Felty's syndrome and hypofibrinogenemia: an unusual target for anti-cyclic citrullinated peptide antibodies?

BACKGROUND: Rheumatoid arthritis (RA) is a risk factor for the development of Felty's syndrome and large granular lymphocyte (LGL) leukemia. Anti-cyclic citrullinated peptide (CCP) antibodies are considered highly specific for RA and are directed against various citrullinated antigens, including citrullinated fibrinogen. Anti-CCP antibodies may interfere with the detection of citrullinated proteins and their function. In this article, we describe the possible inhibition of fibrinogen by anti-CCP antibodies with clinical consequences which have never been reported in the literature to our best knowledge. CASE REPORT: We present the case of a 79-year-old Caucasian woman with a longstanding history of untreated seropositive RA and who had been investigated for severe neutropenia since several months. The association of splenomegaly led to suspicion of Felty's syndrome. Flux cytometry was compatible with T-cell LGL leukemia. In addition, severe hypofibrinogenemia was detected. The later finding has not been consistently associated with the former clinical entities. Further investigations demonstrated that the anti-CCP antibodies of the patient also recognized the P41 peptide of citrullinated fibrinogen. The patient deceased of intracranial hemorrhage. CONCLUSION: It is likely, yet not definite, that high anti-citrullinated fibrinogen titers may contribute to low fibrinogen levels and could have contributed to the fatal hemorrhagic event.

Variability between laboratories performing coagulation tests with identical platforms: a nationwide evaluation study.

BACKGROUND: While the assessment of analytical precision within medical laboratories has received much attention in scientific enquiry, the degree of as well as the sources causing variation between them remains incompletely understood. In this study, we quantified the variance components when performing coagulation tests with identical analytical platforms in different laboratories and computed intraclass correlations coefficients (ICC) for each coagulation test. METHODS: Data from eight laboratories measuring fibrinogen twice in twenty healthy subjects with one out of 3 different platforms and single measurements of prothrombin time (PT), and coagulation factors II, V, VII, VIII, IX, X, XI and XIII were analysed. By platform, the variance components of (i) the subjects, (ii) the laboratory and the technician and (iii) the total variance were obtained for fibrinogen as well as (i) and (iii) for the remaining factors using ANOVA. RESULTS: The variability for fibrinogen measurements within a laboratory ranged from 0.02 to 0.04, the variability between laboratories ranged from 0.006 to 0.097. The ICC for fibrinogen ranged from 0.37 to 0.66 and from 0.19 to 0.80 for PT between the platforms. For the remaining factors the ICC's ranged from 0.04 (FII) to 0.93 (FVIII). CONCLUSIONS: Variance components that could be attributed to technicians or laboratory procedures were substantial, led to disappointingly low intraclass correlation coefficients for several factors and were pronounced for some of the platforms. Our findings call for sustained efforts to raise the level of standardization of structures and procedures involved in the quantification of coagulation factors.

Acquired factor XIII deficiency: a therapeutic challenge.

Less than 60 cases of acquired factor (F)XIII deficiencies have been reported, most having distinct clinical features. To illustrate the therapeutic challenges of acquired FXIII inhibitors, we report a case of a 65-year-old patient with no previous bleeding history who suddenly developed massive haemorrhages associated to a strong and isolated FXIII inhibitor. No underlying disorder has been detected till now after three years of follow-up. Despite aggressive treatment with prednisone, rituximab, cyclophosphamide, immunoglobulin, immunoadsorption and immune tolerance his inhibitor is still present, although at low titre and with a clinical benefit since the patient has no more bleed since more than one year. Moreover the patient had a venous thromboembolic complication. After a review of the management of acquired FXIII deficiency patients and based on the management of acquired haemophilia we discuss a possible strategy for such difficult cases.

Inflammatory and prothrombotic states in obese children of European descent.

Adipose tissue may release mediators that induce a chronic inflammatory state and alterations in coagulation, which contribute to insulin resistance, atherosclerosis, and thrombosis. We investigated whether inflammatory and/or prothrombotic states exist in obese children and assessed their interrelationship. Sixty-one subjects were recruited, aged between 6 and 16 years, to participate in a cross-sectional study at Children's University Hospital of Geneva. Selected pro/anti-inflammatory cytokines/chemokines and hemostasis parameters were measured in obese children and lean controls. Cardiovascular risk factors in the family were indexed. Fasting glucose level, insulin, prothrombin time (PT), fibrinogen, activated partial thromboplastin time (aPTT), D-dimer, endogenous thrombin potential (ETP), C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-γ-inducible-protein (IP-10), monocyte chemoattractant protein 1 (MCP-1), and interleukin-1 receptor antagonist (IL-1Ra) were measured. We estimated insulin resistance by homeostatic model assessment (HOMA). Anti- (IL-1Ra) and proinflammatory cytokines (MCP-1, IL-6) were significantly increased in obese children in comparison to the control group, even before puberty. Hemostasis was also altered in obese children with a significantly increased fibrinogen level, increased D-dimer, a shortened PT, as well as an increased ETP. No correlation was found between cytokine levels and hemostasis parameters, except for IL-6 and fibrinogen. Obese children present with inflammatory and prothrombotic states as early as 6 years of age and these states are similar in prepubertal and pubertal obese children. The cytokines IL-1Ra and MCP-1 were most significantly increased in obese children. Further investigation is necessary to determine if these cytokines, together with ETP, can reliably predict the development of diabetes and atherosclerosis.

Toll-like receptor 2 mediates the activation of human monocytes and endothelial cells by antiphospholipid antibodies.

The presence of antiphospholipid antibodies (aPLAs) is associated with arterial or venous thrombosis and/or recurrent fetal loss. The proposed pathogenic mechanisms for aPLA effects include the inflammatory activation of monocytes and endothelial cells. Toll-like receptors (TLRs) are candidate signaling intermediates. The aim of this study was to investigate the relative contribution of TLR2 and TLR4 in cell activation by aPLAs. Of 32 patient-derived aPLAs, 19 induced an inflammatory activation of human monocytes and umbilical vein endothelial cells (HUVECs). In HUVECs, inflammatory responses to these aPLAs were increased by TNF pretreatment, which increases the expression of TLR2 but not TLR4. Anti-TLR2 but not anti-TLR4 antibodies reduced the aPLA-induced activation of monocytes and HUVECs. aPLAs activated TLR2-expressing human embryonic kidney 293 (HEK293) cells but not TLR4-expressing cells. Binding studies demonstrated an interaction between aPLAs and TLR2 but not TLR4. A role for CD14, a coreceptor for TLR2 and TLR4, can be inferred by observations that anti-CD14 antibodies reduced responses to aPLAs in monocytes, and that responses in HEK293 cells expressing TLR2 and CD14 were greater than in HEK293 cells expressing TLR2 alone. Our results demonstrate a role for TLR2 and CD14 in human endothelial cell and monocyte activation by aPLAs.

A novel frameshift mutation in FGA (c.1846 del A) leading to congenital afibrinogenemia in a consanguineous Syrian family.

Congenital afibrinogenemia is a rare autosomal recessive coagulation disorder characterized essentially by bleeding symptoms, but miscarriages and, paradoxically, thromboembolic events can also occur. Most reported mutations leading to congenital afibrinogenemia are located in FGA encoding the fibrinogen A α-chain. In this study, we analysed 12 individuals from a consanguineous Syrian family with reduced or absent fibrinogen levels: those with fibrinogen levels around 1 g/l (n = 7) were found to be heterozygous for a novel frameshift mutation in FGA exon 5 (c.1846 del A) and those with undetectable fibrinogen levels (n = 5) were homozygous for the same mutation. This novel frameshift mutation is the most C-terminal causative FGA mutation identified to date in afibrinogenemic patients. The resulting aberrant Aα-chain (p.Thr616HisfsX32) is most likely synthesized, but is less efficiently assembled and/or secreted into the circulation given the phenotype of asymptomatic hypofibrinogenemia in heterozygous individuals and bleeding diathesis in homozygous individuals.

[Evaluating the biological efficacy of clopidogrel: genotype or phenotype?]. / Evaluation de l'efficacité biologique du clopidogrel: génotype ou phénotype?

Clopidogrel has a known biological variability that has been consistently associated with recurrence of coronary ischemic events in clinical studies. Among the tests that are currently available, quantification of the phosphorylation status of the vasodilator phosphoprotein (VASP assay) is probably the most specific assay to evaluate the inhibition of the P2Y12 receptor by clopidogrel. A genetic polymorphism of the cytochrome 2C19 has been associated with the biological efficacy of clopidogrel and is also associated with recurrent ischemic events. The VASP assay and the 2C19 genotyping are candidates for the identification of patients at risk; this is the focus of the present review.

Anti-apolipoprotein A-1 IgG as an independent cardiovascular prognostic marker affecting basal heart rate in myocardial infarction.

AIMS: To assess the prognostic value of anti-apolipoprotein A-1 (anti-apoA-1) IgG after myocardial infarction (MI) and its association with major cardiovascular events (MACEs) at 12 months and to determine their association with resting heart rate (RHR), a well-established prognostic feature after MI. Anti-apoA-1 IgG have been reported in MI without autoimmune disease, but their clinical significance remains undetermined. METHODS AND RESULTS: A total of 221 consecutive patients with MI were prospectively included, and all completed a 12-month follow-up. Major cardiovascular events consisted in death, MI, stroke, or hospitalization either for an acute coronary syndrome or heart failure. Resting heart rate was obtained on Holter the day before discharge under the same medical treatment. Neonate rat ventricular cardiomyocytes (NRVC) were used in vitro to assess the direct anti-apoA-1 IgG effect on RHR. During follow-up, 13% of patients presented a MACE. Anti-apoA-1 IgG positivity was 9% and was associated with a higher RHR (P = 0.0005) and higher MACE rate (adjusted OR, 4.3; 95% CI, 1.46-12.6; P = 0.007). Survival models confirmed the significant nature of this association. Patients with MACE had higher median anti-apoA-1 IgG values at admission than patients without (P = 0.007). On NRVC, plasma from MI patients and monoclonal anti-apoA-1 IgG induced an aldosterone and dose-dependent positive chronotropic effect, abrogated by apoA-1 and therapeutic immunoglobulin (IVIG) pre-incubation. CONCLUSIONS: In MI patients, anti-apoA-1 IgG is independently associated with MACE at 1-year, interfering with a currently unknown aldosterone-dependent RHR determinant. Knowing whether anti-apoA-1 IgG assessment could be of interest to identify an MI patient subset susceptible to benefit from apoA-1/IVIG therapy remains to be demonstrated.

Technical aspects in laboratory testing for antiphospholipid antibodies: is standardization an impossible dream?

Anticardiolipin (aCL) and anti-beta2 glycoprotein I (anti-beta2GPI) assays are widely performed because they are part of the laboratory criteria for the antiphospholipid syndrome (APS). Despite several standardization workshops and the availability of a worldwide accepted calibrator material for aCL, a high variability in numerical assay results and in sample classification is still observed in comparative studies and external quality control surveys. For anti-beta2GPI assays, comparison of numerical values is impeded by the absence of a common calibrator material, and external quality surveys similarly show a large overlap in sample classification. Numerous variables impact assay results, among them the source and integrity of beta2GPI, the secondary calibration process, and the assessment and derivation of cutoff values. For both assays, the vast majority of laboratories use commercial kits whose number has risen considerably in the past years. However, many problems persist, and there is a need to improve the comparability in assay results. The use of monoclonal antibodies as reference calibrators has to be especially considered, with their suitability evaluated by future collaborative studies and external quality controls surveys. Manufacturers should provide more precise information on results obtained when testing control groups for establishing reference ranges and cutoff values. From the customers' perspective, it is important that each laboratory, even if using commercial kits, assesses its local cutoff value whenever possible. In the field of autoimmunity, assay standardization is a difficult but nevertheless important task. Much more effort is needed to reduce the high interlaboratory variability in assay results even if absolute standardization cannot be feasibly achieved.

A multicenter evaluation of a new quantitative highly sensitive D-dimer assay for exclusion of venous thromboembolism.

D-dimer testing is widely applied for exclusion of deep-vein thrombosis (DVT) and pulmonary embolism (PE). We report on a multicenter performance evaluation of a new particle-enhanced immunoassay, Innovance D-Dimer. Innovance D-Dimer assay was performed in 1,543 frozen samples from outpatients suspected of DVT and/or PE enrolled in three management studies as well as in a routine clinical practice. Samples were assayed on BCS/BCS XP, BCT as well as Sysmex CA-7000, CA-1500 and CA-560 analyzers (cut-off on all analyzers: 0.5 mg/l). Stratus CS D-Dimer and Vidas D-Dimer Exclusion were used for comparison. The precision study indicated total coefficients of variation ranging from 2.1% to 8.4% depending on the analyzer and on the sample. Sensitivity and negative predictive values were above 99% and their lower 95% confidence interval were equal or above 97.4% and 98.6%, respectively. Specificity ranged from 38.2% to 40.4% and the respective lower 95% confidence intervals from 35.5% to 37.7%. Area under the curve was 0.90 for all assay systems except for Innovance D-Dimer with BCT (0.89). Two samples from patients with distal DVT tested negative with all assay systems. One patient with high pre-test clinical probability and proximal DVT tested negative with Vidas D-Dimer Exclusion. Our data indicate that the performances of Innovance D-Dimer, regardless of the analyzer, are similar to the reference methods, and that this assay can be used for the exclusion of venous thromboembolic disease.

Induction of TLR2 expression by inflammatory stimuli is required for endothelial cell responses to lipopeptides.

Human endothelial cells (EC) express Toll-like receptor 4 (TLR4), a receptor for lipopolysaccharides (LPS), but little or no TLR2, a lipopeptide receptor. The aim of this study was to investigate to what extent inflammatory stimuli modify the expression by EC of TLR4 and TLR2, of the TLR2 co-receptors TLR1 and TLR6 and of the TLR2-accessory proteins CD14 and CD36. Stimulation of umbilical vein derived EC with TNF-alpha, LPS or IL-1beta for 24h induced a strong increase in TLR2 mRNA but not in TLR1, TLR4 and TLR6 mRNA. Inflammatory activation had little effect on CD14 mRNA, but decreased the expression of CD36 mRNA. TLR2 antigen was readily detected by flow cytometry on activated EC, but not on resting EC. A significant proportion of TLR2 was found to be located intracellularly. By using specific signalling pathway inhibitors we established that the induction of TLR2 by inflammatory stimuli was dependent on NF-kappaB, p38-MAP kinase and c-Jun kinase. IRAK-1 phosphorylation after treatment with 10mug/ml of lipoteichoic acid (LTA), a TLR2 agonist, was only observed in TNF-alpha-stimulated EC and not in resting EC. Furthermore, LTA potentiated the increase of the inflammatory markers E-Selectin or IL-8 in EC pre-treated with TNF-alpha, LPS or IL-1beta, but not in resting EC. These results imply that the up-regulated TLR2 is functionally active. Interestingly, LTA had no effect on TLR2 expression, nor maintained TLR2 expression, in activated EC. This suggests that lipopeptide responses of EC are dependent on the continued presence of inflammatory cytokines, provided by other cell types, or LPS. In conclusion, inflammatory stimuli induce a high TLR2 expression in EC, which in turn enables the cells to strongly respond to lipopeptides. The up-regulation of TLR2 may be of relevance for the vascular effects of Gram-positive bacteria.

[Seventy years later: prothrombin time revisited]. / Le temps de prothrombine reyisité 70 ans après.

Introduced by Armand Quick in 1935, the prothrombin time is the test the most frequently performed in haemostasis laboratories. The diversity of origin and purity of the tissue factor contained in the thromboplastin reagents as well as the composition of phospholipids explain the differences in sensitivity to factor(s) deficiency(ies) observed. Unfortunately, the expression of assay results in non anticoagulated patients varies depending on the place the test is performed. For patients under oral anticoagulation treatment, huge standardisation efforts have led to the implementation of the International Normalized Ratio (INR) for the monitoring of the treatment, which has considerably reduced the differences between thromboplastin reagents. It is hoped that a comparable degree of standardization will be achieved for samples from patients with hepatic disease.

Anti-(apolipoprotein A-1) IgGs are associated with high levels of oxidized low-density lipoprotein in acute coronary syndrome.

ApoA-1 (apolipoprotein A-1) is the main component of HDL (high-density lipoprotein) and stabilizes PON-1 (paraoxonase-1), which prevents lipid peroxidation and oxLDL (oxidized low-density lipoprotein) formation. Autoantibodies against apoA-1 [anti-(apoA-1) IgG] have been found in antiphospholipid syndrome and systemic lupus erythematosous, two diseases with an increased risk of thrombotic events, as well as in ACS (acute coronary syndrome). OxLDL levels are also elevated in these diseases. Whether anti-(apoA-1) IgGs exist in other prothrombotic conditions, such as APE (acute pulmonary embolism) and stroke, has not been studied and their potential association with oxLDL and PON-1 activity is not known. In the present study, we determined prospectively the prevalence of anti-(apoA-1) IgG in patients with ACS (n=127), APE (n=58) and stroke (n=34), and, when present, we tested their association with oxLDL levels. The prevalance of anti-(apoA-1) IgG was 11% in the ACS group, 2% in the control group and 0% in the APE and stroke groups. The ACS group had significantly higher median anti-(apoA-1) IgG titres than the other groups of patients. Patients with ACS positive for anti-(apoA-1) IgG had significantly higher median oxLDL values than those who tested negative (226.5 compared with 47.7 units/l; P<0.00001) and controls. The Spearman ranked test revealed a significant correlation between anti-(apoA-1) IgG titres and serum oxLDL levels (r=0.28, P<0.05). No association was found between PON-1 activity and oxLDL or anti-(apoA-1) IgG levels. In conclusion, anti-(apoA-1) IgG levels are positive in ACS, but not in stroke or APE. In ACS, their presence is associated with higher levels of oxLDL and is directly proportional to the serum concentration of oxLDL. These results emphasize the role of humoral autoimmunity as a mediator of inflammation and coronary atherogenesis.

An analysis of experimental conditions influencing the anti-beta2-glycoprotein I ELISA assay results.

Five components of the anti-beta(2)-glycoprotein I (abeta(2)GPI) enzyme-linked immunosorbent assay (ELISA) (coating buffer, microplate brand, blocking buffer, dilution buffer, and conjugate) were analyzed to evaluate how they affect variability in test results. Thirty-two samples from patients with antiphospholipid syndrome (APS) positive for abeta(2)GPI IgG antibodies and three calibrators (a pool of abeta(2)GPI-positive patients, the monoclonal HCAL antibody, and a home-made calibrator) were tested. No differences with regard to the blocking step were noted. Differences were found between the neutral and basic coating buffer when HCAL was used. There were significant differences between Maxisorp and all the other brands of tested microplates. Differences were found between phosphate-buffered saline (PBS) and all the other dilution buffers examined, with exception of TRIS when HCAL or the home-made calibrator was used. There were differences between our routine conjugate and one of the other four conjugates tested when using two of the three calibrators. There were also significant differences between the routine and another conjugate analyzed when using the third calibrator. As variations in abeta(2)GPI ELISA conditions determine significant differences in the results, selecting the appropriate test variables is an important step toward abeta(2)GPI assay standardization.

Influence of antidepressants on hemostasis.

Antidepressants, particularly selective serotonin reuptake inhibitors (SSRIs), are widely used for the treatment of depression and anxious disorders. The observation that depression is an independent risk factor for cardiovascular mortality and morbidity in patients with ischemic heart disease, the assessment of the central role of serotonin in pathophysiological mechanisms of depression, and reports of cases of abnormal bleeding associated with antidepressant therapy have led to investigations of the influence of antidepressants on hemostasis markers. In this review, we summarize data regarding modifications of these markers, drawn from clinical studies and case reports. We observed an association between the type of antidepressant drug and the number of abnormal bleeding case reports, with or without modifications of hemostasis markers. Drugs with the highest degree of serotonin reuptake inhibition--fluoxetine, paroxetine, and sertraline--are more frequently associated with abnormal bleeding and modifications of hemostasis markers. The most frequent hemostatic abnormalities are decreased platelet aggregability and activity, and prolongation of bleeding time. Patients with a history of coagulation disorders, especially suspected or documented thrombocytopenia or platelet disorder, should be monitored in case of prescription of any serotonin reuptake inhibitor (SRI). Platelet dysfunction, coagulation disorder, and von Willebrand disease should be sought in any case of abnormal bleeding occurring during treatment with an SRI. Also, a non-SSRI antidepressant should be favored over an SSRI or an SRI in such a context. Considering the difficulty in performing platelet aggregation tests, which are the most sensitive in SRI-associated bleeding, and the low sensitivity of hemostasis tests when performed in case of uncomplicated bleeding in the general population, establishing guidelines for the assessment of SRI-associated bleeding complications remains a challenge.

Immunization of LDL receptor-deficient mice with beta2-glycoprotein 1 or human serum albumin induces a more inflammatory phenotype in atherosclerotic plaques.

Antiphospholipid antibodies are a risk factor for venous and arterial thrombosis and may contribute to the development of atherosclerosis.The aim of this study was to investigate whether antibodies to human beta2-glycoprotein 1 (beta2GP1), as a model of antiphospholipid antibodies, modify the phenotype of atherosclerotic lesions. LDL receptor-deficient mice were immunized with human beta2GP1, human serum albumin (HSA), or not immunized, and fed a high-cholesterol diet for 14 weeks. Some mice also received pravastatin. Immunization with human beta2GP1 or HSA resulted in formation of autoantibodies recognizing murine beta2GP1 or murine albumin, respectively. We quantified atherosclerotic lesion development and mRNA levels of inflammation associated proteins in the thoraco-abdominal aorta as well as lesion development,cellular composition and collagen content in the aortic roots. Immunization with beta2GP1 or HSA had no effect on lesion size,but modified the expression in plaque areas of several inflammation-associated proteins. Expression of matrix metalloproteinase-9, tissue factor, interferon-gamma and CD25 was highest in the thoraco-abdominal aorta of beta2GP1-immunized mice, lowest in non-immunized mice and intermediate in HSA-immunized animals. Immunization with beta2GP1, but not HSA, resulted in a lower smooth muscle cell and collagen content of lesions in aortic roots. Statin treatment partially reversed the effects of beta2GP1 immunization. We conclude that immunization with beta2GP1, and to a lesser extent with HSA, leads to modifications in the cellular and protein composition of atherosclerotic plaques, which are associated with a more inflammatory phenotype. Statin treatment partially prevents these changes.

The role of TLR2 in the inflammatory activation of mouse fibroblasts by human antiphospholipid antibodies.

Antiphospholipid antibodies (APLAs) promote inflammatory and procoagulant responses in endothelial cells and monocytes. Previous studies have shown that MyD88, TRAF6, and NF-kappaB mediate cell activation by APLAs. These intermediates are also used by toll-like receptors (TLRs). We investigated the role of TLRs in the cellular response to APLAs. IgGs were isolated from the plasma of 5 patients with antiphospholipid syndrome along with immunopurified anti-beta2-glycoprotein 1 IgG from a sixth patient. Control IgG was obtained from a pool of healthy donor plasmas negative for APLAs. Wild-type mouse embryonic fibroblasts (EFs) and EFs deficient in TLR1, TLR2, TLR4, or TLR6 were incubated with APLAs, anti-beta2-glycoprotein 1 IgG, or control IgG. On incubation with the patient IgG, but not control IgG, a significant increase in mRNA levels of the inflammatory marker proteins MCP-1, ICAM-1, and IL-6 as well as IL-6 secretion was observed in wild-type EFs, whereas TLR2-deficient EFs did not respond. Responses in TLR1- and TLR6-deficient EFs were decreased and those in TLR4-deficient EFs comparable to those in wild-type EFs. Overexpression of human TLR2 in the TLR2-deficient EFs restituted the response to patient IgG. Our results imply that TLR2 plays a role in mouse fibroblast activation by APLAs.