Getting to the Outer Leaflet: Physiology of Phosphatidylserine Exposure at the Plasma Membrane.

Phosphatidylserine (PS) is a major component of membrane bilayers whose change in distribution between inner and outer leaflets is an important physiological signal. Normally, members of the type IV P-type ATPases spend metabolic energy to create an asymmetric distribution of phospholipids between the two leaflets, with PS confined to the cytoplasmic membrane leaflet. On occasion, membrane enzymes, known as scramblases, are activated to facilitate transbilayer migration of lipids, including PS. Recently, two proteins required for such randomization have been identified: TMEM16F, a scramblase regulated by elevated intracellular Ca(2+), and XKR8, a caspase-sensitive protein required for PS exposure in apoptotic cells. Once exposed at the cell surface, PS regulates biochemical reactions involved in blood coagulation, and bone mineralization, and also regulates a variety of cell-cell interactions. Exposed on the surface of apoptotic cells, PS controls their recognition and engulfment by other cells. This process is exploited by parasites to invade their host, and in specialized form is used to maintain photoreceptors in the eye and modify synaptic connections in the brain. This review discusses what is known about the mechanism of PS exposure at the surface of the plasma membrane of cells, how actors in the extracellular milieu sense surface exposed PS, and how this recognition is translated to downstream consequences of PS exposure.

Molecular functions of anoctamin 6 (TMEM16F): a chloride channel, cation channel, or phospholipid scramblase?

Anoctamin 6 (Ano6; TMEM16F gene) is a ubiquitous protein; the expression of which is defective in patients with Scott syndrome, an inherited bleeding disorder based on defective scrambling of plasma membrane phospholipids. For Ano6, quite diverse functions have been described: (1) it can form an outwardly rectifying, Ca(2+)-dependent and a volume-regulated Cl(-) channel; (2) it was claimed to be a Ca(2+)-regulated nonselective cation channel permeable for Ca(2+); (3) it was shown to be essential for Ca(2+)-mediated scrambling of membrane phospholipids; and (4) it can regulate cell blebbing and microparticle shedding. Deficiency of Ano6 in blood cells from Scott patients or Ano6 null mice appears to affect all of these cell responses. Furthermore, Ano6 deficiency in mice impairs the mineralization of osteoblasts, resulting in reduced skeletal development. These diverse results have been obtained under different experimental conditions, which may explain some of the contradictions. This review therefore aims to summarize the currently available information on the diverse roles of Ano6 and tries to clear up some of the existing controversies.

Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation.

Scott syndrome, a bleeding disorder caused by defective phospholipid scrambling, has been associated with mutations in the TMEM16F gene. The role of TMEM16F in apoptosis- or agonist-induced phosphatidylserine (PS) exposure was studied in platelets from a Scott syndrome patient and control subjects. Whereas stimulation of control platelets with the BH3-mimetic ABT737 resulted in 2 distinct fractions with moderate and high PS exposure, the high PS-exposing fraction was markedly delayed in Scott platelets. High, but not moderate, PS exposure in platelets was suppressed by chelation of intracellular Ca(2+), whereas caspase inhibition completely abolished ABT737-induced PS exposure in both Scott and control platelets. On the other hand, high PS exposure induced by the Ca(2+)-mobilizing agonists convulxin/thrombin fully relied on mitochondrial depolarization and was virtually absent in Scott platelets. Finally, PS exposure induced by collagen/thrombin was partly affected in Scott platelets, and the residual PS positive fraction was insensitive to inhibition of caspases or mitochondrial depolarization. In conclusion, TMEM16F is not required for, but enhances, caspase-dependent PS exposure; convulxin-/thrombin-induced PS exposure is entirely dependent on TMEM16F, whereas collagen/thrombin-induced PS exposure results from 2 distinct pathways, one of which involves mitochondrial depolarization and is mediated by TMEM16F.

Ribavirin-induced externalization of phosphatidylserine in erythrocytes is predominantly caused by inhibition of aminophospholipid translocase activity.

Ribavirin in combination with interferon-α is the standard treatment for chronic hepatitis C, but often induces severe anemia forcing discontinuation of the therapy. Whereas suppression of bone marrow by interferon may impact on the production of erythrocytes, it has been suggested that accumulation of ribavirin in erythrocytes induces alterations causing an early removal of these cells by the mononuclear phagocytic system. Externalization of phosphatidylserine, which is exclusively present in the cytoplasmic leaflet of the plasma membrane, is a recognition signal for phagocytosis in particular of apoptotic cells. Here, we demonstrate that surface exposure of phosphatidylserine upon prolonged treatment of erythrocytes with ribavirin results mainly from inactivation of the aminophospholipid translocase, an ATP-dependent lipid pump, which specifically transports phosphatidylserine from the outer to the inner leaflet of the plasma membrane. Inactivation is due to severe ATP depletion, although competitive inhibition by ribavirin or its phosphorylated derivatives cannot be excluded. Phospholipid scramblase, responsible for collapse of lipid asymmetry, appears to be of minor importance as erythrocytes of patients with the Scott syndrome, lacking Ca(2+)-induced lipid scrambling, are equally sensitive to ribavirin treatment. Neither the antioxidant N-acetylcysteine nor the pan-caspase inhibitor Q-VD-OPH did affect ribavirin-induced phosphatidylserine exposure, suggesting that oxidative stress or apoptotic-related mechanisms are not involved in this process. In conclusion, we propose that spontaneous loss of lipid asymmetry, not corrected by aminophospholipid translocase activity, is the mechanism for ribavirin-induced phosphatidylserine exposure that may contribute to ribavirin-induced anemia.

Antithrombotic potential of blockers of store-operated calcium channels in platelets.

OBJECTIVE: Platelet Orai1 channels mediate store-operated Ca(2+) entry (SOCE), which is required for procoagulant activity and arterial thrombus formation. Pharmacological blockage of these channels may provide a novel way of antithrombotic therapy. Therefore, the thromboprotective effect of SOCE blockers directed against platelet Orai1 is determined. METHODS AND RESULTS: Candidate inhibitors were screened for their effects on SOCE in washed human platelets. Tested antagonists included the known compounds, SKF96365, 2-aminoethyl diphenylborate, and MRS1845 and the novel compounds, Synta66 and GSK-7975A. The potency of SOCE inhibition was in the order of Synta66>2-aminoethyl diphenylborate>GSK-7975A>SKF96365>MRS1845. The specificity of the first 3 compounds was verified with platelets from Orai1-deficient mice. Inhibitory activity on procoagulant activity and high-shear thrombus formation was assessed in plasma and whole blood. In the presence of plasma, all 3 compounds suppressed platelet responses and restrained thrombus formation under flow. Using a murine stroke model, arterial thrombus formation was provoked in vivo by transient middle cerebral artery occlusion. Postoperative administration of 2-aminoethyl diphenylborate markedly diminished brain infarct size. CONCLUSIONS: Plasma-soluble SOCE blockers such as 2-aminoethyl diphenylborate suppress platelet-dependent coagulation and thrombus formation. The platelet Orai1 channel is a novel target for preventing thrombotic events causing brain infarction.

Structure and function of a recombinant von Willebrand factor drug candidate.

The complex structure, large size, and multiple posttranslational modifications of von Willebrand factor (VWF) presented a technological challenge for the production of recombinant VWF (rVWF). Nonetheless, we developed an rVWF product for treating von Willebrand disease, whereupon rVWF is coexpressed with recombinant factor VIII (rFVIII) in Chinese hamster ovary cells used to produce rFVIII for the treatment of hemophilia A. Here we describe the characterization of the structure and function of the rVWF drug product, with a focus on its in vitro platelet aggregation and matrix protein binding functions. Electron microscopy and multimer analysis revealed a highly organized structure for the rVWF protein, with a homogeneous multimer distribution including ultrahigh molecular weight multimers. The specific activity for binding to collagen and platelets mediated by ristocetin is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. The affinity and binding capacity of rVWF to FVIII is comparable to VWF in plasma. rVWF effectively binds to platelets and promotes platelet adhesion under shear stress similar to VWF in human plasma.

Phospholipid scramblase: an update.

The best understood consequence of the collapse of lipid asymmetry is exposure of phosphatidylserine (PS) in the external leaflet of the plasma membrane bilayer, where it is known to serve at least two major functions: providing a platform for development of the blood coagulation cascade and presenting the signal that induces phagocytosis of apoptotic cells. Lipid asymmetry is collapsed by activation of phospholipid scramblase(s) that catalyze bidirectional transbilayer movement of the major classes of phospholipid. The protein corresponding to this activity is not yet known. Observations on cells from patients with Scott syndrome, a rare hereditary bleeding disorder resulting from impaired lipid scrambling, have shown that there are multiple activation pathways that converge on scramblase activity.

Absence of platelet-dependent fibrin formation in a patient with Scott syndrome.

To gain insight into the contribution of platelet-dependent thrombin formation in haemostasis and thrombosis, we investigated under flow conditions the haemostatic functions of platelets from a patient with Scott syndrome. Scott platelets are characterised by a diminished platelet-dependent thrombin generation. Thrombin generation was determined by calibrated automated thrombography and flow-based experiments were performed to reveal collagen-mediated platelet activation and fibrin deposition. Our studies indicate that adherent Scott platelets do not differ from control platelets in the formation of stable platelet aggregates under static and flow conditions. While for adherent control platelets a shape change, e.g. balloon formation, and externalisation of phosphatidylserine (PS) is associated with an increase in intracellular calcium concentration, this is not the case for Scott platelets. The calcium-induced morphological changes in control platelets are accompanied with a diminished recruitment of free flowing platelets. Scott platelets, not showing a calcium-induced shape change, also lost the ability to recruit free flowing platelets. These findings rebut the hypothesis that the mild bleeding tendency of Scott syndrome patients is due to a preserved adhesive activity of patient's platelets. Perfusion of tissue factor (TF)-activated control blood over immobilised collagen results in the formation of fibrin fibers that radiate from platelet aggregates. Although platelet aggregates were also observed after perfusion with TF-activated Scott blood, fibrin deposition was not observed. In conclusion, our findings indicate that platelet adhesion and spreading on a collagen matrix in the absence of fibrin formation is sufficient to sustain haemostasis under non-traumatic conditions.

Factor Xa-driven thrombin generation in plasma: dependency on the aminophospholipid density of membranes and inhibition by phospholipid-binding proteins.

Phosphatidylserine (PS) externalization of activated platelets plays a pivotal role in haemostasis and thrombosis. In the present study we have explored the relationship between the PS density of membranes and the rate of thrombin generation in plasma. Factor (F)Xa-initiated thrombin generation was measured in platelet-free plasma (PFP) containing either phospholipid vesicles of varying PS-content or non-stimulated platelets (reconstituted PRP). The duration of the initiation phase of FXa-driven thrombin generation decreased dramatically with increasing PS density. Concomitantly, the maximal rate of thrombin generation during the propagation phase (maxR) increased non-linearly, with the steepest incline between 5 and 10 mol% PS. Titration of FVa into plasma containing 2 mol% PS increased maxR proportionally and diminished the lag phase. In contrast, platelet-dependent thrombin generation was not influenced by addition of FVa. With increasing platelet concentration, the duration of the initiation phase drastically decreased, and maxR increased proportionally. At a physiologically relevant platelet concentration, maxR corresponded with the maxR found with 2 microM of 10 mol% PS. Annexin A5 (AnxA5) and lactadherin appeared to be powerful inhibitors of in-situ thrombin generation under all conditions examined, with AnxA5 being three- to four-fold more potent than lactadherin. In conclusion, maximal thrombin generation in plasma requires membranes with a density of 10-20 mol% PS. Our data further indicate that thrombin formed in situ induces externalization of PS to approx 10 mol% in a substantial platelet subpopulation.

Reversible inhibition of the platelet procoagulant response through manipulation of the Gardos channel.

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.

Anti-prothrombin IgG from patients with anti-phospholipid antibodies inhibits the inactivation of factor Va by activated protein C.

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.

Synergistic effect of thrombin on collagen-induced platelet procoagulant activity is mediated through protease-activated receptor-1.

OBJECTIVE: In the blood coagulation process, the rate of thrombin formation is critically dependent on phosphatidylserine (PtdSer) at the surface of activated platelets. Thrombin synergistically enhances the collagen-induced platelet procoagulant response. The objective of this study is to elucidate the mechanism of this synergistic action with a focus on the intracellular Ca2+ concentration ([Ca2+]i) and the various platelet receptors for thrombin. METHODS AND RESULTS: We demonstrate that procoagulant activity is related to a sustained increased [Ca2+]i, which in turn depends on extracellular Ca2+ influx. Increased PtdSer exposure coincides with increased [Ca2+]i and was observed in a subpopulation (approximately 14%) of the platelets after stimulation with thrombin plus collagen. 2D2-Fab fragments against the thrombin binding site on GPIbalpha made clear that this receptor did not signal for platelet procoagulant activity. Inhibition of protease-activated receptor 1 (PAR-1) and PAR-4 by selective intracellular inhibitors and selective desensitization of these receptors revealed that PAR-1, but not PAR-4, activation is a prerequisite for both sustained elevations in [Ca2+]i and procoagulant activity induced by collagen plus thrombin. CONCLUSIONS: The interaction of thrombin with PAR-1 mediates a synergistic effect on collagen-induced procoagulant activity by inducing a sustained elevation in [Ca2+]i in a subpopulation of platelets.

Quantitative determination of the binding of beta2-glycoprotein I and prothrombin to phosphatidylserine-exposing blood platelets.

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.

The effect of phospholipids on the formation of immune complexes between autoantibodies and beta2-glycoprotein I or prothrombin.

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.

Scott syndrome, a bleeding disorder caused by defective scrambling of membrane phospholipids.

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.

Influence of erythrocyte shape on the rate of Ca2+-induced scrambling of phosphatidylserine.

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.

Store-mediated calcium entry in the regulation of phosphatidylserine exposure in blood cells from Scott patients.

Scott syndrome is a bleeding disorder, characterized by impaired surface exposure of procoagulant phosphatidylserine (PS) on platelets and other blood cells, following activation with Ca(2+)-elevating agents. Since store-mediated Ca(2+) entry (SMCE) forms an important part of the Ca(2+) response in various blood cells, it has been proposed that deficiencies in Ca(2+) entry may relate to the impaired PS exposure in the Scott syndrome. Here, we have tested this hypothesis by investigating the relationship between Ca(2+) fluxes and PS exposure in platelets as well as B-lymphoblasts derived from the original Scott patient (M.S.), a newly identified Welsh patient (V.W.) with similar bleeding symptoms, and two control subjects. Procoagulant activity of V.W. platelets in suspension, measured after stimulation with collagen/thrombin or Ca(2+)-ionophore, ionomycin, resulted in 52% or 17%, respectively, compared to that of correspondingly activated control platelets. Procoagulant activity of V.W. erythrocytes treated with Ca(2+)-ionophore resulted in less than 6% of the activity of control erythrocytes. Single-cell Ca(2+) responses of M.S. and V.W. platelets, adhering to collagen, were similar to those of platelets from control subjects, while PS exposure was reduced to 7% and 15%, respectively, compared to controls. Stimulation of non-apoptotic B-lymphoblasts derived from both patients and controls with Ca(2+)-ionophore or agents causing Ca(2+) mobilization and SMCE, resulted in similar Ca(2+) responses. However, in lymphoblasts from M.S. and V.W. Ca(2+)-induced PS exposure was reduced to 7% and 13% of the control lymphoblasts, respectively. We conclude that i. patient V.W. is a new case of Scott syndrome, ii. Ca(2+) entry in the platelets and lymphoblasts from both Scott patients is normal, and iii. elevated [Ca(2+)](i) as caused by SMCE is not sufficient to trigger PS exposure.

Kinetics of prothrombin-mediated binding of lupus anticoagulant antibodies to phosphatidylserine-containing phospholipid membranes: an ellipsometric study.

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.