Antiphospholipid Syndrome IgG Research Articles

IgG fractions from patients with antiphospholipid syndrome and systemic lupus erythematosus bind to platelets, but do not affect collagen-induced platelet activation

ABSTRACT Anti-beta-2 glycoprotein 1 (anti-β2GP1) is an antiphospholipid antibody found in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). Its presence commonly is associated with thrombosis; however, the mechanisms of interaction of anti-β2GP1 antibodies and platelets remain unclear. We investigated the effects of APS and SLE patient-derived IgG fractions on collagen-mediated platelet aggregation and examined the binding of patient-derived IgG to platelets before and after activation by collagen. IgG fractions, 150, 200, 300 or 350 µg/ml, isolated from 11 patients with APS and SLE were incubated with two sets of platelet-rich plasma (PRP) in the incubation wells of an aggregometer. The first set was activated by collagen and the other set was incubated for an additional 10 min. All platelets were collected by centrifugation and fixed in cell blocks. We assessed binding of IgG to platelets using immunocytochemistry (ICC). Patient-derived IgG fractions did not affect collagen-induced platelet aggregation. ICC staining using anti-human IgG antibodies demonstrated that patient-derived IgG fractions had greater affinity for non-activated platelets than those activated by 0.75 µg/ml collagen. Patient-derived IgG fractions bound to the surface of platelets and potentially could be internalized by platelets. IgG fractions from APS and SLE patients may sensitize non-activated platelets, which could increase platelet reactivity and thrombotic risk in patients. We did not detect secondary effects of patient-derived IgG fractions.

Do all antiphospholipid antibodies confer the same risk for major organ involvement in systemic lupus erythematosus patients?

We aimed to investigate the association between the different antiphospholipid antibodies (aPL) and both systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) manifestations. Patients from the RELESSER registry, a Spanish retrospective, cross-sectional, forty-five hospital registry of adult SLE patients, were included. Out of a total of 3,658 SLE patients, 1372 were aPL positive (555 of them fulfilled criteria for APS). All aPL types showed a negative association with cutaneous SLE manifestations. Lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) were both associated with haematological, ophthalmological and neuropsychiatric manifestations. IgG isotypes were associated with a higher risk of lupus manifestations compared with IgM. We found that the risk of neuropsychiatric and ophthalmological manifestations significantly increased with a higher number of positive aPL whereas the risk of cutaneous symptoms showed a negative correlation. All types of aPL, and more strongly LA, were associated with non-criteria antiphospholipid syndrome (APS) manifestations such as thrombocytopenia and haemolytic anaemia. Moreover, LA and aCL (particularly IgG isotype) were also associated with Libman-Sacks endocarditis and cognitive impairment. This association was stronger with more than one positive aPL. All types of aPL were also associated with classic APS manifestations, although LA, IgG isotypes, and patients with more than one aPL displayed a higher risk. There is a hierarchy for aPL and the risk of APS and SLE manifestations. aCL, and especially LA, confer a higher risk for major organ involvement in SLE. IgG isotypes seem to have a more important role. The load of aPL confer a higher risk for APS and certain SLE manifestations.

Increased Adhesive Potential of Antiphospholipid Syndrome Neutrophils Mediated by β2 Integrin Mac-1.

While the role of antiphospholipid antibodies in activating endothelial cells has been extensively studied, the impact of these antibodies on the adhesive potential of leukocytes has received less attention. This study was undertaken to investigate the extent to which antiphospholipid syndrome (APS) neutrophils adhere to resting endothelial cells under physiologic flow conditions and the surface molecules required for that adhesion. Patients with primary APS (n = 43), patients with a history of venous thrombosis but negative test results for antiphospholipid antibodies (n = 11), and healthy controls (n = 38) were studied. Cells were introduced into a flow chamber and perfused across resting human umbilical vein endothelial cells (HUVECs). Surface adhesion molecules were quantified by flow cytometry. Neutrophil extracellular trap release (NETosis) was assessed in neutrophil-HUVEC cocultures. Upon perfusion of anticoagulated blood through the flow chamber, APS neutrophils demonstrated increased adhesion as compared to control neutrophils under conditions representative of either venous (n = 8; P < 0.05) or arterial (n = 15; P < 0.0001) flow. At the same time, APS neutrophils were characterized by up-regulation of CD64, CEACAM1, β2 -glycoprotein I, and activated Mac-1 on their surface (n = 12-18; P < 0.05 for all markers). Exposing control neutrophils to APS plasma or APS IgG resulted in increased neutrophil adhesion (n = 10-11; P < 0.0001) and surface marker up-regulation as compared to controls. A monoclonal antibody specific for activated Mac-1 reduced the adhesion of APS neutrophils in the flow-chamber assay (P < 0.01). The same monoclonal antibody reduced NETosis in neutrophil-HUVEC cocultures (P < 0.01). APS neutrophils demonstrate increased adhesive potential, which is dependent upon the activated form of Mac-1. In patients, this could lower the threshold for neutrophil-endothelium interactions, NETosis, and possibly thrombotic events.

Antiphospholipid antibody‐induced cellular responses depend on epitope specificity : implications for treatment of antiphospholipid syndrome

Background Antiphospholipid antibodies (aPLs) contribute to the pathogenesis of the antiphospholipid syndrome (APS) by induction of an inflammatory and procoagulant state in different cell types, and several signaling pathways have been described. Objectives To investigate whether signaling depends on the epitope specificity of aPLs. Methods Cellular effects of three human monoclonal aPLs with distinctly different epitope specificities were analyzed in vitro. Expression of tumor necrosis factor-α mRNA by mouse and human monocytes was the major readout. Analysis included cells from genetically modified mice, and the use of specific inhibitors in human monocytes. Data were validated with IgG isolated from 20 APS patients. Results Cofactor-independent anticardiolipin aPLs activated monocytes by induction of endosomal NADPH oxidase. Activation could be blocked by hydroxychloroquine (HCQ). Anti-β2 -glycoprotein I aPL activated monocytes by interacting with LDL receptor-related protein 8 (LRP8). This could be blocked by rapamycin. Analysis of 20 APS patients' IgG showed that all IgG fractions activated the same two pathways as the monoclonal aPL, depending on their epitope patterns as determined by ELISA. Monocyte activation by APS IgG could be blocked completely by HCQ and/or rapamycin, suggesting that in most, if not all, APS patients there is no other relevant signaling pathway. Conclusions aPLs activate two major proinflammatory signal transduction pathways, depending on their epitope specificity. HCQ and rapamycin, either alone or in combination, completely suppress signaling by APS IgG. These observations may provide a rationale for specific treatment of APS patients according to their aPL profile.

Activated signature of antiphospholipid syndrome neutrophils reveals potential therapeutic target.

Antiphospholipid antibodies, present in one-third of lupus patients, increase the risk of thrombosis. We recently reported a key role for neutrophils - neutrophil extracellular traps (NETs), in particular - in the thrombotic events that define antiphospholipid syndrome (APS). To further elucidate the role of neutrophils in APS, we performed a comprehensive transcriptome analysis of neutrophils isolated from patients with primary APS. Moreover, APS-associated venous thrombosis was modeled by treating mice with IgG prepared from APS patients, followed by partial restriction of blood flow through the inferior vena cava. In patients, APS neutrophils demonstrated a proinflammatory signature with overexpression of genes relevant to IFN signaling, cellular defense, and intercellular adhesion. For in vivo studies, we focused on P-selectin glycoprotein ligand-1 (PSGL-1), a key adhesion molecule overexpressed in APS neutrophils. The introduction of APS IgG (as compared with control IgG) markedly potentiated thrombosis in WT mice, but not PSGL-1-KOs. PSGL-1 deficiency was also associated with reduced leukocyte vessel wall adhesion and NET formation. The thrombosis phenotype was restored in PSGL-1-deficient mice by infusion of WT neutrophils, while an anti-PSGL-1 monoclonal antibody inhibited APS IgG-mediated thrombosis in WT mice. PSGL-1 represents a potential therapeutic target in APS.

Factor Xa Mediates Calcium Flux in Endothelial Cells and is Potentiated by Igg From Patients With Lupus and/or Antiphospholipid Syndrome

Factor (F) Xa reactive IgG isolated from patients with antiphospholipid syndrome (APS) display higher avidity binding to FXa with greater coagulant effects compared to systemic lupus erythematosus (SLE) non APS IgG. FXa signalling via activation of protease-activated receptors (PAR) leads to increased intracellular calcium (Ca2+). Therefore, we measured alterations in Ca2+ levels in human umbilical vein endothelial cells (HUVEC) following FXa-mediated PAR activation and investigated whether FXa reactive IgG from patients with APS or SLE/APS- alter these responses. We observed concentration-dependent induction of Ca2+ release by FXa that was potentiated by APS-IgG and SLE/APS- IgG compared to healthy control subjects’ IgG, and FXa alone. APS-IgG and SLE/APS- IgG increased FXa mediated NFκB signalling and this effect was fully-retained in the affinity purified anti-FXa IgG sub-fraction. Antagonism of PAR-1 and PAR-2 reduced FXa-induced Ca2+ release. Treatment with a specific FXa inhibitor, hydroxychloroquine or fluvastatin significantly reduced FXa-induced and IgG-potentiated Ca2+ release. In conclusion, PAR-1 and PAR-2 are involved in FXa-mediated intracellular Ca2+ release in HUVEC and FXa reactive IgG from patients with APS and/or SLE potentiate this effect. Further work is required to explore the potential use of IgG FXa reactivity as a novel biomarker to stratify treatment with FXa inhibitors in these patients.

In Vivo Role of Neutrophil Extracellular Traps in Antiphospholipid Antibody-Mediated Venous Thrombosis.

Antiphospholipid syndrome (APS) is a leading acquired cause of thrombotic events. Although antiphospholipid antibodies have been shown to promote thrombosis in mice, the role of neutrophils has not been explicitly studied. The aim of this study was to characterize neutrophils in the context of a new model of antiphospholipid antibody-mediated venous thrombosis. Mice were administered fractions of IgG obtained from patients with APS. At the same time, blood flow through the inferior vena cava was reduced by induction of stenosis. Resulting thrombi were characterized for size and neutrophil content. Circulating factors and the vessel wall were also assessed. As measured by both thrombus weight and thrombosis frequency, mice treated with IgG from patients with APS (APS IgG) demonstrated exaggerated thrombosis as compared with control IgG-treated mice. Thrombi in mice treated with APS IgG were enriched for citrullinated histone H3 (a marker of neutrophil extracellular traps [NETs]). APS IgG-treated mice also demonstrated elevated levels of circulating cell-free DNA and human IgG bound to the neutrophil surface. In contrast, circulating neutrophil numbers and markers of vessel wall activation were not appreciably different between APS IgG-treated mice and control mice. Treatment with either DNase (which dissolves NETs) or a neutrophil-depleting antibody reduced thrombosis in APS IgG-treated mice to the level in control mice. These data support a mechanism whereby circulating neutrophils are primed by antiphospholipid antibodies to accelerate thrombosis. This line of investigation suggests new, immunomodulatory approaches for the treatment of APS.

Pregnancy outcome in patients with systemic lupus erythematosus: A single center study in the High Risk Pregnancy unit

Abstract Background Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that mainly affects females in the reproductive age. Objective To investigate pregnancy course and outcome in females with SLE, to evaluate the effect of pregnancy on SLE disease activity and to investigate the predictors of adverse pregnancy outcomes. Methods A prospective study of 91 pregnancies (84 women) with SLE. Comparisons were done using Chi-square test to evaluate the impact of clinical and laboratory parameters on maternal and fetal outcomes. Logistic regression analysis was used to study the predictors of adverse pregnancy outcome (defined as the occurrence of abortion, pre-eclampsia, prematurity, IUFD or SLE flare). Results The most common maternal manifestations of SLE were cutaneous lesions (93%), articular (92%), lupus nephritis (53%), hypertension (39%) and secondary antiphospholipid syndrome (APS) (38%). The incidence of abortion was (15%), IUGR (32%), prematurity (13%), pre-eclampsia (12%), IUFD (8%), NICU (15%), and LBW (22%). SLE antenatal flares were (44%), with 70% occurring in the second trimester, with renal flares being the most commonly reported (21%). The incidence of postnatal flares was 7%. There was significant association between hypertension and abortion (p = 0.04), pre-eclampsia (p = 0.0001) and SLE flares (p = 0.0001). Lupus nephritis and hypertension were predictors of preeclampsia (p = 0.01 and p = 0.002 respectively) and SLE flares (p = 0.048 and p = 0.003 respectively). Secondary APS and aCL IgG were also predictors of abortion (p = 0.001 and p = 0.04 respectively). Conclusion Careful monitoring of pregnancy and efficient treatment of SLE can decrease the risks for the mother and the fetus. However, despite improvements in pregnancy outcome of SLE pregnant patients, adverse maternal and fetal outcomes may occur. SLE may flare-up with pregnancy.

PReS13-SPK-1240: New aspects on APS pathogenesis

Antiphospholipid antibodies (aPL) are both diagnostic markers for, and pathogenic drivers of, antiphospholipid syndrome (APS). Beta 2 glycoprotein I (Beta2GPI)-dependent aPL, the most important subset, mediate different--and not necessarily alternative--thrombogenic mechanisms, mainly on the basis of their reactivity with Beta2GPI expressed on cells that participate in the coagulation cascade. In vivo experimental models showed that Beta2GPI cannot be detected in vascularized organs in resting conditions, while it can be over-expressed after pro-inflammatory stimulus. In this condition autoantibodies recognize the molecule, fix complement (C') and eventually induce clotting. This finding is in line with the observation that although the presence of aPL is a necessary pre-condition, APS-associated clotting is triggered by an additional 'second hit', frequently related to innate inflammatory immune responses. Recurrent pregnancy complications associated with aPL cannot be explained solely by thrombosis, and alternative pathogenic mechanisms have been reported. Although one in vivo model of fetal loss supports a mechanism of aPL-mediated acute placental inflammation, other models and the histopathological examination of APS placentae do not support an inflammatory signature. Beta2GPI can be detected on endothelial cells of uterine vessels in resting conditions and its presence is increased during pregnancy in uterine endothelium and trophoblast. This finding does support the hypothesis that Beta2GPI -dependent aPL recognize their antigen on placental tissues. There is evidence that the antibodies may inhibit the growth and differentiation of trophoblasts, and eventually cause defective placentation so explaining the APS obstetric manifestations. Why antibodies with similar antigen specificity produce different clinical manifestations is not clear. The formation of immune complexes on the membrane of cells with different biological functions may at least in part explain the diverse effects mediated by the autoantibodies (i.e. clotting versus defective placentation). Altogether this finding strongly supports the pivotal role of Beta2GPI as the main tissue target for aPL. Accordingly, new approaches have been tried in order to interfere with the antibody binding and its consequences. A synthetic peptide displaying a similarity in the PL-binding region of the Vth domain of the Beta2GPI was shown to compete with the molecule binding to the membrane of cells involved in the pathogenesis of the syndrome (i.e. endothelial cells, monocytes and trophoblasts). The passive infusion of the peptide was protective against the effect of polyclonal human APS IgG fractions on experimental thrombus formation and fetal loss induction. Moreover, a human monoclonal antibody against the immunodominant epitope of the Beta2GPI (Domain I) was shown to induce fetal loss and to trigger clotting by fixing C' in naive mice. A similar monoclonal antibody lacking the CH2 fragment in the Fc gamma portion was still reacting with the Beta2GPI but no more able to activate C'. The C' non-fixing monoclonal antibody was protective by competing with the pathogenic one when passively infused in naive mice and evaluated for both the induction of thrombus formation and the induction of fetal loss.

Inhibition Of Anti-b2GPI-Dependent Thrombus Enhancement In The Laser-Induced Thrombosis Model In Mice

B2GPI is the major antigen for pathogenic antibodies in antiphospholipid syndrome (APS). The presence of anti-b2GPI antibodies correlates with increased risk for thrombosis and pregnancy loss in patients with APS. In animal models of thrombosis, anti-b2GPI antibodies cause the increase in thrombus size after injury to the vessel wall. We engineered a molecule, A1-A1, that prevents the binding of b2GPI to anionic phospholipids and ApoER2, and studied how it inhibits prothrombotic properties of anti-b2GPI antibodies in vivo using the laser-induced arterial thrombosis model. Lupus-prone (NZWxBXSB)F1 male mice express chronic autoimmune anti-b2GPI antibodies. In (NZWxBXSB)F1 mice, infusion of A1-A1 at a dose of 4 ug/g of body weight caused a decrease in thrombus size of more than 85% as compared to thrombus size in the absence of A1-A1. Similarly, A1-A1 inhibits prothrombotic properties of b2GPI/anti-b2GPI antibody complexes in wild type mice following acute infusion with anti-b2GPI positive APS IgG. The thrombus reduction was due to the binding of A1-A1 to b2GPI, because infusion of LA6, a structural homologue of A1 that does not bind b2GPI, did not affect thrombus size. We did not observe thrombus inhibition by A1-A1 in the absence of anti-b2GPI antibodies, indicating that A1-A1 does not affect normal thrombus formation in the absence of anti-b2GPI antibodies. Disclosures:No relevant conflicts of interest to declare.

AB0218 Plasma heparanase levels are elevated in patients with antiphospholipid syndrome and correlate with platelet activation

BackgroundOverexpression of tissue factor (TF) and activation of the coagulation system are characteristic pathophysiology of antiphospholipid syndrome (APS). Heparanase (HPSE) is an endo-b-D-glucuronidase that cleaves heparan sulfate chains on cell...

In Vitro Effects of Antiphospholipid Syndrome‐IgG Fractions and Human Monoclonal Antiphospholipid IgG Antibody on Human Umbilical Vein Endothelial Cells and Monocytes

It has been shown that stimulation of endothelial cells and monocytes by antiphospholipid antibodies leads to a prothrombotic state involving upregulation of tissue factor (TF). We examined the in vitro effects of IgG fractions from patients with antiphospholipid syndrome (APS) and of a beta-2-glycoprotein 1-independent human monoclonal antiphospholipid antibody (HL-5B) on human umbilical vein endothelial cells (HUVEC) in comparison to untreated cell controls and to exposure to monoclonal IgG control antibody. We also examined the effect of recombinant monocyte chemoattractant protein-1 (MCP-1) on peripheral blood monocytes. Stimulation of endothelial cells with APS IgG fractions or HL-5B resulted in time-dependent upregulation of MCP-1 mRNA and protein expression. Stimulation with HL-5B also led to time-dependent upregulation of interleukin (IL)-8 and intracellular adhesion molecule-1 (ICAM-1) mRNA and IL-8 protein expressions. Stimulation of monocytes with recombinant MCP-1 resulted in an upregulation of TF mRNA and TF protein. In conclusion these results might represent a mechanism for antiphospholipid antibody-mediated thrombosis in APS patients.

The Pathogenesis of Neural Injury in Animal Models of the Antiphospholipid Syndrome

Circulating antiphospholipid antibodies (aPL) are associated with central nervous system dysfunction in antiphospholipid syndrome (APS) patients and in a mouse model of APS. We propose a logical pathway of how experimental APS (eAPS) causes brain dysfunction: binding of the antibodies to the brain endothelium evoking microthrombosis, endothelial dysfunction, and IgG leakage through the blood-brain barrier (BBB), then secondary inflammatory cell spread around blood vessels and production of cytokines by these inflammatory cells leading to further disruption of the BBB. The diffuse brain endothelial dysfunction would result in extravasation of serum proteins including APS IgG and activated thrombin, which may induce the behavioral changes observed in the APS mice. We have collected data from the mouse eAPS model which supports this hypothesis. Elucidating the mechanism of the pathogenicity of aPL in vitro and in vivo will serve as a much needed basis for developing new therapeutic modalities in this important disorder.

In vivo inhibition of antiphospholipid antibody-induced pathogenicity utilizing the antigenic target peptide domain I of beta2-glycoprotein I: proof of concept.

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 beta(2)-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. C57BL/6 mice (n = 5, each group) were injected with purified IgG derived from APS patients (IgG-APS, 500 microg) 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 microg). 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. 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 <or= 0.0001) and DI(D8S/D9G) (P <or= 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 <or= 0.01). DI also inhibited IgG-APS induction of VCAM-1 on the aortic endothelial surface and TF production by murine macrophages. 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.

Hematological presentation in systemic lupus erythematosus and its relationship with disease activity

Abnormalities of hematological system are very common in systemic lupus erythematosus (SLE). The aim of the study is to evaluate hematological findings in patients with SLE at the time of referral and their relationship with disease activity and organ involvement.The study was carried consecutive 115 patients, including 20 males and 85 females. Most of the cases had anemia at the time of presentation due to various etiologies. Anemia of chronic disease was seen in 46% and it was the most common encountered picture. The rate of hemolytic anemia was 28%, and Coombs' positivity rate was 22% among our patients. Leukopenia <4.0 × 109/l), neutropenia <1.8 × 109/l), and lymphopenia <1.5 × 109/l) rates were 57, 20, and 82%, respectively. Thrombocytopenia was seen in 40% for <150 × 109/l, 26% for <100 × 109/l, and 8% for <50 × 109/l. Of our patients, 10% had antiphospholipid syndrome (APS) at the time of diagnosis. Increased fibrinogen levels were observed in 35% without relating to disease activity. The rates of C3 and C4 hypocomplementemia were 86 and 64%, respectively, and both are closely correlated with the disease activity. Moreover, C3 hypocomplementemia was more prominent in cases with renal or serosal involvement. Leukopenia and hyperfibrinogenemia were more common in patients with skin/mucosal involvements. C3 hypocomplementemia, APS and elevated IgG levels were more common in our patients with renal involvement. SLE should be kept in mind while evaluating patients with mentioned hematological findings, especially in those with accompanying organ involvements.

Antiphospholipid antibodies, thrombin and LPS activate brain endothelial cells and Ras-dependent pathways through distinct mechanisms

Background The antiphospholipid syndrome (APS) commonly affects the central nervous system through mechanisms that may include small vessel pathology and activation of thrombin. Antiphospholipid antibodies (aPL) activate endothelial cells but the specific activation of brain vascular endothelial cells (BVEC) and the receptors and signaling pathways involved have not been fully characterized. Objective To examine whether aPL, the inflammatory stimulant lipopolysacharide (LPS) and thrombin activate BVECs through a Ras-dependent pathway. Methods Rat BVEC (G8) were grown to confluence on 24-well plates. IgG was purified from 8 APS patients on a protein G column. Phosphorylation of ERK in the BVEC was measured by immunoblot utilizing a specific antibody. Results Significant phosphorylation of ERK was measured following exposure of the cells to LPS and thrombin and this was blocked by the Ras inhibitor farnesylthiosalicylate (FTS). aPL IgG (1:100 relative to serum) from 7/8 patients also induced phosphorylation of ERK. Conclusions Activation of the Ras-ERK pathway is an effect of both APS IgG and thrombin. This pathway is potentially amenable to drugs such as FTS and may serve as a therapeutic target in APS.