Some plasmin-induced antibodies bind to cardiolipin, display lupus anticoagulant activity and induce fetal loss in mice

The combined presence of anti-phospholipid Ab (aPL), thrombosis, and/or fetal loss is recognized as the antiphospholipid syndrome (APS). aPL include anti-cardiolipin Ab (aCL) and/or lupus anticoagulants (LAC, detected as Ig that prolong certain in vitro phospholipid (PL)-restricted blood clotting tests); both aCL and LAC are the diagnostic Ab for APS. Studies show that aPL represent a heterogeneous group of Ab, which recognize various PL, PL-binding plasma proteins, and/or PL-protein complexes. Recently, we found that five of seven patient-derived IgG monoclonal aCL react with thrombin, activated protein C, and plasmin. All three proteins are trypsin-like serine proteases (SP), and are highly homologous in their catalytic domains. Importantly, among these SP autoantigens, the reactive aCL bind to plasmin with the highest affinity, suggesting that plasmin may serve as a major driving autoantigen for some aCL in approximately 30% of APS patients who are positive for IgG anti-plasmin Ab. To test this hypothesis, we immunized BALB/c mice with human plasmin and analyzed immune sera for aCL activity and reactivity with relevant SP. We found that some immune sera displayed aCL activity and/or bound to test SP. Subsequently, eight mAb were obtained and studied. The results revealed that one mAb displayed the aCL and the LAC activities and induced fetal loss when injected into pregnant mice. Immunohistological analyses of placentas revealed extensive deposits of activated C3 components. Combined, these data demonstrate that plasmin may serve as a driving Ag for some pathogenic aPL.     

Identification of anti-plasmin antibodies in the antiphospholipid syndrome that inhibit degradation of fibrin

The combined presence of anti-phospholipid Ab (aPL) and thrombosis is recognized as the antiphospholipid syndrome (APS). The aPL represent a heterogeneous group of Ab that recognize various phospholipids (PL), PL-binding plasma proteins, and/or PL-protein complexes. Recently, we found the presence of antithrombin Ab in some APS patients and that some of these anti-thrombin Ab could inhibit thrombin inactivation by antithrombin. Considering that thrombin is homologous to plasmin, which dissolves fibrin, we hypothesize that some APS patients may have Ab that react with plasmin, and that some anti-plasmin Ab may interfere with the plasmin-mediated lysis of fibrin clots. To test this hypothesis, we searched for anti-plasmin Ab in APS patients and then studied those found for their effects on the fibrinolytic pathway. The results revealed that seven of 25 (28%) APS patients have IgG anti-plasmin Ab (using the mean OD plus 3 SD of 20 normal controls as the cutoff) and that six of six patient-derived IgG anti-thrombin mAb bind to plasmin with relative K(d) values ranging from 5.6 x 10(-8) to 1 x 10(-6) M. These K(d) values probably represent affinities in the higher ranges known for human IgG autoantibodies against protein autoantigens. Of these mAb, one could reduce the plasmin-mediated lysis of fibrin clots. These findings suggest that plasmin may be an important driving Ag for some aPL B cells in APS patients, and that the induced anti-plasmin Ab may act either directly, by binding to plasmin and inhibiting its fibrinolytic activity, or indirectly, by cross-reacting with other homologous proteins in the coagulation cascade to promote thrombosis.     

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.     

Prothrombin binds to the surface of apoptotic,but not viable,cells and serves as a target of lupus anticoagulant autoantibodies

Anti-phospholipid Ab (aPL) are a heterogeneous group of autoantibodies directed against various combinations of phospholipids (PL) and PL-binding proteins. Lupus anticoagulant (LA) Ab, a subset of aPL, exhibit anticoagulant properties in vitro, but are procoagulant in vivo. Most LA Ab are specific for either beta(2)-glycoprotein I (beta(2)GPI) or prothrombin (PT), two PL-binding proteins. We have previously shown that beta(2)GPI and beta(2)GPI-dependent aPL bind specifically to apoptotic, but not viable, thymocytes. In this study, we demonstrate that PT, like beta(2)GPI, binds selectively to the surface of apoptotic, but not viable, Jurkat cells. Furthermore, PT supports the binding of systemic lupus erythematosus-derived polyclonal and murine monoclonal LA Ab to apoptotic cells. Two LA mAb, which differed dramatically in their relative affinities for PT, were studied. Although one mAb (29J3-62) had a high affinity for PT alone, the other (29I4-24) showed minimal reactivity with PT alone and required PL for elevated binding. Monovalent fragments of 29I4-24 reacted with PL-bound PT with high affinity, suggesting that this mAb recognizes a PL-dependent epitope. Despite these differences, PT-dependent binding of both mAb to apoptotic cells was 30-fold greater than that to viable cells. Moreover, binding of PT to apoptotic cells was, itself, increased in the presence of bivalent, but not monovalent, forms of either mAb. In summary, our data demonstrate the following: 1) specific binding of PT to apoptotic cells, an effect enhanced by PT-dependent LA Ab; 2) heterogeneity of PT-dependent LA Ab; and 3) potential pathogenicity of Ab of either low or high affinity for PT.     

Plasma gelsolin facilitates interaction between β2 glycoprotein I and α5β1 integrin

Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL) that directly recognizes plasma β₂‐glycoprotein I (β₂GPI). Tissue factor (TF), the major initiator of the extrinsic coagulation system, is induced on monocytes by aPL in vitro, explaining in part the pathophysiology in APS. We previously reported that the mitogen‐activated protein kinase (MAPK) pathway plays an important role in aPL‐induced TF expression on monocytes. In this study, we identified plasma gelsolin as a protein associated with β₂GPI by using immunoaffinity chromatography and mass spectrometric analysis. An in vivo binding assay showed that endogenous β₂GPI interacts with plasma gelsolin, which binds to integrin a₅β₁ through fibronectin. The tethering of β₂GPI to monoclonal anti‐β₂GPI autoantibody on the cell surface was enhanced in the presence of plasma gelsolin. Immunoblot analysis demonstrated that p38 MAPK protein was phosphorylated by monoclonal anti‐β₂GPI antibody treatment, and its phosphorylation was attenuated in the presence of anti‐integrin a₅β₁ antibody. Furthermore, focal adhesion kinase, a downstream molecule of the fibronectin‐integrin signalling pathway, was phosphorylated by anti‐β₂GPI antibody treatment. These results indicate that molecules including gelsolin and integrin are involved in the anti‐β₂GPI antibody‐induced MAPK pathway on monocytes and that integrin is a possible therapeutic target to modify a prothrombotic state in patients with APS.     

Thrombotic risk assessment in antiphospholipid syndrome: the role of new antibody specificities and thrombin generation assay

Antiphospholipid syndrome (APS) is an autoimmune condition characterized by the presence of antiphospholipid antibodies (aPL) in subjects presenting with thrombosis and/or pregnancy loss. The currently used classification criteria were updated in the international consensus held in Sidney in 2005. Vascular events seem to result of local procoagulative alterations upon triggers influence (the so called “second-hit theory”), while placental thrombosis and complement activation seem to lead to pregnancy morbidity. The laboratory tests suggested by the current classification criteria include lupus anticoagulant, a functional coagulation assay, and anticardiolipin and anti-β2-glycoprotein-I antibodies, generally detected by solid phase enzyme-linked immunosorbent assay. The real challenge for treating physicians is understanding what is the actual weight of aPL in provoking clinical manifestations in each case. As thrombosis has a multi-factorial cause, each patient needs a risk-stratified approach. In this review we discuss the role of thrombotic risk assessment in primary and secondary prevention of venous and arterial thromboembolic disease in patients with APS, focusing on new antibody specificities, available risk scoring models and new coagulation assays.     

A role for toll-like receptor mediated signals in neutrophils in the pathogenesis of the anti-phospholipid syndrome

The anti-phospholipid syndrome (APS) is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL). aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN) and Toll-like receptors (TLR) to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonist LPS monitoring neutrophil effector functions, namely the oxidative burst, phagocytosis, L-Selectin shedding and IL-8 production. aPL alone were only able to induce minor activation of PMN effector functions at high concentrations. However, in the additional presence of LPS the activation threshold was markedly lower indicating a synergistic activation pathway of aPL and TLR in PMN. In summary, our results indicate that PMN effector functions are directly activated by aPL and boosted by the additional presence of microbial products. This highlights a role for PMN as important innate immune effector cells that contribute to the pathophysiology of APS.     

A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli

Background  

The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (β₂GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL.     

Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation

The antiphospholipid syndrome (APS) is defined by thrombosis and recurrent pregnancy loss in the presence of antiphospholipid (aPL) antibodies and is generally treated with anticoagulation therapy. Because complement activation is essential and causative in aPL antibody-induced fetal injury, we hypothesized that heparin protects pregnant APS patients from complications through inhibition of complement. Treatment with heparin (unfractionated or low molecular weight) prevented complement activation in vivo and in vitro and protected mice from pregnancy complications induced by aPL antibodies. Neither fondaparinux nor hirudin, other anticoagulants, inhibited the generation of complement split products or prevented pregnancy loss, demonstrating that anticoagulation therapy is insufficient protection against APS-associated miscarriage. Our data indicate that heparins prevent obstetrical complications in women with APS because they block activation of complement induced by aPL antibodies targeted to decidual tissues, rather than by their anticoagulant effects.     

Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins

Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity in targeting phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or β2-glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by–among other factors–viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL with prothrombin (PT). The strength of the antibody response against SARS-CoV-2 was further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity, and aPL against PT in patients with SARS-CoV-2.     

Arginine residues are important in determining the binding of human monoclonal antiphospholipid antibodies to clinically relevant antigens

In the antiphospholipid syndrome (APS), antiphospholipid Abs (aPL) bind to anionic phospholipids (PL) and various associated proteins, especially beta(2)-glycoprotein I (beta2GPI) and prothrombin. In the present study, we show that altering specific Arg residues in the H chain of a human pathogenic beta2GPI-dependent aPL, IS4, has major effects on its ability to bind these clinically important Ags. We expressed whole human IgG in vitro by stable transfection of Chinese hamster ovary cells with expression plasmids containing different V(H) and V(L) sequences. V(H) sequences were derived from IS4 by altering the number of Arg residues in CDR3. V(L) sequences were those of IS4, B3 (anti-nucleosome Ab), and UK4 (beta2GPI-independent aPL). Binding of the expressed H/L chain combinations to a range of anionic, neutral, and zwitterionic PL, as well as prothrombin, beta2GPI, dsDNA, and chicken OVA, was determined by ELISA. Of four Arg residues in IS4VH CDR3 substituted to Ser, two at positions 100 and 100g, reduced binding to all Ags, while two at positions 96 and 97 reduced binding to beta2GPI but increased or decreased binding to different PL. Eleven of 14 H/L chain combinations displayed weak binding to OVA with Arg to Ser replacements of all four Arg residues enhancing binding to this Ag. Only one H/L chain combination bound neutral PL and none bound dsDNA; hence, these effects are particularly relevant to Ags important in antiphospholipid syndrome. We hypothesize that these four Arg residues have developed as a result of somatic mutations driven by an Ag containing both PL and beta2GPI.     

An allosteric redox switch in domain V of β2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition

β₂-glycoprotein I (β₂GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that β₂GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to β₂GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate β₂GPI''s structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of β₂GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating β₂GPI''s physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.     

Effect of Low Molecular Weight Heparins (LMWHs) on antiphospholipid Antibodies (aPL)-mediated inhibition of endometrial angiogenesis

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by vascular thrombosis and/or pregnancy morbidity in the presence of circulating antiphospholipid antibodies (aPL). Different pathogenic mechanisms for aPL-mediated pregnancy failure have been proposed. In particular a direct effect of aPL on both maternal and fetal side of the placental tissue has been reported, since their reactivity with β2-glycoprotein I (β2GPI) makes them adhere to trophoblast and human endometrial endothelial cell (HEEC) membranes. β2GPI can be recognized by aPL that, once bound, interfere with both trophoblast functions and with the HEEC differentiation.APS patients can be successfully treated with Low Molecular Weight Heparin (LMWH). Recent reports suggest that LMWH acts through mechanisms alternative to its well known anticoagulant effect, because of its ability to bind β2GPI. In our previous studies, we showed that LMWH is able to reduce the aPL binding to trophoblasts and restore cell invasiveness and differentiation. So far, however, no study has described its effects on endometrial angiogenesis.The aim of our research was to evaluate whether two LMWHs, tinzaparin and enoxaparin, have an effect on the aPL-inhibited endometrial angiogenesis. This prompted us to investigate: (i) in vitro HEEC angiogenesis through a Matrigel assay; (ii) VEGF secretion by ELISA; (iii) matrix metalloproteinase-2 (MMP-2) activity by gelatin zymography; (iv) Nuclear Factor-κB (NF-κB) DNA binding activity by colorimetric assay; (v) STAT-3 activation by a sandwich-ELISA kit. Furthermore, using an in vivo murine model we investigated the LMWHs effects on angiogenesis.We demonstrated that the addition of LMWHs prevents aPL-inhibited HEEC angiogenesis, both in vitro and in vivo, and is able to restore the aPL inhibited NF-κB and/or STAT-3 activity, the VEGF secretion and the MMPs activity.The demonstration of a beneficial role for LMWHs on the aPL-inhibited HEEC angiogenesis might provide additional mechanisms whereby this treatment protects early pregnancy in APS.     

Polymorphism of the plasminogen activator inhibitor type 1 gene, plasminogen level and thromboses in patients with the antiphospholipid syndrome

The frequency of venous and arterial thromboses and plasminogen level have been investigated in 78 patients with the antiphospholipid syndrome (APS), including 35 patients with systemic lupus erythematosus (SLE + APS) and 43 patients with primary APS (PAPS). The levels and genotypes of plasminogen activator inhibitor type 1 (PAI-1) were determined in 45 patients with APS (21 patients with SLE + APS and 24 patients with PAPS). A control group included 10 individuals without autoimmune disease signs and thromboses during the observation period and in anamnesis. It has been shown for the first time that for one third of 67 patients with APS and thromboses, high-positive levels of antiphospholipid antibodies (aPL) are associated with low plasminogen levels. The levels of PAI-1 antigen measured by the ELISA method, which detects active, latent and bound to plasminogen activator PAI-1, were compared with frequency of thromboses in APS patients. In one third of 43 patients with APS and thromboses the high and increased levels of PAI-1 were associated with high-positive aPL levels. One of possible mechanisms of this interrelationship was considered. It was shown that arterial and, to a greater extent, venous thromboses are associated with the 4G/5G polymorphism of the PAI-1 gene and high plasma level of the inhibitor in 79% of APS patients. In the presence of the 4G allele SLE + APS patients had higher PAI-1 levels than PAPS patients. The data obtained show that measuring the levels of plasminogen and PAI-1 as well as the 4G/5G polymorphism of the PAI-1 gene associated with thromboses may have the practical importance for identification of high risk of thrombosis in APS patients.     

Detection of anti-phosphatidylethanolamine antibodies using flow cytometry.

BACKGROUND: The finding that lupus anticoagulant (LA) is significantly associated with anti-phosphatidylethanolamine (PE) activity has led to great interest in its relation to the clinical features of the antiphospholipid syndrome (APS). Considerable variability has, however, been reported in the prevalence of anti-PE antibodies in APS patients using enzyme-linked immunosorbent assay (ELISA) methodology. The lack of standardization and differences in technique may in part explain these discrepancies. PE binds variably to different types of microtiter wells, reflected in the consequent detection, or lack of detection, of anti-PE antibodies. This study describes the use of flow cytometry as an alternative method for the detection of anti-PE antibodies. METHODS: Six LA-positive plasma samples were used in this original study. Polystyrene beads were coated with PE overnight. These were subsequently incubated with patient plasma. Both IgG and IgM binding were detected by flow cytometry using a cocktail of fluorescently labelled anti-human Ig isotypes. RESULTS: When these results were compared with those from ELISA, flow cytometric analysis provided an apparent enhanced detection of anti-PE antibodies. It was found that 6/6 were IgM anti-PE positive by flow cytometry, whereas 5/6 were IgM by ELISA; 2/6 negative for anti-cardiolipin antibodies by ELISA were positive by flow cytometry; and 2/6 positive for antiphosphatidylcholine antibodies in cytometry were negative by ELISA. CONCLUSIONS: With appropriate quantification, this method may be more sensitive than ELISA in detecting anti-PE antibodies in plasma samples of patients with APS.     

Platelets promote coagulation factor XII-mediated proteolytic cascade systems in plasma

Blood coagulation factor XII (FXII, Hageman factor) is a plasma serine protease which is autoactivated following contact with negatively charged surfaces in a reaction involving plasma kallikrein and high-molecular-weight kininogen (contact phase activation). Active FXII has the ability to initiate blood clotting via the intrinsic pathway of coagulation and inflammatory reactions via the kallikrein-kinin system. Here we have determined FXII-mediated bradykinin formation and clotting in plasma. Western blotting analysis with specific antibodies against various parts of the contact factors revealed that limited activation of FXII is sufficient to promote plasma kallikrein activation, resulting in the conversion of high-molecular-weight kininogen and bradykinin generation. The presence of platelets significantly promoted FXII-initiated bradykinin formation. Similarly, in vitro clotting assays revealed that platelets critically promoted FXII-driven thrombin and fibrin formation. In summary, our data suggest that FXII-initiated protease cascades may proceed on platelet surfaces, with implications for inflammation and clotting.     

Identification of anti-thrombin antibodies in the antiphospholipid syndrome that interfere with the inactivation of thrombin by antithrombin.

The combined presence of anti-phospholipid (PL) Ab, including lupus anticoagulants (LAC) and/or anticardiolipin Ab (aCL), and thrombosis is recognized as the antiphospholipid syndrome (APS). LAC are detected as an inhibitory effect on PL-restricted in vitro blood coagulation tests, and are comprised mainly of Ab against beta(2) glycoprotein I and prothrombin (PT). Recently, anti-PT Ab (aPT) were found to be associated with thrombosis by some investigators, although this is not confirmed by others. Considering that aPT are heterogeneous in patients and that PT is converted into thrombin, we hypothesize that certain aPT in patients may bind to thrombin, and that some of such anti-thrombin Ab may interfere with thrombin-antithrombin (AT) interaction and thus reduce the AT inactivation of thrombin. To test this hypothesis, we searched for anti-thrombin Ab in APS patients and then studied those found for their effects on the AT inactivation of thrombin. The results revealed that most, but not all, aPT-positive patient plasma samples contained anti-thrombin Ab. To study the functional significance of these Ab, we identified six patient-derived mAb that bound to both PT and thrombin. Of these mAb, three could reduce the AT inactivation of thrombin, whereas others had minimal effect. These findings indicate that some aPT in patients react with thrombin, and that some of such anti-thrombin Ab could inhibit feedback regulation of thrombin. Because the latter anti-thrombin Ab are likely to promote clotting, it will be important to develop specific assays for such Ab and study their roles in thrombosis in APS patients.     

A Role for Uric Acid and the Nalp3 Inflammasome in Antiphospholipid Antibody-Induced IL-1β Production by Human First Trimester Trophoblast

Women with antiphospholipid syndrome (APS) are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR). Antiphospholipid antibodies (aPL) directly target the placenta by binding beta₂-glycoprotein I (β₂GPI) expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-β₂GPI antibodies (Abs) induce the secretion of IL-1β in a Toll-like receptor 4 (TLR4)-dependent manner. IL-1β secretion requires processing of pro-IL-1β and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. The objective of this study was to determine if aPL induce IL-1β production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-β₂GPI mAb and human polyclonal aPL-IgG induce IL-1β processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-β₂GPI Ab-induced IL-1β secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1β production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1β processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS.     

TNF-alpha is a critical effector and a target for therapy in antiphospholipid antibody-induced pregnancy loss

The antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, intrauterine growth restriction, and vascular thrombosis in the presence of antiphospholipid (aPL) Abs. Our studies in a murine model of APS induced by passive transfer of human aPL Abs have shown that activation of complement and recruitment of neutrophils into decidua are required for fetal loss, and emphasize the importance of inflammation in aPL Ab-induced pregnancy loss. In this study, we examine the role of TNF-alpha in pregnancy complications associated with aPL Abs in a murine model of APS. We show that aPL Abs are specifically targeted to decidual tissue and cause a rapid increase in decidual and systemic TNF-alpha levels. We identify the release of TNF-alpha as a critical intermediate that acts downstream of C5 activation, based on the fetal protective effects of TNF-alpha deficiency and TNF blockade and on the absence of increased TNF-alpha levels in C5-deficient mice treated with aPL Abs. Our results suggest that TNF-alpha links pathogenic aPL Abs to fetal damage and identify TNF blockade as a potential therapy for the pregnancy complications of APS.     

Monocyte tissue factor induction by activation of beta 2-glycoprotein-I-specific T lymphocytes is associated with thrombosis and fetal loss in patients with antiphospholipid antibodies

Antiphospholipid (aPL) syndrome (APS) is characterized by thromboembolic events, thrombocytopenia, or recurrent miscarriage associated with aPL Abs with specificity for beta2-glycoprotein-I (beta2GPI). We recently reported that at least 44% of patients with the APS possess circulating type 1 (Th1) CD4+ T cells that proliferate and secrete IFN-gamma when stimulated with beta2GPI in vitro. In this study, we show that stimulation of PBMCs from 20 APS patients with beta2GPI induced substantial monocyte tissue factor (TF) (80 +/- 11 TF stimulation index (TF-SI)), whereas no induction was observed using PBMCs from 13 patients with aPL Abs without APS (6 +/- 1 TF-SI) or 7 normal and 7 autoimmune controls (5 +/- 1 and 3 +/- 1 TF-SI, respectively) (p < 0.0001). TF induction on monocytes by beta2GPI was dose dependent and required CD4+ T lymphocytes and class II MHC molecules. Because monocyte TF induction by beta2GPI was observed in all patients with APS, but not in any patient with aPL Abs without APS, this response is a potentially useful predictor for APS in patients with aPL Abs, as well as providing mechanistic insight into thrombosis and fetal loss in these patients.