Prothrombin and β₂-glycoprotein I frequently contribute to antiphospholipid antibody interactions with phospholipids and the generation of abnormal waveform profiles in coagulation assays

Summary

Transmittance waveforms are generated during clot formation on photo-optical coagulation analyzers. We previously showed that 61.5% of patients with antiphospholipid antibodies (APLA) exhibited a negative deflection in the pre-coagulation phase of the prothrombin time (PT slope 1). The current studies investigated the ‘molecular basis’ of this abnormal parameter. We found that the negative PT slope 1 is IgG-mediated and is not dependent on the presence of fibrinogen or thrombin activity. We also found that IgG from most of the patients required a specific thromboplastin and the presence of prothrombin or β₂-glycoprotein I (β₂GPI) to produce an abnormal IgG waveform assay. In addition, the abnormal IgG waveform required cofactor binding to phospholipids when β₂GPI was the cofactor, and annexin V could partially block this interaction. In conclusion, these results showed that the interactions of IgG with phospholipids via β₂GPI or prothrombin constitute the core mechanisms of the abnormal waveforms.

Keywords

Antiphospholipid antibodies - prothrombin - β₂-glycoprotein I - transmittance waveforms

^* Present address: Division of Proteomics Research, Institute of Medical Science, University of Tokyo Tokyo, Japan. Presented in part at the 9^th International Symposium on Antiphospholipid Antibodies, Tours, France, 12-16 September 2000, and at the XVIII Congress of the International Society on Thrombosis and Haemostasis, Paris, France, 6-12 July 2001

• References

• 1 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: β₂-glycoprotein I (apolipoprotein H). Proc Natl Acad Sci USA 1990; 87: 4120-4.
• 2 Oosting JD, Derksen RHWM, Bobbink IWG, Hackeng TM, Bouma BN, de Groot PG. Antiphospholipid antibodies directed against a combination of phospholipids with prothrom-bin, protein C, or protein S: An explanation for their pathogenic mechanism?. Blood 1993; 81: 2618-25.
• 3 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.
• 4 Levine JS, Branch DW, Rauch J. The antiphospholipid syndrome. N Engl J Med 2002; 346: 752-63.
• 5 Permpikul P, Rao LVM, Rapaport SI. Functional and binding studies of the roles of pro-thrombin and β₂-glycoprotein I in the expression of lupus anticoagulant activity. Blood 1994; 83: 2878-92.
• 6 Galli M, Finazzi G, Bevers EM, Barbui T. Kaolin clotting time and dilute Russell’s viper venom time distinguish between prothrombin-dependent and β₂-glycoprotein I-dependent antiphospholipid antibodies. Blood 1995; 86: 617-23.
• 7 Tsutsumi A, Matsuura E, Ichikawa K. et al. Antibodies to β₂-glycoprotein I and clinical manifestations in patients with systemic lupus erythematosus. Arthritis Rheum 1996; 39: 1466-74.
• 8 Forastiero RR, Martinuzzo ME, Cerrato GS, Kordich LC, Carreras LO. Relationship of anti β₂-glycoprotein I and anti prothrombin antibodies to thrombosis and pregnancy loss in patients with antiphospholipid antibodies. Thromb Haemost 1997; 78: 1008-14.
• 9 Braun PJ, Givens TB, Stead AG. et al. Properties of optical data from activated partial thromboplastin time and prothrombin time assays. Thromb Haemost 1997; 78: 1079-87.
• 10 Su Z, Braun PJ, Klemp KF, Baker KR, Thames EH, Ortel TL. Abnormal optical profiles in coagulation assays from patients with anti-phospholipid antibodies. Blood Coagul Fibrinolysis 2002; 13: 7-17.
• 11 Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: an update. Thromb Haemost 1995; 74: 1185-90.
• 12 Izumi T, Pound ML, Su Z, Iverson GM, Ortel TL. Anti-β₂-glycoprotein I antibody-mediated inhibition of activated protein C requires binding of β₂-glycoprotein I to phospholipids. Thromb Haemost 2002; 88: 620-6.
• 13 Ortel TL, Neal MC, Thames EH, Moore KD, Lawson JH. Immunologic impact and clinical outcomes after surgical exposure to bovine thrombin. Ann Surg 2001; 233: 88-96.
• 14 Su Z, Izumi T, Thames EH, Lawson JH, Ortel TL. Antiphospholipid antibodies after surgical exposure to topical bovine thrombin. J Lab Clin Med 2002; 139: 349-56.
• 15 Smirnov MD, Triplett DT, Comp PC, 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.
• 16 Willems GM, Janssen MP, Pelsers MMAL. et al. Role of divalency in the high-affinity binding of anticardiolipin antibody-β₂-glyco-protein I complexes to lipid membranes. Biochemistry 1996; 35: 13833-42.
• 17 Field SL, Chesterman CN, Dai Y-P, Hogg PJ. Lupus antibody bivalency is required to enhance prothrombin binding to phospholipid. J Immunol 2001; 166: 6118-25.
• 18 Sanmarco M, Alessi MC, Harle JR. et al. Antibodies to phosphatidylethanolamine as the only antiphospholipid antibodies found in patients with unexplained thromboses. Thromb Haemost 2001; 85: 800-5.
• 19 Branch DW, Rote NS, Dostal DA, Scott JR. Association of lupus anticoagulant with anti-body against phosphatidylserine. Clin Immunol Immunopathol 1987; 42: 63-75.
• 20 Wu JR, Lentz BR. Phospholipid-specific conformational changes in human prothrombin upon binding to procoagulant acidic lipid membranes. Thromb Haemost 1994; 71: 596-604.
• 21 Harper MF, Hayes PM, Lentz BR, Roubey RAS. Characterization of β₂-glycoprotein I binding to phospholipid membranes. Thromb Haemost 1998; 80: 610-4.
• 22 Pei G, Powers DD, Lentz BR. Specific contribution of different phospholipid surfaces to the activation of prothrombin by the fully assembled prothrombinase. J Biol Chem 1993; 268: 3226-33.
• 23 Smirnov MD, Ford DA, Esmon CT, Esmon NL. The effect of membrane composition on the hemostatic balance. Biochemistry 1999; 38: 3591-8.
• 24 Horbach DA, van Oort E, Derksen RH, de Groot PG. The contribution of anti-prothrom-bin-antibodies to lupus anticoagulant activity—discrimination between functional and non-functional anti-prothrombin-antibodies. Thromb Haemost 1998; 79: 790-5.
• 25 Downey C, Kazmi R, Toh CH. Novel and diagnostically applicable information from optical waveform analysis of blood coagulation in disseminated intravascular coagulation. Br J Haematol 1997; 98: 68-73.
• 26 Downey C, Kazmi R, Toh CH. Early identification and prognostic implications in disseminated intravascular coagulation through transmittance waveform analysis. Thromb Haemost 1998; 80: 65-9.
• 27 Toh CH, Samis J, Downey C, Walker J. et al. Biphasic transmittance waveform in the APTT coagulation assay is due to the formation of a Ca(++)-dependent complex of C-reactive protein with very-low-density lipoprotein and is a novel marker of impending disseminated intravascular coagulation. Blood 2002; 100: 2522-9.