New targeted therapies for treatment of thrombosis in antiphospholipid syndrome.

Antiphospholipid (aPL) antibodies (Abs) are associated with thrombosis and pregnancy loss in antiphospholipid syndrome (APS), a disorder initially characterised in patients with systemic lupus erythematosus (SLE) but now known to occur in the absence of other autoimmune disease. There is strong evidence that aPL Abs are pathogenic in vivo, from studies of animal models of thrombosis, endothelial cell activation and pregnancy loss. In recent years, progress has been made in characterising the molecular basis of this pathogenicity, which includes direct effects on platelets, endothelial cells and monocytes as well as activation of complement. This review summarises the clinical manifestations of APS and current modalities of treatment, and explains recent advances in understanding the molecular events triggered by aPL Abs on target cells in coagulation pathways as well as effects of aPL Abs on complement activation. Based on this information and on additional scientific evidence using in vitro and in vivo models, new potential targeted therapies for treatment and/or prevention of thrombosis in APS are proposed and discussed.

Mechanisms of aPL-mediated thrombosis: effects of aPL on endothelium and platelets.

Antiphospholipid antibodies (aPL) are associated with thrombosis and pregnancy loss in patients with systemic lupus erythematosus and antiphospholipid syndrome. Strong evidence demonstrates that aPL are pathogenic in vivo from studies that utilized animal models of thrombosis, endothelial cell activation, and pregnancy loss. However, the mechanisms by which aPL mediate disease are only partially understood, and our knowledge is limited by the polyspecificity of the antibodies, the multiple potential end-organ targets, and the variability of the clinical context in which the disease may present. This review discusses and summarizes the most current data available on molecular interactions and pathogenic mechanisms in antiphospholipid syndrome.

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-beta(2)glycoprotein 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.

A human monoclonal antiprothrombin antibody is thrombogenic in vivo and upregulates expression of tissue factor and E-selectin on endothelial cells.

Prothrombin is now accepted as one of the target antigens recognised by antiphospholipid (aPL) antibodies. However, it is not clear whether anti-prothrombin antibodies are pathogenic in vivo and if so, the possible mechanism(s) involved. Here, we examined the pathogenic effects of the IS6 monoclonal anti-prothrombin antibody isolated from a patient with Antiphospholipid Syndrome (APS). IS6 antibody was purified from hybridoma supernatant. Its pathogenic potentials were studied in an in vivo model of induced thrombosis. The expression of tissue factor (TF) and E-selectin on human umbilical vein endothelial cells (HUVEC) was determined by cyto-enzyme-linked immunosorbent assay. Transcription of TF mRNA was determined by quantitative real time-polymerase chain reaction (RT-PCR). In vivo, the thrombus size increased significantly when treated with IS6 compared with control-treated mice (5388 +/- 1035 microm2 vs. 2845 +/- 1711 microm2). In vitro, IS6 induced significant expression of TF and E-selectin on HUVEC, when compared with control preparation (3.1- and 5.1-fold increase compared with the control-treated cells). RT-PCR analysis of TF mRNA transcription showed a 2.5-fold increase of IS6-treated cells over the value obtained with control-treated cells. Taken together, these data show that IS6 monoclonal anti-prothrombin antibody promotes thrombosis and this is associated with TF and E-selectin expression.

Complement activation: a novel pathogenic mechanism in the antiphospholipid syndrome.

Antiphospholipid antibodies (aPLs) have been shown to induce thrombosis, activate endothelial cells, and induce fetal loss. The pathogenesis of aPL-induced thrombosis is incompletely understood, but it is thought to involve platelet and endothelial cell activation as well as pro-coagulant effects of aPL antibody directly on clotting pathway components. Recent studies have shown that uncontrolled complement activation leads to fetal death in aPL-antibody treated mice. We hypothesized that aPL antibodies activate complement, generating split products that induce thrombosis. To test this hypothesis, we used an in vivo model of thrombosis in which aPL antibodies induce a significant increase in thrombus size and a mouse model of endothelial cell activation in which aPLs induce significant adhesion of leukocytes (WBCs) to endothelial cells. We found that mice deficient in complement components C3 and C5 were resistant to enhanced thrombosis and endothelial cell activation induced by aPL antibodies. Furthermore, inhibition of C5 activation using anti-C5 mAb prevented thrombophilia induced by aPL antibodies. Our data show that complement activation mediates two important effectors of aPL antibodies: induction of thrombosis and endothelial activation.

Requirement of activation of complement C3 and C5 for antiphospholipid antibody-mediated thrombophilia.

OBJECTIVE: Antiphospholipid antibodies (aPL) have been shown to induce thrombosis, activate endothelial cells, and induce fetal loss. The pathogenesis of aPL-induced thrombosis, although not completely understood, may involve platelet and endothelial cell activation as well as procoagulant effects of aPL directly on clotting pathway components. Recent studies have shown that uncontrolled complement activation leads to fetal death in aPL-treated mice. In this study, we tested the hypothesis that aPL are responsible for activation of complement, thus generating split products that induce thrombosis. METHODS: To study thrombus dynamics and adhesion of leukocytes we used in vivo murine models of thrombosis and microcirculation, in which injections of aPL were used. RESULTS: Mice deficient in complement components C3 and C5 were resistant to the enhanced thrombosis and endothelial cell activation that was induced by aPL. Furthermore, inhibition of C5 activation using anti-C5 monoclonal antibodies prevented thrombophilia induced by aPL. CONCLUSION: These data show that complement activation mediates 2 important effectors of aPL, induction of thrombosis and activation of endothelial cells.

Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antibodies.

OBJECTIVE: To study the intracellular mechanism involved in the up-regulation of tissue factor (TF) on endothelial cells (ECs) by antiphospholipid antibodies (aPL), we examined the effects of aPL on the transcription, expression, and function of TF, the expression of interleukin-6 (IL-6) and IL-8, the induction of inducible nitric oxide synthase (iNOS), and the phosphorylation of p38 MAPK on human umbilical vein ECs (HUVECs). METHODS: Cultured HUVECs were treated with IgG aPL (from patients with antiphospholipid syndrome [APS]) or with control IgG (from normal human serum). Phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS) were used as positive controls. TF expression was determined on the surface of HUVECs using an enzyme-linked immunosorbent assay (ELISA). TF activity was determined with the use of a chromogenic assay in cell lysates, and TF messenger RNA (mRNA) was determined by real-time quantitative polymerase chain reaction. Phosphorylation of p38 MAPK and induction of iNOS were determined by Western blotting, and levels of IL-6 and IL-8 were determined by ELISA. RESULTS: PMA, LPS, and aPL significantly increased the expression of TF compared with controls. This up-regulation was significantly inhibited by SB203580 (a specific inhibitor of p38 MAPK) and by MG132 (a specific inhibitor of NF-kappaB). TF activity was significantly increased by treatment with IgG aPL and this effect was also inhibited by SB203580. Incubation of HUVECs with aPL increased TF mRNA 2-15-fold; these effects were abrogated by SB203580. IgG aPL induced significant phosphorylation of p38 MAPK and produced iNOS on HUVECs in a time-dependent manner. Treatment with IgG aPL also induced increased expression of IL-6 and IL-8 on HUVECs. CONCLUSION: Our data show that aPL induces significant increases in TF transcription, function, and expression, in IL-6 and IL-8 up-regulation, and in iNOS expression on HUVECs and that these processes involve phosphorylation of p38 MAPK and activation of NF-kappaB. Understanding intracellular events in aPL-mediated EC activation may help in designing new targeted therapies for thrombosis in APS.

Intracellular signaling triggered by antiphospholipid antibodies in platelets and endothelial cells: a pathway to targeted therapies.

Understanding the intracellular events triggered by antiphospholipid (aPL) antibodies in platelets and endothelial cells (ECs) is important in designing new modalities of targeted therapies for the treatment of thrombosis in Antiphospholipid Syndrome (APS). A recent study showed a significant increase in the expression of GPIIb/IIIa on platelets treated with aPL antibodies and a thrombin receptor peptide agonist (TRAP), and these effects were abrogated by hydroxychloroquine (HQ). Hydroxychloroquine has also been shown to reduce in vivo aPL-induced thrombus formation. Furthermore, aPL-enhanced thrombosis in vivo can be abrogated by infusions of a GPIIb/IIIa antagonist (1B5) monoclonal antibody, and aPL-mediated thrombophilia is not observed in GPIIb/IIIa-deficient mice. Treatment of platelets with aPL antibodies has resulted in a significant increase in p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and aPL-induced platelet aggregation and thromboxane B2 (TXB2) production was abrogated by SB203580 (a p38MAPK inhibitor). aPL antibodies induce increased expression, function and transcription of tissue factor (TF) on EC. Activation of ECs and thrombogenicity of aPL in vivo can be reversed by treatment of the animals with statins. Upregulation of TF on ECs can also be abrogated by treatment of the cells with fluvastatin. There is also indication of activation of nuclear factor kappa B (NFkappaB), increase in phosphorylation of p38MAPK in ECs by aPL antibodies that can be reversed by specific inhibitors MG132 and SB203580, respectively. The data open the possibility to new treatment modalities that may include the use of hydroxychloroquine, statins, specific antagonists of GPIIb/IIIa (such as abciximab or equivalent) and specific p38MAPK inhibitors, after the completion of well-designed clinical studies.

Intracellular events in platelet activation induced by antiphospholipid antibodies in the presence of low doses of thrombin.

OBJECTIVE: Thrombosis and thrombocytopenia are features of the antiphospholipid syndrome (APS), suggesting that antiphospholipid antibodies (aPL) may bind platelets, causing activation and aggregation of platelets and thrombosis. The intracellular events involved in aPL-mediated platelet activation are not fully understood and are therefore the subject of this study. METHODS: IgG fractions and their F(ab')(2) fragments were purified from the sera of 7 patients with APS and from the pooled sera of 10 healthy subjects (as controls). Phosphorylation of p38 MAPK, ERK-1/2, and [Ca(2+)]-dependent cytosolic phospholipase A(2) (cPLA(2)) was determined in lysates of washed platelets pretreated with low doses of thrombin and aPL or control IgG or their F(ab')(2) fragments, by immunoblot. The effects of aPL on platelet aggregation in the presence or absence of a p38 MAPK inhibitor, SB203580, were examined. Thromboxane B(2) (TXB(2)) production was detected by enzyme-linked immunosorbent assay on gel-filtered platelets treated with aPL and thrombin, with or without SB203580. Calcium mobilization studies were done utilizing a fluorometric assay. RESULTS: Treatment of platelets with IgG aPL, or their F(ab')(2) fragments, in conjunction with subactivating doses of thrombin resulted in a significant increase in phosphorylation of p38 MAPK. Neither the IgG aPL nor their F(ab')(2) fragments increased significantly the phosphorylation of ERK-1/2 MAPKs. Furthermore, pretreatment of platelets with SB203580 completely abrogated the aPL-mediated enhanced aggregation of the platelets. Platelets treated with F(ab')(2) aPL and thrombin produced significantly larger amounts of TXB(2) when compared with controls, and this effect was completely abrogated by treatment with SB203580. In addition, cPLA(2) was also significantly phosphorylated in platelets treated with thrombin and F(ab')(2) aPL. There were no significant changes in intracellular [Ca(2+)] when platelets were treated with aPL and low doses of thrombin. CONCLUSION: The data strongly indicate that aPL in the presence of subactivating doses of thrombin induce the production of TXB(2) mainly through the activation of p38 MAPK and subsequent phosphorylation of cPLA(2). The ERK-1/2 pathway does not seem to be involved in this process, at least in the early stages of aPL-mediated platelet activation.