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From www.bloodjournal.org by guest on June 18, 2017. For personal use only. ● ● ● THROMBOSIS & HEMOSTASIS Comment on Ağar et al, page 1336 A---------------------------------------------------------------------------------------------------------------snappy new concept for APS Jacob H. Rand ALBERT EINSTEIN COLLEGE OF MEDICINE In this issue of Blood, Ağar and colleagues present data for a novel explanation of how an antigenic target on 2GPI, a central protein in the APS disease process, can become available for binding by antibodies.1 DV + + DI DI + + DII DIV DII DIII Arg 39 & 34 Lys 19 Binding site of antibodies DIII DIV DV - - - - + + - Lys 305 & 317 - An adaptation of Ağar et al’s Figure 71 showing the transition between the 2 conformations of 2GPI. Professional illustration by Paulette Dennis. he antiphospholipid syndrome (APS), an autoimmune thrombophilic disorder, was recognized as a diagnostic entity by astute clinical observations of the coincidence of thrombosis and/or recurrent miscarriages with empirically derived clinical tests.2 These include immunoassays that were derived from the biologic falsepositive syphilis test and blood tests that detect inhibitors of phospholipid-dependent coagulation reactions, known as lupus anticoagulant assays. In retrospect, the “antiphospholipid” terminology is erroneous and reflects the initial belief that phospholipids themselves are the targets of the antibodies. This misconception— but not the name of the syndrome— was corrected approximately 20 years ago, when it was discovered that the actual target antigens are phospholipid-binding proteins, T blood 2 6 A U G U S T 2 0 1 0 I V O L U M E 1 1 6 , N U M B E R 8 particularly 2-glycoprotein I (2GPI), a relatively abundant plasma protein whose biologic function(s) has not been established. X-ray crystallographic studies3,4 revealed that the protein, with its 5 homologous domains, has a J-shaped structure that is analogous to a fishhook with a “barb” consisting of a hydrophobic loop with surrounding positively charged residues near the carboxyterminus on domain V. This region allows the protein to bind bilayers containing anionic phospholipids via affinity for negatively charged polar heads and insertion of the loop within the hydrophobic middle of the bilayer (see figure). While several investigators have reported different specificities for the antibodies, there is significant evidence that patients having antibodies recognizing an epitope in domain I are at an increased risk for thrombosis.5 It has been proposed that the antibodies may dimerize or perhaps multimerize the protein and that the multivalency of these antibody2GPI complexes for phospholipids would increase their affinity/avidity for the bilayers (and potentially other receptors), which in turn would amplify the putative downstream thrombogenic effects of the antibodies. The latter might occur via disruption of endogenous anticoagulant mechanisms on vascular endothelial cells or by triggering signaling events that induce prothrombotic programs (eg, expression of tissue factor or cell-adhesion molecules). Ağar et al address the specific question of why these antibodies can only recognize domain I after the 2GPI has become bound to phospholipid6 but not unbound 2GPI in solution. The specifics of this process were not previously understood, but were presumed caused by conformational change(s). Ağar et al provide convincing evidence for a simple and elegant mechanism: unbound 2GPI present in solution exists in a closed circular conformation—in effect, as a coiled fishhook—in which domain V is noncovalently bound to domain I and thereby shields the domain I epitope from availability for antibody recognition. The binding of 2GPI to a phospholipid bilayer via the “barb” on domain V unsnaps this coiled protein into its open fishhook conformation, thereby exposing the epitope (see figure). The authors present a convincing body of evidence for this idea, including electron microscopic images, differential trypsin digestion profiles, surface plasmon resonance binding studies of the affinity of recombinant domains for each other, and functional studies that compare the anticoagulant effects of the conformations. These results add a significant detail in our understanding of the APS disease process, which can be outlined as follows: 2GPI is present in plasma where it has been suggested to play a role in the clearance of apoptotic cells and microparticles. The circular protein then undergoes a conformational change when it comes into contact with membranes of cells that have entered the apoptotic program and express anionic phospholipids; domain V unsnaps from domain I and inserts into the bilayer and the fishhooks agglomerate into disc-like clusters,7,8 a process that is probably required for the protein’s biologic role. In patients who have a genetic susceptibility for 1193 From www.bloodjournal.org by guest on June 18, 2017. For personal use only. developing APS, the exposure of the neoepitope on domain I may trigger an autoimmune response and stimulate formation of aPL antibody2GPI immune complexes on the surface of the cell membranes.8 In turn, these antiphospholipid antibody-2GPI immune complexes may exert a variety of effects on the cell membranes, including interference with membrane-mediated antithrombotic mechanisms such as A5 crystallization, protein C activation, and annexin A2– mediated fibrinolysis, as well as stimulation of a prothrombotic and proadhesive phenotype, activation of complement, and perhaps effects on the rigidity of the cell membranes. There were a few limitations in this study. Having the direct evidence of EM images of 2GPI bound to phospholipid would have been helpful; however, the authors report that this was not technically feasible because the protein aggregated lipid vesicles. In addition, the 2 conformations of purified 2GPI were prepared by dialyzing the purified protein against nonphysiologic buffers: alkaline pH and a high salt concentration for the open conformation and an acidic buffer for the closed conformation. Finally, IgG fractions from only a small group of 3 patients were used to show that the antibodies recognize the open, but not the closed, conformation of 2GPI; it would be interesting to know whether analysis of a larger number of APS patients would confirm this to be a consistent finding or show evidence for heterogeneity. Nevertheless, this work is an important contribution that advances the understanding of an early step in the APS disease process and may provide new insights for improved diagnosis and treatment of this disorder. Learning whether this “coiled fishhook” model for conformational change is found to occur with other binding proteins will also be interesting. Conflict-of-interest disclosure: The author declares no competing financial interests. ■ REFERENCES 1. Ağar C, van Os GM, Mörgelin M, et al. {beta}2-Glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome. Blood. 2010;116(8):1336-1343. 2. Hughes GR, Harris NN, Gharavi AE. The anticardiolipin syndrome. J Rheumatol. 1986;13(3):486-489. 3. Bouma B, de Groot PG, van den Elsen JM, et al. Adhesion mechanism of human beta(2)-glycoprotein I to phospholipids based on its crystal structure. EMBO J. 1999;18 (19):5166-5174. 4. Schwarzenbacher R, Zeth K, Diederichs K, et al. Crystal structure of human beta2-glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome. EMBO J. 1999;18(22):6228-6239. 5. de Laat B, Derksen RH, Urbanus RT, de Groot PG. IgG antibodies that recognize epitope Gly40-Arg43 in domain I of beta 2-glycoprotein I cause LAC, and their presence correlates strongly with thrombosis. Blood. 2005;105 (4):1540-1545. 6. de Laat B, Pengo V, Pabinger I, et al. The association between circulating antibodies against domain I of beta2-glycoprotein I and thrombosis: an international multicenter study. J Thromb Haemost. 2009;7(11): 1767-1773. 7. Gamsjaeger R, Johs A, Gries A, et al. Membrane binding of beta2-glycoprotein I can be described by a two-state reaction model: an atomic force microscopy and surface plasmon resonance study. Biochem J. 2005;389(Pt 3):665673. 8. Rand JH, Wu XX, Quinn AS, et al. Hydroxychloroquine directly reduces the binding of antiphospholipid antibody-beta2-glycoprotein I complexes to phospholipid bilayers. Blood. 2008;112(5):1687-1695. ● ● ● VASCULAR BIOLOGY Comment on Zhang et al, page 1377 PLDing a case for angiogenesis ---------------------------------------------------------------------------------------------------------------Anne Hamik and Mukesh K. Jain CASE WESTERN RESERVE UNIVERSITY In this issue of Blood, Zhang et al identify the Src-PLD1-PKC␥ axis as critically involved in the process that causes ROP, highlighting new potential targets for therapy.1 ascular endothelial growth factor (VEGF) is acknowledged as the predominant regulator of angiogenesis; blockade of VEGF signaling is central in therapy for numerous cancers and the vascular retinopathies of diabetes, age-related macular degeneration, and retinopathy of prematu- V 1194 rity (ROP). Although anti-VEGF monotherapy shows substantial results, in many cases it is becoming increasingly appreciated that combination therapy will be necessary.2 Tumors have demonstrated various degrees of intrinsic refractoriness or the development of treatment-related resistance. For age-related macular degeneration, expected to soon affect nearly 3 million people in the United States, the optimal therapy of longterm monthly intraocular injections of antiVEGF agents will likely prove unsustainable for practical and clinical reasons. Thus, effective treatment will require the combination of anti-VEGF therapy with conventional chemotherapeutic agents, radiotherapy or phototherapy, or the targeting of multiple components of VEGF-activated processes. The breadth of disease states in which VEGF-induced angiogenesis plays a central role correlates to a large and incompletely defined population of regulatory molecules of VEGF signaling, many likely to be tumor/ context-specific. The Zhang paper defines the players in a model of ROP and thus identifies potential new specific targets for therapy. In their report, Zhang and colleagues demonstrate that an intact VEGF-signaling axis— constituted by the sequential activation of Src, phospholipase 1 (PLD1), and protein kinase C␥ (PKC␥)—mediates the pathologic neovascularization seen in the oxygen-induced retinopathy model of ROP. This axis was delineated in vitro using chemical inhibitors (1-butanol and propranolol) and in vivo using intraocular administration of siRNAs specific to individual components of the pathway. Previous work has identified the protein tyrosine kinase activity of Src as a regulator of both VEGF expression and of responses to VEGF stimulation.3 Zhang et al are the first to report activation of PLD1 by Src. Furthermore, they demonstrate that Srcdependent PLD1 activation is required for subsequent activation of PLC␥. The recent development of selective small molecule inhibitors that target Src and the demonstration that Src inhibition can attenuate chemoresistance of some solid tumors suggests a possible clinical use of Src inhibition in vascular retinopathy. Investigation of the role of bioactive lipids in regulation of angiogenesis is a burgeoning area of research likely to result in a new class of therapeutic agents.4-6 Of particular topical interest are the bioactive lipids PLD1, phosphatidic acid (PA), lysophosphatidic acid (LPA), and sphingosine1-phosphate (S1P). After activation by any of a variety of intracellular factors (including 26 AUGUST 2010 I VOLUME 116, NUMBER 8 blood From www.bloodjournal.org by guest on June 18, 2017. For personal use only. 2010 116: 1193-1194 doi:10.1182/blood-2010-06-288209 A snappy new concept for APS Jacob H. Rand Updated information and services can be found at: http://www.bloodjournal.org/content/116/8/1193.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.