Antiphospholipid antibody: functional specificity for inhibition of prothrombin activation by the prothrombinase complex S ILVIA S. P IERANGELI, 1 G EORGE H. G OLDSMITH, 2 D. WARE B RANCH 3 AND E. N IGEL H ARRIS 1 1 Departments of Microbiology/Immunology and Medicine, Morehouse School of Medicine, Atlanta, Georgia, 2 Division of Hematology-Oncology, Department of Medicine, University of Louisville, Louisville, Kentucky, and 3 Department of Obstetrics and Gynecology, University of Utah, Salt Lake City, Utah, U.S.A. Received 21 June 1996; accepted for publication 19 March 1997 Summary. The antiphospholipid syndrome (APS) is associated with production of autoantibodies with lupus anticoagulant (LA) activity. These antibodies cause prolong- ation of in vitro clotting tests by inhibition of the conversion of prothrombin to thrombin in the presence of anionic phospholipid (PL). The extent to which this inhibition reflects antibody binding to, or functional inhibition of, phospho- lipids alone, prothrombin alone, or a prothrombin– phospholipid complex is pertinent to our understanding of the pathophysiology of this syndrome. Immunoglobulin fractions (IgG) from 18 patients with LA activity were tested for inhibitory activity against prothrombin activation by either factor Xa, in a purified prothrombinase system, or by purified fractions of snake venoms (E. carinatus, E. multisquamatus) which cleave prothrombin at the same initial site as the prothrombinase complex but do not require anionic phospholipid as a cofactor. Parallel testing of the same IgG samples for prothrombin binding by immunoassay was performed. Although all IgG samples inhibited the prothrombinase reaction, only three exhibited any inhibition of venom protease prothrombin activation in either the presence or absence of PL. Only one sample exhibited prothrombin binding by Western blot. These results suggest that lupus anticoagulant antibodies are heterogenous and that many, if not most, of the autoantibody populations responsible for LA activity impair prothrombin activation by interaction either with phospholipid alone or with a restricted range of prothrombin–phospholipid epitopes expressed by prothrombin only as part of the intact prothrombin–prothrombinase complex. Keywords: lupus anticoagulant, antiphospholipid anti- bodies, snake venom proteases, antiphospholipid syndrome. Antiphospholipid syndrome (APS) is a disorder of recurrent thrombosis, pregnancy losses and thrombocytopenia asso- ciated with positive anticardiolipin and lupus anticoagulant tests (Harris & Pierangeli, 1994). Both anticardiolipin and lupus anticoagulant tests detect antibodies which have been shown to induce pregnancy loss (Branch et al, 1990; Blank et al, 1991) and to enhance thrombosis in mice (Pierangeli et al, 1994a, 1995; Pierangeli & Harris, 1994). The mechan- ism by which these antibodies might induce thrombosis and pregnancy loss is unknown. It is generally believed that determination of the specifities of these antibodies may provide clues to their mechanism of action. There are several postulates with respect to the specificities of these antibodies. In the anticardiolipin test, binding activity is increased by the serum protein b 2 -glycoprotein 1 (b 2 GP1) and various investigators believe this is to be due to antibodies binding b 2 GP1 (Roubey, 1994), a conformationally altered b 2 GP1 molecule (Matsuura et al, 1994), epitopes shared by b 2 GP1 and cardiolipin (McNeil et al, 1990, 1991), or cardiolipin that is conformationally altered by b 2 GP1 (Harris & Pierangeli, 1990; Pierangeli et al, 1992; Sammaritano et al, 1992; Gharavi et al, 1993; Stewart et al, 1995). The lupus anticoagulant test is a functional measure of antibody activity assessed by their ability to prolong in vitro clotting tests. This activity is the result of inhibition of conversion of prothrombin to thrombin or activation of factor X (Brandt, 1991; Galli et al, 1989; Goldsmith et al, 1994). Various groups attribute this inhibitory activity to British Journal of Haematology , 1997, 97, 768–774 768  1997 Blackwell Science Ltd Correspondence: Dr Silvia S. Pierangeli, Department of Microbiology and Immunology, Rm 1236, Morehouse School of Medicine, 720 Westview Dr. S.W., Atlanta, GA 30310-1495, U.S.A.