Phosphatidylserine Asymmetry and Cell Survival
Posted July 16, 2012
Over the last century, the cell membrane has progressed, in the corpus of scientific knowledge, from a vague boundary between the cell's interior and exterior to become a complex, dynamic bilayer of lipid molecules. A growing body of research points to the sweeping and subtle roles that phospholipid molecules play in biology and medicine. In 1925 Gorter and Grendel determined that the cell membrane consisted of a lipid bilayer, but only in the 1950s did scientists confirm that idea with electron microscopy. In the last 40 years, researchers have shown that the outside and inside of those lipid bilayers contain vastly different distributions of various lipid molecules. Plasma membranes can include lipid rafts, clusters of lipids whose composition differs from those surrounding them. These structures are constantly changing, and their dynamics alongside integral membrane proteins regulate signaling, transport, enzyme activity, adhesion, and cell shape, structure, and movement.
Phosphatidylserine (PS)—a phospholipid—tends to be sequestered in the inner leaflet of the plasma membrane, and that asymmetry and any changes in the distribution of PS lead to various biological consequences. That distribution may also play a role in apoptotic signaling and immunogenic responses. So studies in this area not only answer important functional biological questions, but may also be important to treating cancer and infectious diseases.
On May 1, 2012 in a symposium titled Phosphatidylserine Asymmetry and Cell Survival: Therapeutic Applications in Cancer and Infectious Disease, organized by Kenneth Olive of Columbia University Medical Center, Philip Thorpe of UT Southwestern Medical Center, George Zavoico of MLV & Co., and Jennifer Henry of the Academy, researchers gathered to discuss PS and how knowledge of this lipid and its dynamics is shaping our understanding of biology and medicine. Alan Schroit from UT Southwestern Medical Center reviewed for the audience how asymmetry is maintained in the lipid bilayer and the role of that asymmetry in physiological processes. David Ucker of the University of Illinois at Chicago looked at the link between PS and the externalization of glycolytic enzymes and at how this process provides innate immunity against apoptosis. Ari Helenius of the University of Helsinki talked about the role of PS in viral entry to cells. Chris Reutelingsperger of Maastricht University described the use of annexin A5 to target PS and to diagnose and treat PS-related diseases. Finally, Philip Thorpe of UT Southwestern Medical Center described studies of bavituximab, an antibody-based drug that targets PS and that is currently in clinical testing.
Use the tabs above to find a meeting report and multimedia from this event.
Presentations available from:
George Zavoico, PhD (MLV & Co.)
Alan Schroit, PhD (UT Southwestern Medical Center)
David Ucker, PhD (University of Illinois at Chicago)
Ari Helenius, PhD (ETH Hönggerberg, Switzerland)
Chris Reutelingsperger, PhD (Maastricht University, The Netherlands)
Philip E. Thorpe, PhD (UT Southwestern Medical Center)
- 00:011. Introduction
- 0:502. Typical virus entry pathway into cell
- 02:413. Surface virus receptors
- 03:554. Mechanism of endocytosis
- 06:245. Poxvirus
- 10:046. Cellular genes involved in virus entry
- 16:437. Virus uptake by Macropinocytosis
- 19:258. Phosphatydlserine role on vaccinia virus (vv)
- 25:309. Ligand receptor capturing
- 28:3410. Receptor candidates for VV
- 29:3211. Is Gas6 a bridging factor?
- 33:3712. Q and
- 00:011. Introduction
- 01:152. Cell surface expression on PS
- 02:473. Domains that bind to PS
- 04:584. Annexin 5 as imaging agent
- 10:485. Annexin 5 in vivo study
- 15:026. Fusion protein of annexin (anxA5-ETA)
- 17:577. AnaxA5-ETA depend on PS binding
- 19:518. Enhancement strategy of efferocytosis (vv)
- 23:079. In vivo study of RGT-anxA5 with RGD-anxA5
- 30:0510. Q and
- 00:011. Introduction
- 01:022. Symmetry breakdown exposing PS
- 04:363. Tumor blood vessels
- 06:034. Anti-tumor effects in mice
- 09:215. Mechanism by ADCC
- 11:476. Mechanism2 via blockade of PS immunosuppression
- 14:347. M2 to M1 switch
- 20:008. Generation of specific cytotoxic T-cells
- 23:079. Clinical trials (bavituximab)
- 34:2610. Q and
- 00:011. Introduction
- 01:122. Apoptotic cells inhibit cytokine release
- 05:113. Suppresive effect of apoptotic cells
- 09:354. Discrimination of viable cells
- 12:555. PLB-985 do not externalize PS
- 16:546. The cytopathic effect
- 19:187. What are the determinants of cell death?
- 21:578. Proteomic analysis of apoptotic cells
- 24:589. The early process of apoptotic event
- 28:5610. Q and
Mercer J, Helenius A. 2010. Apoptotic mimicry: phosphatidylserine-mediated macropinocytosis of vaccinia virus. Ann. NY Acad. Sci. 1209: 49-55.
Mercer J, Knébel S, Schmidt FI, et al. 2010. Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry. Proc. Natl. Acad. Sci. USA 107: 9346-51.
Mercer J, Helenius A. 2008. Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells. Science 320: 531-5.
Schutters K, Reutelingsperger C. 2010. Phosphatidylserine targeting for diagnosis and treatment of human diseases. Apoptosis 15: 1072-82.
Kenis H, Reutelingsperger C. 2009. Targeting phosphatidylserine in anti-cancer therapy. Curr. Pharm. Des. 15: 2719-23.
Mirnikjoo B, Balasubramanian K, Schroit AJ. 2009. Suicidal membrane repair regulates phosphatidylserine externalization during apoptosis. J. Biol. Chem. 284: 22512-6.
Mirnikjoo B, Balasubramanian K, Schroit AJ. 2009. Mobilization of lysosomal calcium regulates the externalization of phosphatidylserine during apoptosis. J. Biol. Chem. 284: 6918-23.
Balasubramanian K, Mirnikjoo B, Schroit AJ. Regulated externalization of phosphatidylserine at the cell surface: implications for apoptosis. J. Biol. Chem. 282: 18357-64.
Luster TA, He J, Huang X, et al. 2006. Plasma protein beta-2-glycoprotein 1 mediates interaction between the anti-tumor monoclonal antibody 3G4 and anionic phospholipids on endothelial cells. J. Biol. Chem. 281: 29863-71.
Balasubramanian K, Schroit AJ. 2003. Aminophospholipid asymmetry: A matter of life and death. Annu. Rev. Physiol. 65:701-34.
DeRose P, Thorpe PE, Gerber DE. 2011. Development of bavituximab, a vascular targeting agent with immune-modulating properties, for lung cancer treatment. Immunotherapy 3: 933-44.
He J, Yin Y, Luster TA, et al. 2009. Antiphosphatidylserine antibody combined with irradiation damages tumor blood vessels and induces tumor immunity in a rat model of glioblastoma. Clin. Cancer. Res. 15:6871-80.
Mitchell JE, Cvetanovic M, Tibrewal N, et al. 2006. The presumptive phosphatidylserine receptor is dispensable for innate anti-inflammatory recognition and clearance of apoptotic cells. J. Biol. Chem. 281: 5718-25.
Ucker DS, Jain MR, Pattabiraman G, et al. 2012. Externalized glycolytic enzymes are novel, conserved, and early biomarkers of apoptosis. J. Biol. Chem. 287: 10325-43.
Kenneth P. Olive, PhD
Kenneth P. Olive began his doctoral studies in 1998 with Tyler Jacks at the MIT Center for Cancer Research, investigating the neomorphic effects of mutant p53 in a mouse model of Li-Fraumeni Syndrome. While at MIT, he also helped develop a conditional mutant model of advanced lung adenocarcinoma. After graduating in 2005, Olive began a postdoctoral fellowship in the laboratory of David Tuveson at the University of Pennsylvania, later moving with the lab to the University of Cambridge in England. There he built a translational research facility for studying novel anticancer therapeutics in genetically engineered mouse models of pancreatic cancer. His studies into chemoresistance and the effects of Hh pathway inhibitors on drug delivery in pancreatic cancer were published in Science in 2009, and have led to multiple clinical trials to evaluate the approach in patients with metastatic pancreatic cancer. In 2010, Olive joined the faculty of the Columbia University Herbert Irving Comprehensive Cancer Center, where he has established a laboratory dedicated to translational science and experimental therapeutics in pancreatic ductal adenocarcinoma.
Philip E. Thorpe, PhD
George Zavoico, PhD
George B. Zavoico, PhD, is Managing Director, Research, and a Senior Equity Research Analyst at MLV & Co., a boutique investment bank and institutional broker-dealer based in New York. He has over 7 years of experience as a life sciences analyst writing research on publicly traded equities. Prior to MLV, he was an equity analyst with Westport Capital Markets and Cantor Fitzgerald. Prior to working as an analyst, Zavoico established his own consulting company serving the biotech and pharmaceutical industries by providing competitive intelligence and marketing research, due diligence services, and guidance in regulatory affairs. He also wrote extensively on healthcare and the biotech and pharmaceutical industries for periodicals targeting the general public and industry executives. Zavoico began his career as a Senior Research Scientist at Bristol-Myers Squibb Co., moving on to management positions at Alexion Pharmaceuticals, Inc. and T Cell Sciences, Inc. (now Celldex Therapeutics, Inc.). He has a BS in Biology from St. Lawrence University and PhD in Physiology from the University of Virginia and has held post-doctoral positions at the University of Connecticut Health Sciences Center and Brigham and Women's Hospital and Harvard Medical School.
Jennifer Henry, PhD
The New York Academy of Sciences
Jennifer Henry received her PhD in plant molecular biology from the University of Melbourne, Australia, with Paul Taylor at the University of Melbourne and Phil Larkin at CSIRO Plant Industry in Canberra, specializing in the genetic engineering of transgenic crops. She was then appointed as Associate Editor, then Editor, of Functional Plant Biology at CSIRO Publishing. She moved to New York for her appointment as a Publishing Manager in the Academic Journals division at Nature Publishing Group, where she was responsible for the publication of biomedical journals in nephrology, clinical pharmacology, hypertension, dermatology, and oncology. Henry joined the Academy in 2009 as Director of Life Sciences and organizes 35–40 seminars each year. She is responsible for developing scientific content in coordination with the various life sciences Discussion Group steering committees, under the auspices of the Academy's Frontiers of Science program. She also generates alliances with outside organizations interested in the programmatic content.
Ari Helenius, PhD
Educated as a biochemist, Ari Helenius obtained his PhD in 1973 in the University of Helsinki, Finland. After a six year period in the newly founded European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, he became Professor in the Department of Cell Biology, Yale School of Medicine. In 1997, he moved to his current position as professor of biochemistry in ETH Zurich. His research has involved membrane biology, cell biology, virology, infectious disease biology, protein biochemistry, and glycobiology. In most of the studies, animal viruses—their structure, function, and host cell interactions—have been a central theme.
Philip Thorpe, PhD
Philip Thorpe is a professor of pharmacology and holds the Serena S. Simmons Distinguished Chair at the University of Texas Southwestern Medical Center in Dallas, Texas. His expertise is in drug targeting, vascular targeting and most recently, antiviral research; his work has been published more than 200 times, including publications in Nature, Nature Medicine, Science, and Proceedings of the Natural Academy of Sciences. He is an inventor on more than 240 issued and pending U.S. and international patents. His long-standing collaboration with Peregrine Pharmaceuticals Inc, Tustin, CA, has culminated in the company developing bavituximab, a therapeutic antibody directed against the lipid, phosphatidylserine. Thorpe has been awarded the American Cancer Society Award of Excellence, the Texas State Legislature Award, and the Pierce Immunotoxin Award. He received his PhD from the Clinical Research Centre, London, UK. He was formerly director of the Drug Targeting Laboratory at the Imperial Cancer Research Fund, London.
Chris Reutelingsperger, PhD
Chris Reutelingsperger studied Biochemistry at the University of Utrecht and received his PhD in Biochemistry of Thrombosis and Haemostasis from the Maastricht University (1987). He discovered annexin A5 and is inventor of the annexin A5 affinity assay to determine apoptosis in vitro and in vivo in animal models and in patients. He was senior investigator of the Royal Dutch Academy of Sciences. Currently he is appointed Professor of Biochemistry and Apoptosis at Maastricht University. He founded and is chairman of the Board of the Virtual Laboratory Euregional PACT II, which is a cross-border collaboration between Universities of Maastricht, Leuven, Antwerp, Ghent and Aachen to develop innovative cancer diagnostics and therapeutics. He (co)-authored more than 150 peer-reviewed scientific papers and is inventor of 8 patents.
Alan Schroit, PhD
Alan Schroit is Adjunct Professor of Pharmacology at the University of Texas Southwestern Medical Center in Dallas, Texas. He received his PhD degree in Immunology from the Hadassah-Hebrew University Medical, Jerusalem, Israel and completed his postdoctotal training in lipid biochemistry at the Carnegie Institution of Washington in Baltimore, Maryland. His research has focused on the chemistry, biology and pathology of phosphatidylserine (PS) exposure in the outer leaflet of cells. His studies have led to the first observations that PS is constitutively expressed on the surface of many tumor cells and serves as a recognition ligand for phagocytosis by macrophages. Schroit was a founding Director of the Gulf Coast Consortia for Membrane Biology and was formerly John Q. Gaines Professor for Cancer Biology and Deputy Chair of the Department of Cancer Biology at the M. D. Anderson Cancer Center in Houston, Texas.
David Ucker, PhD
David Ucker is Professor of Microbiology and Immunology at the University of Illinois College of Medicine in Chicago. He received his PhD in Biochemistry and Biophysics from the University of California at San Francisco in 1981, and focused on studies of Molecular Immunology during his postdoctoral training at M.I.T. and Harvard. He has a long-standing interest in the molecular and cellular biology of physiological cell death (apoptosis) and in the immunological consequences of apoptosis. Those interests have led to the characterization of the phenomenon of "innate apoptotic immunity," and to the identification of novel apoptotic recognition determinants involved in triggering that response. Ucker is the elected chair of the upcoming Gordon Research Conference on Apoptotic Cell Recognition and Clearance. He has served as a member of several NIH and VA grant review panels, as a reviewer and editor for numerous scientific journals, and has received honors and recognition from a variety of scientific and educational societies.
Sarah Webb, PhD
Before hanging up her labcoat, Sarah Webb earned a PhD in bioorganic chemistry from Indiana University in Bloomington. Based in Brooklyn, NY, she writes about science, health, and technology for many publications including Scientific American, Discover, Science Careers, Science News, Nature Biotechnology, and ACS Chemical Biology.
- In young, healthy cells almost all PS molecules face the cytoplasm within the two layers of the cell membrane.
- Disruption of PS asymmetry is an important signal in clotting and in clearing aging cells from the bloodstream.
- Red blood cells (RBCs) lose the ability to internalize PS as they age. PS externalization serves as a tolerizing signal that prevents the body from triggering an immune response to these cells when they undergo apoptosis.
Understanding PS asymmetry via red blood cells
Much of our scientific understanding about PS and its asymmetric distribution in the cell has come from studies of red blood cells (RBCs). An average of one trillion of these cells are cleared from the human body each day. An important biological question about RBCs has always been how the body knows to clear these cells without triggering an immune response, said Alan Schroit of UT Southwestern Medical Center. A better understanding of PS asymmetry has helped researchers answer this question.
Because the networks of lipids and proteins within lipid membranes are associated non-covalently, studies to understand their distribution have required careful design. Early studies by Maddy probed the membrane distribution of lipids by incubating molecules that would react with amines on the outside surface of the membranes of RBCs to see which molecules were modified. Molecules facing the cytoplasm of the cell would be protected. Nearly all of the PS and most of the phosphatidylethanolamine (PE) was not modified. Other studies confirmed these initial findings that PS was primarily on the inner leaflet of the plasma membrane. But researchers did not understand how and why cells maintained this asymmetry, what stimuli perturbed it, and what consequences might arise when the distribution was perturbed.
About a decade later, in 1982, researchers showed that stimuli such as calcium ions and thrombin could prompt platelets to scramble their lipids and to distribute them evenly across both membranes. PS on the surfaces of these cells bound to activated Factor V and Factor X, which bound to prothrombin and fibrinogen and converted them to their active forms. Therefore the movement of PS to the surface had functional significance in specific biological processes, such as clotting.
Chemical tools that add a fluorescent or spin-labeled probe to lipid molecules have provided ways to investigate the dynamics of lipid distribution within the cell. The incubation of cells with labeled lipids under various conditions allowed researchers to follow the movement of the labeled lipids from the outside to the inside of the bilayer. Almost all PS will move from the outside to the inside of the cell, along with 25% of the PC and most of the PE, and that movement depends on both ATP and temperature.
So why would cells invest energy in maintaining this distribution of lipids? Studies of a range of RBCs of different ages showed that young cells readily internalize PS, but that older cells gradually lose this ability. This waning ability to internalize PS also correlates with the phagocytosis, or "devouring," of these cells by macrophages. Imaging studies show that macrophages form rosettes around RBCs that have PS on their membrane's exterior. Electron microscopy shows that the interaction between macrophages and RBCs is precise and does not distort cell shape particularly when compared with binding between antibodies and RBCs.
In other experiments researchers injected labeled RBCs with PS on their surfaces into live mice. Within 30 seconds most of those cells had disappeared from the blood. Within minutes the labeled cells were detected in the liver and spleen of the mice, indicating that they had been cleared from circulation.
ATP-dependent flippase enzymes facilitate the movement of PS from the outer to the inner leaflet of the plasma membrane. ATP binding cassette (ABC) transporters can serve as floppases that move lipid molecules from the inner leaflet to the outer leaflet. Calcium-dependent scramblases completely disrupt the asymmetry of the plasma membrane in coagulation, to clear apoptotic cells and as a signal that the immune system to tolerate these cells (a tolerizing signal) rather than considering them as foreign bodies.
This tolerizing signal for the immune system has a variety of implications in cancer and in infectious disease. Tumor cells express PS on the surface which explains how these cells shut down the body's autoimmune response. Viruses and parasites also take advantage of PS to evade the immune system.
David Ucker, University of Illinois at Chicago
- Although the RBC studies have pointed to PS as an important factor for immune tolerance during apoptosis, detailed studies of the steps of early apoptosis ruled out PS as the triggering factor for the dampened immune response.
- Glycolytic enzymes that function within the cytosol can trigger early apoptosis when they are externalized to the outer surface of the cell.
Examining the biology of early apoptosis
In their studies of the events that happen early in apoptosis, David Ucker and his colleagues at the University of Illinois at Chicago wondered whether the movement of PS to the exterior of the membrane might dampen the immune response to these cells. But in a series of experiments, they showed that PS was not the culprit. Instead, these cells shuttle glycolytic enzymes to their surfaces, which triggers immunity.
Consistent with earlier results, Ucker observed that apoptotic cells, unlike viable or necrotic cells, inhibit the release of two pro-inflammatory cytokines, IL-6 and TNF-α. Those cells don't just block the synthesis of those proteins, but they also block the expression of the genes themselves. Therefore the expression of these genes during apoptosis was not a factor in the suppression of the immune response in these studies.
The researchers decided to look at the engulfment of apoptotic cells by macrophages. When they treated apoptotic cells with cytochalasin D, a molecule that blocks the reorganization of the cytoskeleton, necessary for cell–cell recognition, they showed that the apoptotic cells and macrophages need to recognize each other to block the inflammatory response. But the macrophages did not need to engulf the apoptotic cells to block the inflammatory response.
In studies that compared apoptotic and necrotic cells, macrophages recognized these cells by distinctly different mechanisms. When the researchers used propidium iodide, an intercalating and staining agent to examine the overall membrane integrity of these cells types, the membranes in necrotic cells fell apart almost immediately. Viable cells showed little infiltration from the propidium iodide, and early apoptotic cells maintained much of the membrane integrity.
Cells across species show similar non-immunogenic responses to apoptotic cells, but to understand whether PS was involved the researchers examined PLB-985 cells, a cell line that does not externalize PS. Surprisingly, however, those cells showed the same patterns of membrane integrity and non-immunogenic activity as seen in cells that can externalize this lipid. So PS on the outside of the cell could not be causing the suppression in immune response.
Comparable immune suppression occurred in a variety of organisms, including in flies, frogs, mice, and humans, which suggested a common molecular cause found in all these organisms. So Ucker and his colleagues were looking for another factor that might mitigate the immune response.
Two dimensions of immunity
Building on some of the ideas in Schroit's talk, Ucker described the two dimensions of immunity. Classical immunity is the process by which an organism differentiates between itself and an invading, potentially threatening pathogen. A different dimension of immunity comes from the process by which a cell ages from a young viable cell toward apoptosis. The elimination of these older cells occurs through an immune process distinct from that of classical immunity.
Some viral pathogens take advantage of the same immunity tricks that aging, apoptotic cells use. Cells that die in response to an infection from adenovirus produce cytopathic effect (CPE) bodies that mimic the immunosuppression observed in apoptotic cells.
But with evidence that the chemical trigger for this process was not PS, the researchers treated apoptotic cells with proteases, to see if the protective factor might be a protein. Their results showed that apoptotic cells are enriched in proteins in the plasma membrane and are protease sensitive. So Ucker and his colleagues now knew that they were looking for evolutionarily conserved proteins. Because glycolytic enzymes are the largest group of proteins overexpressed on the membranes of apoptotic cells, they were potential candidates. The researchers showed both with proteomic studies and with assays that these glycolytic proteins are enriched on the surfaces of early apoptotic cells and that this externalization does not occur in viable cells. This process happens at the same time that PS externalization typically occurs.
The externalization of glycolytic enzymes accounts for the binding of plasminogen to apoptotic cells. Caspase activity appears to be one of the triggers of this process because blocking caspase activity prevented the externalization of these enzymes.
Ari Helenius, ETH Hönggerberg, Switzerland
- Viruses interact with surface proteins to trigger macropinocytosis, and PS in the viral membrane can trigger this process.
- The activation of surface proteins does not necessarily occur via direct interaction between PS in the viral membrane and proteins on the surface of host cells. Adaptor proteins bridge some of these interactions.
- PS on the surface of viruses furnishes them with the immunity conferred to apoptotic cells.
PS in the viral membrane
Viruses enter the cell through endocytosis, a cascade of signals that allow viruses to bind to the surface of the hosts' cells and become internalized by the membrane. Eventually a virus's nucleic acids are transported to the nucleus where they hijack the activity of a host cell. Ari Helenius of ETH in Hönggerberg described recent studies to uncover the role of PS in viral entry.
A variety of attachment factors can concentrate viruses on the surface of any given host cell and interactions with various receptor proteins can facilitate viral entry through a number of mechanisms: endocytosis, conformational changes, or membrane fusion. Endocytosis occurs through a variety of visually distinct mechanisms where the cells form protrusions or indentations that surround the external material. In macropinocytosis a finger-like projection extends from the plasma membrane and engulfs the target viral particle. This mechanism is active in the endocytosis of a variety of viral particles including HIV, Ebola, and vaccinia virus particles.
To study viral entry, researchers can use microscopy and biochemical techniques to view the cells and to track different molecules. Inhibitors and siRNA silencing allow researchers to perturb viral entry to understand the proteins that facilitate these processes, Helenius said.
Identifying the infectome
In their recent studies of viral entry, Helenius and postdoctoral researcher Jason Mercer have focused on vaccinia virus, one of the most complex animal viruses. As the virus infects cells, the cells extend blebs (bulges in the membrane) and filopodia and then retract the blebs as particles are internalized through macropinocytosis. Mercer and Helenius wanted to better understand the genes in the host cells that assist in this process of infection, called Trojan genes. To do so they used high-throughput screening techniques to identify the infectome, the body of genes that facilitate infection, of these viruses.
They initially screened HeLa cells with vaccinia virus particles against a Qiagen library of 7000 druggable genes, and in every case they tested 3 or 4 siRNAs that targeted each gene. The researchers identified an initial set of hits based on whether 2 or 3 of the siRNAs targeting these genes reduced viral infection by 50% or more. They then narrowed this group of primary hits by screening against a secondary library available from Ambion, which produced 300 secondary hits.
Using bioinformatics, the team analyzed the identified genes and found that many are in pathways associated with the epidermal growth factor receptor (EGFR) and with the proteins that facilitate macropinocytosis. The researchers then studied EGFR inhibitors against vaccinia virus in mixtures of cells. The inhibitors blocked entry of and infection by the virus.
The role of PS
The vaccinia virus expresses PS on its surface as a way of evading the classical immune response, and surface expression of PS is required for the virus to enter cells. Treatment with annexin V (a protein that binds PS) interferes with infection of and blebbing on the host cells, but the viral particles still bind to the cell surface. If PS is eliminated from the virus and then added back via new liposomes, viral activity is restored. The virus also regains its infectivity if another negatively charged phospholipid is added.
To identify the surface proteins that recognize PS directly or indirectly, Helenius and Mercer collaborated with Alexander Frei and Bernd Wollscheid, at ETH in Zurich. Frei and Wollscheid had developed ligand-based receptor capturing (LCR), a technique that takes advantage of a crosslinking molecule with three functions—one functional component can conjugate to carbohydrates on the surface of cells, another part of the molecule reacts with protein side chains, and the molecule's biotin moiety aids purification of the "captured" receptor.
Using this technique the researchers could bind cellular receptors, purify the receptors from a digested cell lysis mixture, and identify them from the peptide sequences in the mixture using mass spectrometry. In this way, the team identified seven candidate receptors likely involved in viral entry and used siRNA to monitor perturbations in viral entry in response to the elimination of five of those receptors: Axl, the mannose 6-phosphate receptor, dystroglycan-1, chondroitin sulfate proteoglycan-4, and cadherin 13.
Many of the receptor tyrosine kinases that recognize PS on the surface of viruses act through an additional untethered connector protein. Gas6 may serve as one of these proteins that bridge PS and the identified surface receptors.
Chris Reutelingsperger, Maastricht University, The Netherlands
- Proteins that bind to PS, such as annexin A5, provide tools for imaging and the basis for new treatments for disease.
- A fusion protein of annexin A5 with a cytotoxic protein can kill tumor cells.
- A mutation that changes the affinity of annexin A5 binding can prevent the formation of atherosclerotic plaques in rats.
PS on the outer membrane surfaces of viruses or of diseased cells, such as those in tumors, can differentiate those cells from healthy ones. Therefore, proteins that bind to PS could provide tools for both diagnosis and treatment of diseases, says Chris Reutelingsperger of Maastricht University. Expression of PS on the surface of cells serves several purposes in the body, both to cue the removal of aging or dying cells from the system and to activation of various clotting and immune responses.
The proteins annexin A5, synaptotagmin I, and lactadherin all bind to membranes that contain PS. For the first two proteins, binding also requires calcium ions. Reutelingsperger presented his work with annexin A5, a 36 kDa protein with a convex binding face that interacts with phospholipids. Annexin A5 also has a concave binding face.
When annexin A5 is labeled with various types of chemical tags, researchers can use optical imaging, magnetic resonance imaging, nuclear imaging, and ultrasound imaging to highlight the tissues it binds within the body. Researchers have used annexin A5 as a non-invasive imaging agent both in animal models and in human studies to examine diseased tissue from a range of diseases including Crohn's disease, rheumatoid arthritis, atherosclerosis, and cancer.
Tumor imaging with annexin A5
Reutelingsperger focused most of his talk on the application of these techniques to studies of cancer. He demonstrated how his team employed labeled annexin A5 to image tumor cells in the neck of an individual with lymphoma. They could follow the uptake of the annexin A5 into the tumor after treatment with radiation.
One challenge in using annexin A5 is the delivery of the protein to the tumor cells. Because of the leaky vasculature in tumors, the uptake of these proteins depends on the fluid pressure. Annexin A5 falls into a size and affinity gap that limits its ability to infiltrate tumor cells efficiently. To avoid this problem the researchers either needed a smaller molecule with higher affinity for PS or a larger molecule with similar affinity. This second option was easier to develop.
To construct a larger, macromolecular variant of the protein, Reutelingsperger and his colleagues built in several sites labeled with biotin that bind tightly to streptavidin, increasing the overall bulk of the annexin A5 complex to 200 kDa. They also designed control structures that included the backbone of the annexin protein but without the calcium binding residues so that they would not bind to PS. But this bigger complex did not give better results in studies with nude mice that had colon cancer tumors. The larger complex had a long half-life, and therefore the researchers could not distinguish a tumor that had been treated from one that hadn't. Though it will be more difficult to construct the smaller annexin-type derivatives with higher affinity, the researchers are currently working on this strategy.
Annexin A5 to treat tumors
The researchers also looked at treatment strategies using these annexin A5-based molecules. They developed a fusion protein that linked annexin A5 with ETA, an exotoxin from Pseudomonas aeruginosa that kills through interactions with a cytosolic protein. The linkage between the two proteins includes a protease cleavage site. Cell culture studies showed that the fusion protein still showed calcium-dependent binding to PS. The fusion protein was taken into the cells and was cleaved, and PS binding enhanced the fusion protein's cytotoxicity.
Reutelingsperger and his colleagues then looked at the effect of the fusion protein on nude mice that had human colon cancer tumors. The treatment appears to be PS dependent, and the inhibition of tumor growth increased when the fusion protein was given with another agent, cyclophosphamide, a chemotherapy drug.
Annexin A5 to treat inflammation
The researchers also investigated looked at another potential therapeutic application of annexin A5. Efferocytosis, the phagocytosis of apoptotic cells, doesn't occur appropriately in inflammatory diseases such as atherosclerosis. To enhance this process, they developed a new annexin A5-based molecule that included an RGD (arginine-glycyl-aspartic acid) motif that enhances the binding of phagocytes.
Although wild-type annexin A5 inhibited efferocytosis as expected, the modified protein enhanced this process, suggesting that the protein was oriented as predicted. In initial in vivo studies, the effect varied depending on the population of the phagocytes, but the team did see enhancement of one inflammatory factor, IL-10, in the presence of the modified annexin protein. These results suggested that this therapeutic strategy could work.
Reutelingsperger and his team then tested these proteins in rats that were fed a Western-type diet. The wild-type annexin A5 protein had no effect on the plaques measured in the carotid arteries of the rats. However, the RGD-annexin A5 inhibited plaque formation in rats. In animals where plaques had already formed, RGD-annexin A5 stabilized plaques those plaques so that they did not continue to grow.
Philip Thorpe, UT Southwestern Medical Center
- Bavituximab, an antibody drug in development by Peregrine Pharmaceuticals, targets PS in tumor blood vessels and can reactivate immunity to cancer cells.
- This antibody can serve as both an imaging agent and as an adjuvant treatment with other therapies.
- Bavituximab can induce tumor-specific T-cell immunity in some situations.
Targeting tumor vasculature
Taking advantage of the externalization of PS on tumor cells could provide new treatment strategies for cancer treatments. Philip Thorpe of UT Southwestern Medical Center described preclinical and clinical studies of bavituximab, an antibody drug developed by Peregrine Pharmaceuticals that targets PS in the vasculature of tumors.
As tumors develop, they create a stressful, hypoxic cellular environment that results in the formation of reactive oxygen species and the release of inflammatory cytokines. In this environment, the asymmetry of lipid distribution in the membrane of the endothelial cells that line tumor blood vessels breaks down, leading to the externalization of PS. In addition, PS is often exposed on the surface of tumor cells.
As a strategy for targeting PS externalization, Peregrine Pharmaceuticals initially developed a mouse monoclonal antibody that targets PS by binding to two molecules of β2-glycoprotein-I that in turn bind to PS on the surface of cells. With only a single molecule of the protein, binding to the surface of cells is relatively weak, but the affinity increases by a factor of 30,000 with the addition of a second molecule of β2-glycoprotein-I. The researchers have constructed a series of antibodies that targets the tumor vasculature. Two are mouse antibodies, 3G4 and 2aG4. Bavituximab is a chimeric antibody with mouse PS binding regions fused to human IgG. The fully human antibody is known as PGN635.
These antibodies localize specifically to the blood vessels in tumors, which makes them useful as imaging agents. Studies with normal tissues and various tumor types reveal that these antibodies show affinity for a range of tumor types but minimal binding to a variety of normal tissues.
Bavituximab as an anti-tumor agent
In rodent studies, antibodies alone inhibit tumor growth by 30%–90%. That variation most likely reflects the differences in PS expression on blood vessels in different tumors, Thorpe said. However, combining the antibody treatment with other treatments, including chemotherapy, radiation, or androgen deprivation, improved these results. In studies of prostate tumors in mice, the combination of the mouse antibody 2aG4 and docetaxel slowed tumor growth by 98% compared with 90% for docetaxel alone and 80% for antibody alone. The PS-targeting antibody in combination with intense radiation can shrink lung cancer tumors that are resistant to radiation alone.
How does the antibody work? As one proposed mechanism of action, these PS-targeting antibodies damage tumor vessels by a process known as antibody-dependent cellular cytotoxicity. As poor blood flow and hypoxia lead cells in the tumor vasculature to expose PS on their surfaces, bavituximab targets those cells and shuts down blood flow, which starves those tumor cells of nutrients.
As a second mechanism, bavituximab can overcome the immunosuppressive processes observed with cells that externalize PS. In tumors the expression of PS on cell surfaces keeps macrophages in their M2 state, an activation state that suppresses inflammation and supports angiogenesis. It also prevents myeloid-derived suppressor cells (MDSCs) from differentiating into macrophages and dendritic cells (DCs). In addition, PS inhibits the maturation of DCs, which prevents the presentation of antigens to T cells. Bavituximab repolarizes macrophages to their M1 state, which allows them to kill cells that express PS, including the tumor vascular endothelium. It also leads to differentiation of the MDSCs and prompts DCs to mature and to present tumor antigens to T cells.
Tumor-specific immunity and clinical trials
Bavituximab can also prompt the development of tumor-specific immunity. In experiments with rats with treatment-resistant glioma tumors, 15% of the animals treated both with radiation and with antibody therapies did not die from those tumors. When the researchers reintroduced glioma tumor cells into these surviving rats, the rats were immune to the cancer. The team determined that spleen cells from these animals could kill gliomas, which indicated the antibody treatment had induced T cell immunity.
Bavituximab is currently being tested clinically. More than 400 people have been treated with the drug, which is well tolerated and appears not to show combined toxicity when used with chemotherapy, Thorpe said. A variety of single-arm, early phase II studies have been completed as a second line or first line treatment in people with advanced breast cancer and as a front line treatment for non-small cell lung cancers (NSCLC). Randomized phase II studies in NSCLC and pancreatic cancer are also underway.
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Will bavituximab or related drugs show efficacy in large-scale clinical trials?