintaining normal haemostasis when platelet function is impaired or reduced, as in thrombocytopenic patients.

Asunto(s)

Coagulación Sanguínea , Plaquetas/fisiología , Colágeno Tipo I/fisiología , Fibrinógeno/fisiología , Factor de von Willebrand/fisiología , Animales , Antitrombina III/fisiología , Capilares , Hemorreología , Humanos , Masculino , Ratones , Ratones Endogámicos , Microscopía Fluorescente , Péptido Hidrolasas/fisiología , Estrés Mecánico , Trombocitopenia/sangre , Trombocitopenia/terapia , Adherencias Tisulares

RESUMEN

BACKGROUND: Blockade of the thrombin receptors protease-activated receptor (PAR)1 and PAR4 with pepducins, cell-penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen-induced externalization of phosphatidylserine (PS) at the platelet plasma membrane. OBJECTIVE: In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)-initiated thrombin generation in platelet-rich plasma (PRP) and the interaction between PAR4 pepducin-loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data. RESULTS: Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF-initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin-induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus. CONCLUSIONS: Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet-platelet interactions.

Asunto(s)

Anticoagulantes/farmacología , Plaquetas/efectos de los fármacos , Fibrinolíticos/farmacología , Lipoproteínas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Proteinasa-Activados/antagonistas & inhibidores , Animales , Anticoagulantes/metabolismo , Anticoagulantes/uso terapéutico , Plaquetas/metabolismo , Arteria Carótida Común/cirugía , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fibrinolíticos/metabolismo , Fibrinolíticos/uso terapéutico , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Técnicas In Vitro , Lipoproteínas/metabolismo , Lipoproteínas/uso terapéutico , Masculino , Ratones , Microscopía por Video , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/metabolismo , Inhibidores de Agregación Plaquetaria/uso terapéutico , Receptor PAR-1/antagonistas & inhibidores , Receptores Proteinasa-Activados/metabolismo , Receptores de Trombina/antagonistas & inhibidores , Trombina/metabolismo , Tromboplastina/metabolismo , Trombosis/sangre , Trombosis/metabolismo , Trombosis/prevención & control , Factores de Tiempo

RESUMEN

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.

Asunto(s)

Plaquetas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Anexina A5/metabolismo , Antígenos de Superficie/metabolismo , Colágeno/farmacología , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Humanos , Ionomicina/farmacología , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Leche/metabolismo , Proteínas de Transferencia de Fosfolípidos/agonistas , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Activación Plaquetaria , Trombina/farmacología , Tromboplastina/metabolismo

RESUMEN

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.

Asunto(s)

Células Eucariotas/metabolismo , Fosfatidilserinas/metabolismo , Síndrome Antifosfolípido/metabolismo , Apoptosis/fisiología , Membrana Celular/metabolismo , Células Eucariotas/patología , Enfermedades Hematológicas/metabolismo , Humanos , Infecciones/metabolismo , Enfermedades Renales/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/fisiología , Enfermedades Metabólicas/metabolismo , Neoplasias/metabolismo , Fosfatidilserinas/fisiología , Enfermedades Respiratorias/metabolismo

Asunto(s)

Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/diagnóstico , Síndrome Antifosfolípido/inmunología , Adulto , Anticuerpos Anticardiolipina/análisis , Anticuerpos Anticardiolipina/inmunología , Anticuerpos Antifosfolípidos/análisis , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Femenino , Glicoproteínas/inmunología , Humanos , Lupus Eritematoso Sistémico , Masculino , Tamizaje Masivo/métodos , Persona de Mediana Edad , Estudios Retrospectivos , Medición de Riesgo , Muestreo , beta 2 Glicoproteína I

RESUMEN

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.

Asunto(s)

4-Cloro-7-nitrobenzofurazano/análogos & derivados , Proteínas Portadoras/metabolismo , Linfocitos/enzimología , Proteínas de la Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos , Fosfolípidos/metabolismo , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animales , Apoptosis/genética , Linfocitos B/enzimología , Linfocitos B/patología , Trastornos de la Coagulación Sanguínea/enzimología , Trastornos de la Coagulación Sanguínea/genética , Trastornos de la Coagulación Sanguínea/patología , Calcio/metabolismo , Proteínas Portadoras/genética , Línea Celular Transformada , Activación Enzimática/genética , Citometría de Flujo , Humanos , Hibridomas , Células Jurkat , Linfocitos/citología , Proteínas de la Membrana/genética , Ratones , Mutación , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Espectrometría de Fluorescencia , Síndrome

RESUMEN

There is increasing evidence that endogenously generated aldehydes formed as a result of lipid peroxidation are involved in the pathophysiological effects associated with oxidative stress in cells and tissues. Malondialdehyde (MDA), a major product of lipid peroxidation, can modify amines present on the cell surface and thereby introduce negative charges that can affect the interfacial ionic layer. We show that lipid peroxidation of RBC generates MDA adducts that, similar to phosphatidylserine (PS), bind annexin V in a Ca(2+)-dependent manner. Like PS, these adducts also promote the "PS-dependent" prothrombinase assays, albeit to lower levels. These results indicate that annexin V binding cannot be used as an exclusive indicator of cell surface PS and raise the possibility that some phenomenon attributed to PS may, in fact, also involve aldehyde-lipid adducts.

Asunto(s)

Anexina A5/metabolismo , Peroxidación de Lípido , Lípidos de la Membrana/metabolismo , Animales , Anexina A5/sangre , Bovinos , Eritrocitos/metabolismo , Humanos , Malondialdehído/sangre , Malondialdehído/metabolismo , Lípidos de la Membrana/sangre , Microscopía Fluorescente , Fosfatidiletanolaminas/sangre , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/sangre , Fosfatidilserinas/metabolismo , Unión Proteica

RESUMEN

We investigated the mechanism by which anti-prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity-purified anti-prothrombin antibodies from LAC-positive plasma samples (alpha-FII-LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified alpha-FII-LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when alpha-FII-LAC+ were added to prothrombin-deficient plasma or when purified anti-prothrombin antibodies from LAC-negative plasma samples (alpha-FII-LAC-) were added. When a combination of prothrombin and alpha-FII-LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The alpha-FII-LAC+ alone or a combination of prothrombin and alpha-FII-LAC- did not show inhibition. Ellipsometry studies showed that, in the presence of alpha-FII-LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with alpha-FII-LAC-. Our results show that complexes of prothrombin and anti-prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti-beta2-glycoprotein I antibodies.

Asunto(s)

Síndrome Antifosfolípido/metabolismo , Autoanticuerpos/metabolismo , Inhibidor de Coagulación del Lupus/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Proteínas de Neoplasias , Fosfolípidos/metabolismo , Protrombina/inmunología , Síndrome Antifosfolípido/inmunología , Cisteína Endopeptidasas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Factor Xa/metabolismo , Femenino , Humanos , Membrana Dobles de Lípidos/metabolismo , Lupus Eritematoso Sistémico/inmunología , Masculino , Unión Proteica , Protrombina/metabolismo , Tromboplastina/metabolismo

RESUMEN

In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca(2+) signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca(2+)](i). Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.

Asunto(s)

Plaquetas/fisiología , Proteínas Portadoras , Lectinas Tipo C , Activación Plaquetaria/fisiología , Adhesividad Plaquetaria/fisiología , Plaquetas/citología , Plaquetas/efectos de los fármacos , Western Blotting , Calcio/metabolismo , Calcio/fisiología , Calpaína/metabolismo , Calpaína/farmacología , Venenos de Crotálidos/metabolismo , Venenos de Crotálidos/farmacología , Activación Enzimática , Citometría de Flujo , Humanos , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/farmacología , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacología , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/farmacología , Proteínas/metabolismo , Proteínas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Platelets in an advanced stage of activation change from coagulation-inactive to coagulation-promoting cells. This procoagulant response is characterised by exposure of aminophospholipids, such as phosphatidylserine, to the platelet surface and by formation of microvesicles. Under specific conditions, when both signalling and adhesive platelet receptors are occupied, collagen and also thrombin are able to trigger this response. Thus, platelets express high coagulation-promoting activity only after interacting with multiple receptors.

Asunto(s)

Coagulación Sanguínea/fisiología , Plaquetas/metabolismo , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Humanos

RESUMEN

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.

Asunto(s)

4-Cloro-7-nitrobenzofurazano/análogos & derivados , Transportadoras de Casetes de Unión a ATP/metabolismo , Aminocaproatos , Membrana Celular/metabolismo , Eritrocitos/metabolismo , 4-Cloro-7-nitrobenzofurazano/farmacología , Ácido Aminocaproico/farmacología , Antiinflamatorios no Esteroideos/farmacología , Eritrocitos/ultraestructura , Colorantes Fluorescentes/farmacología , Humanos , Hidrólisis , Indometacina/farmacología , Membrana Dobles de Lípidos/metabolismo , Microscopía Electrónica , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Fosfatidilcolinas/metabolismo , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Fosfolípidos/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Factores de Tiempo , Vasodilatadores/farmacología , Verapamilo/farmacología

Asunto(s)

Anexina A5/metabolismo , Anticuerpos Antifosfolípidos/metabolismo , Síndrome Antifosfolípido/sangre , Enfermedades Autoinmunes/sangre , Trombina/biosíntesis , Adsorción , Síndrome Antifosfolípido/inmunología , Enfermedades Autoinmunes/inmunología , Unión Competitiva , Humanos , Lípidos de la Membrana/metabolismo , Modelos Inmunológicos , Unión Proteica , Tiempo de Protrombina , Tromboplastina/metabolismo

RESUMEN

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.

Asunto(s)

Anexina A5/metabolismo , Anticuerpos/metabolismo , Cardiolipinas/inmunología , Anexina A5/química , Síndrome Antifosfolípido/metabolismo , Unión Competitiva , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Humanos , Ionóforos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Factores de Tiempo , beta 2 Glicoproteína I

RESUMEN

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.

Asunto(s)

Membrana Celular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Metabolismo de los Lípidos , Proteínas de Transferencia de Fosfolípidos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Apoptosis , Proteínas Portadoras/metabolismo , Línea Celular , Humanos , Proteínas de la Membrana/metabolismo , Fosfatidilserinas/metabolismo

RESUMEN

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.

Asunto(s)

Lípidos de la Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos , Fosfolípidos/metabolismo , Animales , Aniones , Anticuerpos Antifosfolípidos/inmunología , Anticuerpos Antifosfolípidos/metabolismo , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/inmunología , Apoptosis , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/inmunología , Coagulación Sanguínea , Señalización del Calcio , Proteínas Portadoras/metabolismo , Activación Enzimática , Humanos , Membrana Dobles de Lípidos , Mamíferos/metabolismo , Proteínas de la Membrana/metabolismo , Sistema Mononuclear Fagocítico/fisiología , Fosfatidilserinas/metabolismo

RESUMEN

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.

Asunto(s)

Factores de Coagulación Sanguínea/química , Coagulación Sanguínea , Lípidos de la Membrana/química , Animales , Sitios de Unión , Humanos , Fluidez de la Membrana , Fosfolípidos/química , Trombomodulina/química , Tromboplastina/química

RESUMEN

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.

Asunto(s)

4-Cloro-7-nitrobenzofurazano , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/sangre , Membrana Eritrocítica/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfolípidos/sangre , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/sangre , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Transporte Biológico , Resistencia a Múltiples Medicamentos/genética , Activación Enzimática , Membrana Eritrocítica/enzimología , Humanos , Membrana Dobles de Lípidos/sangre , Ratones , Ratones Noqueados , Tromboplastina/genética , Tromboplastina/metabolismo

RESUMEN

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.

Asunto(s)

Células Sanguíneas/metabolismo , Lípidos de la Membrana/metabolismo , Fosfolípidos/sangre , Animales , Humanos , Membrana Dobles de Lípidos