Functional Analyses of Patient-derived IgG Monoclonal Anticardiolipin Antibodies Using In Vivo Thrombosis and In Vivo Microcirculation Models

Summary

Antiphospholipid antibodies (aPL) have been associated with thrombosis and pregnancy losses in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. It has been proposed that aPL may affect endothelial cell (EC) function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. It has been proposed that aPL may affect EC cell function and/or induce their activation, transforming their anticoagulant surface into procoagulant, thus predisposing to thrombosis. This study proposes to test the hypotheses that some IgG anticardiolipins (IgG aCL) with thrombogenic properties in mice, exert their effects through activation of endothelium. We studied seven patient-derived monoclonal aCL for their thrombogenic properties in an in vivo pinch-induced thrombosis model, and their functional activities in activating EC by analyzing in vivo leukocyte adhesion to endothelium in microcirculation in venules in exposed murine cremaster muscle and in vitro adhesion molecule expression in cultured EC. The binding of the monoclonal aCL to EC was also tested. In addition to the previous identified thrombogenic IS2, four of the five new more IgG monoclonal aCL (from two patients) were found to be thrombogenic. Of these five thrombogenic aCL, three caused more in vivo leukocyte adhesion to EC in microcirculation, as compared to that induced by the H2 control human monoclonal IgG, and enhanced expression of adhesion molecules (particularly VCAM-1) on cultured EC. These data show that about 2/3 patientderived IgG monoclonal aCL are thrombogenic and suggest that some thrombogenic IgG aCL exert their effects through activating EC.

Abbreviations: aCL, anticardiolipin antibodies; aPL, antiphospholipid antibodies; APS, antiphospholipid syndrome; CL, cardiolipin; dRVVT, dilute Russell’s viper venom time; EC, endothelial cells; ELISA, enzyme-linked immunosorbent assay; GP, glycoprotein; h, hour; HUVEC, human umbilicalvein endothelial cells; ICAM, intercellular adhesion molecule; KCT, kaolin clotting time; LA, lupus anticoagulant; PBS, phosphate-buffered saline; PL, phospholipid; sq, square; TBS, Tris-buffered saline; VCAM, vascular cell adhesion molecule; PMBC, peripheral mononuclear blood cells

Key words

Antiphospholipid antibodies/syndrome - thrombosis - endothelial cell activation

• References

• 1 Harris EN. Syndrome of the Black Swan. Br J Rheumatol 1987; 26: 324-326.
• 2 Harris EN, Pierangeli SS. Antiphospholipid antibodies and the antiphospholipid syndrome. Spinger Semin Immunopathol 1994; 16: 223-45.
• 3 Khamashta MA, Hughes GRV. Antiphospholipid antibodies: a marker for thrombosis and recurrent abortion. Clin Rev Allergy 1994; 12: 287-96.
• 4 Sammaritano LR, Gharavi AE, Lockshin MD. Antiphospholipid antibody syndrome: immunologic and clinical aspects. Semin Arth Rheum 1990; 20: 81-96.
• 5 Petri M. Pathogenesis and treatment of the antiphospholipid antibody syndrome. Med Clin North Am 1997; 81: 151-77.
• 6 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 Int Med 1990; 12: 682-98.
• 7 Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 1990; 336: 177-8.
• 8 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: β₂glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990; 87: 4120-4.
• 9 Galli M, Comfurius P, Maasen C, Hemker HC, deBaets MH, van Breda-Vriesman PJC, Barbui T, Zwaal RFA, Bevers EM. Anticardiolipin (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990; 335: 1544-7.
• 10 Fleck RA, Rapaport SI, Rao LV. Anti-prothrombin antibodies and the lupus anticoagulant. Blood 1988; 72: 512-9.
• 11 Bevers EM, Galli M, Barbui T, Comfurius P, Zwaal RFA. Lupus anticoagulant IgG’s (LA) are not directed to phospholipids only, but to a complex of lipid-bound human prothrombin. Thromb Haemost 1991; 66: 629-32.
• 12 Oosting JD, Derksen RHWM, Bobbink IWG, Hackeng TM, Bouma BN, de Groot PG. Antiphospholipid antibodies directed against a combination of phospholipids with prothrombin, protein C or protein S: an explanation for their pathogenic mechanism?. Blood 1993; 81: 2618-25.
• 13 Triplett DA. Antiphospholipid-protein antibodies: a paradigm for antibody-mediated thrombosis. J Clin Ligand Assay 1998; 21: 214-7.
• 14 Roubey RAS. Immunology of the antiphospholipid antibody syndrome. Arthritis Rheum 1996; 39: 1444-54.
• 15 Roubey RAS, Pratt CW, Buyon JP, Winfield JB. Lupus anticoagulant activity of autoimmune antiphospholipid antibodies is dependent upon β₂glycoprotein I. J Clin Invest 1992; 90: 1100-4.
• 16 Permpikul P, Rao LVM, Rapaport SI. Functional and binding studies of the roles of prothrombin and b2glycoprotein I in the expression of lupus anticoagulant activity. Blood 1994; 83: 2878-92.
• 17 De Groot PG, Horbach DA, Simmelink MJS, Van Ort E, Derksen RHWM. Antiprothrombin antibodies and their relation with thrombosis and lupus anticoagulant. Lupus 1998; 07: S32-6.
• 18 Del Papa N, Guidali L, Sala A, Buccellati C, Khamashta MA, Ichikawa K, Koike T, Balestrieri G, Tincani A, Hughes GRV. Human polyclonal and monoclonal anti- β₂glycoprotein 1 antibodies react in vitro with endothelial cells through adherent β₂glycoprotein I and induced endothelial activation. Arthritis Rheum 1997; 40: 551-61.
• 19 Simantov E, LaSala J, Lo SK, Gharavi AW, Sammaritano LR, Salmon JE, Silverstein RL. Activation of cultured vascular endothelial cells by antiphospholipid antibodies. J Clin Invest 1995; 86: 2211-9.
• 20 Pierangeli SS, Colden-Stanfield M, Liu X, Barker JH, Anderson GJ, Harris EN. Antiphospholipid antibodies from Antiphospholipid Syndrome patients activate endothelial cells in vitro and in vivo. Circulation 1999; 99: 1997-2002.
• 21 Kornberg AS, Blank M, Kaufman S, Shoenfeld Y. Induction of tissue factor-like activity in monocytes by anti-cardiolipin antibodies. J Immunol 1994; 153: 1328-32.
• 22 Cuadrado MJ, Lopez-Pedrera C, Khamashta MA, CAMps MT, Tinahones F, Torres A, Hughes GRV, Velasco F. Thrombosis in primary antiphospholipid syndrome – a pivotal role for monocyte tissue factor expression. Arthritis Rheum 1997; 40: 834-41.
• 23 Amengual O, Atsumi T, Khamashta MA, Hughes GRV. The role of tissue factor pathway in the hypercoagulable state in patients with the antiphospholipid syndrome. Thromb Haemost 1998; 79: 276-81.
• 24 Bachman RL, Silverstein RL. Hypercoagulable states. Ann Intern Med 1993; 119: 819-27.
• 25 Hasselaar P, Derksen RHWM, Oosting JD. et al. Synergistic effect of low doses of tumor necrosis factor and sera from patients with systemic lupus erythematosus on the expansion of procoagulant activity by cultured endothelial cells. Thromb Haemost 1989; 62: 654-60.
• 26 Rand JH, Wu XX, Andree HAM, Lockwood CJ, Guller S, Scher J, Harpel PC. Pregnancy loss in the antiphospholipid-antibody syndrome – A possible thrombogenic mechanism. N Engl J Med 1997; 337: 154-60.
• 27 Pierangeli SS, Kozima K, Fernandez JH, Griffin JH, Harris EN. Inhibition of protein C activation by antiphospholipid antibodies in vitro is dependent on the composition of phospholipid (PL) liposomes used as template. Arthritis Rheum 1996; 39 (09) S94.
• 28 Malia RG, Kitchen S, Greavesd M, Prston FE. Inhibition of activated protein C and its cofactor protein S by antiphospholipid antibodies. Br J Haematol 1990; 76: 101-7.
• 29 Smirnov MD, Triplett DT, Comp PV, Esmon NL, Esmon CT. On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies. J Clin Invest 1995; 95: 309-16.
• 30 Olee TW, Pierangeli SS, Handley HH, Le DT, Wei X, Lai CJ, En J, Novotny W, Harris EN, Woods Jr. VL, Chen PP. A monoclonal IgG anticardiolipin antibody from a patient with the antiphospholipid syndrome is thrombogenic in mice. Proc Natl Acad Sci USA 1996; 93: 8606-11.
• 31 Zhu M, Olee T, Le DT, Roubey RAS, Hahn BH, Woods VL, Chen PP. Characterization of IgG monoclonal anti-cardiolipin/anti- β₂GP1 anti bodies from two patients with the anti-phospholipid syndrome reveals three species of antibodies. Br J Haematol 1999; 105 (01) 102-9.
• 32 Pierangeli SS, Liu Xwei, Anderson GH, Barker JH, Harris EN. Induction of thrombosis in a mouse model by IgG, IgM and IgA immunoglobulins from patients with the Antiphospholipid Syndrome. Thromb Haemost 1995; 74: 1361-7.
• 33 Peter FW, Schuschke DA, Wand WZ, Anderson GH, Franken RJPM, Gu JM, Pierangeli SS, Baker JH. Do leukocytes contribute to impaired microvascular tissue perfusion after arterial repair?. Microsurgery 1998; 18: 23-8.
• 34 Barker JH, Gu JM, Anderson GL. et al. The effects of heparin and dietary fish oil on embolic events and the microcirculation downstream from a small artery repair. Plastic Recontr Surg 1993; 91: 335-42.
• 35 Damianovich M, Gilburd B, George J, Del Papa N, Akeka A, Goldberg I, Kopolovic Y, Roth D, Barkai G, Meroni PL, Shoenfeld Y. Pathogenic Role of anti-endothelial cell antibodies in vasculitis. J Immunol 1996; 156: 4946-51.
• 36 Exner T, Rickard KA, Kronenberg H. A sensitve test demonstrating lupus anticoagulant and its behavioral patterns. Br J Haemato;l 1978; 40: 143-50.
• 37 Derksen RH, Hasselaar P, Blokzijl L, Gmelig FHMeyling, deGroot PG. Coagulation screen is more specific than the anticardiolipin antibody ELISA in defining a thrombotic subset of lupus patients. Ann Rheum Dis 1988; l47: 364-71.
• 38 Galli M, Finazzi G, Bevers EM, Barbui T. Kaolin clotting time and dilute Russell’s viper venom time distinguish between prothrombin-dependent and β₂glycoprotein -I dependent antiphospholipid antibodies. Blood 1995; 86 (02) 617-23.
• 39 Gharavi AE, Pierangeli SS, Hua Tang, Liu Xwei, Harris EN. Thrombogenic properties of antiphospholipid antibodies do not depend on their binding to β₂glycoprotein 1 (β₂GP1) alone. Lupus 1998; 07: 341-6.
• 40 Roubey RAS, Hoffman M. From antiphospholipid antibody syndrome to antibody-mediated thrombosis. Lancet 1997; 350: 1491-3.
• 41 Ferro D, Pittoni V, Quintarelli C, Basili S, Saliola M, Caroselli C, Valesini G, Viloi F. Coexistence of antiphospholipid antibodies and endothelial perturbation in systemic lupus erythematosus patients with on-going prothrombotic state. Thromb Haemost 1997; 01 75 (suppl): 359.
• 42 George J, Blank M, Levy Y, Meroni P, Damianovich M, Tincani A, Shoenfeld Y. Differential experimental antiphospholipid syndrome. Circulation 1998; 97: 900-6.
• 43 Del Papa N, Guidali L, Spatola L, Bonara P, Borghi MO, Tincani A, Balestrieri G, Meroni PL. Relationship between anti-phospholipid and anti-endothelial cell antibodies: β₂glycoprotein I mediates the antibody binding to endothelial membranes and induced the expression of adhesion molecules. Clin Exp Rheumatol 1995; 13: 179-85.
• 44 Cariou R, Tobelem G, Bellucci S, Soria J, Soria C, Maclouf J, Cane J. Effect of lupus anticoagulant on antithrombogenic properties of endothelial cells – inhibition of thrombomodulin-dependent protein C activation. Thromb Haemost 1988; 60: 54-8.
• 45 Marciniak E, Romond EH. Impaired catalytic function of activated protein C: a new in vitro manifestation of lupus anticoagulant. Blood 1989; 74: 2426-32.
• 46 Malia RG, Kitchen S, Greaves M, Preston FE. Inhibition of activated protein C and its cofactor protein S by antiphospholipid antibodies. J Haematol 1990; 76: 101-7.
• 47 Borrell M, Sala N, de Castellenau C, Lopez S, Gari M, Foncuberta J. Immunoglobulin fractions isolated from patients with antiphospholipid antibodies prevent the inactivation of factor Va by activated protein C on human endothelial cells. Thromb Haemost 1992; 68: 268-72.