Evaluation of phosphatidylserine-dependent antiprothrombin antibody testing for the diagnosis of antiphospholipid syndrome: results of an international multicentre study.

Objective A task force of scientists at the International Congress on Antiphospholipid Antibodies recognized that phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) might contribute to a better identification of antiphospholipid syndrome (APS). Accordingly, initial and replication retrospective, cross-sectional multicentre studies were conducted to ascertain the value of aPS/PT for APS diagnosis. Methods In the initial study (eight centres, seven countries), clinical/laboratory data were retrospectively collected. Serum/plasma samples were tested for IgG aPS/PT at Inova Diagnostics (Inova) using two ELISA kits. A replication study (five centres, five countries) was carried out afterwards. Results In the initial study ( n = 247), a moderate agreement between the IgG aPS/PT Inova and MBL ELISA kits was observed ( k = 0.598). IgG aPS/PT were more prevalent in APS patients (51%) than in those without (9%), OR 10.8, 95% CI (4.0-29.3), p < 0.0001. Sensitivity, specificity, positive (LR+) and negative (LR-) likelihood ratio of IgG aPS/PT for APS diagnosis were 51%, 91%, 5.9 and 0.5, respectively. In the replication study ( n = 214), a moderate/substantial agreement between the IgG aPS/PT results obtained with both ELISA kits was observed ( k = 0.630). IgG aPS/PT were more prevalent in APS patients (47%) than in those without (12%), OR 6.4, 95% CI (2.6-16), p < 0.0001. Sensitivity, specificity, LR + and LR- for APS diagnosis were 47%, 88%, 3.9 and 0.6, respectively. Conclusions IgG aPS/PT detection is an easily performed laboratory parameter that might contribute to a better and more complete identification of patients with APS.

Immunoreactivity and avidity of IgG anti-ß2-glycoprotein I antibodies from patients with autoimmune diseases to different peptide clusters of ß2-glycoprotein I.

The pathogenicity of antibodies against ß2-glycoprotein I (anti-ß2GPI) depends on multiple factors such as subclass type, epitope binding and avidity. Due to their large heterogeneity, their impact on antiphospholipid syndrome (APS) onset is still not fully clarified. We studied the binding characteristics of IgG anti-ß2GPI with known avidity from sera of 201 autoimmune patients (87 with APS, 67 with APS associated with systemic lupus erythematosus (SLE), 47 with only SLE) to six ß2GPI peptides corresponding to amino acid clusters on domains I-II, II, III and III-IV by indirect ELISA and evaluated their association with clinical features of APS. Peptides A (LKTPRV; domain I-II), B (KDKATF; domain IV) and C (TLRVYK; domain III) were derived from a hexapeptide phage display library previously shown to react with pathogenic monoclonal anti-ß2GPI. Peptides D (NGPANSK; domain III), E (YNPLWFV; domain II) and F (KMDGNHP; domain III-IV) represent surface amino acid clusters on ß2GPI. The percentage of patients positive for peptides were observed as follows: 30.3% for peptide D, 28.90% for B, 25.9% for C, 24.9% for E, 24.4% for F and 10.0% for A. The anti-peptide antibodies in studied serum samples were predominantly of heterogeneous avidity, followed by law avidity anti-peptide antibodies, whereas only a few were of high avidity. Positive and negative correlations were found between several anti-peptide antibodies and the rate of thrombosis. Our results indicated diverse reactivity of IgG anti-ß2GPI to different epitopes on ß2GPI. Classification of IgG anti-ß2GPI into subgroups regarding epitope specificity and avidity could represent an additional tool in understanding their pathogenicity in APS.

Summary of the 9th meeting of the European Forum on Antiphospholipid Antibodies.

The 9th meeting of the European Forum on Antiphospholipid Antibodies (Euro aPL Forum) was held in Krakow, Poland, on 16-18 May 2013. This was an excellent occasion for the exchange of information on current research in the area of antiphospholipid syndrome (APS), as well as a starting point for many new research projects. About 120 physicians and researchers from various medical specialities representing 15 European countries, USA, Argentina and Israel attended the event. This report summarizes the major studies and new research projects presented during the Forum.

Avidity of anti-ß2-glycoprotein I antibodies in patients with antiphospholipid syndrome.

Antibodies against ß(2)-glycoprotein I (anti-ß(2)GPI) are one of the hallmarks of the antiphospholipid syndrome (APS). However, they are heterogenic regarding their epitope specificity, pathogenic mechanisms and their avidity. In the current study we present some outstanding issues about avidity of anti-ß(2)GPI antibodies. Our results confirmed that high avidity anti-ß(2)GPI are associated with thrombosis and APS, while in low avidity anti-ß(2)GPI group non-APS (predominantly systemic lupus erythematosus) patients prevailed.

The avidity of anti-beta2-glycoprotein I antibodies in patients with or without antiphospholipid syndrome: a collaborative study in the frame of the European forum on antiphospholipid antibodies.

OBJECTIVE: The objective of this study was to extend the findings of the preliminary study by measuring the avidity of IgG anti-ß2-glycoprotein I antibodies (anti-ß2-GPI) on a larger group of patients with primary or secondary antiphospholipid syndrome (APS) and anti-ß2-GPI positive patients without APS in the frame of the European Forum on antiphospholipid antibodies (aPL). METHODS: Serum from 137 patients with primary APS, APS associated with autoimmune diseases, and patients with autoimmune diseases other than APS from five EU rheumatology centres were tested for anti-ß2-GPI antibodies. The 109 patients who were sera positive for anti-ß2-GPI by the in-house anti-ß2-GPI enzyme-linked immunosorbent assay (ELISA) at the Immunology Laboratory, UMC Ljubljana were selected for further testing on avidity with chaotropic anti-ß2-GPI ELISA. RESULTS: High, low and heterogeneous avidity IgG anti-ß2-GPI was found in 32/109, 17/109 and 60/109 patients respectively. Significantly more patients with APS were in the high avidity than in the low avidity anti-ß2-GPI group, while the opposite was observed for non-APS (both p < 0.001). The most common clinical feature among patients with high avidity anti-ß2-GPI was thrombosis, mainly due to venous thrombosis (p < 0.01 and p < 0.001, versus low avidity anti-ß2-GPI group). CONCLUSION: Patients with or without APS had anti-ß2-GPI of high, low or heterogeneous avidity. High avidity anti-ß2-GPI was associated with thrombosis and APS, while in the low avidity anti-ß2-GPI group non-APS (predominantly SLE) patients prevailed. Determination of anti-ß2-GPI avidity should be considered in the analytical strategies for further differentiation of patients with anti-ß2-GPI antibodies.

Lupus anticoagulant: performance of the tests as recommended by the latest ISTH guidelines.

OBJECTIVES: Lupus anticoagulant (LA) is clinically the most relevant among all antiphospholipid antibody tests. Recently, new guidelines for LA detection were published. The objective of this retrospective cohort study was to compare tests recommended under these guidelines with other methods used for LA detection. METHODS: The study group consisted of 336 subjects suffering from various autoimmune diseases. We used activated partial thromboplastin time (aPTT), diluted Russell viper venom time (dRVVT) and diluted prothrombin time (dPT) tests for LA detection together with a ratio between sensitive and insensitive aPTT reagent. We also tested if LA was dependent on ß(2) glycoprotein I (ß(2) GPI) using one of the recently described methods. RESULTS: All LA tests performed were associated with a history of thrombosis. The highest odds ratio (OR) for thrombosis was found for ß(2) GPI-dependent LA but sensitivity was low (OR = 8.4; specificity/sensitivity = 98%/15%). All LA tests showed a much stronger association with thrombosis than with pregnancy failure. CONCLUSIONS: LA tested by aPTT and/or dRVVT (at least one out of two tests positive), as recommended by the guidelines, was associated less strongly with a history of thrombosis (OR = 4.1) than either of these tests separately (OR = 5.0 and 4.3, respectively). With both tests positive ('double LA positivity') the association with thrombosis was stronger (OR = 6.5) compared with only one positive test. In fact, 'double LA positivity', detected by combinations of any of the tests studied, was markedly associated with a history of thrombosis.

An international multicentre-laboratory evaluation of a new assay to detect specifically lupus anticoagulants dependent on the presence of anti-beta2-glycoprotein autoantibodies.

BACKGROUND: Antiphospholipid syndrome (APS) is diagnosed by the simultaneous presence of vascular thrombosis and/or pregnancy morbidity and detection of antiphospholipid antibodies in plasma. OBJECTIVES: We have shown that prolongation of clotting time by anti-beta2-glycoprotein I (beta2GPI) antibodies correlates better with thrombosis than a positive classic lupus anticoagulant (LAC) assay in a single center study. To confirm or falsify this finding we have conducted a multicenter study. METHODS AND RESULTS: In 325 LAC-positive samples, we found that the beta2GPI-dependent LAC correlated 2.0 times better with thrombosis than the classic LAC assay. Although significant, this was a minimal improvement compared with the 'classic' LAC. It was published that calcium influences the behavior of anti-beta2GPI antibodies in coagulation assays. To investigate whether calcium plays a role in the present study, we divided the patient population into two groups: (i) blood was collected in 0.109 m sodium citrate and (ii) blood was drawn in 0.129 m sodium citrate as anticoagulant. We found that a positive result with the beta2GPI-dependent LAC assay correlated better with thrombosis [odds ratio (OR): 3.3, 95% confidence interval (CI) 1.9-5.8] when 0.109 m sodium citrate was used compared with 0.129 m sodium citrate (OR: 0.4, 95% CI 0.1-1.1). CONCLUSION: We were able to confirm in an international multicenter study that a positive result in a beta2GPI-dependent LAC assay correlates better with thrombosis than the classic LAC assay, but that the assay needs further study as it is sensitive to external factors such as the sodium citrate concentration used as anticoagulant in the test sample.

Revised classification criteria for antiphospholipid syndrome and the thrombotic risk in patients with autoimmune diseases.

BACKGROUND: The classification criteria for antiphospholipid syndrome (APS) were updated in 2006. OBJECTIVE: The aim of the study was to analyze associations between clinical complications and laboratory test abnormalities typical for APS in a group of patients with autoimmune diseases, based on the recently updated criteria. PATIENTS/METHODS: Three hundred and thirty-six patients were enrolled into the study, with the majority (n = 235) suffering from systemic lupus erythematosus. Laboratory determinations included: lupus anticoagulant (LA), anticardiolipin (aCL) and anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies (ABs) [of both immunoglobulin G (IgG) and IgM class]. RESULTS: A significant association was found between laboratory and clinical features of APS; odds ratios (ORs) for thrombosis associated with the presence of LA, aCL, and anti-beta(2)GPI Abs were 4.04 [95% CI: 2.44-6.68], 3.71 (95% CI 2.32-5.92) and 2.57 (95% CI 1.60-4.1), respectively. Detailed analysis showed marked differences between the risk of clinical complications associated with the presence of an antibody in the IgG class (OR 4.15, 95% CI 2.42-7.12, and OR 4.77, 95% CI 2.37-9.61 for aCL and anti-beta(2)GPI, respectively) and in the IgM class (OR 2.2, 95% CI 1.31-3.70, and OR 1.9, 95% CI 1.15-3.14 for aCL and anti-beta(2)GPI, respectively). The postulated inclusion of anti-beta(2)GPI antibody positivity into the previous laboratory criteria changed only slightly the number of patients diagnosed with APS (from 112 to 117). CONCLUSIONS: The updated APS classification criteria clearly represent a step forward. However, our results argue against the use of overall positivity for aCL or anti-beta(2)GPI, and favor a clear distinction between the IgG and IgM classes of antiphospholipid ABs. Patients with both LA and anti-beta(2)GPI IgG or LA and aCL IgG positivity may represent the subgroups at the highest risk of thrombotic complications.

Valine/Leucine247 polymorphism of beta2-glycoprotein I in patients with antiphospholipid syndrome: lack of association with anti-beta2-glycoprotein I antibodies.

In antiphospholipid syndrome (APS) the presence of anti-beta2-glycoprotein I (beta2GPI) antibodies is strongly associated with thromboembolic complications. It has been suggested that the common beta2GPI Valine/Leucine247 (Val/Leu247) polymorphism could be found more commonly in APS and might influence the generation of anti-beta2GPI antibodies. Therefore we studied beta2GPI Val/Leu247 single-nucleotide polymorphism (SNP) by PCR in 338 patients with various autoimmune diseases (46 with secondary and 84 with primary APS) and 147 sex and age-matched healthy controls. In all patients lupus anticoagulant, anticardiolipin and anti-beta2GPI antibodies (both IgG and IgM) were also determined. All patients and controls were Caucasians. Frequencies of the SNP genotypes in patients did not depart from genetic equilibrum and did not differ from those found in controls. There was also no association between the presence of beta2GPI Val/Leu247genotypes and the presence or absence of lupus anticoagulant, anticardiolipin antibodies, anti-beta2GPI antibodies or clinical APS symptoms in all patients studied. In conclusion, among the exclusively Caucasian, Polish population of autoimmune patients beta2GPI Val/Leu247SNP has the same distribution as in healthy subjects and does not influence the production of anti-beta2GPI antibodies.

Hereditary hemorrhagic telangiectasia, factor V Leiden and antiphospholipid syndrome: a case report.

We report on a 57-year-old woman with three episodes of ischemic strokes and hereditary hemorrhagic telangiectasia (HHT). Tests for inherited and acquired thrombophilia showed elevated anticardiolipin immunoglobulin (Ig)M antibodies (on three separate occasions), anti-prothrombin IgG antibodies, and the heterozygous form of factor V Leiden. This is the first case of HHT, a primary antiphospholipid syndrome, combined with factor V Leiden. No detectable arteriovenous malformation was found and ischemic episodes, documented by computer tomography, were related to the presence of antiphospholipid antibodies and possibly the carriership of factor V Leiden mutation. Since aspirin provoked severe nasal hemorrhages, treatment with ticlopidine was initiated after the third stroke. Over an 18-month follow-up, ischemic episodes were absent and we regarded oral anticoagulation as unjustifiable.

Antithrombotic actions of statins.

Aspirin depresses thrombin generation, probably through a mechanism independent of the cyclooxygenase inhibition, but rather related to acetylation of the platelet membrane macromolecules. This action of aspirin is blunted in hypercholesterolemia. In men with marked hypercholesterolemia, lowering serum cholesterol by a three-month simvastatin treatment is accompanied by a reduction of thrombin generation both at basal conditions in venous blood and after activation of hemostasis by microvascular injury. Similar results are obtained in patients with coronary heart disease and borderline - high cholesterol levels. We assessed tissue-factor initiated coagulation in blood samples collected every 30-seconds from bleeding time wounds in patients with advanced coronary artery disease and total cholesterol levels of 224 mg/dL. Three-month simvastatin treatment depressed blood clotting, leading to reduced rates of prothrombin activation, FVa generation, fibrinogen cleavage, FXIII activation, and an increased rate of FVa inactivation. Such a concerted influence of statins on the clotting cascade seems to be independent of their lipid-lowering action and may be the result of depressed isoprenoid production.

Inhibition of thrombin generation by simvastatin and lack of additive effects of aspirin in patients with marked hypercholesterolemia.

OBJECTIVES: To assess the effects of aspirin compared with simvastatin on thrombin generation in hypercholesterolemic men, and to establish whether the reduction of elevated blood cholesterol by simvastatin would affect the action of aspirin on thrombin formation. BACKGROUND: Aspirin inhibits thrombin formation, but its performance is blunted in hypercholesterolemia. By virtue of altering lipid profile, statins could be expected to influence thrombin generation. METHODS: Thirty-three men, aged 34 to 61 years, with minimal or no clinical symptoms, serum total cholesterol >6.5 mmol/liter and serum triglycerides <4.6 mmol/liter, completed the study consisting of three treatment phases. First, they received 300 mg of aspirin daily for two weeks (phase I), which was then replaced by simvastatin at the average dose of 24 mg/d for three months (phase II). In phase III, aspirin, 300 mg/day, was added for two weeks to simvastatin, the dose of which remained unchanged. Thrombin generation was assessed: 1) in vivo, by measuring levels of fibrinopeptide A (FPA) and prothrombin fragment 1+2 (F1+2) in venous blood; and 2) ex vivo, by monitoring the rates of increase of FPA and F1+2 in blood emerging from standardized skin incisions of a forearm. A mathematical model was used to describe the kinetics of thrombin formation at the site of microvascular injury. RESULTS: Two-week treatment with aspirin had no effect on thrombin markers in vivo, while ex vivo it depressed the total amount of thrombin formed, though not the reaction rate. After simvastatin treatment, serum cholesterol decreased by 31% and LDL cholesterol by 42%, while thrombin generation became markedly depressed. In venous blood, FPA was significantly reduced. Concomitantly, the initial thrombin concentration and total amount of thrombin generated decreased significantly. Addition of aspirin to simvastatin (phase III) had no further effect on any of these parameters. CONCLUSIONS: In men with hypercholesterolemia, lowering serum cholesterol level by a three-month simvastatin treatment is accompanied by a marked reduction of thrombin generation both at basal conditions in venous blood and after activation of hemostasis by microvascular injury. Once blood cholesterol became reduced, adding aspirin to simvastatin did not enhance dampening of thrombin formation.

[Three episodes of acute multiorgan failure in a woman with secondary antiphospholipid syndrome]. / Trzy epizody ostrej niewydolnosci wielonarzadowej u chorej z wtórnym zespolem antyfosfolipidowym.

We report the case of a 43-year-old woman with systemic lupus erythematosus who survived three episodes of catastrophic antiphospholipid syndrome. During the first episode symptoms involved predominantly the central nervous system, whilst during the second episode of multiorgan failure, the cardiovascular system, lungs and kidneys were particularly affected. Twenty months later, the patient experienced an acute exacerbation of chronic renal failure and later, died of massive pulmonary embolism. The characteristic findings of antiphospholipid syndrome included persistently high titers of IgG anticardiolipin antibodies, positive lupus anticoagulant, and microcytic anaemia with a distinct haemolytic component.

Thrombin generation measured ex vivo following microvascular injury is increased in SLE patients with antiphospholipid-protein antibodies.

Antiphospholipid-protein antibodies (APA) include lupus-type anticoagulant (LA) and antibodies recognizing complexes of anionic phospholipids (e.g. cardiolipin) and proteins (e.g. prothrombin and beta2-glycoprotein I). The presence of APA is associated with an increased risk of both arterial and venous thrombosis. However, the pathogenic mechanism leading to thrombosis in patients with APA remains unclear. We studied 32 patients with systemic lupus erythematosus (SLE) who were divided into two groups depending on the presence (n = 19) or absence (n = 13) of APA. Healthy volunteers (n = 12) matched by age and sex served as controls. In all subjects LA and IgG class anticardiolipin antibodies (ACA) were determined. Thrombin generation was monitored ex vivo measuring fibrinopeptide A (FPA) and prothrombin fragment F1 + 2 (F1 + 2) in blood emerging from a skin microvasculature injury, collected at 30 second intervals. In subjects with antiphospholipid antibodies mean FPA and F1 + 2 concentrations were significantly higher at most blood sampling times than in controls. In some SLE patients with APA the process of thrombin generation was clearly disturbed and very high concentrations of fibrinopeptide A were detected already in the first samples collected. Two minutes after skin incision SLE patients without APA produced slightly more FPA, but not F1 + 2, as compared to healthy subjects. Mathematical model applied to analyze the thrombin generation kinetics revealed that APA patients generated significantly greater amounts of thrombin than healthy controls (p = 0.02 for either marker). In contrast, in the same patients generation of thrombin in recalcified plasma in vitro was delayed pointing to the role of endothelium in the phenomenon studied. In summary, these data show for the first time that in SLE patients with antiphospholipid-protein antibodies thrombin generation after small blood vessel injury is markedly increased. Enhanced thrombin generation might explain thrombotic tendency observed in these patients.

Anticardiolipin, anti-beta(2)-glycoprotein I, antiprothrombin antibodies, and lupus anticoagulant in patients with systemic lupus erythematosus with a history of thrombosis.

OBJECTIVE: To determine the clinical importance of anticardiolipin (aCL), anti-beta(2)-glycoprotein I (a beta(2)-GPI), antiprothrombin (aPT), and lupus anticoagulant (LAC) antibodies in patients with systemic lupus erythematosis (SLE) with and without a history of thrombosis. METHODS: We studied 100 patients with SLE (32 with a history of thrombosis), 27 patients with a lupus-like disease (9 with a history of thrombosis), and 41 healthy volunteers, IgG and IgM aCL, antibodies to the protein cofactors (a beta(2)-GPI and aPT) were determined by ELISA. Eighty-six of 127 patients were also tested for LAC. RESULTS: IgG aCL and LAC were associated with thrombosis but sensitivity (63 and 61%) and specificity (66 and 66%, respectively) of these tests were low. IgG and IgM a beta(2)-GPI were, respectively, 85 and 86% specific for a thrombotic history, but sensitivity was very low (32 and 29%). High IgG aCL were associated with the presence of a beta(2)-GPI and were 85% specific for thrombosis. There was no significant association between IgG or IgM aPT and thrombosis. CONCLUSION: Only high levels of IgG aCL and presence of LAC and/or a beta(2)-GPI are relevant in defining a thrombotic subset of patients with SLE. Longitudinal prospective studies are needed to investigate the predictive value of the different antiphospholipid and protein cofactor antibodies.

[The role of magnesium in the pathogenesis and therapy of bronchial asthma]. / Rola magnezu w patogenezie i terapii astmy oskrzelowej.

Magnesium is the fourth most abundant metal found in the body. It plays a crucial role in numerous biological processes. It is a natural calcium antagonist. New experimental data suggest that Mg+2 influences a variety of lung structures. Intracellular Mg+2 is thought to modulate smooth muscle contractions and it is known to have a direct effect on calcium uptake, resulting in smooth muscle relaxation. Magnesium has been forgotten cation from the therapeutical point of view, but now several clinical reports point to the salutary actions of Mg+2 in various lung diseases. Many reports suggests that magnesium sulfate and aspartate has certainly a role as an adjunct to traditional therapy in asthma and asthma-like conditions and have been helpful in the treatment of acute exacerbations of asthma.