Clinical relevance of nitrated beta 2-glycoprotein I in antiphospholipid syndrome: Implications for thrombosis risk.

Β2-Glycoprotein I (ß2GPI) is an important anti-thrombotic protein and is the major auto-antigen in the antiphospholipid syndrome (APS). The clinical relevance of nitrosative stress in post translational modification of ß2GPI was examined.The effects of nitrated (n)ß2GPI on its anti-thrombotic properties and its plasma levels in primary and secondary APS were determined with appropriate clinical control groups. ß2-glycoprotein I was nitrated at tyrosines 218, 275 and 309. ß2-glycoprotein I binds to lipid peroxidation modified products through Domains IV and V. Nitrated ß2GPI loses this binding (p < 0.05) and had diminished activity in inhibiting platelet adhesion to vWF under high shear flow (p < 0.01). Levels of nß2GPI were increased in patients with primary APS compared to patients with either secondary APS (p < 0.05), autoimmune disease without APS (p < 0.05) or non-autoimmune patients with arterial thrombosis (p < 0.01) and healthy individuals (p < 0.05).In conclusion tyrosine nitration of plasma ß2GPI is demonstrated and has important implications with regards to the pathophysiology of platelet mediated thrombosis in APS. Elevated plasma levels of nß2GPI in primary APS may be a risk factor for thrombosis warranting further investigation.

In vivo modulation of angiogenesis by beta 2 glycoprotein I.

Beta 2 glycoprotein I (ß2GPI) is the major auto antigen in the antiphospholipid syndrome but also interacts with fibrinolytic and angiogenic proteins. The aim of this study was to examine the angiogenic potential of ß2GPI in vivo in ß2GPI deficient mice utilizing angiogenic assays. ß2GPI deficient mice show increased microvessel formation in comparison to ß2GPI replete controls when injected with growth factor free-matrigel implants. However, microvessel formation in matrigel plugs of ß2GPI deficient mice was less than in ß2GPI replete mice when basic fibroblast growth factor (bFGF) was included in the matrigel. Hemoglobin content was higher in vascular endothelial growth factor (VEGF) containing-matrigel plugs in the ß2GPI deficient mouse demonstrating that the lack of ß2GPI led to increased extravasation by VEGF. Melanoma B16F10 tumour growth was enhanced in ß2GPI deficient mice. Melanoma microvessel density was increased in ß2GPI deficient mice but the proliferation rate of tumour cells (determined by Ki67 immunohistochemistry) was unaffected by the presence or absence of ß2GPI. Subcutaneous delivery of native human ß2GPI by the ALZET osmotic pump did not affect melanoma tumour growth in ß2GPI deficient mice. We conclude that the in vivo unopposed action of ß2GPI is anti-angiogenic however this function is modified in the presence of a strong angiogenic stimulus into stabilization of vessel formation. Although the presence of ß2GPI attenuates vessel sprouting in certain tumours, no survival benefit is conferred to tumour bearing animals. This does not preclude the potential benefit of modified or fragments of ß2GPI in anti-angiogenesis research.

Quantitation of Total and Free Thiol ß2-Glycoprotein I Levels for Diagnostic and Prognostic Purposes in the Antiphospholipid Syndrome.

ß2-Glycoprotein I is the major autoantigen in the antiphospholipid syndrome (APS), a prothrombotic disorder characterized by the occurrence of either venous or arterial thrombosis. In women it is also associated with an increased risk of obstetric complications such as recurrent miscarriages. We have identified that the plasma protein ß2-glycoprotein I in healthy individuals exists in an optimal ratio between two distinct forms, an oxidized and free thiol, reduced form. This ratio is disrupted in pathophysiological conditions associated with increased oxidative stress such as the APS, but also in the setting of age-related macular degeneration and gram-negative sepsis. We have developed assays that quantify plasma/serum levels of total and free thiol ß2-glycoprotein I which can potentially be used for risk stratification and prognostic purposes in the early stages of the aforementioned conditions.

Quantitation of Total and Free Thiol Angiotensinogen as a Prognostic Marker for Preeclampsia.

Angiotensinogen mediates an important role in the pathophysiology of preeclampsia, a disorder of pregnancy characterized by hypertension and proteinuria usually after 20 weeks of gestation. Angiotensinogen is found in two distinct posttranslational forms in the plasma, an oxidized and a reduced (free thiol) form. Higher levels of the oxidized form are associated with an increased risk of preeclampsia. We have developed novel ELISA assays to quantitate the levels of total and free thiol angiotensinogen allowing for calculation of the amount of oxidized angiotensinogen species. We describe the methodology for performing these assays.

International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).

New clinical, laboratory and experimental insights, since the 1999 publication of the Sapporo preliminary classification criteria for antiphospholipid syndrome (APS), had been addressed at a workshop in Sydney, Australia, before the Eleventh International Congress on antiphospholipid antibodies. In this document, we appraise the existing evidence on clinical and laboratory features of APS addressed during the forum. Based on this, we propose amendments to the Sapporo criteria. We also provide definitions on features of APS that were not included in the updated criteria.

Immunology and clinical importance of antiphospholipid antibodies.

Having reviewed the literature on the association of aPL antibodies with clinical manifestations, it is clear that this group of autoantibodies are of considerable importance. The presence of aPL antibodies in some but not all individuals confers a risk of a clinical syndrome characterized by recurrent arterial or venous thrombosis, thrombocytopenia, hemolytic anemia, or positive Coombs' test, and in females, recurrent idiopathic fetal loss. In SLE, the risk is approximately 40%, compared with a risk of 15% in the absence of aPL antibodies. However, only one half of persons possessing these antibodies have SLE, and overall the risk is around 30%. In some circumstances, such as in chlorpromazine or infection-associated aPL antibodies, there appears to be no increased risk. At the other end of the spectrum are seen patients whose only clinical manifestations comprise features of this clinical syndrome, and this entity has been designated the primary antiphospholipid syndrome (PAPS). aPL antibodies are also important because they are not uncommon. They have been found frequently in women with idiopathic recurrent fetal loss (30%), in non-autoimmune patients with ischemic heart disease (20%), or venous thrombosis (up to 30%), or stroke (4-47%), and in chronic immune thrombocytopenia (30%). These autoantibodies can be detected using sensitive solid-phase immunoassays employing the CL antigen, or in appropriate coagulation tests to detect LA activity. These assays are simple to perform but require care in selection of the best test and in interpretation of results. Current tests do not distinguish between those persons at risk of the clinical events and those not at risk. Detection of specific isotypes (especially IgG) and antibody level may aid in such a designation. Treatment of aPL antibody-associated syndromes remains a controversial subject. Since thromboses are associated with significant morbidity and potential mortality, there is a good argument for long-term preventive antithrombotic therapy, at least for as long as the antibodies are detectable, in those patients in whom clinical complications have previously occurred. It is not generally recommended that this treatment be offered to individuals in whom aPL antibodies are detected but who have not suffered previous thromboses, since the risk of such events does not appear to be equal within a group of aPL antibody-positive persons. This particularly applies to pregnant women, since live births and uncomplicated pregnancies are observed regularly in the presence of aPL antibodies without specific treatment. A previous history of at least one unexplained, late fetal loss is considered a prerequisite before intervention in subsequent pregnancies.(ABSTRACT TRUNCATED AT 400 WORDS)

Current insights into the "antiphospholipid" syndrome: clinical, immunological, and molecular aspects.

Advances in defining the target antigen(s) for the autoantibodies in the APS highlight the inadequacies of the current classification of these autoantibodies into anticardiolipin and LA antibodies. The discovery that beta 2GPI is the target antigen for the autoantibodies detected in solid-phase immunoassays has opened a number of areas of research linking these autoantibodies to atherogenesis and thrombus formation. Although the role of beta 2GPI in the regulation of blood coagulation in unclear, current evidence suggests that anti-beta 2GPI antibodies interfere with its "normal" role and appear to promote a procoagulant tendency. The expansion of research in this area and the diversity of the clinical manifestations of patients with APS have resulted in the inclusion of molecular biologists and pharmaceutical companies joining immunologists, hematologists, rheumatologists, obstetricians, neurologists, vascular surgeons, and protein and lipid biochemists in attempting to understand the pathophysiology of this condition. Although the published literature may result in conflicting results and introduce new controversies, developing standardized laboratory methods and extrapolation of in vitro experimental results to the vivo situation will advance our understanding of the regulation of the immune system and its interaction with normal hemostatic mechanisms. Since the authors' last review in 1991, the study and understanding of the pathophysiology of APS have evolved from lipid biochemistry to molecular techniques that may eventually provide specific therapies for the clinical manifestations of this condition. Although current treatment has improved the morbidity associated with this condition, especially in improving pregnancy outcomes, future therapies, as outlined in this review, may specifically address the biological abnormalities and have fewer side effects. Better diagnostic tools, such as magnetic resonance imaging with perfusion studies, will allow the study of the true incidence and prevalence of vascular flow changes/tissue ischemia and infarction associated with aPL antibodies and help determine treatment and prophylaxis for APS patients. APS is still the only hypercoagulable condition where both arterial and venous beds can be affected independently or in the same individual.

Increased plasma beta-thromboglobulin in patients with coronary artery vein graft occlusion: response to low dose aspirin.

The therapeutic effect of aspirin on vein graft patency was studied in patients undergoing coronary artery bypass graft surgery. The study design enabled the prospective evaluation of the relation of platelet activation, as measured by plasma beta-thromboglobulin concentration, to subsequent coronary vein graft occlusion. Serial beta-thromboglobulin levels were measured in 105 patients randomized to receive aspirin (324 mg/day) or placebo beginning within 1 h after surgery. Graft patency was assessed angiographically at 1 week and 1 year after surgery. Of 49 patients receiving placebo, 17 (34.7%) had one or more graft occlusions, 6 early, 10 late and 1 with both early and late occlusion. Of 56 patients receiving aspirin, 7 (12.5%) had one or more occlusions, 3 early and 4 late (p less than 0.01). Preoperatively, the beta-thromboglobulin level in surgical patients (29 +/- 13.5 ng/ml) was significantly higher than that of 51 control subjects (22.6 +/- 11.1 ng/ml) (p less than 0.004). Plasma beta-thromboglobulin levels remained comparatively constant at 3 and 12 months after surgery in the 43 patients who had both samples available (p less than 0.001, r = 0.65). The reduction in beta-thromboglobulin concentration from the preoperative level to 12 months postoperatively was greater in the aspirin-treated group (p less than 0.001). Multivariate logistic regression analysis demonstrated a significant association between preoperative beta-thromboglobulin concentration and graft occlusion (p less than 0.02), and aspirin treatment was effective in preventing occlusion when adjusted for the preoperative beta-thromboglobulin level (p less than 0.005). Plasma beta-thromboglobulin concentrations are elevated in patients with coronary artery disease, suggesting ongoing platelet activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of lupus anticoagulant (LA) antibodies: LA activity in dilute Russell's Viper Venom Time and dilute Kaolin Clotting Time detect different populations of antibodies in patients with the "antiphospholipid" syndrome.

"Antiphospholipid" (aPL) antibodies comprise two main groups of antibodies, lupus anticoagulant (LA) antibodies and "anticardiolipin" (aCL) antibodies which can be separated by certain chromatographic techniques. In this study we analysed the plasma of 10 patients with aPL antibodies, and were able to demonstrate that in four patients with both clotting test reactivities, the dilute Russell's Viper Venom Time (dRVVT) activity can be separated from the dilute Kaolin Clotting Time (dKCT) reactivity by using a polyacrylamide-immobilised phosphatidylserine column but not with phospholipid liposomes. The differential reactivity of the autoantibodies in this patient population is not due to binding to beta2GPI, prothrombin or protein C in solid-phase immunoassays. Hence LA antibodies detected in different phospholipid-dependent clotting tests detect different populations of antibodies in some APS patients and the routine detection of LA antibodies should be performed with at least two clotting tests looking at different coagulation reactions.

Distinguishing features of anti-beta2 glycoprotein I antibodies between patients with leprosy and the antiphospholipid syndrome.

Anticardiolipin (ACA), anti-beta2 glycoprotein I (beta2GPI), and antiprothrombin antibodies of IgG and IgM classes were quantitated by enzyme-linked immunosorbent assays in 176 untreated leprosy patients across the histopathological spectrum. Positivity rates ranged from 21% (IgG ACA) to 30% (IgM anti-prothrombin) versus 4% in healthy controls (p <10(-2) to 10(-3)). Levels of IgM anti-beta2GPI and IgG ACA were significantly higher in lepromatous leprosy and multibacillary patient subgroups. IgG3 was the most common subclass reactive to both beta2GPI and prothrombin in selected high-titer leprosy sera, unlike antibodies from patients with the antiphospholipid syndrome (APS) largely restricted to IgG2. In leprosy patients, but not in the APS control group, there was no statistical correlation between ACA and anti-beta2GPI antibody levels. Likewise, a large fraction of anti-beta2GPI positive sera (36/45 and 28/44 for IgG and IgM, respectively) were unreactive in the standard ACA assay. Most assayed anti-beta2GPI antibodies from leprosy patients showed (i) ability to recognize both human and bovine beta2GPI immobilized on non-irradiated polystyrene plates, (ii) concentration-dependent inhibition of binding by cardiolipin, and (iii) relatively high avidity binding to fluid-phase beta2GPI, thereby differing from those found in APS. Finally, the location of the major epitopic region on the beta2GPI molecule targeted by autoantibodies was different in leprosy and APS, as assessed by direct binding to domain I- and V-deleted mutants and competition with the mouse monoclonal antibody 8C3, directed at domain I. Thus, leprosy-related antiphospholipid antibodies comprise persistent IgG and IgM anti-beta2GPI that differ from APS-related ones with respect to IgG subclass, avidity and epitope specificity, possibly reflecting distinct pathophysiological significance.

Antibodies to phosphatidylethanolamine in antiphospholipid syndrome and systemic lupus erythematosus: their correlation with anticardiolipin antibodies and beta 2 glycoprotein-I plasma levels.

The sera patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) were tested, by ELISA, for antibodies to phosphatidylethanolamine (aPE), as well as to cardiolipin (aCL) and compared to healthy blood donors (HBD). Both, SLE and APS patients presented a higher titre of IgM-aPE antibodies than normals, while the IgG and IgA aPE reactivity did not differ. APS patients were characterized by higher IgM-aPE antibody titres than SLE patients. In contrast, the predominant isotype of aCL antibodies in APS patients was IgG. The IgM aPE reactivity was correlated with IgM aCL reactivity, while no correlation was observed between the total IgM values and IgM-aPE binding units of sera tested. Since it was shown that beta 2-glycoprotein-I (beta 2-GPI) contributes to a complex antigen by binding to phospholipids and that this antigen is recognized by antiphospholipid antibodies from autoimmune patients, sera beta 2-GPI levels were measured and correlated to aCL and APE activity. Although APS patients had higher beta 2-GPI levels than SLE patients, no correlation was found between the beta 2-GPI levels and IgG/IgM aCL and IgM-aPE reactivities a finding suggesting that in addition to beta 2-GPI, other cofactors for aPE antibodies may exist. These findings indicate that aPE and aCL antibodies co-exist and that the IgM-isotype is predominant in APS. In addition, the IgA and IgG aPE antibodies appear to occur in low titres in these patients, as well as in normals and may exist as natural autoantibodies. We suggest that the high IgM-aPE antibodies may be viewed as a thymus independent process.

Binding specificity of lupus anticoagulants and anticardiolipin antibodies.

Antiphospholipid antibodies have been found to be strongly associated with syndromes characterised by spontaneous arterial and venous thromboses, recurrent miscarriage, immune thrombocytopenia, and occasionally neurological manifestations. These antibodies can be detected using solid phase immunoassays, and by their effect on prolonging phospholipid dependent clotting tests. This latter phenomenon is termed the lupus anticoagulant (LA). The relationship between anticardiolipin antibodies (ACA) and the LA activity of plasma was investigated in 14 patients. Plasma of these patients exhibited both LA activity and high levels of ACA. The patients included 7 with systemic lupus erythematosus, 6 without and 1 chlorpromazine induced lupus anticoagulant. 7 patients had a history of thrombosis and 7 did not, despite high antibody levels. Plasma was incubated in a serial fashion with solid phase cardiolipin and the residual ACA level and LA activity were monitored using a solid phase enzyme linked immunoassay, and the kaolin clotting time (KCT) and activated partial thromboplastin time (APTT) respectively. There was no correlation between baseline ACA levels and parameters of LA activity (dKCT or dAPTT) in contrast to previous reports. However, there was a concurrent reduction in both LA and ACA levels over 24 hours during incubation with cardiolipin in all patients. The rate of reduction of both parameters was highly correlated (r = 0.99. p less than 0.001). The relative reduction of LA activity versus ACA level varied between patients, and may represent different affinities for phospholipid in thromboplastin versus phospholipid in solid phase. Thus, despite the lack of concordance between LA and ACA in many patients, the two activities can be removed concurrently in vitro, suggesting similar binding specificities of the antibodies. The incomplete concordance could be explained by varying affinities for different structural presentations of the lipid antigen.

New basic aspects of the antiphospholipid syndrome.

This article reviews recent changes in our understanding of the basic aspects of the Antiphospholipid syndrome (APS) with special emphasis on interactions with phospholipid binding proteins. We survey the history of the development of tests for the APS, and discuss current methods of detection of antiphospholipid antibodies, clinical events associated with APS, and new concepts regarding the immunological specificity of the antibodies.

Association of mast cells with fibrosis and fatty infiltration in the minor salivary glands of patients with Sjögren's syndrome.

OBJECTIVE: To determine the distribution and density of mast cells in the minor salivary glands of patients with primary Sjögren's syndrome (pSS) and of normal controls. METHODS: Minor salivary gland biopsies were obtained from 19 patients with pSS, 9 with systemic lupus erythematosus, one each with rheumatoid arthritis, sarcoidosis, and Hodgkin's disease, and from 10 individuals who had subjective xerostomia with normal salivary gland biopsies. Biopsy specimens were evaluated for the degree of inflammation according to Tarpley's classification. Sections were analysed for staining with Toluidine blue and with the mast cell specific marker c-kit. The data obtained were correlated with the histological findings of fatty infiltration, fibrosis and lymphocytic infiltration. RESULTS: There was a significant correlation between the number of mast cells identified and the degrees of fibrosis and fatty infiltrates. There was no correlation between the intensity of lymphoid infiltration and the number of mast cells. c-kit staining showed a high correlation when compared to Toluidine blue staining. CONCLUSION: Mast cells in the minor salivary glands of patients with pSS are strongly associated with fibrosis and cell acid infiltration. However, there is no correlation with parameters of disease activity such as lymphoid infiltration.

Sneddon's syndrome, anti-cardiolipin antibody and glomerular thrombosis.

Sneddon's syndrome, cerebrovascular thrombosis and livedo reticularis, is often a variant of the "primary" anti-phospholipid syndrome (PAPS). We report a woman with PAPS, presenting as Sneddon's syndrome, with renal impairment and glomerular thrombosis on renal biopsy. An IgG anti-cardiolipin antibody (aCL) was identified. The aCL was purified by affinity chromatography, gel filtration chromatography and ion-exchange chromatography, assayed in a modified ELISA and found to be of the type that requires the plasma protein beta 2-GPI to bind aCL. As beta 2-GPI has anticoagulant properties it is postulated that its interaction with aCL has a pathogenic role in the thrombotic lesions associated with aCL.

Molecular pathophysiology of the antiphospholipid syndrome: the role of oxidative post-translational modification of beta 2 glycoprotein I.

It has been well established that antiphospholipid antibodies and specifically those directed against beta 2 glycoprotein I (ß2GPI) are pathogenic for the development of thrombosis in the antiphospholipid syndrome (APS). Several groups have shown that anti-ß2GPI antibodies, in complex with ß2GPI, elicit effects on blood cells and coagulation-fibrinolysis proteins, which prime the arterial and venous vasculature for the development of thrombosis. However, much less is known about the mechanism initiating the production of autoantibodies against ß2GPI, a physiological abundant protein of blood. In the current review, novel findings are presented regarding the structure and oxidative post-translational modifications of ß2GPI, which trigger the immune response. The majority of circulating ß2GPI exists in a form containing unpaired cysteines (free thiols), which constitutes the reduced form of ß2GPI. The free thiols exposed on ß2GPI are involved in the interaction with platelets and endothelial cells. We propose that this abundant pool of free thiols may serve as an antioxidant reservoir protecting cells or critical molecules from oxidative stress. Oxidation of ß2GPI confers an increase in its immunogenicity through a Th1 immunological mechanism. The clinical significance of these observations is that serum from patients with APS, assessed by a novel ELISA assay, have a significant increase in oxidised ß2GPI. These findings hold promise, not only for the delineation of the role of ß2GPI as an immunological target, but also for the development of improved diagnostic and prognostic assays for APS.

Risk stratification and outcome of cellulitis admitted to hospital.

OBJECTIVES: To identify risk factors associated with mortality and adverse outcome of community acquired cellulitis/erysipelas requiring hospital admission. METHODS: A retrospective analysis of 395 episodes of cellulitis/erysipelas admitted to a tertiary referral hospital between January 1999 and December 2006. RESULTS: Mortality was 2.5% (10/395). There were 112 complications (28.4%). Median hospitalisation was 5 days. Factors independently associated with mortality, adverse outcome and prolonged stay (>7 days) were bacteraemia and albumin <30 g/L. A risk stratification model was designed based on factors independently associated with adverse outcome: altered mental status, neutrophilia/paenia, discharge from the cellulitic area, hypoalbuminaemia and history of congestive cardiac failure. Adverse outcome risk among patients with scores <4, 6-9 and >9 was <20%, 55% and 100%, respectively. All patients who died had admission score >or=4. Factors independently associated with prolonged hospitalisation were: age >60, symptom duration >4 days, hypoalbuminaemia, bacteraemia, isolation of MRSA and time to effective antibiotics >8 h. MRSA was more frequent among patients admitted during 2003-2006 (OR 2.43, 95% CI: 1-12-5.27). Streptococci accounted for most bacteraemia (11/20). Infectious Disease physician input was independently associated with shorter hospitalisation. CONCLUSIONS: Cellulitis/erysipelas requiring hospitalisation confers considerable morbidity and mortality. Clinical markers present on admission can be used to stratify patient risk of mortality and adverse outcome.

Redox control of ß2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1.

BACKGROUND: ß(2) -Glycoprotein I (ß(2) GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of ß(2) GPI in thrombus formation is unknown. We have recently shown that ß(2) GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin-1 and protein disulfide isomerase, and that reduction of ß(2) GPI can take place on the platelet surface. METHODS: ß(2) GPI, reduced by thioredoxin-1, was labeled with the selective sulfhydryl probe N(a)-(3-maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced ß(2) GPI for von Willebrand factor (VWF) and the effect of reduced ß2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin-activated VWF was studied in the presence of reduced ß(2) GPI. RESULTS: We demonstrate that the Cys288-Cys326 disulfide in domain V of ß(2) GPI is the predominant disulfide reduced by thioredoxin-1. Reduced ß(2) GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. ß(2) GPI reduced by thioredoxin-1, in comparison with non-reduced ß(2) GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. CONCLUSIONS: Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol-dependent interaction of ß(2) GPI with VWF may contribute to the redox regulation of platelet adhesion.