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J Clin Invest. Aug 1, 1996; 98(3): 815–825.
PMCID: PMC507492

Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids. Recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low density lipoprotein.


The optimal clinical management of patients with antiphospholipid antibody syndrome (APS) is uncertain because of a lack of an underlying hypothesis to explain why antiphospholipid autoantibodies (aPL) form to such ubiquitous compounds as phospholipids (PL). In this paper, we demonstrate that many, if not most, aPL are actually directed at neoepitopes of oxidized PL, or neoepitopes generated by adduct formation between breakdown products of oxidized PL and associated proteins. Each cardiolipin (CL) molecule contains four unsaturated fatty acids and is highly susceptible to oxidation, particularly upon exposure to air. Yet, standard anticardiolipin antibodies (aCL) immunoassays routinely bind CL to microtiter wells by evaporation of the ethanol solvent overnight at 4 degrees C. Using a variety of techniques, we demonstrated that rapid oxidation occurs when CL is plated and exposed to air. Sera from apo E-deficient mice, which have high autoantibody titers to oxidized low density lipoprotein, showed a striking time-dependent increase in binding to CL that was exposed to air for increasing periods of time. Monoclonal antibodies to oxidized LDL, cloned from the apo E-deficient mice, also bound to oxidized CL. Both sera and affinity-purified aCL-IgG from APS patients bound to CL progressively as it was oxidized. However, the monoclonal antibodies from apo E-deficient mice, or sera or aCL-IgG from APS patients did not bind to a reduced CL analog that was unable to undergo peroxidation. These data demonstrate that many aPL are directed at neoepitopes of oxidized phospholipids, and suggest that oxidative events may be important in the pathophysiology of APS. In turn, this suggests new therapeutic strategies, possibly including intensive antioxidant therapy.

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Selected References

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  • Alarcón-Segovia D, Delezé M, Oria CV, Sánchez-Guerrero J, Gómez-Pacheco L, Cabiedes J, Fernández L, Ponce de León S. Antiphospholipid antibodies and the antiphospholipid syndrome in systemic lupus erythematosus. A prospective analysis of 500 consecutive patients. Medicine (Baltimore) 1989 Nov;68(6):353–365. [PubMed]
  • Asherson RA, Khamashta MA, Ordi-Ros J, Derksen RH, Machin SJ, Barquinero J, Outt HH, Harris EN, Vilardell-Torres M, Hughes GR. The "primary" antiphospholipid syndrome: major clinical and serological features. Medicine (Baltimore) 1989 Nov;68(6):366–374. [PubMed]
  • Harris EN, Pierangeli SS. Antiphospholipid antibodies and the antiphospholipid syndrome. Springer Semin Immunopathol. 1994;16(2-3):223–245. [PubMed]
  • Silver RM, Draper ML, Scott JR, Lyon JL, Reading J, Branch DW. Clinical consequences of antiphospholipid antibodies: an historic cohort study. Obstet Gynecol. 1994 Mar;83(3):372–377. [PubMed]
  • Lockshin MD. Thrombosis in the antiphospholipid-antibody syndrome. N Engl J Med. 1995 Sep 7;333(10):667–667. [PubMed]
  • Vaarala O, Mänttäri M, Manninen V, Tenkanen L, Puurunen M, Aho K, Palosuo T. Anti-cardiolipin antibodies and risk of myocardial infarction in a prospective cohort of middle-aged men. Circulation. 1995 Jan 1;91(1):23–27. [PubMed]
  • Peaceman AM, Silver RK, MacGregor SN, Socol ML. Interlaboratory variation in antiphospholipid antibody testing. Am J Obstet Gynecol. 1992 Jun;166(6 Pt 1):1780–1787. [PubMed]
  • Witztum JL, Steinbrecher UP, Fisher M, Kesaniemi A. Nonenzymatic glucosylation of homologous low density lipoprotein and albumin renders them immunogenic in the guinea pig. Proc Natl Acad Sci U S A. 1983 May;80(9):2757–2761. [PMC free article] [PubMed]
  • Steinbrecher UP, Fisher M, Witztum JL, Curtiss LK. Immunogenicity of homologous low density lipoprotein after methylation, ethylation, acetylation, or carbamylation: generation of antibodies specific for derivatized lysine. J Lipid Res. 1984 Oct;25(10):1109–1116. [PubMed]
  • Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989 Apr 6;320(14):915–924. [PubMed]
  • Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991 Dec;88(6):1785–1792. [PMC free article] [PubMed]
  • Palinski W, Ylä-Herttuala S, Rosenfeld ME, Butler SW, Socher SA, Parthasarathy S, Curtiss LK, Witztum JL. Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Arteriosclerosis. 1990 May-Jun;10(3):325–335. [PubMed]
  • Palinski W, Rosenfeld ME, Ylä-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1372–1376. [PMC free article] [PubMed]
  • Palinski W, Ord VA, Plump AS, Breslow JL, Steinberg D, Witztum JL. ApoE-deficient mice are a model of lipoprotein oxidation in atherogenesis. Demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Arterioscler Thromb. 1994 Apr;14(4):605–616. [PubMed]
  • Haberland ME, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science. 1988 Jul 8;241(4862):215–218. [PubMed]
  • Boyd HC, Gown AM, Wolfbauer G, Chait A. Direct evidence for a protein recognized by a monoclonal antibody against oxidatively modified LDL in atherosclerotic lesions from a Watanabe heritable hyperlipidemic rabbit. Am J Pathol. 1989 Nov;135(5):815–825. [PMC free article] [PubMed]
  • Holvoet P, Perez G, Zhao Z, Brouwers E, Bernar H, Collen D. Malondialdehyde-modified low density lipoproteins in patients with atherosclerotic disease. J Clin Invest. 1995 Jun;95(6):2611–2619. [PMC free article] [PubMed]
  • Hammer A, Kager G, Dohr G, Rabl H, Ghassempur I, Jürgens G. Generation, characterization, and histochemical application of monoclonal antibodies selectively recognizing oxidatively modified apoB-containing serum lipoproteins. Arterioscler Thromb Vasc Biol. 1995 May;15(5):704–713. [PubMed]
  • Houglum K, Filip M, Witztum JL, Chojkier M. Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload. J Clin Invest. 1990 Dec;86(6):1991–1998. [PMC free article] [PubMed]
  • Yan SD, Schmidt AM, Anderson GM, Zhang J, Brett J, Zou YS, Pinsky D, Stern D. Enhanced cellular oxidant stress by the interaction of advanced glycation end products with their receptors/binding proteins. J Biol Chem. 1994 Apr 1;269(13):9889–9897. [PubMed]
  • Neale TJ, Ojha PP, Exner M, Poczewski H, Rüger B, Witztum JL, Davis P, Kerjaschki D. Proteinuria in passive Heymann nephritis is associated with lipid peroxidation and formation of adducts on type IV collagen. J Clin Invest. 1994 Oct;94(4):1577–1584. [PMC free article] [PubMed]
  • Libby P, Hansson GK. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991 Jan;64(1):5–15. [PubMed]
  • Martin BA, Branch DW, Rodgers GM. The preparation of a sensitive partial thromboplastin reagent from bovine brain. Blood Coagul Fibrinolysis. 1992 Jun;3(3):287–294. [PubMed]
  • Martin BA, Branch DW, Rodgers GM. Sensitivity of the activated partial thromboplastin time, the dilute Russell's viper venom time, and the kaolin clotting time for the detection of the lupus anticoagulant: a direct comparison using plasma dilutions. Blood Coagul Fibrinolysis. 1996 Jan;7(1):31–38. [PubMed]
  • Branch DW, Silver RM, Blackwell JL, Reading JC, Scott JR. Outcome of treated pregnancies in women with antiphospholipid syndrome: an update of the Utah experience. Obstet Gynecol. 1992 Oct;80(4):614–620. [PubMed]
  • Harris EN. Special report. The Second International Anti-cardiolipin Standardization Workshop/the Kingston Anti-Phospholipid Antibody Study (KAPS) group. Am J Clin Pathol. 1990 Oct;94(4):476–484. [PubMed]
  • Harris EN, Pierangeli S, Birch D. Anticardiolipin wet workshop report. Fifth International Symposium on antiphospholipid antibodies. Am J Clin Pathol. 1994 May;101(5):616–624. [PubMed]
  • Palinski W, Hörkkö S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK, Witztum JL. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma. J Clin Invest. 1996 Aug 1;98(3):800–814. [PMC free article] [PubMed]
  • Cabral AR, Cabiedes J, Alarcón-Segovia D. Tween 20 detaches cardiolipin from ELISA plates and makes anticardiolipin antibodies undetectable regardless of the presence of beta 2-glycoprotein-I. J Immunol Methods. 1994 Sep 30;175(1):107–114. [PubMed]
  • Reaven P, Parthasarathy S, Grasse BJ, Miller E, Steinberg D, Witztum JL. Effects of oleate-rich and linoleate-rich diets on the susceptibility of low density lipoprotein to oxidative modification in mildly hypercholesterolemic subjects. J Clin Invest. 1993 Feb;91(2):668–676. [PMC free article] [PubMed]
  • Ezaki M, Witztum JL, Steinberg D. Lipoperoxides in LDL incubated with fibroblasts that overexpress 15-lipoxygenase. J Lipid Res. 1995 Sep;36(9):1996–2004. [PubMed]
  • Levy RA, Gharavi AE, Sammaritano LR, Habina L, Lockshin MD. Fatty acid chain is a critical epitope for antiphospholipid antibody. J Clin Immunol. 1990 May;10(3):141–145. [PubMed]
  • Pierangeli SS, Harris EN, Gharavi AE, Goldsmith G, Branch DW, Dean WL. Are immunoglobulins with lupus anticoagulant activity specific for phospholipids? Br J Haematol. 1993 Sep;85(1):124–132. [PubMed]
  • Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994 Sep 17;344(8925):793–795. [PubMed]
  • Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL, Edwards PA, Watson AD, Lusis AJ. Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation. 1995 May 1;91(9):2488–2496. [PubMed]
  • Branch DW, Mitchell MD, Miller E, Palinski W, Witztum JL. Pre-eclampsia and serum antibodies to oxidised low-density lipoprotein. Lancet. 1994 Mar 12;343(8898):645–646. [PubMed]
  • Hubel CA, Roberts JM, Taylor RN, Musci TJ, Rogers GM, McLaughlin MK. Lipid peroxidation in pregnancy: new perspectives on preeclampsia. Am J Obstet Gynecol. 1989 Oct;161(4):1025–1034. [PubMed]
  • Sheppard BL, Bonnar J. An ultrastructural study of utero-placental spiral arteries in hypertensive and normotensive pregnancy and fetal growth retardation. Br J Obstet Gynaecol. 1981 Jul;88(7):695–705. [PubMed]
  • Steinbrecher UP, Parthasarathy S, Leake DS, Witztum JL, Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3883–3887. [PMC free article] [PubMed]
  • Parthasarathy S, Steinbrecher UP, Barnett J, Witztum JL, Steinberg D. Essential role of phospholipase A2 activity in endothelial cell-induced modification of low density lipoprotein. Proc Natl Acad Sci U S A. 1985 May;82(9):3000–3004. [PMC free article] [PubMed]
  • Stremler KE, Stafforini DM, Prescott SM, Zimmerman GA, McIntyre TM. An oxidized derivative of phosphatidylcholine is a substrate for the platelet-activating factor acetylhydrolase from human plasma. J Biol Chem. 1989 Apr 5;264(10):5331–5334. [PubMed]
  • Itabe H, Takeshima E, Iwasaki H, Kimura J, Yoshida Y, Imanaka T, Takano T. A monoclonal antibody against oxidized lipoprotein recognizes foam cells in atherosclerotic lesions. Complex formation of oxidized phosphatidylcholines and polypeptides. J Biol Chem. 1994 May 27;269(21):15274–15279. [PubMed]
  • Watson AD, Navab M, Hama SY, Sevanian A, Prescott SM, Stafforini DM, McIntyre TM, Du BN, Fogelman AM, Berliner JA. Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein. J Clin Invest. 1995 Feb;95(2):774–782. [PMC free article] [PubMed]
  • Heery JM, Kozak M, Stafforini DM, Jones DA, Zimmerman GA, McIntyre TM, Prescott SM. Oxidatively modified LDL contains phospholipids with platelet-activating factor-like activity and stimulates the growth of smooth muscle cells. J Clin Invest. 1995 Nov;96(5):2322–2330. [PMC free article] [PubMed]
  • Asherson RA, Khamashta MA, Gil A, Vazquez JJ, Chan O, Baguley E, Hughes GR. Cerebrovascular disease and antiphospholipid antibodies in systemic lupus erythematosus, lupus-like disease, and the primary antiphospholipid syndrome. Am J Med. 1989 Apr;86(4):391–399. [PubMed]
  • Love PE, Santoro SA. Antiphospholipid antibodies: anticardiolipin and the lupus anticoagulant in systemic lupus erythematosus (SLE) and in non-SLE disorders. Prevalence and clinical significance. Ann Intern Med. 1990 May 1;112(9):682–698. [PubMed]
  • Ginsburg KS, Liang MH, Newcomer L, Goldhaber SZ, Schur PH, Hennekens CH, Stampfer MJ. Anticardiolipin antibodies and the risk for ischemic stroke and venous thrombosis. Ann Intern Med. 1992 Dec 15;117(12):997–1002. [PubMed]
  • Khamashta MA, Cuadrado MJ, Mujic F, Taub NA, Hunt BJ, Hughes GR. The management of thrombosis in the antiphospholipid-antibody syndrome. N Engl J Med. 1995 Apr 13;332(15):993–997. [PubMed]
  • Hamsten A, Norberg R, Björkholm M, de Faire U, Holm G. Antibodies to cardiolipin in young survivors of myocardial infarction: an association with recurrent cardiovascular events. Lancet. 1986 Jan 18;1(8473):113–116. [PubMed]
  • Morton KE, Gavaghan TP, Krilis SA, Daggard GE, Baron DW, Hickie JB, Chesterman CN. Coronary artery bypass graft failure--an autoimmune phenomenon? Lancet. 1986 Dec 13;2(8520):1353–1357. [PubMed]
  • Klemp P, Cooper RC, Strauss FJ, Jordaan ER, Przybojewski JZ, Nel N. Anti-cardiolipin antibodies in ischaemic heart disease. Clin Exp Immunol. 1988 Nov;74(2):254–257. [PMC free article] [PubMed]
  • Matsuura E, Igarashi Y, Yasuda T, Triplett DA, Koike T. Anticardiolipin antibodies recognize beta 2-glycoprotein I structure altered by interacting with an oxygen modified solid phase surface. J Exp Med. 1994 Feb 1;179(2):457–462. [PMC free article] [PubMed]
  • McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Anti-phospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci U S A. 1990 Jun;87(11):4120–4124. [PMC free article] [PubMed]
  • Galli M, Comfurius P, Maassen C, Hemker HC, de Baets MH, van Breda-Vriesman PJ, Barbui T, Zwaal RF, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet. 1990 Jun 30;335(8705):1544–1547. [PubMed]
  • Pierangeli SS, Harris EN, Davis SA, DeLorenzo G. Beta 2-glycoprotein 1 (beta 2GP1) enhances cardiolipin binding activity but is not the antigen for antiphospholipid antibodies. Br J Haematol. 1992 Nov;82(3):565–570. [PubMed]
  • Gharavi AE, Sammaritano LR, Wen J, Elkon KB. Induction of antiphospholipid autoantibodies by immunization with beta 2 glycoprotein I (apolipoprotein H). J Clin Invest. 1992 Sep;90(3):1105–1109. [PMC free article] [PubMed]
  • Pierangeli SS, Harris EN. Induction of phospholipid-binding antibodies in mice and rabbits by immunization with human beta 2 glycoprotein 1 or anticardiolipin antibodies alone. Clin Exp Immunol. 1993 Aug;93(2):269–272. [PMC free article] [PubMed]
  • Steinkasserer A, Estaller C, Weiss EH, Sim RB, Day AJ. Complete nucleotide and deduced amino acid sequence of human beta 2-glycoprotein I. Biochem J. 1991 Jul 15;277(Pt 2):387–391. [PMC free article] [PubMed]
  • Vaarala O, Palosuo T, Kleemola M, Aho K. Anticardiolipin response in acute infections. Clin Immunol Immunopathol. 1986 Oct;41(1):8–15. [PubMed]
  • Vaarala O, Alfthan G, Jauhiainen M, Leirisalo-Repo M, Aho K, Palosuo T. Crossreaction between antibodies to oxidised low-density lipoprotein and to cardiolipin in systemic lupus erythematosus. Lancet. 1993 Apr 10;341(8850):923–925. [PubMed]
  • Amara A, Constans J, Chaugier C, Sebban A, Dubourg L, Peuchant E, Pellegrin JL, Leng B, Conri C, Geffard M. Autoantibodies to malondialdehyde-modified epitope in connective tissue diseases and vasculitides. Clin Exp Immunol. 1995 Aug;101(2):233–238. [PMC free article] [PubMed]
  • Savolainen A, von Essen R, Leikola J, Alfthan G, Vaarala O, Palosuo T, Aho K. Antibodies against oxidised low-density lipoprotein in juvenile chronic arthritis. Scand J Rheumatol. 1995;24(4):209–211. [PubMed]
  • Hashimoto Y, Kawamura M, Ichikawa K, Suzuki T, Sumida T, Yoshida S, Matsuura E, Ikehara S, Koike T. Anticardiolipin antibodies in NZW x BXSB F1 mice. A model of antiphospholipid syndrome. J Immunol. 1992 Aug 1;149(3):1063–1068. [PubMed]
  • Mizutani H, Kurata Y, Kosugi S, Shiraga M, Kashiwagi H, Tomiyama Y, Kanakura Y, Good RA, Matsuzawa Y. Monoclonal anticardiolipin autoantibodies established from the (New Zealand white x BXSB)F1 mouse model of antiphospholipid syndrome cross-react with oxidized low-density lipoprotein. Arthritis Rheum. 1995 Oct;38(10):1382–1388. [PubMed]
  • Mironova M, Virella G, Lopes-Virella MF. Isolation and characterization of human antioxidized LDL autoantibodies. Arterioscler Thromb Vasc Biol. 1996 Feb;16(2):222–229. [PubMed]
  • Alving CR. Antibodies to liposomes, phospholipids and phosphate esters. Chem Phys Lipids. 1986 Jun-Jul;40(2-4):303–314. [PubMed]
  • Rauch J, Janoff AS. Phospholipid in the hexagonal II phase is immunogenic: evidence for immunorecognition of nonbilayer lipid phases in vivo. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4112–4114. [PMC free article] [PubMed]
  • Swartz GM, Jr, Gentry MK, Amende LM, Blanchette-Mackie EJ, Alving CR. Antibodies to cholesterol. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1902–1906. [PMC free article] [PubMed]
  • Bashir S, Harris G, Denman MA, Blake DR, Winyard PG. Oxidative DNA damage and cellular sensitivity to oxidative stress in human autoimmune diseases. Ann Rheum Dis. 1993 Sep;52(9):659–666. [PMC free article] [PubMed]
  • Frenkel K, Karkoszka J, Kim E, Taioli E. Recognition of oxidized DNA bases by sera of patients with inflammatory diseases. Free Radic Biol Med. 1993 May;14(5):483–494. [PubMed]

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