SummaryAntiphospholipid syndrome is a disorder of recurrent thrombosis and pregnancy losses associated with production of anticardiolipin antibodies and lupus anticoagulant positivity. Recently, we have adapted a mouse model of induced venous thrombosis to study the role of autoantibodies in thrombus formation. To determine whether immunoglobulins from patients with the antiphospholipid syndrome play a role in thrombosis, we injected groups of CDI mice either with immunoglobulins purified from seven patients with the antiphospholipid syndrome (nine preparations studied: four IgG, three IgM and two IgA) or with immunoglobulins of the same isotype from healthy controls. Seventy- two h after injection, a non-occlusive thrombus was induced in the femoral veins of experimental mice by a pinch injury; the thrombus areas as well as times of formation and disappearance of the thrombi were measured. Eight of the nine antiphospholipid syndrome immunoglobulin preparations caused a significant increase in mean thrombus area and a significant delay in mean thrombus disappearance time as compared with normal controls. To determine whether anticardiolipin antibodies might be involved, separate groups of mice were injected with affinity-purified IgG (n = 2) or IgM (n = 2) anticardiolipin antibodies or with normal immunoglobulins of the same isotype, and the effects on thrombus formation compared. Mean thrombus area and mean disappearance times were again significantly increased in all four groups injected with affinity-purified antibodies. This is the first study to show that anticardiolipin antibodies of IgG, IgM and IgA isotypes may play a role in thrombosis in vivo.
SummaryHigh levels of IgG antiphospholipid antibodies (aPL) have been associated with clinical thrombosis. It is uncertain however whether these antibodies play a direct role in thrombosis or are merely epiphenomena. To investigate whether antiphospholipid antibodies might play a role in thrombosis, we utilized a novel mouse model in which the dynamics of in vivo thrombosis can be studied. CD1 mice (26-30 g) were passively immunized with 25 mg of human IgG from a patient with the Antiphospholipid Syndrome (IgG-APS) (n = 17), IgG from normal pooled sera (TgG-NHS) (n = 9). or saline solution (n = 17), followed by 40 mg of the same preparations at 48 h. At 72 h, levels of human aPL antibodies, detected using the anticardiolipin ELISA test (aCL ELISA test), in mice immunized with IgG-APS, were 50-100 GPL units. Each animal was anesthetized, femoral vein minimally mobilized and subjected to a standardized “pinch” injury to induce thrombosis. The vessel was transilluminated using acrylic optical fibers connected to a light source, and clot formation and dissolution were visualized by a standard surgical microscope equipped with a video camera, video recorder, and computer assisted analysis system. Results showed that average clot size was significantly larger in mice immunized with IgG-APS compared to those treated with saline (p <0.037). In addition, the thrombus persisted longer in a significantly higher number of mice immunized with IgG-APS (10/17) compared to mice immunized with IgG-NHS (1/9) or saline (2/12) (p <0.02). These data suggest that IgG-APS may play a role in thrombus formation in humans. In addition, this study shows the feasibility of using this in vivo murine thrombus model for study of the effects of aPL antibodies.
Immunization of mice with beta2glycoprotein 1 (beta2GP1) induces production of antiphospholipid (aPL) antibodies, which were shown to have thrombus enhancing properties in an experimental mouse model, indicating that these antibodies are thrombogenic in vivo. To determine whether the thrombogenic effect of murine antiphospholipid antibodies is due to their aPL or their anti-beta2GP1 activity, we injected mice with murine monoclonal anticardiolipin (aCL) and anti-beta2GP1 antibodies. Effects of these antibodies on thrombus formation, was evaluated utilizing a mouse model which enables kinetics of thrombus formation to be studied. The results of this study showed that the size of the thrombus in animals injected with murine aCL antibodies was larger than that in control groups. There was no difference in thrombus kinetics between anti-beta2GP1 injected mice and controls, suggesting that the thrombogenic effect of aPL antibodies is not related to their anti-beta2GP1 activity alone. Mice receiving monoclonal antibodies with both aCL and anti-beta2GP1 activity, also increase thrombus size when compared with controls. These data indicate that murine aCL, but not anti-beta2GP1, antibodies are thrombogenic in vivo.
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