Alan J. Schroit

Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase

Influx of calcium in platelets and red cells produces formation of vesicles shed from the plasma membrane. The time course of the shedding process closely correlates with the ability of both cells to stimulate prothrombinase activity when used as a source of phospholipid in the prothrombinase assay. This reflects increased surface exposure of phosphatidylserine, presumably resulting from a loss in membrane asymmetry. Evidence is presented that the shed vesicles have a random phospholipid distribution, while the remnant cells show a progressive loss of membrane phospholipid asymmetry when more shedding occurs. Removal of intracellular calcium produces a decrease of procoagulant activity of the remnant cells but not of that of the shed vesicles. This is consistent with reactivation of aminophospholipid translocase activity, being first inhibited by intracellular calcium and subsequently reactivated upon calcium removal. Involvement of aminophospholipid translocase is further supported by the observation that reversibility of procoagulant activity is also dependent on metabolic ATP and reduced sulfhydryl groups. The finding that this reversibility process is not apparent in shed vesicles may be ascribed to the absence of translocase or to a lack of ATP. These data support and extend the suggestion made by Sims et al. [1989) J. Biol. Chem. 264, 17049-17057) that membrane fusion, which is required for shedding to occur, produces transient flip-flop sites for membrane phospholipids. Furthermore, the present results indicate that scrambling of membrane phospholipids can only occur provided that aminophospholipid translocase is inactive.

Immune Clearance of Phosphatidylserine-expressing Cells by Phagocytes THE ROLE OF β2-GLYCOPROTEIN I IN MACROPHAGE RECOGNITION

The function of β2-glycoprotein I (β2GPI), a 50-kDa serum glycoprotein, is not completely understood but has been suggested to be involved in the regulation of thrombosis (Brighton, T. A., Hogg, P. J., Dai, Y.-P., Murray, B. H., Choing, B. H., and Chesterman, C. N. (1996) Br. J. Haematol. 93, 185–194) and the clearance of phosphatidylserine (PS)-expressing cells (Chonn, A., Semple S. C., and Cullis P. R. (1995)J. Biol. Chem. 270, 25845–25849). To further understand the role of this protein, we characterized the ability of β2GPI to interact with PS vesicles and influence their uptake by macrophages in vitro. β2GPI bound to and precipitated vesicles containing anionic but not zwitterionic phospholipids in a gel diffusion assay. β2GPI also inhibited the procoagulant activity of PS liposomes. In vitro phagocytosis studies showed 20-fold greater uptake of PS liposomes over phosphatidylcholine liposomes. This enhanced uptake was maintained even after PS was “shielded” with β2GPI and further increased upon the addition of β2GPI antibodies. Similar to liposomes, PS-expressing apoptotic thymocytes and lipid symmetric red blood cell ghosts bound β2GPI. Macrophage uptake of these cells was also maintained or enhanced in the presence of β2GPI and further increased upon the addition of β2GPI antibodies. It is concluded that β2GPI can play a critical role in hemostasis by influencing both thrombosis and the clearance of PS-expressing cells.

Regulated Externalization of Phosphatidylserine at the Cell Surface IMPLICATIONS FOR APOPTOSIS

The regulated loss of plasma membrane phosphatidylserine (PS) asymmetry is critical to many biological processes. In particular, the appearance of PS at the cell surface, a hallmark of apoptosis, prepares the dying cell for engulfment and elimination by phagocytes. While it is well established that PS externalization is regulated by activation of a calcium-dependent phospholipid scramblase activity in concert with inactivation of the aminophospholipid translocase, there is no evidence indicating that these processes are triggered and regulated by apoptotic regulatory mechanisms. Using a novel model system, we show that PS externalization is inducible, reversible, and independent of cytochrome c release, caspase activation, and DNA fragmentation. Additional evidence is presented indicating that the outward movement of plasma membrane PS requires sustained elevation in cytosolic Ca2+ in concert with inactivation of the aminophospholipid translocase and is inhibited by calcium channel blockers.

Characterization of Phosphatidylserine-dependent β2-Glycoprotein I Macrophage Interactions IMPLICATIONS FOR APOPTOTIC CELL CLEARANCE BY PHAGOCYTES

The binding and uptake of phosphatidylserine (PS)-expressing cells appears to involve multiple receptor-mediated systems that recognize the lipid either directly or indirectly through intermediate proteins that form a molecular bridge between the cells. Here we show that β2-glycoprotein I (β2GPI), a 50-kDa serum glycoprotein, binds PS-containing vesicles and serves as an intermediate for the interaction of these vesicles with macrophages. Chemical modification of lysines and cysteines abolished β2GPI-dependent PS uptake by inhibiting the binding of PS to β2GPI and the binding of PS·β2GPI complex to macrophages, respectively. Recognition was mediated by β2GPI and not by the lipid because antibodies to β2GPI inhibited binding of the complex to macrophages. These results indicate that human (THP-1-derived) macrophages bind β2GPI only after it is bound to its lipid ligand. Competition experiments with monosaccharides that inhibit lectin-dependent interactions, and PS·β2GPI binding experiments using deglycosylated β2GPI, suggested that carbohydrate residues were not required for macrophage recognition of the complex. Antibodies to putative macrophage PS receptors (CD36, CD68, and CD14) did not inhibit uptake of the complex. These data suggest that β2GPI can bind cells that fail to maintain membrane lipid asymmetry and generate a specific bridging moiety that is recognized for clearance by a phagocyte receptor that is distinct from CD36, CD68, and CD14.

ROLE OF TRANSLOCASES IN THE GENERATION OF PHOSPHATIDYLSERINE ASYMMETRY

The distribution of phospholipids across the plasma membrane of eukaryotic cells is not random, and certain phospholipids are distributed asymmetrically across the lipid bilayer. This is particularly true both for the choline-containing phospholipids, phosphatidylcholine (PC) and sphingomyelin, which are primarily located in the cell’s outer leaflet, and for the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE) which are found at the membrane’s cytoplasmic face. Although it has been known for more than two decades that membranes are asymmetric (Bretscher, 1972) the biological significance of this likely ubiquitous phenomenon has only recently been addressed. While our understanding is still limited, it is clear that transmembrane orientation of lipids influences membrane structure and the function of various membrane-bound enzymatic systems. There is also evidence to suggest that certain lipids localized at specific locations within the cell membrane participate in processes as diverse as cell-cell recognition and blood coagulation. Because spontaneous transbilayer movement of charged phospholipids is very slow in artificial phospholipid bilayers (Kornberg & McConnell, 1971; Pagano & Sleight, 1985), the generation of lipid asymmetry in cells must be controlled and regulated by specific lipid transport processes. Indeed, the movement of aminophospholipids from the cells outer-to-inner leaflet has been shown to be dependent upon an ATP-driven aminophospholipid translocase/flipase which transports PS and PE across the cell membrane (Seigneuret & Devaux, 1984). In this review, we summarize data supporting the concept that specific membrane proteins constitute the machinery that generates transbilayer aminophospholipid movement, thereby controlling and regulating the equilibrium distribution of lipids between both membrane leaflets. Important recent results concerning the identification of the aminophospholipid flipase from red blood cells will be discussed. Its possible cooperativity with other lipid flopases/scramblases that ultimately regulate the membrane sidedness of PS and determine the equilibrium distribution of lipids across the cell’s plasma membrane will also be discussed. This review focuses on data obtained with red cells and platelets. The reader is also referred to other recent reviews on lipid flipases and related topics (Devaux, 1992; Schroit, 1994; Devaux & Zachowski, 1994; Menon, 1995).

Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer.

Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes. Over the past decades, the molecular and cell biological events associated with efferocytosis have been rigorously studied, and many eat-me signals and receptors have been identified. The externalization of phosphatidylserine (PS) is arguably the most emblematic eat-me signal that is in turn bound by a large number of serum proteins and opsonins that facilitate efferocytosis. Under physiological conditions, externalized PS functions as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. Pathologically, the innate immunosuppressive effect of externalized PS has been hijacked by numerous viruses, microorganisms, and parasites to facilitate infection, and in many cases, establish infection latency. PS is also profoundly dysregulated in the tumor microenvironment and antagonizes the development of tumor immunity. In this review, we discuss the biology of PS with respect to its role as a global immunosuppressive signal and how PS is exploited to drive diverse pathological processes such as infection and cancer. Finally, we outline the rationale that agents targeting PS could have significant value in cancer and infectious disease therapeutics.

Lipid antioxidant, etoposide, inhibits phosphatidylserine externalization and macrophage clearance of apoptotic cells by preventing phosphatidylserine oxidation.

Apoptosis is associated with the externalization of phosphatidylserine (PS) in the plasma membrane and subsequent recognition of PS by specific macrophage receptors. Selective oxidation of PS precedes its externalization/recognition and is essential for the PS-dependent engulfment of apoptotic cells. Because etoposide is a potent and selective lipid antioxidant that does not block thiol oxidation, we hypothesized that it may affect PS externalization/recognition without affecting other features of the apoptotic program. We demonstrate herein that etoposide induced apoptosis in HL-60 cells without the concomitant peroxidation of PS and other phospholipids. HL-60 cells also failed to externalize PS in response to etoposide treatment. In contrast, oxidant (H2O2)-induced apoptosis was accompanied by PS externalization and oxidation of different phospholipids, including PS. Etoposide potentiated H2O2-induced apoptosis but completely blocked H2O2-induced PS oxidation. Etoposide also inhibited PS externalization as well as phagocytosis of apoptotic cells by J774A.1 macrophages. Integration of exogenous PS or a mixture of PS with oxidized PS in etoposide-treated HL-60 cells reconstituted the recognition of these cells by macrophages. The current data demonstrate that lipid antioxidants, capable of preventing PS peroxidation, can block PS externalization and phagocytosis of apoptotic cells by macrophages and hence dissociate PS-dependent signaling from the final common pathway for apoptosis.

Plasma Protein β-2-Glycoprotein 1 Mediates Interaction between the Anti-tumor Monoclonal Antibody 3G4 and Anionic Phospholipids on Endothelial Cells

A promising target on tumor vasculature is phosphatidylserine (PS), an anionic phospholipid that resides exclusively on the inner leaflet of the plasma membrane of resting mammalian cells. We have shown previously that PS becomes exposed on the surface of endothelial cells (EC) in solid tumors. To target PS on tumor vasculature, the murine monoclonal antibody 3G4 was developed. 3G4 localizes to tumor vasculature, inhibits tumor growth, and enhances anti-tumor chemotherapies without toxicity in mice. A chimeric version of 3G4 is in clinical trials. In this study, we investigated the basis for the interaction between 3G4 and EC with surface-exposed PS. We demonstrate that antibody binding to PS is dependent on plasma protein β-2-glycoprotein 1 (β2GP1). β2GP1 is a 50-kDa glycoprotein that binds weakly to anionic phospholipids under physiological conditions. We show that 3G4 enhances binding of β2GP1 to EC induced to expose PS. We also show that divalent 3G4-β2GP1 complexes are required for enhanced binding, since 3G4 Fab′ fragments do not bind EC with exposed PS. Finally, we demonstrate that an artificial dimeric β2GP1 construct binds to EC with exposed PS in the absence of 3G4, confirming that antibody binding is mediated by dimerization of β2GP1. Together, these data indicate that 3G4 targets tumor EC by increasing the avidity of β2GP1 for anionic phospholipids through formation of multivalent 3G4-β2GP1 complexes.

Crosstalk between protease-activated receptor 1 and platelet-activating factor receptor regulates melanoma cell adhesion molecule (MCAM/MUC18) expression and melanoma metastasis.

The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis.

β-2-Glycoprotein 1-dependent Macrophage Uptake of Apoptotic Cells BINDING TO LIPOPROTEIN RECEPTOR-RELATED PROTEIN RECEPTOR FAMILY MEMBERS

The recognition and removal of apoptotic cells is critical to development, tissue homeostasis, and the resolution of inflammation. Many studies have shown that phagocytosis is regulated by signaling mechanisms that involve distinct ligand-receptor interactions that drive the engulfment of apoptotic cells. Studies from our laboratory have shown that the plasma protein β-2-glycoprotein 1 (β2GP1), a member of the short consensus repeat superfamily, binds phosphatidylserine-containing vesicles and apoptotic cells and promotes their bridging and subsequent engulfment by phagocytes. The phagocyte receptor for the protein/apoptotic cell complex, however, is unknown. Here we report that a member of the low density lipoprotein receptor-related protein family on phagocytes binds and facilitates engulfment of β2GP1-phosphatidylserine and β2GP1-apoptotic cell complexes. Using recombinant β2GP1, we also show that β2GP1-dependent uptake is mediated by bridging of the target cell to the phagocyte through the protein C- and N-terminal domains, respectively.