Evidence for Anticoagulant Activity and β₂-GPI Accumulation in Late Endosomes of Endothelial Cells Induced by Anti-LBPA Antibodies

Summary

This investigation was undertaken to test whether anti-LBPA antibodies and IgG from patients with APS interfere with intracellular β₂GPI distribution in EAhy926 endothelial cells and with the coagulation system. Cell incubation with anti-LBPA MoAb or with patients’ IgG resulted in antibody binding to late endosomes and caused β₂GPI redistribution and accumulation within perinuclear vesicular structures reminiscent of late endosomes. This finding suggests that aPl may contribute to the pathogenic mechanisms of APS by modifying the intracellular traffic of proteins, by interactions between aPl and LBPA, β₂GPI and/or LBPA- β₂GPI complexes. The anticoagulant activity of anti-LBPA MoAb was analyzed in a sensitized activated partial thromboplastin time (aPTT) system and in a dilute Russell’s viper venom time (dRVVT). A significant, concentration-dependent effect of the antibody on both aPTT and dRVVT prolongation was found. These observations suggest that LBPA is an important lipid target for aPl with potential functional implications for the immunopathogenesis of APS.

Keywords

β₂-glycoprotein I - late endosomes - endothelial cells - anti-phospholipid antibodies

• References

• 1 Hughes GRV. The anticardiolipin syndrome. Clin Exp Rheumatol 1985; 03: 285-6.
• 2 Hughes GRV, Harris EN, Gharavi AE. The anticardiolipin syndrome. J Rheumatol 1986; 13: 486-9.
• 3 Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC, Brey R, Derksen R, Harris EN, Hughes GR, Triplett DA, Khamashta MA. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. Arthritis Rheum 1999; 42: 1309-11.
• 4 Harris EN, Gharavi AE, Loizou S. Crossreactivity of antiphospholipid antibodies. J Clin Lab Immunol 1985; 16: 1-6.
• 5 Gharavi AE, Harris EN, Asherson RA, Hughes GV. Anticardiolipin antibodies: Isotype, distribution and phospholipid specificity. Ann Rheum Dis 1987; 46: 1-6.
• 6 Oosting JD, Derksen RHWM, Bobbink IWG, Hackengt TM, Bouma BN, De Groot PG. Antiphospholipid antibodies directed against a combination of phospholipids with prothrombin, protein C and protein S – an explanation for their pathogenetic mechanism?. Blood 1993; 81: 2618-25.
• 7 Pengo VA, Biasiolo A, Brocco T, Tonetto S, Ruffatti A. Autoantibodies to phospholipid-binding plasma proteins in patients with thrombosis and phospholipid-reactive antibodies. Thromb Haemost 1996; 75: 721-4.
• 8 Mc Neil HP, Simpson RJ, Chesterman CN, Krilis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta-2 glycoprotein-I (apolipoprotein II). Proc Natl Acad Sci USA 1990; 87: 4120-4.
• 9 Arvieux J, Darnige L, Caron C, Reber G, Bensa JC, Colomb MG. Development of an ELISA for autoantibodies to prothrombin showing their prevalence in patients with lupus anticoagulants. Thromb Haemost 1995; 74: 1120-5.
• 10 Sorice M, Griggi T, Circella A, Lenti L, Arcieri P, Di Nucci GD, Mariani G. Protein S antibodies in acquired protein S deficiencies. Blood 1994; 83: 2383-4.
• 11 Sorice M, Arcieri P, Griggi T, Circella A, Misasi R, Lenti L, Di Nucci GD, Mariani G. Inhibition of protein S by autoantibodies in patients with acquired protein S deficiency. Thromb Haemost 1996; 75: 555-9.
• 12 Kaburaki J, Kuwana M, Yamamoto M, Kawai S, Ikeda Y. Clinical significance of anti-annexin V antibodies in patients with systemic lupus erythematosus. Am J Hematol 1997; 54: 209-13.
• 13 Kobayashi T, Stang E, Fang KS, de Moerloose P, Parton RG, Gruenberg J. A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature 1998; 392: 193-7.
• 14 Gruenberg J, Maxfield FR. Membrane transport in the endocytic pathway. Curr Biol Cell Biol 1995; 07: 552-63.
• 15 Dowhan W. Molecular basis for membrane phospholipid diversity: why are there so many lipids?. Annu Rev Biochem 1997; 66: 199-232.
• 16 Schmid SL, Cullis PR. Membrane sorting. Endosome marker is fat not fiction. Nature 1998; 392: 135-6.
• 17 Besterman JM, Low RB. Endocytosis: a rewiew of mechanisms and plasma membrane dynamics. Biochem J 1983; 210: 1-13.
• 18 Kobayashi T, Beuchat MH, Lindsay M, Frias S, Palmiter RD, Sacuraba H, Parton RG, Gruenberg J. Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nature Cell Biol 1999; 01: 113-8.
• 19 Galve-de Rochemonteix B, Kobayashi T, Rosnoblet C, Lindsay M, Parton RG, Reber G, de Maistre E, Wahl D, Kruithof EKO, Gruenberg J, de Moerloose P. Interaction of anti-phospholipid antibodies with late endosomes of human endothelial cells. Arterioscler Thromb Vasc Biol 2000; 20: 563-74.
• 20 Edgell CJS, McDonald CC, Graham JB. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Natl Acad Sci USA 1983; 80: 3734-7.
• 21 Caronti B, Calderaro C, Alessandri C, Conti F, Tinghino R, Palladini G, Valesini G. β₂-glycoprotein I (β₂GPI) mRNA is expressed by several cell types involved in antiphospholipid syndrome-related tissue damage. Clin Exp Immunol 1999; 115: 214-9.
• 22 Kotani M, Ozawa H, Kawashima I, Ando S, Tai T. Generation of one set of monoclonal antibodies specific for a-pathway ganglio-series gangliosides. Biochim Biophys Acta 1992; 1117: 97-103.
• 23 Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem 1951; 193: 265-75.
• 24 Sammons DW, Adams LD, Nishizawa EE. Ultrasensitive silver-based color staining of polypeptides in polyacrilamide gels. Electrophoresis 1981; 02: 135-41.
• 25 Ferro D, Saliola M, Quintarelli C, Valesini G, Basili S, Grandilli MA, Bonavita MS, Violi F. Methods for detecting lupus anticoagulants and their relation to thrombosis and miscarriage in patients with systemic lupus erythematosus. J Clin Pathol 1992; 45: 332-8.
• 26 Armitage P, Berry G. Statistical methods in medical research. 2nd ed. Blackwell Scientific; 1990
• 27 Dunoyer-Geindre S, Kruithof EKO, Galve-de Rochemonteix B, Rosnoblet C, Gruenberg J, Reber G, de Moerloose P. Localization of β₂-glycoprotein I in late endosomes of human endothelial cells. Thromb Haemost 2001; 85: 903-7.
• 28 Sorice M, Griggi T, Circella A, Garofalo T, d’Agostino F, Pittoni V, Pontieri GM, Lenti L, Valesini G. Detection of antiphospholipid antibodies by immunostaining on thin layer chromatography plates. J Immunol Methods 1994; 173: 49-54.
• 29 Galli M, Comfurius P, Maassen C, Hemker HC, De Baets MH, van Breda-Vriesman PJC, Barbue T, Zwaal RFA, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990; 355: 1544-7.
• 30 Valesini G, Shoenfeld Y. A new player in the antiphospholipid syndrome: the β₂-glycoprotein I cofactor. Autoimmunity 1992; 14: 105-10.
• 31 Ferro D, Pittoni V, Quintarelli C, Basili S, Saliola M, Caroselli C, Valesini G, Violi F. Coexistence of anti-phospholipid antibodies and endothelial perturbation in systemic lupus erythematosus patients with ongoing prothrombotic state. Circulation 1997; 95: 1425-32.
• 32 Del Papa N, Guidali L, Sala A, Buccellati C, Khamashta MA, Iebikawa K, Koike T, Balestrieri G, Tincani A, Hughes GRV, Meroni PL. Endothelial cell are target of antiphospholipid antibodies: human polyclonal and monoclonal anti-β₂-glycoprotein I antibodies react in vitro with endothelial cells through adherent β₂-glycoprotein I and induce endothelial cell activation. Arthritis Rheum 1997; 40: 551-61.
• 33 Alarcon-Segovia D, Ruiz-Arguelles A, Llorente L. Broken dogma: penetration of autoantibodies into living cells. Trend Immunol Today 1996; 17: 163-4.
• 34 Cheng HM. Cell penetration by antiphospholipid antibodies. Immunology Today 1998; 19: 49.
• 35 Del Papa N, Guidali L, Spatola L, Bonra P, Borghi MO, Tincani A, Balestrieri G, Meroni PL. Relationship between anti-phospholipid and antiendothelial antibodies. III: β₂-glycoprotein I mediates the antibody binding to endothelial membranes and induces the expression of adhesion molecules. Clin Exp Rheumatol 1995; 13: 179-85.
• 36 Simantov R, La Sala JM, Lo SK, Gharavi AE, Sammaritano LR, Salmon JE, Silverstein RL. Activation of cultured vascular endothelial cells by antiphospholipid antibodies. J Clin Invest 1995; 96: 2211-9.
• 37 George J, Blank M, Levy Y, Meroni P, Damianovich M, Tincani A, Shoenfeld Y. Differential effects of anti-β₂-glycoprotein I antibodies on endothelial cells and on the manifestation of experimental antiphospholipid syndrome. Circulation 1998; 97: 900-6.
• 38 Galli M, Ruggeri L, Barbui T. Differential effects of anti-β₂-glycoprotein I and anti-prothrombin antibodies on the anticoagulant activity of activated protein C. Blood 1998; 91: 1999-2004.
• 39 Potzsch B, Kawamura H, Preissner KT, Schmidt M, Seelig C, Muller-Bergaus G. Acquired protein C dysfunction but not decreased activity of thrombomodulin is a possible marker of thrombophilia in patients with lupus anticoagulant. J Lab Clin Med 1995; 125: 56-65.
• 40 Ma K, Simantov R, Zhang JC, Silverstein R, Hajjar KA, McCrae KR. High affinity binding of beta 2-glycoprotein I to human endothelial cells is mediated by annexin II. J Biol Chem 2000; 275: 15541-8.
• 41 Chung CY, Murphy-Ullrich JE, Erickson HP. Mitogenesis, cell migration and loss of focal adhesions induced by tanascin C interacting with its cell surface receptor, annexin II. Mol Biol Cell 1996; 07: 883-92.
• 42 Valesini G, Violi F, Ferro D, Quintarelli C, Falco M, Tincani A, Balestrieri G, Balsano F. Anticardiolipin antibodies possess anticoagulant activity which is blocked by a monoclonal antibody against a cross reacting idiotype of antiphospholipid antibodies. Clin Exp Rheumatol 1989; 07: 615-8.
• 43 Violi F, Valesini G, Ferro D, Tincani A, Balestrieri G, Balsano F. Anticoagulant activity of anticardiolipin antibodies. Thromb Res 1986; 44: 543-7.
• 44 Takeya H, Mori T, Gabazza EC, Kuroda K, Deguchi H, Matsuura E, Ichicawa K, Koike T, Suzuki K. Anti-β₂GPI-Glycoprotein I (β₂GPI) Monoclonal antibodies with lupus anticoagulant-like activity enhance the β₂GPI binding to phospholipids. J Clin Invest 1997; 99: 2260-8.