Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

Summary

Lupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

• References

• 1 Thiagarajan P, Shapiro SS, De Marco L. Monoclonal immunoglobulin Mλ coagulation inhibitor with phospholipid specificity. Mechanism of a lupus anticoagulant. J Clin Invest 1980; 66: 397-405
• 2 Pengo V, Thiagarajan P, Shapiro SS, Heine MJ. Immunologic specificity and mechanism of action of IgG lupus anticoagulants. Blood 1987; 70: 69-76
• 3 Rauch J, Janoff AS. Phospholipid in the hexagonal II phase is immunogenic: Evidence for immunorecognition on nonbilayer lipid phases in vivo. Proc Natl Acad Sci USA 1990; 87: 4112-4
• 4 Triplett DA, Brandt JT. Laboratory identification of the lupus anticoagulant. Br J Haematol 1989; 73: 139-42
• 5 Espinoza LR, Hartmann RC. Significance of the lupus anticoagulant. Am J Hematol 1986; 22: 331-7
• 6 Triplett DA. Antiphospholipid antibodies and recurrent pregnancy loss. Am J Reprod Immunol 1989; 20: 52-67
• 7 Lechner K, Pabinger-Fasching I. Lupus anticoagulants and thrombosis. A study of 25 cases and review of the literature. Haemostasis 1985; 15: 254-62
• 8 Levine SR, Welch KMA. The spectrum of neurologic disease associated with antiphospholipid antibodies: lupus anticoagulants and anticardiolipin antibodies. Arch Neurol 1987; 44: 876-83
• 9 Carreras LO, Vermylen JG. "Lupus" anticoagulant and thrombosis – possible role of inhibition of prostacyclin formation. Thromb Haemostas 1982; 48: 38-40
• 10 Freyssinet J-M, Cazenave J-P. Lupus like anticoagulants, modulation of the protein C pathway and thrombosis. Thromb Haemostas 1987; 58: 679-81
• 11 Marciniak E, Romond EHx. Impaired catalytic function of activated protein C: a new in vitro manifestation of lupus anticoagulant. Blood 1989; 74: 2426-32
• 12 Amer L, Kisiel W, Searles RP, Williams RC. Impairment of the protein C anticoagulant pathway in a patient with systemic lupus erythematosus, anticardiolipin antibodies and thrombosis. Thromb Res 1990; 57: 247-58
• 13 Rauch J, Tannenbaum M, Tannenbaum H, Ramelson H, Cullis PR, Tilcock CPS, Hope MJ, Janoff AS. Human hybridoma lupus anticoagulants distinguish between lamellar and hexagonal phase lipid systems. J Biol Chem 1986; 261: 9672-7
• 14 McNeil HP, Chesterman CN, Krilis SA. Binding specificity of lupus anticoagulants and anticardiolipin antibodies. Thromb Res 1988; 52: 609-19
• 15 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 Rheum 1989; 7: 615-8
• 16 Steinbuch M, Audran R. The isolation of IgG from mammalian sera with the aid of caprylic acid. Arch Biochem Biophys 1969; 134: 279-84
• 17 Prowse C, Hornsey V, McKay G, Waterson Y. Room temperature, microtray chromogenic assay of factor VIII: C. Vox Sang 1986; 50: 21-5
• 18 Brandt JT, Triplett DA. The effect of phospholipid on the detection of lupus anticoagulants by the dilute Russell viper venom time. Arch Pathol Lab Med 1989; 113: 1376-8
• 19 Kasper CK, Aledort LM, Counts RB, Edson JE, Fratantoni J, Green D, Hampton JW, Hilgartner MW, Lazerson J, Levine PH, McMillan CW, Pool JG, Shapiro SS. A more uniform measurement of factor VIII inhibitors. Thromb Diathes Haemorrh 1975; 34: 869-71
• 20 Triplett DA, Brandt JT, Musgrave KA, Orr CA. Relationship between lupus anticoagulants and antiphospholipid antibodies. JAMA 1988; 259: 550-4
• 21 Coots MC, Miller MA, Glueck HI. The lupus inhibitor: a study of its heterogeneity. Thromb Haemostas 1981; 46: 734-9
• 22 Triplett DA, Brandt JT, Maas RL. The laboratory heterogeneity of lupus anticoagulants. Arch Pathol Lab Med 1985; 109: 946-51
• 23 Lillicrap DP, Pinto M, Benford K, Ford PM, Ford S. Heterogeneity of laboratory test results for antiphospholipid antibodies in patients treated with chlorpromazine and other phenothiazines. Am J Clin Pathol 1990; 93: 771-5
• 24 Skjonsberg OH, Wisloff F, Godal HC. Inhibition of the tissue factor-factor VII mediated activation of coagulation factor X by a monoclonal IgM antibody expressing lupus anticoagulant activity. Thromb Res 1990; 58: 349-52
• 25 Edson JR, Vogt JM, Hasegawa DK. Abnormal prothrombin crossed-immune-electrophoresis in patients with lupus inhibitors. Blood 1984; 64: 807-16
• 26 Fleck RA, Rapaport SI, Rao VM. Anti-prothrombin antibodies and the lupus anticoagulant. Blood 1988; 72: 512-9
• 27 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
• 28 Galli M, Comfurius P, Maasen C, Hemker HC, DeBaets MH, van Breda-Vriesman PJC, Barbui T, Zwaal RFA, Bevers EM. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990; 1: 1544-7
• 29 Nimpf J, Bevers EM, Bomans PHH, Till U, Wurm H, Kostner GM, Zwaal RFA. Prothrombinase activity of human platelets is inhibited by β₂-glycoprotein-I. Biochim Biophys Acta 1986; 884: 142-9