Zurina Romay-Penabad

Toll‐like receptor and antiphospholipid mediated thrombosis: in vivo studies

Objective: A study was undertaken to investigate the in vivo pathogenic role of Toll-like receptor 4 (TLR-4) in the antiphospholipid syndrome (APS) by studying the thrombogenic antiphospholipid (aPL) activity in lipopolysaccharide (LPS) non-responsive (LPS–/–) mice and the association between tlr4 gene polymorphisms and APS in patients. Methods: IgGs from two patients with APS, one with aPL negative systemic lupus erythematosus (SLE) and one with normal human serum (NHS), were evaluated for thrombosis, tissue factor (TF) activity and endothelial cell activation in LPS–/– mice displaying a tlr4 spontaneous mutation vs LPS responsive (LPS+/+) mice. Human tlr4 Asp299Gly and Thr399Ile polymorphisms were evaluated by allele-specific PCR in 110 patients with APS with arterial/venous thrombosis and in 220 controls of the same ethnic origin. Results: IgG-APS produced significantly larger thrombi and more leucocytes (WBC) adhering to endothelial cells in the cremaster muscle microcirculation of LPS+/+ mice than IgG-NHS or aPL negative SLE-IgG. These effects were abrogated after absorption of the anti-β2glycoprotein I activity by an affinity column. The two IgG-APS induced significantly smaller thrombi and fewer WBC adhering to endothelial cells in LPS−/− mice than in LPS+/+ mice. IgG-APS induced higher TF activity in carotid artery homogenates of LPS+/+ mice than in LPS−/− mice. The prevalence of Asp299Gly and Thr399Ile tlr4 polymorphisms was significantly lower than in controls. Conclusions: These findings in LPS−/− mice and the reduction in the “protective” polymorphism in patients with APS with thrombosis suggest that TLR-4 is involved in the interaction of aPL with endothelial cells in vivo.

Annexin A2 is involved in antiphospholipid antibody-mediated pathogenic effects in vitro and in vivo.

Antiphospholipid (aPL) antibodies recognize receptor-bound beta(2) glycoprotein I (beta(2)GPI) on target cells, and induce an intracellular signaling and a procoagulant/proinflammatory phenotype that leads to thrombosis. Evidence indicates that annexin A2 (A2), a receptor for tissue plasminogen activator and plasminogen, binds beta(2)GPI on target cells. However, whether A2 mediates pathogenic effects of aPL antibodies in vivo is unknown. In this work, we studied the effects of human aPL antibodies in A2-deficient (A2(-/-)) mice. A2(-/-) and A2(+/+) mice were injected with immunoglobulin G (IgG) isolated from either a patient with antiphospholipid syndrome (IgG-APS), a healthy control subject (IgG-normal human serum), a monoclonal anti-beta(2)GPI antibody (4C5), an anti-A2 monoclonal antibody, or monoclonal antibody of irrelevant specificity as control. We found that, after IgG-APS or 4C5 injections and vascular injury, mean thrombus size was significantly smaller and tissue factor activity was significantly less in A2(-/-) mice compared with A2(+/+) mice. The expression of vascular cell adhesion molecule-1 induced by IgG-APS or 4C5 in explanted A2(-/-) aorta was also significantly reduced compared with A2(+/+) mice. Interestingly, anti-A2 monoclonal antibody significantly decreased aPL-induced expression of intercellular cell adhesion molecule-1, E-selectin, and tissue factor activity on cultured endothelial cells. Together, these data indicate for the first time that A2 mediates the pathogenic effects of aPL antibodies in vivo and in vitro APS.

In vivo inhibition of antiphospholipid antibody‐induced pathogenicity utilizing the antigenic target peptide domain I of β2‐glycoprotein I: proof of concept

Summary.  Objectives: In the antiphospholipid syndrome (APS), the immunodominant epitope for the majority of circulating pathogenic antiphospholipid antibodies (aPLs) is the N‐terminal domain I (DI) of β2‐glycoprotein I. We have previously shown that recombinant DI inhibits the binding of aPLs in fluid phase to immobilized native antigen, and that this inhibition is greater with the DI(D8S/D9G) mutant and absent with the DI(R39S) mutant. Hence, we hypothesized that DI and DI(D8S/D9G) would inhibit aPL‐induced pathogenicity in vivo. Methods: C57BL/6 mice (n = 5, each group) were injected with purified IgG derived from APS patients (IgG‐APS, 500 μg) or IgG from normal healthy serum (IgG‐NHS) and either recombinant DI, DI(R39S), DI(D8S/D9G), or an irrelevant control peptide (at 10–40 μg). Outcome variables measured were femoral vein thrombus dynamics in treated and control groups following standardized vessel injury, expression of vascular cell adhesion molecule‐1 (VCAM‐1) on the aortic endothelial surface, and tissue factor (TF) activity in murine macrophages. Results: IgG‐APS significantly increased thrombus size as compared with IgG‐NHS. The IgG‐APS thrombus enhancement effect was abolished in mice pretreated with recombinant DI (P ≤ 0.0001) and DI(D8S/D9G) (P ≤ 0.0001), but not in those treated with DI(R39S) or control peptide. This inhibitory effect by DI was dose‐dependent, and at lower doses DI(D8S/D9G) was a more potent inhibitor of thrombosis than wild‐type DI (P ≤ 0.01). DI also inhibited IgG‐APS induction of VCAM‐1 on the aortic endothelial surface and TF production by murine macrophages. Conclusion: Our findings in this proof‐of‐concept study support the development of recombinant DI or the novel variant DI(D8S/D9G) as a potential future therapeutic agent for APS.

Role of p38 mitogen‐activated protein kinase in antiphospholipid antibody‐mediated thrombosis and endothelial cell activation

Summary.  Background: The purpose of this study was to examine whether SB 203580, a p38 mitogen‐activated protein kinase (MAPK) inhibitor, is effective in reversing the pathogenic effects of antiphospholipid antibodies. Methods: The adhesion of THP‐1 monocytes to cultured endothelial cells (EC) treated with immunoglobulin G (IgG) from a patient with antiphospholipid syndrome (IgG‐APS) or control IgG (IgG‐NHS) in the presence and absence of SB 203580 was examined. The size of an induced thrombus in the femoral vein, the adhesion of leukocytes to EC of cremaster muscle, tissue factor (TF) activity in carotid artery and in peritoneal macrophages, the ex vivo expression of vascular cell adhesion molecule‐1 (VCAM‐1) in aorta preparations and platelet aggregation were studied in mice injected with IgG‐APS or control IgG‐NHS and with or without SB 203580. Results: SB 203580 significantly reduced the increased adhesion of THP‐1 to EC in vitro, the number of leukocytes adhering to EC, the thrombus size, the TF activity in carotid arteries and in peritoneal mononuclear cells, and the expression of VCAM‐1 in aorta of mice, and completely abrogated platelet aggregation induced by IgG‐APS. Conclusion: These data suggest that targeting the p38 MAPK pathway may be valuable in designing new therapy modalities for treating thrombosis in patients with APS.

In Vivo Effects of an Inhibitor of Nuclear Factor-Kappa B on Thrombogenic Properties of Antiphospholipid Antibodies

Abstract:  It has been shown that endothelial cell (EC) activation and tissue factor (TF) upregulation in EC and monocytes by antiphospholipid antibodies (aPL Abs) leads to a prothrombotic state and involves translocation of nuclear factor‐kappa B (NF‐κB). Here we examined the effects of an NF‐κB inhibitor on aPL‐induced thrombosis, TF activity, and EC in vivo. We treated CD1 mice with IgG from a patient with antiphospholipid syndrome (IgG‐APS) or with control IgG (IgG‐NHS). The adhesion of leukocytes (number of white blood cells) to EC in cremaster muscle (as an indication of EC activation) as well as the size of an induced thrombus in the femoral vein of the mice were examined. Some mice in each group were infused with 10 μM MG132 (an inhibitor of NF‐κB). TF activity was determined using a chromogenic assay in homogenates of carotid arteries and in peritoneal cells of mice. In vivo, IgG‐APS increased significantly the number of white blood cells adhering to ECs (4.7 ± 2.2) when compared to control mice (1.5 ± 0.8), and these effects were significantly reduced when mice were pretreated with MG132 (0.8 ± 0.2). IgG‐APS increased significantly the thrombus size and MG132 inhibited that effect (93%). Treatment of the mice with IgG‐APS also induced significantly increased TF function in peritoneal cells and in homogenates of carotid arteries. Pretreatment of the mice with MG132 abrogated those effects significantly. Mice injected with IgG‐APS or with IgM‐APS with or without the inhibitor had medium–high titers of anticardiolipin antibodies in serum at the time of the surgical procedures. The data show that prothrombotic and proinflammatory properties of IgG‐APS and IgM‐APS are downregulated in vivo by an NF‐κB inhibitor. These findings may be important in designing new modalities of targeted therapies to treat thrombosis in patients with APS.

C5a receptor-deficient mice are protected from thrombophilia and endothelial cell activation induced by some antiphospholipid antibodies.

Abstract:  Recent findings indicate that complement activation—involving specifically C3 and C5—contributes to antiphospholipid (aPL)‐mediated thrombosis. Two complement effector pathways are initiated by the cleavage of C5, C5a and C5b, which leads to the formation of the C5b‐9 membrane attack complex. To delineate and distinguish the role of C5a from the C5b‐9 membrane attack complex seeded by C5b, we examined the in vivo effects (thrombosis) of aPL on C5a receptor‐deficient (C5aR−/−) mice. C5aR−/− and C5aR+/+ mice were injected with IgM or with IgG from two different patients with APS (IgM‐APS or IgG‐APS) or with control IgM or IgG (IgM‐NHS or IgG‐NHS) twice. Complement fixing activity of the Ig fractions and anticardiolipin activity in the sera of the mice were determined by enzyme‐linked immunosorbent assay. Surgical procedures to study thrombus dynamics were performed. IgM‐APS but not IgG‐APS fixed C1q to cardiolipin‐coated plates. IgM‐APS significantly enhanced thrombus size in C5aR+/+ mice compared to C5aR+/+ mice treated with IgM‐NHS (3198 ± 2361 μm2 versus 585 ± 460 μm2). C5aR−/− mice treated with IgM‐APS showed a significant reduction in thrombi size as compared with C5aR+/+ mice (676 ± 690 μm2 versus 3198 ± 2361 μm2; P= 0.001). IgG‐APS enhanced thrombus formation significantly in C5aR+/+ when compared to IgG‐NHS‐treated mice (3507 ± 965 μm2 versus 1321 ± 798 μm2), and these effects were not altered in C5aR−/− mice (3400 ± 1681 μm2). The data indicate that C5aR−/− mice are protected from the thrombogenic effects of some aPL antibodies.

Statins for the treatment of antiphospholipid syndrome

Fluvastatin has been shown to revert proinflammatory/prothrombotic effects of antiphospholipid antibodies (aPL) in vitro and in mice. Here, we examined whether fluvastatin affects the levels of proinflammatory/prothrombotic markers in antiphospholipid syndrome (APS) patients. Vascular endothelial growth factor (VEGF), soluble tissue factor (sTF), tumor necrosis factor‐α (TNF‐α), soluble intercellular adhesion molecule‐1 (sICAM‐1), sE‐selectin (E‐sel), C‐reactive protein (CRP), and soluble vascular cell adhesion molecule (sVCAM‐1), were measured in the sera of 93 APS patients and 60 controls and in the sera of nine patients with APS before and after 30 days of treatment with fluvastatin. Elevated levels of VEGF, sTF, and TNF‐α were found in APS patients. Fluvastatin significantly reduced those markers in the majority of treated subjects. The data from this study show that statins may be beneficial in aPL‐positive patients and warrant larger clinical trials to confirm the efficacy of the drug for the treatment of APS clinical manifestations.

Proof-of-concept study demonstrating the pathogenicity of affinity-purified IgG antibodies directed to domain I of β2-glycoprotein I in a mouse model of anti-phospholipid antibody-induced thrombosis

Objective. IgG aPL against domain I of β2-glycoprotein I (β2GPI) [anti-DI (aDI)] is associated with the pathogenesis of APS, an autoimmune disease defined by thrombosis and pregnancy morbidity. To date, however, no study has demonstrated direct pathogenicity of IgG aDI in vivo. In this proof-of-concept study, we designed a novel system to affinity purify polyclonal aDI aPL in order to assess its prothrombotic ability in a well-characterized mouse microcirculation model for APS. Methods. Two polyclonal IgG fractions were isolated from serum of a patient with APS, both with high aPL activity but differing in aDI activity (aDI-rich and aDI-poor). These IgG fractions were tested for their pathogenic ability in an in vivo mouse model of thrombosis. Male CD1 mice were injected intraperitoneally with either aDI-rich or aDI-poor IgG; as a control, IgG isolated from healthy serum was used. A pinch injury was applied to the right femoral vein and thrombus dynamics and tissue factor activity in isolated tissue were evaluated. Results. Both aDI-rich and aDI-poor IgG retained aCL and anti-β2GPI activity, while only aDI-rich IgG displayed high aDI activity, as defined by our in-house cut-offs for positivity in each assay. aDI-rich IgG induced significantly larger thrombi in vivo compared with aDI-poor IgG (P < 0.0001). Similarly, aDI-rich IgG significantly enhanced the procoagulant activity of carotid artery endothelium and peritoneal macrophages isolated from experimental animals (P < 0.01). Conclusion. These data directly demonstrate that the ability to cause thrombosis in vivo is concentrated in the aDI fraction of aPL.

C6 knock-out mice are protected from thrombophilia mediated by antiphospholipid antibodies

Background: Complement activation plays a role in pathogenesis of the antiphospholipid syndrome (APS), but the involvement of the C5b-9 membrane attack complex (MAC) is unknown. Here we studied the effects of human polyclonal antiphospholipid (aPL) antibodies on thrombosis and tissue factor (TF) up-regulation in C6 deficient (C6−/−) mice. Methods: C6−/− mice or the wild-type C3H/HeJ (C6+/+) mice were injected twice with IgG-APS (n = 2) or IgM-APS (n = 1) isolated from APS patients or with the corresponding control immunoglobulins (Igs) of normal human serum, (NHS) (IgG-NHS or IgM-NHS). Then, the sizes of induced thrombi in the femoral vein were determined 72 hours after the first injection. Tissue factor was determined in homogenates of carotid arteries and in peritoneal macrophages. Results: Thrombus sizes were significantly larger in C6+/+ treated with IgG-APS1 or with IgG-APS2 or with IgM-APS when compared with C6+/+ mice treated with IgG-NHS or with IgM-NHS, respectively. The sizes of thrombi were significantly smaller in the C6−/− mice injected with IgG-APS1, IgG-APS2 or IgM-APS (p < 0.001), compared to their C6+/+ counterparts showing an important abrogation of thrombus formation in mice lacking C6. The TF expression and activity in the C6−/− mice treated with IgG-APS or IgM-APS were diminished when compared to C3H/HeJ (C6+/+) mice treated with the same Igs. All mice injected with IgG-APS and IgM-APS had medium-high titers of anticardiolipin (aCL) and anti-β2glycoprotein I (aβ2GPI) antibodies. Conclusions: These data indicate that the C6 component of the complement system mediates aPL-thrombogenic effects, underscoring an important pathogenic mechanism and indicating the possibility of inhibiting complement to ameliorate APS-related manifestations.

Complement C5-inhibitor rEV576 (coversin) ameliorates in-vivo effects of antiphospholipid antibodies.

Activation of the complement cascade is an important mechanism for antiphospholipid antibody-mediated thrombosis. We examined the effects of rEV576 (coversin), a recombinant protein inhibitor of complement factor 5 activation, on antiphospholipid antibody-mediated tissue factor up-regulation and thrombosis. Groups of C57BL/6J mice (n = 5) received either IgG from a patient with antiphospholipid syndrome (APS) or control IgG from normal human serum (NHS). Each of these groups of mice had IgG administration preceded by either rEV576, or phosphate buffer control. For each of the four treatment groups, the size of induced thrombus, tissue factor activity in carotid homogenates, anticardiolipin and anti-β2glycoprotein I (anti-β2GPI) levels were measured 72 h after the first injection. Mice treated with IgG-APS had significantly higher titers of anticardiolipin antibodies and anti-β2GPI at thrombus induction compared with those treated with IgG-NHS. The IgG-APS/phosphate buffer treatment induced significantly larger thrombi and tissue factor activity compared with other groups. Mice treated with IgG-APS/rEV576 had significantly smaller thrombi and reduced tissue factor activity than those treated with IgG-APS/phosphate buffer. The data confirm involvement of complement activation in antiphospholipid antibody-mediated thrombogenesis and suggest that complement inhibition might ameliorate this effect.