Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-ß2-glycoprotein I (anti-ß2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of ß2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

'Criteria' aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, Texas, April 2010.

Current classification criteria for definite antiphospholipid syndrome (APS) mandate the use of one or more of three positive 'standardized' laboratory assays to detect antiphospholipid antibodies (aPL) (viz: anticardiolipin [aCL] IgG and IgM; anti-ß(2)glycoprotein I [anti-ß(2)GPI] antibodies IgG and IgM; and/or a lupus anticoagulant [LAC]), when at least one of the two major clinical manifestations (thrombosis or pregnancy losses) are present. Although, efforts of standardization for these 'criteria' aPL tests have been conducted over the last 27 years, reports of inconsistencies, inter-assay and inter-laboratory variation in the results of aCL, LAC, and anti-ß(2)GPI, and problems with the interpretation and the clinical value of the tests still exist, which affect the consistency of the diagnosis of APS. A Task Force of scientists and pioneers in the field from different countries, subdivided in three working groups, discussed and analyzed critical questions related to 'criteria' aPL tests in an evidence-based manner, during the 13(th) International Congress on Antiphospholipid Antibodies (APLA 2010, April 13-16, 2010, Galveston, TX). These included: review of the standardization and the need for international consensus protocol for aCL and anti-ß(2)GPI tests; the use of monoclonal and/or polyclonal standards in the calibration curve of those tests; and the need for establishment of international units of measurement for anti-ß(2)GPI tests. The group also reviewed the recently updated guidelines for LAC testing, and analyzed and discussed the possibility of stratification of 'criteria' aPL tests as risk factors for APS, as well as the clinical value of single positive vs. multiple aPL positivity. The group members presented, discussed, analyzed data, updated and re-defined those critical questions at a preconference workshop that was open to congress attendees. This report summarizes the findings, conclusions, and recommendations of this Task Force.

Antiphospholipid Syndrome Clinical Research Task Force report.

The Antiphospholipid Syndrome (APS) Clinical Research Task Force (CRTF) was one of six Task Forces developed by the 13(th) International Congress on Antiphospholipid Antibodies (aPL) organization committee with the purpose of: a) evaluating the limitations of APS clinical research and developing guidelines for researchers to help improve the quality of APS research; and b) prioritizing the ideas for a well-designed multicenter clinical trial and discussing the pragmatics of getting such a trial done. Following a systematic working algorithm, the Task Force identified five major issues that impede APS clinical research and the ability to develop evidence-based recommendations for the management of aPL-positive patients: (1) aPL detection has been based on partially or non-standardized tests, and clinical (and basic) APS research studies have included patients with heterogeneous aPL profiles with different clinical event risks; (2) clinical (and basic) APS research studies have included a heterogeneous group of patients with different aPL-related manifestations (some controversial); (3) thrombosis and/or pregnancy risk stratification and quantification are rarely incorporated in APS clinical research; (4) most APS clinical studies include patients with single positive aPL results and/or low-titer aPL ELISA results; furthermore, study designs are mostly retrospective and not population based, with limited number of prospective and/or controlled population studies; and (5) lack of the understanding the particular mechanisms of aPL-mediated clinical events limits the optimal clinical study design. The Task Force recommended that there is an urgent need for a truly international collaborative approach to design and conduct well-designed prospective large-scale multi-center clinical trials of patients with persistent and clinically significant aPL profiles. An international collaborative meeting to formulate a good research question using 'FINER' (Feasible; Interesting; Novel; Ethical; and Relevant) criteria took place in November 2010.

Risky business: the interpretation, use, and abuse of antiphospholipid antibody tests in clinical practice.

Antiphospholipid antibodies (aPL) are best considered as risk factors. aPL are not diagnostic tests and considering them as such can be misleading and may direct attention away from the more important clinical issue of risk modification and management. When considering aPL as risk factors, quantitative aPL tests such enzyme-linked immunosorbent assay (ELISA) for anticardiolipin (aCL) and anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies, should be interpreted carefully. Risk for clinical manifestations appears to be associated with moderate to high levels of these autoantibodies. Lower levels may be statistically abnormal compared with a control population, but may not be associated with the risk of thrombosis or pregnancy loss. Lupus anticoagulants (LA) are generally thought to be more strongly associated with the risk of clinical manifestation of antiphospholipid syndrome (APS) than aCL and anti-beta(2)GPI antibodies. One reason for the stronger association may be related to patients' antibody titers. LA assays are not very analytically sensitive, i.e. a relatively high concentration of antibodies is required to prolong the clotting time in these tests. Thus, the presence of LA indicates a high titer of aPL and this, rather than the intrinsic functional characteristics of LA antibodies, may explain the high risk of clinical manifestations associated with LA.

Serum complement activation on heterologous platelets is associated with arterial thrombosis in patients with systemic lupus erythematosus and antiphospholipid antibodies.

Complement plays a major role in inflammation and thrombosis associated with systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS). A cross-sectional retrospective analysis was performed to evaluate serum complement fixation on platelets and thrombotic incidence using banked sera and clinical data from patients with SLE (n = 91), SLE with antiphospholipid antibodies (aPL) or APS (n = 78) and primary aPL (n = 57) or APS (n = 96). In-situ complement fixation was measured as C1q and C4d deposition on heterologous platelets using an enzyme-linked immunosorbent assay approach. Platelet activation by patient serum in the fluid phase was assessed via serotonin release assay. Enhanced in-situ complement fixation was associated with the presence of IgG aPL and IgG anti-beta2 glycoprotein 1 antibodies (P < 0.05) and increased platelet activation (P < 0.005). Moreover, enhanced complement fixation, especially C4d deposition on heterologous platelets, was positively associated with arterial thrombotic events in patients with SLE and aPL (P = 0.039). Sera from patients with aPL possess an enhanced capacity for in-situ complement fixation on platelets. This capacity may influence arterial thrombosis risk in patients with SLE.

Tissue factor in the antiphospholipid syndrome.

Antiphospholipid (aPL) antibodies are clinically important acquired risk factors for thrombosis and pregnancy loss and are thought to have a direct prothrombotic effect in vivo. Data suggest that a major mechanism by which aPL antibodies contribute to thrombophilia is the upregulation of tissue factor (TF) (CD142) on blood cells and vascular endothelium. TF is the physiological trigger of normal blood coagulation and thrombosis in many hypercoagulable conditions. This article reviews the physiology of TF, the molecular regulation of TF expression and the effects of aPL antibodies on intravascular TF regulation and expression. Inhibition of TF and the pathways by which aPL antibodies induce TF expression are potentially attractive therapeutic targets in the antiphospholipid syndrome.

Gamma-tocopherol prevents airway eosinophilia and mucous cell hyperplasia in experimentally induced allergic rhinitis and asthma.

BACKGROUND: Traditional therapies for asthma and allergic rhinitis (AR) such as corticosteroids and antihistamines are not without limitations and side effects. The use of complementary and alternative approaches to treat allergic airways disease, including the use of herbal and dietary supplements, is increasing but their efficacy and safety are relatively understudied. Previously, we have demonstrated that gamma-tocopherol (gammaT), the primary form of dietary vitamin E, is more effective than alpha-tocopherol, the primary form found in supplements and tissue, in reducing systemic inflammation induced by non-immunogenic stimuli. OBJECTIVE: We used allergic Brown Norway rats to test the hypothesis that a dietary supplement with gammaT would protect from adverse nasal and pulmonary responses to airway allergen provocation. METHODS: Ovalbumin (OVA)-sensitized Brown Norway rats were treated orally with gammaT before intranasal provocation with OVA. Twenty-four hours after two challenges, histopathological changes in the nose, sinus and pulmonary airways were compared with gene expression and cytokine production in bronchoalveolar lavage fluid and plasma. RESULTS: We found that acute dosing for 4 days with gammaT was sufficient to provide broad protection from inflammatory cell recruitment and epithelial cell alterations induced by allergen challenge. Eosinophil infiltration into airspaces and tissues of the lung, nose, sinus and nasolacrimal duct was blocked in allergic rats treated with gammaT. Pulmonary production of soluble mediators PGE(2), LTB(4) and cysteinyl leukotrienes, and nasal expression of IL-4, -5, -13 and IFN-gamma were also inhibited by gammaT. Mucous cell metaplasia, the increase in the number of goblet cells and amounts of intraepithelial mucus storage, was induced by allergen in both pulmonary and nasal airways and decreased by treatment with gammaT. CONCLUSIONS: Acute treatment with gammaT inhibits important inflammatory pathways that underlie the pathogenesis of both AR and asthma. Supplementation with gammaT may be a novel complementary therapy for allergic airways disease.

Real world experience with antiphospholipid antibody tests: how stable are results over time?

OBJECTIVE: To determine the stability and the degree of variation of antiphospholipid antibody (aPL) results over time in a large cohort of well evaluated aPL positive patients; and to analyse factors contributing to aPL variation and the validity of aPL in a real world setting in which aPL tests are done in multiple laboratories. METHODS: The clinical characteristics, drug treatment, and 1652 data points for lupus anticoagulant (LA), anticardiolipin antibodies (aCL), and anti-beta2 glycoprotein I antibodies (anti-beta2GPI) were examined in 204 aPL positive patients; 81 of these met the Sapporo criteria for antiphospholipid syndrome (APS) and 123 were asymptomatic bearers of aPL. RESULTS: 87% of initially positive LA results, 88% of initially negative to low positive aCL results, 75% of initially moderate to high positive aCL results, 96% of initially negative to low positive anti-beta2GPI results, and 76% of initially moderate to high positive anti-beta2GPI results subsequently remained in the same range regardless of the laboratory performing the test. Aspirin, warfarin, and hydroxychloroquine use did not differ among patients whose aCL titres significantly decreased or increased or remained stable. On same day specimens, the consistency of aCL results among suppliers ranged from 64% to 88% and the correlation ranged from 0.5 to 0.8. Agreement was moderate for aCL IgG and aCL IgM; however, for aCL IgA agreement was marginal. CONCLUSIONS: aPL results remained stable for at least three quarters of subsequent tests, regardless of the laboratory performing the test; the small amount of variation that occurred did not appear to be caused by aspirin, warfarin, or hydroxychloroquine use.

Prophylaxis of the antiphospholipid syndrome: a consensus report.

Hypothetical circumstances that may require prophylaxis for a potential antiphospholipid syndrome (primary prophylaxis), or in some instances when there already had been some manifestations ofthe syndrome (secondary prophylaxis), were presented to a panel of experts for their consideration on potential prophylactic intervention. These were subsequently presented to the participants in the First International Consensus on Treatment of the Antiphospholipid Syndrome. In most instances there was consensus in adding low dose aspirin, an exception being aspirin allergy when other antiaggregants could be used in nonpregnant subjects. General measures to prevent thrombosis and other vasoprotective actions should also be provided. Higher risk of fetal loss or thrombosis called for anticoagulation with coumadin in nonpregnant subjects or subcutaneous low molecular weight heparin in pregnant ones. When indicated, prophylaxis of the antiphospholipid syndrome should be provided in systemic lupus erythematosus patients who are being treated for their disease. In no instance should corticosteroids or immunosuppresants be given as prophylactic of an antiphospholipid syndrome.

Differences by race, sex and age in the clinical and immunologic features of recently diagnosed systemic lupus erythematosus patients in the southeastern United States.

We examined the prevalence of clinical and immunologic features of systemic lupus erythematosus (SLE) by race, sex and age in a population-based study of 265 SLE patients. Patients fulfilled the American College of Rheumatology classification criteria. The median time between diagnosis and study enrollment was 13 months. The clinical and hematologic data were limited to occurrences up to 6 months after the diagnosis date, as documented in medical records. We used sera collected at study enrollment from 244 (92%) patients for serologic testing of autoantibodies. The associations between clinical and immunological features of SLE and age, sex and race were examined using logistic regression. The effect of each of these variables was examined adjusting for the other two demographic factors. Mean age at diagnosis was 6 years younger among African-Americans and other minorities compared with white patients (P < 0.01). Discoid lupus, proteinuria, anti-Sm and anti-RNP autoantibodies were more commonly seen in African-American patients, with odds ratios higher than 3.0. Photosensitivity and mucosal ulcers were noted less often in African-American patients. Proteinuria, leukopenia, lymphopenia and thrombocytopenia were approximately three times more common in men compared with women. The prevalence of oral or nasal ulcers and anti-DNA autoantibodies declined with age. The extent to which the differences we observed reflect genetic or environmental influences on the disease process should be investigated.

Update on antiphospholipid antibodies.

The association of antibodies with an apparent specificity for anionic phospholipids with thrombosis, fetal loss, thrombocytopenia, and certain other clinical manifestations is now well-recognized as the antiphospholipid syndrome (APS). Recent advances in our understanding of the antibodies and antigens involved include discovery of the crystal structure of beta2-glycoprotein I, (beta2GPI), genetic studies of beta2GPI polymorphisms, and the development of anti-beta2GPI and antiprothrombin immunoassays as clinical laboratory tests. The identification of antigen-specific T cells in APS patients has stimulated interest in the role of the cellular immune response in the syndrome. Clinical research in APS will also benefit from the development of preliminary classification criteria.

Tissue factor pathway and the antiphospholipid syndrome.

Expression of tissue factor activity on cells in contact with flowing blood is the trigger for physiological coagulation as well as many types of thrombosis. A number of older observations and considerable recent data suggest that increased tissue factor activity is an important cause of hypercoagulability in the antiphospholipid syndrome. Potential mechanisms contributing to upregulation of the tissue factor pathway include increased expression of tissue factor due to increased transcription, increased functional activity of tissue factor molecules due to de-encryption and decreased activity of tissue factor pathway inhibitor. Autoantibodies and/or immune complexes appear to play a major role in enhanced tissue factor activity, although increased levels of inflammatory cytokines may also contribute. Anti-beta 2-glycoprotein I autoantibodies have been specifically implicated in the antibody-mediated enhancement of tissue factor activity.

Antiphospholipid syndrome: antibodies and antigens.

Elucidation of the antibodies and antigens involved in the antiphospholipid syndrome has provided many new insights and research opportunities. The major autoantibodies associated with the syndrome and detected in clinical laboratory assays for antiphospholipid antibodies are directed against prothrombin and beta2-glycoprotein I beta2GPI), a phospholipid-binding plasma protein whose physiological function is unknown. Recent advances in our understanding of these antibodies and antigens include discovery of the crystal structure of beta2GPI, identification of a plasmin cleavage site in beta2GPI, genetic studies of beta2GPI polymorphisms, development of clinical laboratory assays using purified protein antigens, and the identification of antigen specific T cells.

Links between the immune and coagulation systems: how do "antiphospholipid antibodies" cause thrombosis?

Inflammation and immune activation have been associated with thrombosis in a number of settings. We have been interested in the question of how the presence of a type of autoantibody, so-called "antiphospholipid" antibody, leads to thrombosis. Several mechanisms have been proposed including modulation of tissue factor expression, enhancement of procoagulant binding to platelets, and interference with antithrombotic mechanisms. We developed a cell-based model of coagulation that, unlike current coagulation assays, reflects some of the in vivo activities of "antiphospholipid" antibodies. "Antiphospholipid" antibodies against the phospholipid-binding protein beta-2-glycoprotein-1 enhance thrombin generation in this model system, primarily by enhancing procoagulant reactions on tissue factor-bearing cells.

Characterization of IgG monoclonal anti-cardiolipin/anti-beta2GP1 antibodies from two patients with antiphospholipid syndrome reveals three species of antibodies.

Antiphospholipid antibodies (aPL), including antibodies detected in anti-cardiolipin (aCL) enzyme-linked immunosorbent assays and in lupus anticoagulant (LA) tests, are strongly associated with recurrent thrombosis and recurrent fetal loss, i.e. the antiphospholipid syndrome (APS). Although recent studies suggest that most APS-associated aCL are directed against the phospholipid (PL)-binding plasma protein beta2-glycoprotein 1 (beta2GP1), the precise nature of aCL binding specificities remains controversial. To address the issue of aCL specificity we generated five new monoclonal IgG aCL from two patients with APS. Characterization of these five aCL, as well as two previously published IgG aCL, revealed three patterns of reactivity: (1) four antibodies reacted strongly with human beta2GP1-cardiolipin (CL) complexes and weakly with human beta2GP1 alone; (2) two antibodies recognized bovine beta2GP1, but not human beta2GP1; (3) one antibody reacted with complexes of human beta2GP1 and CL, but not with human beta2GP1 alone. Only one monoclonal displayed weak LA activity. These patient-derived IgG monoclonal antibodies, and additional ones to be generated, may help define varying species of antibodies detected in aCL assays and identify the specific antibodies that may be pathogenic.

Familial antiphospholipid antibody syndrome: criteria for disease and evidence for autosomal dominant inheritance.

OBJECTIVE: To develop diagnostic criteria for a familial form of antiphospholipid antibody syndrome (APS), identify families with >1 affected member, examine possible modes of inheritance, and determine linkage to potential candidate genes. METHODS: Family members of probands with primary APS were analyzed for clinical and laboratory abnormalities associated with APS. Families with > or =2 affected members were analyzed by segregation analysis and typed for candidate genetic markers. RESULTS: Seven families were identified. Thirty of 101 family members met diagnostic criteria for APS. Segregation studies rejected both environmental and autosomal recessive models, and the data were best fit by either a dominant or codominant model. Linkage analysis showed independent segregation of APS and several candidate genes. CONCLUSION: Clinical and laboratory criteria are essential to identify the spectrum of disease associated with APS. We believe a set of criteria was developed that can precisely define affected family members with APS. Modeling studies utilizing these criteria strongly support a genetic basis for disease in families with APS and suggest that a susceptibility gene is inherited in an autosomal dominant pattern. However, in these families, APS was not linked with HLA, Fas, or other candidate genes, including beta2-glycoprotein 1, HLA, T cell receptor beta chain, Ig heavy chain, antithrombin III, Fas ligand, factor V, complement factor H, IgK, and Fas.

Mechanisms of autoantibody-mediated thrombosis.

It is widely hypothesized that autoantibodies directly contribute to the prothrombotic state in the antiphospholipid syndrome (APS). The discovery that antiphospholipid autoantibodies are specific for phospholipid-binding plasma proteins (beta2-glycoprotein I, prothrombin, etc.) has allowed a much more precise investigation of the interactions of autoantibodies and antigens, and the effects of these interaction on hemostasis. Recent studies suggest that two types of interactions may be important in the pathophysiology of APS: (1) antibody cross-linking of membrane bound antigens may alter the kinetics of phospholipid-dependent reactions; and (2) antibody cross-linking of antigens bound to cell surface receptors may trigger signal transduction and cellular activation. In light of these findings, previous reports implicating various mechanisms of autoantibody-mediated thrombosis are being re-evaluated.