Biochemical Society Transactions

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1 Biochemical Society Transactions 248 Mechanism and function of changes in membrane-phospholipid asymmetry in platelets and erythrocytes Robert F. A. Zwaal, Paul Comfurius and Edouard M. Bevers Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, P.O. Box 6 16, Maastricht, The Netherlands Introduction In the two decades since it was first proposed by Hretscher [ 11, ample evidence has accumulated to support the general view that both leaflets of the lipid bilayer of most, if not all, biological membranes have distinctly different lipid compositions (see [2] for a recent review). Whereas the outer leaflet is generally rich in cholinephospholipids, sphingomyelin and phosphatidylcholine, the inner leaflet is preferentially occupied by aminophospholipids, phosphatidylethanolamine and phosphatidylserine. Despite the fact that membrane-phospholipid asymmetry has become widely appreciated as a ubiquitous phenomenon, the mechanisms involved in its regulation and maintenance have begun to be addressed only recently. The discovery of an ATP-dependent aminophospholipid-specific transporter, which shuttles phosphatidylserine and phosphatidylethanolamine across the bilayer membrane towards the inner leaflet [3], suggests that a specific transmembrane orientation of these lipids is of major importance for cell function. Although our understanding of the physiological role of membrane lipid asymmetry is still fragmentary, there is evidence to suggest that exposure of phosphatidylserine at the cell surface - which can occur during cell activation - provides a signal for cell-cell recognition [4, 51 and is instrumental in promoting blood coagulation [6,7]. In this article we address the possible mechanisms which may play a role in the loss of membrane-phospholipid asymmetry that happens upon activation of blood platelets or upon CaZ+entry into red blood cells. In addition we will highlight an experiment of nature by featuring a rare bleeding disorder in which these phenomena do not occur. Loss of membrane-phospholipid asymmetry Influx of Caz+ into cells represents the key event of cellular activation and initiates a variety of cellular responses. In blood platelets and red blood cells these responses include characteristic shape Abbreviation used: LIMIT, dimyristoylphosphatidylcholine. changes with evaginations of the cell surface and the shedding of small membrane vesicles with a diameter of approx. 0.2 pm from the plasma membrane [8, 91. The formation of these microvesicles, which requires fusion between apposing segments of plasma membrane, is accompanied by a progressive loss of phospholipid asymmetry both in the cell membrane and in that of the microvesicles [ Scrambling of the lipids over the membrane bilayer is presumably caused by rapid trans-bilayer movement (flip-flop) of all the phospholipids [7, 12, 131. This increases exposure of aminophospholipids at the outer surface, particularly when the aminophospholipid translocase (which may generate and maintain phospholipid asymmetry by specifically transporting aminophospholipids to the inner leaflet) becomes inhibited by a rise in cytoplasmic Ca2+ [ 14, 151. Remarkably subsequent removal of intracellular + CaZ restores translocase activity, provided that proteolytic attack on translocase by intracellular calpain is prevented. As a result, phosphatidylserine exposed at the cell surface of activated platelets or Caz+-loaded red blood cells is pumped back to the inner membrane leaflet upon reactivation of translocase by extrusion of Ca2+ [ 111. Since this reversibility process depends on sufficient availability of intracellular ATP, it does not occur in the shed microvesicles, which lack this nucleotide. While the mechanisms by which the surface exposure of phosphatidylserine promotes cell-cell recognition are still unresolved, its pivotal role in promoting assembly on the membrane of two enzyme-substrate complexes of the coagulation cascade, the tenase and the prothrombinase complexes, is much better understood [16]. In the tenase reaction, the pro-enzyme factor X is activated to factor Xa by a complex of the proteolytic enzyme factor IXa and its protein cofactor VIIIa. In the prothrombinase reaction, prothrombin is converted to thrombin by a complex of the enzyme factor Xa and its protein cofactor Va. Membrane assembly of these complexes in the presence of Ca2+ critically depends on the exposure of anionic phosphatidylserine; the assembly dramatically enhances the rate of thrombin formation, accounting for the procoagulant activity of activated blood platelets [ 171. Calcium-ionophore treatment of red Volume 21

2 Phospholipid Translocation, Asymmetry and Membrane Fusion blood cells or their resealed ghosts also leads to surface exposure of phosphatidylserine [18] and to expression of membrane-procoagulant activity [ 1 11 and microvesicle formation [8, 10, 191, albeit more slowly than in platelets. The tight association between Caz+-induced loss of membrane-phospholipid asymmetry and shedding of microvesicles from the plasma membrane has been illustrated in platelets and in red blood cells from a patient with a rare bleeding disorder, known as Scott syndrome, stemming from the inability of the platelets to acquire procoagulant activity upon cell activation. Observations on Scott syndrome Scott syndrome is an extremely rare, moderately severe bleeding disorder, first described by Weiss et al. [20] to be related to an isolated deficiency of platelet-procoagulant activity. Although these platelets exhibit normal secretion and aggregation in response to various agonists, they have a considerably lower capacity to support both tenase and prothrombinase activity [21]. Impaired assembly of both enzyme-substrate complexes on the platelet membrane is likely, since these platelets express a significantly reduced number of binding sites for factors Va [9] and VIIIa [22] relative to normal platelets. Moreover the platelets of the patient upon stimulation have been shown to be appreciably impaired in their ability to expose phosphatidylserine at the outer surface [Zl], while they are also markedly deficient in their capability to generate microvesicles [9]. It is intriguing that this aberrant behaviour can be evoked also in the red blood cells of the patient. In response to a calcium ionophore, these erythrocytes do not expose phosphatidylserine on the outer surface with concurrent shedding of microvesicles from the plasma membrane [19]. Moreover experiments using fluorescently-labelled phospholipids suggest strongly that the calcium ionophoreinduced trans-bilayer flip-flop of the phospholipids does not occur in the red blood cells of the patient. When examined by scanning electron microscopy, untreated Scott-syndrome red blood cells are indistinguishable from normal cells. However, while treatment of normal erythrocytes with calcium ionophore induces marked echinocytosis and spiculation, the cells of the patient show a remarkable ability to retain a biconcave structure (Figure 1). These aberrant responses to intracellular Caz+ are equally apparent in resealed ghosts from Scottsyndrome erythrocytes, irrespective of whether red ghosts or white ghosts, from which the cytoplasm has been completely removed, were examined. Since rigorous examination of the lipid composition has not revealed any differences with respect to normal cells, the aberrant behaviour of the cells may be related to a defect in a membrane protein or in the membrane-associated cytoskeleton. The observation that the erythrocytes and resealed ghosts of the patient share many of the membrane abnormalities found for Scott-syndrome platelets suggests that this defect is common to both cell lines and may reflect a mutation in an early stem cell before differentiation into erythrocytes and platelets. Possible mechanisms involved in scrambling of membrane phospholipids The findings with Scott-syndrome platelets and red blood cells may imply that loss of membranephospholipid asymmetry upon Ca +-influx is inextricably related to the formation of plasma membrane-derived microvesicles. A possible involvement of a soluble cytoplasmic component that would transduce the Ca2+ signal to the membrane can be ruled out, as resealed erythrocyte ghosts respond virtually identically to CaZ+ -influx as do the parent red blood cells, including the aberrant behaviour in Scott syndrome [19]. The remarkable ability of Scott-syndrome red blood cells to retain a discoid shape upon Caz+-influx, in contrast to the echinocyte formation usually observed in normal cells, may point to an involvement of a membrane protein or a cytoskeletal protein that responds to Ca + and is present both in erythrocytes and in platelets. Since blebbing and shedding of microvesicles from the cell surface must entail eversion and fusion of apposing segments of plasma membrane, this process has been suggested to lead to the transient formation of nonbilayer structures at the point where the plasma membrane fuses to form the budding vesicle [9] (Figure 2a). These fusion sites would facilitate rapid flip-flop of lipids leading to a localized collapse of membrane-phospholipid asymmetry, both in the emerging vesicles and in the remnant cell. In this view it is implicitly assumed that microvesicle formation is driven by Ca +-regulated rearrangement of membrane cytoskeleton and that lipid scrambling is facilitated by (or is even the result of) the fusion events required for shedding of vesicles to occur. Alternatively the possible involvement of a putative integral-membrane protein (clearly distinct from aminophospholipid translocase) that, in response to intracellular Ca2+, produces bidirectional flip-flop of all phospholipids cannot be excluded. If protein- 249 I993

3 Biochemical Society Transactions 250 Figure I Scanning electron-micrographs of Ca2+-ionophore-treated red blood cells and resealed ghosts obtained from normal controls and from a patient with Scott syndrome Whereas the Ca2+-ionophore induces marked echinocytosis and spiculation of normal erythrocytes (a) and resealed ghosts (c), in general Scott syndrome erythrocytes (b) and resealed ghosts (d) retain a biconcave structure under these conditions. catalysed outward transport of aminophospholipids were to take place faster than inward transport of cholinephospholipids (Figure Zb), the outermembrane leaflet would accumulate more mass at the expense of the inner-membrane leaflet. Following the coupled bilayer theory of Sheetz and Singer [23], the membrane responds by bending outward to form protrusions, a phenomenon which has been observed frequently upon intercalation of lipids or lipophilic drugs in the outer monolayer as well. In this way membrane eversion can be envisaged to facilitate microvesicle formation, in which case shedding may be driven by unequal flip-flop rates of the different membrane-phospholipid classes. It should be emphasized however that the mechanisms presented in Figure 2 are not mutually exclusive and there is no compelling reason a priori to exclude the possibility that they operate simultaneously to propagate lipid scrambling and microvesicle formation. Although direct evidence for the existence of a membrane protein that scrambles the lipids over the bilayer in response to intracellular CaZ+ is lacking, we have recently designed experiments that suggest that such a protein might be present in redblood-cell membranes. Incubation of intact erythrocytes with dimyristoylphosphatidylcholine (DMPC) is known to result in the release of plasma membrane-derived microvesicles with a diameter of about 150 nm, which completely lack spectrin, the major component of the membrane skeleton [24]. While these vesicles have nearly normal amounts of band 3 protein and of glycophorin, they are markedly enriched in a minor membrane protein of approx. 30 kda. When these vesicles are formed under appropriate conditions to maintain high levels of ATP, both the vesicles and the remnant cells from which they are derived retain an asymmetric phospholipid distribution. Interestingly, treatment of the vesicles with a Ca2+-ionophore Volume 21

4 Phospholipid Translocation, Asymmetry and Membrane Fusion Figure 2 Two hypothetical models that may explain the tight association between Ca2+-induced scrambling of plasma membrane phospholipids and the shedding of microvesicles. (0) Fusion-induced flip-flop facilitates lipid scrambling. (b) Flip-flop-induced eversion facilitates microvesicle formation 25 I (a) Formation of microvesicles as a result of Ca2+-induced rearrangements of membrane skeletal proteins entails eversion and fusion of apposing segments of the plasma membrane. These fusion events may be accompanied by the formation of non-bilayer structures, which facilitate rapid trans-bilayer movement of phospholipids leading to a progressive loss of membrane-phospholipid asymmetry (see [9]). (b) Trans-bilayer movement and scrambling of phospholipids as a result of Ca2+-influx may be mediated by a putative membrane protein. Provided that outward transport of aminophospholipids is catalysed at a faster rate than inward transport of cholinephospholipids, the resulting mass imbalance leads to the eversion of plasma membrane which may facilitate microvesicle formation. (0) Emerging + Ca2 Emerging membrane results in the surface exposure of phosphatidylserine, as detected by the ability of the vesicles to promote prothrombinase activity, whereas the remnant cells have almost completely lost this ability (Figure 3). This suggests that the Ca2+-induced scrambling activity has been preferentially sorted into the vesicles released upon DMPC treatment. Since the size of these vesicles is not appreciably changed upon ionophore treatment, excluding fusion events, it is tempting to speculate that these vesicles have virtually completely collected a protein-catalysed activity that induces rapid flip-flop and scrambling of the phospholipids in response to intracellular Ca2+. I993

5 Biochemical Society Transactions 252 Figure 3 Generation of prothrombinase activity upon Ca2+ionophore treatment of DMPC-induced vesicles from red blood cells The ability of the cell preparations to stimulate prothrombinase reflects surface exposure of phosphatidylserine. DMPC-induced release of membrane vesicles was accomplished as described in [24], using appropriate conditions to maintain high levels of ATP to preserve membrane-phospholipid asymmetry, both in the released vesicles and in the remnant cells. lonophore treatment of released vesicles leads to an approx. ten-fold more rapid time-dependent increase in prothrombinase activity (that is phosphatidylserine exposure) than observed with control red cells, whereas the remnant cells from which the vesicles have been released have virtually completely lost this ability Remnant red cells Time (min) f ~ 2I a7 3 Finally it should be noted that the CaZfinduced rapid bidirectional flip-flop of phospholipids is not only unrelated to aminophospholipid translocase activity, but also differs from the outward movement of phospholipids observed in red cells in the absence of CaZf [25, 261. This latter process, which is lipid-species independent, is appreciably slower than translocase-catalysed inward movement of aminophospholipids, but has similar requirements in that it is dependent on ATP and reduced membrane sulphydryls as well [27, 281. In conjunction both of these processes lead to a situation where all lipids are being continuously, albeit slowly, moved to the outer leaflet, while the aminophospholipids are being concomitantly rapidly transported to the inner leaflet. Whereas this activity contributes to establishing membranephospholipid asymmetry, the rapid Caz+ -induced flip-flop, which does not require ATP and is, at least in platelets, enhanced by sulphhydryl oxidation [ 111, produces lipid randomization resulting in a more symmetric distribution of lipids across the membrane bilayer. Although this + CaZ -induced scrambling of phospholipids requires that aminophospholipid-translocase activity is inhibited, mere inhibition of translocase (for example by ATP depletion) does not by itself result in rapid exposure of aminophospholipids on the cell surface [2] Bretscher, M. S. (1972) Nature (London) New Biol. 236,Il-12 Schroit, A. J. and Zwaal, R. F. A. (1991) Biochim. Biophys. Acta 1071, Seigneuret, M. and Devaux, P. F. (1984) Proc. Natl. Acad. Sci. USA. 81, Schroit, A. J., Madsen, J. W. and Tanaka, Y. (1985) J. Biol. Chem. 260,s Schwartz, R. S., Tanaka, Y., Fidler, I. J., Chiu, D. T., Lubin, B. and Schroit, A. J. (1985) J. Clin. Invest. 75, Zwaal, R. F. A. (1978) Biochim. Biophys. Acta 515, Bevers, E. M., Comfurius, P. and Zwaal, K. F. A. (1983) Biochim. Biophys. Acta 736,57-66 Allan, L). and Michell, R. H. (1 975) Nature (London) 258, Sims, 1. J., Wiedmer, T., Esmon, C. T., Weiss, H. J. and Shattil, S. J. (1989) J. Hiol. Chem. 264, Zwaal, K. F. A., Comfurius, 1. and Bevers, E. M. (1992) Biochim. Biophys. Acta 1180, 1-8 Comfurius, P., Senden, J. M. G.. Tilly, K. H. J., Schroit, A. J., Bevers, E. M. and Zwaal, R. F. A. (1990) Biochim. Biophys. Acta 1026, Bevers, E. M., Verhallen, P. I;. J., Visser, A. J. W. G., Comfurius, P. and Zwaal, R. F. A. (1990) Biochemistry 29,s Williamson, P., Kulick, A., Zachowski, A,, Schlegel, R. A. and Devaux, P. F. (1992) Biochemistry 31, Bitbol, M., Fellmann, P., Zachowski, A. and Lkvaux, P. F. (1987) Biochim. Biophys. Acta Tilly, K. H. J., Senden, J. M. G., Comfurius, P., Revers, E. M. and Zwaal, R. F. A. (1990) Biochim. Biophys. Acta 1029, Mann, K. G., Nesheim, M. E., Church, W. R., Haley, P. and Krishnaswamy, S. ( 1990) Blood 76, Rosing, J., Van Rijn. J. I,. M. I,.. Bevers, E. M., Van Dieijen, G., Comfurius, P. and Zwaal, R. F. A. (1985) Blood Chandra, K., Joshi, P. C., Bajpai, V. K. and Gupta, C. M. (1987) Biochim. Biophys. Acta 902, Bevers, E. M., Wiedmer, T., Comfurius, P., Shattil, S. J.? Weiss, H. J., Zwaal, R. F. A. and Sims, P. J. (1992) Blood 79, Weiss, H. J.? Vivic, W. J.$ Lages, B. A. and Rogers, J. ( 1979) Am. J. Med. 67, Rosing, J., Bevers, E. M., Comfurius, P.. Hemker, H. C., Van Dieijen, G., Weiss, H. J. and Zwaal, R. F. A. (1985) Blood 65, Volume 2 I

6 Phospholipid Translocation, Asymmetry and Membrane Fusion 22. Ahmad, S. S., Rawala-Sheikh, R., Ashby, B. and 26. Connor, J., Gillum, K. and Schroit, A. J. (1990) Walsh, P. N. (1989) J. Clin. Invest. 84, Sheetz, M. P. and Singer, S. J. (1974) Proc. Natl. Acad. Biochim. Biophys. Acta 1025, Connor, J. and Schroit, A. J. (1990) Biochemistry 29. Sci. USA. 71, Ott, P., Hope, M. J.? Verkleij, A. J.? Roelofsen, B., 28. Connor, J., Pak, C. H., Zwaal, R. F. A. and Schroit, Brodbeck. U. and Van Deenen, I,. I,. M. (1981) A. J. (1992) J. Biol. Chem. 267, Biochim. Biophys. Acta 641, Bitbol, M. and Devaux, P. F. (1988) Proc. Natl. Acad. Sci. USA. 85, Received 20 November 1992 Trans-bilayer distribution of phosphatidylinositol 4,5-bisphosphate and its role in the changes of lipid asymmetry in the human erythrocyte membrane P. Gascard$, J. C. Sulpice, D. Tran*, M. Sauvage, M. Claret*, A. Zachowskit, P. F. Devauxt and F. Giraud CNRS URA I I 16, Biomembranes et Messagers Cellulaires, Bat 447, *lnserm U 274, Bat 443, Universite Paris XI, 9 I405 Orsay Cedex and tcnrs URA 526, lnstitut de Biologie Physico-chirnique, 75005, Paris, France Phospholipid asymmetry is clearly established in erythrocyte membranes. The aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), are concentrated in the inner leaflet, while the choline-containing phospholipids, sphingomyelin (SM) and phosphatidylcholine (PC), are mainly located in the external leaflet [ 11. This lipid asymmetry, for PE and PS, is maintained by an ATP- and Mg'+-dependent enzyme, the aminophospholipid translocase [2, 31. Although physiologically relevant, polyphosphoinositides, phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,s-bisphosphate (PIP?) and phosphatidic acid (PA) have never been localized. These phospholipids, especially PIP,, play a major role as a precursor of second messengers in many cells [4]. They also regulate a number of membrane properties, such as controlling the activity of membranebound enzymes [S-7], protein-protein interactions [8] and the anchoring of polar proteins by covalent linkage [O]. While lipid asymmetry is normally maintained during the lifetime of red cells, an increase in the cytosolic CaL+ concentration can induce a rapid lipid randomization in the human erythrocyte mem- Abbreviations used: PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIP, phosphatidylinositol 4-phosphate; PIP,, phosphatidylinositol 4,5-bisphosphate; PLA,, phospholipase A,; PS, phosphatidylserine; SM, sphingomyelin. $Present address: Department of Biomedical Research, St. Elizabeth's Hospital, Boston, MA 02135, USA. brane [ This lipid scrambling is even more effective in platelets with exposure of PS on the outer leaflet, where it plays a physiological role during blood clotting [18]. The mechanism by which lipids are redistributed between both leaflets is not understood yet. + CaL -dependent proteins (transglutaminase, calpain, phospholipase A, (PLA,), calmodulin, phosphoinositidase C) do not seem to play a role in the phospholipid scrambling [ll, 12, 15, 191. The interaction of calcium with acidic phospholipids in artificial vesicles or in erythrocytes is known to induce phospholipid segregation [ which could participate in the membrane destabilization. More specifically, PIP, binds Calf with a high affinity when compared with MgL+ or with monovalent cations [23, 241. Under physiological conditions of intracellular ph and free-cation content, unless it is complexed with positively charged regions of proteins, PIP, is negatively charged [24, 251. An increase in cytosolic CaL+ concentration will neutralize the negative charges of the PIPz and this more hydrophobic complex might be responsible for the phospholipid reorganization in the membrane bilayer. In this short review, evidence will be provided for the localization of a small fraction of PIP,, phosphatidylinositol (PI) and PA but no PIP in the outer layer of the erythrocyte membrane [26]. Subsequently it will be shown that exogenously added PIP,, in the presence of Ca'+ or Mg", induces a rapid scrambling of the lipid bilayer and a shape change of the erythrocyte. Finally the possible role of the asymmetrically distributed endogenous PIP, in promoting transient changes in lipid asymmetry upon an increase in cytosolic Ca2+ will be discussed. I993