SummarySome lupus anticoagulants (LA) have been shown to be directed against phospholipid-bound prothrombin. While developing an ELISA to detect anti-prothrombin autoantibodies in patient serum or plasma, no or very low signal was observed using human prothrombin immobilized on plain polystyrene plates. In contrast, the same LA-positive samples bound specifically to prothrombin coated on γ-irradiated plates, depending on the radiation dose, in the absence of added calcium and phospholipid. Optimization of the assay required the addition of 0.1% Tween 20 to the buffers. Antibody specificity for immobilized prothrombin was ascertained by competition using liposome-bound prothrombin, since fluid-phase prothrombin competed poorly. Seventy-seven of 139 patients (55.4%) with LA related to a variety of underlying diseases possessed anti-prothrombin antibodies (27 IgG, 35 IgM and 15 both isotypes), either isolated or more often associated with anti-(β2 glycoprotein I (β2GPI) antibodies. These included 67-71% of the patients with systemic lupus erythematosus and related disorders, primary antiphospholipid antibody syndrome or drug-induced LA (autoimmune groups), but only 19-20% of those with infection or malignancy (p <0.001). As previously shown for anti-β2GPI antibodies, IgG2 was the predominant IgG subclass reactive with prothrombin. Thus, autoimmune patients with LA have a high incidence of antibodies to β2GPI and prothrombin, the binding of which could similarly require high antigen density and/or exposure of cryptic epitopes resulting from protein interaction with an irradiated (i. e. more anionic) polystyrene surface.

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The serine protease subcomponents of the ac- (12)(13)(14). The (Clr, Cls)2 subunit is a very elongated structure of dimensions 51-59 nm, as first shown by electron microscopy (15) and confirmed by neutron-scattering studies in solution (16). However, the domain structure of (Clr, Cls)2 remains to be elucidated. The present study allows a detailed interpretation of the domain structure of Cir, Cls, and their associations Clr2, Cls2, and (Clr, Cls)2. MATERIALS AND METHODSProenzymic and activated forms of Clr and Cls were purified from human serum by using insoluble immune aggregates as described (5, 12). Calcium-dependent interactions of Clr and Cls before or after limited proteolysis were measured by sucrose gradient ultracentrifugation as described (17).Limited Proteolytic Cleavages. Autolytic cleavage of Cir (12, 18) was obtained by incubation of the protein (0.4-0.5 mg/ml) for 7 hr at 370C in 145 mM NaCl/5 mM triethanolamine HCl, pH 7.4. Limited cleavage of Cis was carried out using plasmin: Cis (1-1.5 mg/ml) in 145 mM NaCI/5 mM triethanolamine HCl, pH 7.4, was incubated with human plasmin (KABI, 25 units/mg) for up to 1 hr at 370C [enzyme/ substrate ratio (wt/wt), 1:50]. When indicated in the text, Clr and Cls were labeled with [1,3-3H]diisopropyl phosphorofluoridate (Amersham) either before or after proteolytic cleavage, as described (17). Fragments from limited proteolysis of Clr and Cis were separated by high-pressure gel permeation on a TSK-G3000 SW column (LKB) equilibrated in the same buffer as used for cleavage.Electron Microscopy. Proteins were dialyzed against 50 mM ammonium bicarbonate and glycerol was added to a final concentration of 75% (vol/vol). Protein samples (40 ,ug/ml) were sprayed onto freshly cleaved mica slides at room temperature. Samples were desiccated in an Edwards evaporator for 0.5-1 hr under a vacuum of at least 1o-, torr(1 torr = 133 Pa). Rotary shadowing with tantalum/tungsten was done with a 7°angle; the resulting replicas were coated with carbon, floated onto distilled water, and picked up on a 400-mesh copper grid. They were examined in 100 CX and 100 S Jeol electron microscopes at 80 kV. Dimensions of the molecules were corrected by subtracting 20 A to account for metal thickness (Clq observed both after rotary shadowing and negative staining was taken as a reference). RESULTSElectron Microscopy of Native Proteins. Pictures of Cir ( Fig. 1 A and B) 4477The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The capacity of cultured human monocytes to synthesize and to secrete the subcomponents of C1 and C1 inhibitor was examined. Non-stimulated monocytes secreted C1q and C1s from day 5 of culture. C1s reached a plateau immediately at its maximum level, whereas C1q secretion increased progressively until the end of the second week. Between day 12 and day 25, C1q secretion remained nearly constant (1-15 fmol/day per microgram of DNA, depending on the donor), whereas C1s secretion decreased and even in some cases stopped. C1r and C1 inhibitor were not secreted in detectable amounts by these resting cells. Stimulation of monocytes by yeasts, immunoglobulin G-opsonized sheep red blood cells or latex beads did not modify consistently C1q and C1s secretion. Activation by conditioned media from mitogen-, antigen- or allogeneic-stimulated lymphocyte cultures increased C1q production from 2 to 7 times and re-activated C1s secretion. Under the same conditions of activation, C1 inhibitor was secreted (up to 300 fmol/day per microgram of DNA) and C1r became detectable in culture supernatants. Isolated human monocytes are thus able to synthesize the whole C1 subcomponents; C1, if assembled, could be protected from non-immunological activation by locally produced C1 inhibitor. Activated monocytes appear to be a good tool for studying the assembly of C1 subcomponents and the role of C1 inhibitor in this process.

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