Fibrinogen Mahdia: A congenitally abnormal fibrinogen characterized by defective fibrin polymerization.

INTRODUCTION: Congenital dysfibrinogenemia is a rare qualitative fibrinogen deficiency. Molecular defects that result in dysfibrinogenemia are usually caused by mutations which affect fibrinopeptide release, fibrin polymerization, fibrin cross-linking or fibrinolysis. AIM: Here, we investigated the genetic basis of hypodysfibrinogenemia in two Tunisian siblings with major bleeding. METHODS: Coagulation-related tests were performed on the patients and their family members. Functional analysis was performed in plasma fibrinogen to characterize fibrin polymerization. The sequences of fibrinogen genes were amplified and analysed by sequencing. RESULTS: Coagulation studies revealed a reduced functional and a borderline low antigenic fibrinogen plasma levels with prolonged thrombin and activated partial thromboplastin times. The fibrinogen is also characterized by a markedly impaired polymerization and could incorporate into fibrin fibres to a smaller extent (22%). Mutational screening disclosed a heterozygous single nucleotide deletion (G) at c.1025, resulting in a frameshift mutation (AαGly323GlufsX79) that is predicted to delete a part of the αC-domain containing some of the FXIII cross-linking sites. Both the normal and the aberrant Aα-chain (approximately 43 kDa) were detected by electrophoretic analysis in the patients. CONCLUSION: The new dysfunctional fibrinogen, Mahdia variant, describes its impact on fibrin assembly after the loss of the αC domains which are involved in the lateral aggregation of protofibrils. The study confirms that the truncated Aα-chain could be incorporated into mature fibrinogen molecules.

Protein modelling to understand FGB mutations leading to congenital hypofibrinogenaemia.

INTRODUCTION: Congenital hypofibrinogenaemia is a quantitative fibrinogen disorder characterized by proportionally decreased levels of functional and antigenic fibrinogen. Mutations accounting for quantitative fibrinogen disorders are relatively frequent in the conserved COOH-terminal globular domains of the γ and Bß chains. The latter mutations are of particular interest since the Bß-chain is considered the rate-limiting chain in the hepatic production of the fibrinogen hexamer. AIM: The aim of this study was to study the molecular pattern of four patients with congenital hypofibrinogenaemia. METHODS: Four novel fibrinogen Bß-chain mutations leading to congenital hypofibrinogenaemia were identified in four women with heterogeneous symptoms. The human fibrinogen beta chain precursor protein sequence (P02675) was obtained from the UniProt database. The resulting models were analysed using swisspdbviewer 4.1.0. RESULTS: Three patients were heterozygous for different missense mutations located in the highly conserved ß nodule: c.882G>C:Arg294Ser (Arg264Ser), c.1298G>T:Trp433Leu (Trp403Leu) and c.1329C>G:Asn443Lys (Asn413Lys). Modelling analyses predicted major structural modifications likely to result in impaired fibrinogen secretion. One patient was heterozygous for an intron 7 donor splice mutation (c.1244 + 1G>A), leading to the complete abolishment of the donor site. CONCLUSIONS: Protein modelling of new causative mutations and comparison of molecular, biochemical and clinical data continue to yield valuable information on the development and course of fibrinogen disorders as well as on the choice of the most appropriate treatments.

Genetics, diagnosis and clinical features of congenital hypodysfibrinogenemia: a systematic literature review and report of a novel mutation.

Essentials Hypodysfibrinogenemia is rarely reported among the congenital fibrinogen disorders. This first systematic literature review led to identification of 51 hypodysfibrinogenemic cases. Diagnosis based only on functional/antigenic fibrinogen ratio may be insufficient. Family studies show an incomplete segregation of mutation with the clinical phenotypes. SUMMARY: Background Hypodysfibrinogenemia is a rare disease characterized by decreased levels of a dysfunctional fibrinogen. It shares features with both hypo- and dysfibrinogenemia, although with specific molecular patterns and clinical phenotypes. Objectives To better define the genetics, the diagnosis and the clinical features of hypodysfibrinogenemia. Patients/Methods A systematic literature search led to 167 records. After removal of duplicates, abstract screening and full-text reviewing, 56 molecular and/or clinical studies were analyzed, including a novel FGB missense mutation in a woman with a mild bleeding phenotype. Results A total of 32 single causative mutations were reported, mainly in the COOH-terminal region of the γ or Aα chains at heterozygous or homozygous state. Seven additional hypodysfibrinogenemias were due to compound heterozygosity. The hypofibrinogenemic phenotypes were a result of an impaired assembly or secretion or an increased clearance of the fibrinogen variant, whereas the dysfibrinogenemic phenotype was mainly a result of a defective fibrin polymerization and an abnormal calcium or tPA binding. Among 51 identified index cases, a functional/antigenic fibrinogen ratio < 0.7 had a sensitivity of 86% for the diagnosis of hypodysfibrinogenemia. Eleven patients (22%) were asymptomatic at time of diagnosis, 23 (45%) had a mild bleeding phenotype with mainly obstetrical or gynecologic-related hemorrhage and 22 (43%) had experienced at least one thrombotic event, including 23 venous and eight arterial thromboses. Conclusions This first systematic review on hypodysfibrinogenemia shows the heterogeneity of causative mutations and that misdiagnosis could occur in relation to the functional and antigenic fibrinogen levels. Family studies reveal an incomplete segregation of the mutation with the clinical phenotype.

Outcome measures in European patients with haemophilia: Survey of implementation in routine clinical practice, perception of relevance and recommendations by European treaters in the EHTSB.

INTRODUCTION: This study was conducted to evaluate the current implementation of outcome measures in routine clinical haemophilia practice and to explore and appreciate the perception of the relevance of such measures by treaters. METHODS: A survey was completed by 19 of the 26 physicians involved in the European Haemophilia Therapy Strategy Board (EHTSB). Employing an extensive inventory of outcome measures used in patients with haemophilia, information was collected about the frequency of data collection and the subjective appreciation of their importance during clinic review. RESULTS: The survey revealed that most treaters currently collect data that are mainly related to the haemostatic treatment (consumption of concentrates) and the bleeding symptoms (number and location of bleeds) in a non-uniform and non-standardized way. By contrast, functional, physical and quality of life scorings are rarely used and show considerable heterogeneity between treaters. Also, many disparities emerged between practice and perception, in particular quality of life data that are perceived as being important but for most of the time are not collected. CONCLUSIONS: This survey represents, in our view, the first attempt to evaluate the actual utilization of outcome measures in haemophilia care. While the value of outcome measures is appreciated, they are not assessed regularly. Therefore, there is a need to include appropriate performance indicators (outcome measures) of haemophilia care in routine clinical practice. Consensus recommendations to provide a framework for achieving this aim are provided.

A multicenter study to assess the reproducibility of antiphospholipid antibody results produced by an automated system.

Essentials Inter-lab variation studies for antiphospholipid antibodies (aPL) with the same assay are lacking. We carried out an assessment of repeatability and reproducibility of an automated aPL assay. High intra-center repeatability for anticardiolipin and aß2 GPI makes duplicate testing unnecessary. Inter-lab reproducibility was high except for aß2GPI IgG. SUMMARY: Background Inter-assay variability is a well-known problem in antiphospholipid antibody testing, because of the lack of standardization. Inter-laboratory reproducibility for the same assay is similarly important. Objectives Testing repeatability and reproducibility of HemosIL® AcuStar for anticardiolipin (aCL) and antiß2-glycoprotein I antibodies (aß2GPI) IgG and IgM. Patients/Methods In this observational study, out of 420 samples from the thrombophilia centers of Ghent and Geneva, 100 samples were randomly selected and successively analyzed in three centers: Ghent (C1, in duplicate for repeatability evaluation), Geneva (C2) and Frankfurt (C3). Results Results from 99 samples were available, including 25 from patients with antiphospholipid syndrome (APS) and 74 from non-APS patients. The intra-center repeatability expressed as intra-class correlation coefficient (ICC) was higher than 0.99 for each parameter. Differences between two measurements rarely exceeded 1 U mL-1 for values below 100 U mL-1 , except for aß2GPI IgG, where differences varied from -4 to 4 U mL-1 . The inter-center ICCs were higher than 0.99, except for aCL IgM (ICC = 0.961). These ICCs remained high even when considering values below 100 U mL-1 (0.943, 0.964 and 0.977 for aCL IgG, aCL gM and aß2GPI IgM, respectively), except for aß2GPI IgG (ICC = 0.652). Qualitative comparison showed less than 5% discordant classification between centers, with somewhat more discordant results for aß2GPI IgG. Conclusions In terms of discriminating properties, the HemosIL® AcuStar has excellent intra-center repeatability and a good inter-center reproducibility for aCL IgG, aCL IgM and aß2GPI IgM. Some concern may arise for aß2GPI IgG.

Hypertension, haematuria and renal functioning in haemophilia - a cross-sectional study in Europe.

BACKGROUND AND OBJECTIVES: This cross-sectional, epidemiological study sought to assess the prevalence and extent of potential risk factors for hypertension, particularly renal function related to haematuria and their associations in people with haemophilia. METHODOLOGY: Demographic and medical data were collected at a single time-point in patients with haemophilia over 40 years of age from 16 European centres. Associations with diagnosis of hypertension were tested in univariate and multivariate analyses. RESULTS: We enrolled 532 patients (median age 52 years, range 40-98) with haemophilia A (n = 467) or haemophilia B (n = 65). Haemophilia was severe (<0.01 IU mL-1 ) in 313 patients (59%). Hypertension was diagnosed in 239 patients (45%). In multivariate analyses, age and body mass index (BMI) were significantly and independently associated with hypertension (adjusted odds ratio (OR) 18.1, P < 0.001, in elderly patients and OR = 25.1, P < 0.001, in patients with BMI >30 kg m-2 ). Estimated glomerular filtration rate (eGFR) <70 mL min-1 (OR = 2.7, P = 0.047) was significantly associated with hypertension, but mean eGFR was significantly higher for severe than mild haemophilia. Further variables with OR > 2.8 were diabetes (OR = 2.8, P = 0.04), coronary artery disease (OR = 3.3, P = 0.052) and family history of hypertension (OR = 4.4, P < 0.001). Neither severity of haemophilia nor history of haematuria was significantly associated with hypertension in univariate or multivariate analyses. CONCLUSION: As in the general population, age and BMI were major risk factors for hypertension in people with haemophilia. Renal dysfunction was associated with hypertension, but the prevalence of renal dysfunction was not extensive and furthermore not significantly correlated with haematuria. The associations of other variables with hypertension require further studies to confirm causal relationships over time.

Management of congenital quantitative fibrinogen disorders: a Delphi consensus.

INTRODUCTION: No evidence-based guidelines for the management of patients suffering from afibrinogenaemia and hypofibrinogenaemia are available. AIM AND METHOD: The aim of this study was to harmonize patient's care among invited haemophilia experts from Belgium, France and Switzerland. A Delphi-like methodology was used to reach a consensus on: prophylaxis, bleeding, surgery, pregnancy and thrombosis management. RESULTS: The main final statements are as follows: (i) a secondary fibrinogen prophylaxis should be started after a first life-threatening bleeding in patients with afibrinogenaemia; (ii) during prophylaxis the target trough fibrinogen level should be 0.5 g L-1 ; (iii) if an adaptation of dosage is required, the frequency of infusions rather than the fibrinogen amount should be modified; (iv) afibrinogenaemic patients undergoing a surgery at high bleeding risk should receive fibrinogen concentrates regardless of the personal or family history of bleeding; (v) moderate hypofibrinogenaemic patients (i.e. ≥0.5 g L-1 ) without previous bleeding (despite haemostatic challenges) undergoing a surgery at low bleeding risk may not receive fibrinogen concentrates as prophylaxis; (vi) monitoring the trough fibrinogen levels should be performed at least once a month throughout the pregnancy and a foetal growth and placenta development close monitoring by ultrasound is recommended; (vii) fibrinogen replacement should be started concomitantly to the introduction of anticoagulation in afibrinogenaemic patients suffering from a venous thromboembolic event; and (viii) low-molecular-weight heparin is the anticoagulant of choice in case of venous thromboembolism. CONCLUSION: The results of this initiative should help clinicians in the difficult management of patients with congenital fibrinogen disorders.

Rare coagulation disorders: fibrinogen, factor VII and factor XIII.

Rare coagulation disorders (RCDs) include the inherited deficiencies of fibrinogen, factor (F) II, FV, combined FV and VIII, FVII, FX, combined FVII and X, FXI, FXIII and combined congenital deficiency of vitamin K-dependent factors (VKCFDs). Despite their rarity, a deep comprehension of all these disorders is essential to really understand haemostasis. Indeed, even if they share some common features each RCD has some particularity which makes it unique. In this review, we focus on three disorders: fibrinogen, FVII and FXIII.

Can the phenotype of inherited fibrinogen disorders be predicted?

Congenital fibrinogen disorders are rare diseases affecting either the quantity (afibrinogenaemia and hypofibrinogenaemia) or the quality (dysfibrinogenaemia) or both (hypodysfibrinogenaemia) of fibrinogen. In addition to bleeding, unexpected thrombosis, spontaneous spleen ruptures, painful bone cysts and intrahepatic inclusions can complicate the clinical course of patients with quantitative fibrinogen disorders. Clinical manifestations of dysfibrinogenaemia include absence of symptoms, major bleeding or thrombosis as well as systemic amyloidosis. Although the diagnosis of any type of congenital fibrinogen disorders is usually not too difficult with the help of conventional laboratory tests completed by genetic studies, the correlation between all available tests and the clinical manifestations is more problematic in many cases. Improving accuracy of diagnosis, performing genotype, analysing function of fibrinogen variants and carefully investigating the personal and familial histories may lead to a better assessment of patients' phenotype and therefore help in identifying patients at increased risk of adverse clinical outcomes. This review provides an update of various tests (conventional and global assays, molecular testing, fibrin clot analysis) and clinical features, which may help to better predict the phenotype of the different types of congenital fibrinogen disorders.

Antiphospholipid antibodies and the risk of severe and non-severe pre-eclampsia: the NOHA case-control study.

BACKGROUND: pre-eclampsia (PEecl) can be defined as non-severe (NS-PEecl) or severe (S-PEecl). Our study aimed to determine the incidence of antiphospholipid antibodies (aPLs) in women with a past history of NS-PEecl or S-PEecl. PATIENTS AND METHODS: This case-control study includes 195 control women, 199 NS-PEecl patients and 143 S-PEecl patients whose plasma samples were collected 6 months after their first delivery. Each plasma was tested for lupus anticoagulant (LA), anticardiolipin (aCL) and antiß2GP1 antibodies, as well as antibodies against phosphatidylserine/prothrombin complex (aPS/PT) and domain I of the ß2GP1. RESULTS: When compared with the control group no significant associations were found for the NS-PEecl group after adjustment of confounding variables. For the S-PEecl group, there was an association with antiß2GP1 immunoglobulin G (IgG) (OR 16.91, 95% CI 3.71-77.06), as well as age, obesity, smoking and multiparity. Antiß2GP1-domain I IgG was associated with aCL, antiß2GP1 and aPS/PT IgG in the three groups. aPS/PT IgG was associated with aCL IgG, and aPS/PT IgM was associated with aCL and antiß2GP1 IgM in the three groups. CONCLUSION: S-PEecl is a distinct entity from NS-PEecl and is mainly associated with the presence of antiß2GP1 IgG. Antiß2GP1 domain I correlates with other aPL IgG tests, and aPS/PT may be promising in patients for whom LA tests cannot be interpreted.

Hypofibrinogenemia and liver disease: a new case of Aguadilla fibrinogen and review of the literature.

INTRODUCTION: Fibrinogen storage disease (FSD) is characterized by hypofibrinogenemia and hepatic inclusions due to impaired release of mutant fibrinogen which accumulates and aggregates in the hepatocellular endoplasmic reticulum. Liver disease is variable. AIM: We studied a new Swiss family with fibrinogen Aguadilla. In order to understand the molecular peculiarity of FSD mutations, fibrinogen Aguadilla and the three other causative mutations, all located in the γD domain, were modelled. METHOD: The proband is a Swiss girl aged 4 investigated because of fatigue and elevated liver enzymes. Protein structure models were prepared using the Swiss-PdbViewer and POV-Ray software. RESULTS: The proband was found to be heterozygous for fibrinogen Aguadilla: FGG Arg375Trp. Familial screening revealed that her mother and maternal grandmother were also affected and, in addition, respectively heterozygous and homozygous for the hereditary haemochromatosis mutation HFE C282Y. Models of backbone and side-chain interactions for fibrinogen Aguadilla in a 10-angstrom region revealed the loss of five H-bonds and the gain of one H-bond between structurally important amino acids. The structure predicted for fibrinogen Angers showed a novel helical structure in place of hole 'a' on the outer edge of γD likely to have a negative impact on fibrinogen assembly and secretion. CONCLUSION: The mechanism by which FSD mutations generate hepatic intracellular inclusions is still not clearly established although the promotion of aberrant intermolecular strand insertions is emerging as a likely cause. Reporting new cases is essential in the light of novel opportunities of treatment offered by increasing knowledge of the degradation pathway and autophagy.

Dysfibrinogenemia: from molecular anomalies to clinical manifestations and management.

Congenital dysfibrinogenemia is a qualitative congenital fibrinogen disorder characterized by normal antigen levels of a dysfunctional fibrinogen. The diagnosis is usually based on discrepancies between fibrinogen activity and antigen levels, but could require more specialized techniques for the assessment of fibrinogen function, owing to some limitations in routine assays. Molecular abnormalities, which are frequently heterozygous missense mutations localized in exon 2 of FGA and exon 8 of FGG, lead to defects in one or more phases of fibrinogen to fibrin conversion, fibrin network formation, and other important functions of fibrinogen. The clinical phenotype is highly heterogeneous, from no manifestations to bleeding and/or thrombotic events. Asymptomatic propositi and relatives with the predisposing genotype are at risk of developing adverse outcomes during the natural course of the disease. Correlations between genotype and phenotype have not yet been clearly established, with the exception of some abnormal fibrinogens that severely increase the risk of thrombosis. Functional analysis of polymerization and fibrinolysis, structural studies of the fibrin network and the viscoelastic properties of fibrin clot could help to predict the phenotype of congenital dysfibrinogenemia, but have not yet been evaluated in detail. The management is essentially based on personal and family history; however, even individuals who are still asymptomatic and without a family history should be carefully assessed and monitored. Particular situations, such as pregnancy, delivery, and surgery, require a multidisciplinary approach.

Recombinant human FVIIa for reducing the need for invasive second-line therapies in severe refractory postpartum hemorrhage: a multicenter, randomized, open controlled trial.

BACKGROUND: Case reports on recombinant human factor VIIa (rhuFVIIa) use in women with severe postpartum hemorrhage (PPH) showed encouraging results, but no randomized controlled trial (RCT) is available. PATIENTS AND METHODS: Eighty-four women with severe PPH unresponsive to uterotonics were randomized to receive one early single rhuFVIIa infusion (n = 42) or standard care (no rhuFVIIa; n = 42). The primary efficacy outcome measure was the reduction of the need for specific second-line therapies, such as interventional hemostatic procedures, for blood loss and transfusions. The primary safety outcome measure was the number of deaths and thrombotic events during the 5 days following rhuFVIIa infusion. RESULTS: rhuFVIIa was associated with a reduction in the number of patients who needed second-line therapies compared with controls (standard care). Specifically, 39/42 (93%) patients in the standard care arm received second-line therapies and 22/42 (52%) patients in the rhuFVIIa arm (absolute difference, 41%; range, 18-63%; relative risk RR, 0.56 [0.42-0.76]). The delivery mode (vaginal or Cesarean section) did not affect the primary outcome. No death occurred. Two venous thrombotic events were recorded in the rhuFVIIa arm: one ovarian vein thrombosis and one deep vein thrombosis with a non-severe pulmonary embolism. CONCLUSION: This open RCT in women with severe PPH refractory to uterotonics shows that rhuFVIIa reduces the need for specific second-line therapies in about one in three patients, with the occurrence of non-fatal venous thrombotic events in one in 20 patients.

Congenital combined deficiency of coagulation factors VII and X--different genetic mechanisms.

Combined coagulation factor VII (FVII) and factor X (FX) deficiency (combined FVII/FX deficiency) belongs to the group of bleeding disorders in which both factors show reduced plasma activity. It may arise from coincidental inheritance of separate coagulation factor deficiencies or a common cause as large deletions comprising both gene loci. The F7 and F10 genes are located on the long arm of chromosome 13. Here, we describe 10 cases with combined FVII/FX deficiency representing both genetic mechanisms of occurrence. Genetic analyses included direct sequencing of the F7 and F10 genes and MLPA (multiplex ligation-dependent probe amplification) for detection of heterozygous large deletions. In four patients, the combined deficiency was due to a large deletion within the terminal end of chromosome 13. In the remaining six cases the deficiency resulted from coincidental inheritance of different genetic alterations affecting both genes independently. In most cases, the genetic defects were heterozygous, presenting with prolonged PT, normal aPTT and mild or no bleeding symptoms. Only in one case compound heterozygous mutations were detected in the F10, resulting in prolonged aPTT and a more severe bleeding phenotype. To avoid a misdiagnosis of combined FVII/FX deficiency, analyses of single factor activities have to be performed in all cases with prolonged PT even if aPTT is normal. Genetic analyses are substantial for correct prediction of an inheritance pattern and a proper genetic counselling.

Refinement of the cutoff values of the HemosIL AcuStar assay for the detection of anticardiolipin and anti-beta2 glycoprotein-1 antibodies.

BACKGROUND: The HemosIL AcuStar antiphospholipid assay (Instrumentation Laboratory, Bedford, MA, USA) is a fully automated assay using chemiluminescent technology for the detection of anticardiolipin and anti-beta2 glycoprotein-1 antibodies. This assay showed excellent agreement between results of different laboratories. The cutoff values to define positivity were calculated in 250 healthy blood bank donors but were associated with large confidence intervals (CIs). OBJECTIVE: The objective of this study was to more precisely determine the cutoff values of the HemosIL AcuStar antiphospholipid assay by increasing the number of healthy blood bank donors through a multicenter study and by applying a normalization procedure of the distribution of each antibody. METHODS: Five laboratories participated to this study, allowing the inclusion of 626 samples. We used a Box-Cox power transformation method to normalize the distribution and calculate the 99th percentile and the corresponding 95%CI for each antibody. RESULTS: The revised cutoff values were overall lower than those initially calculated with more stringent CIs and yielded a 4.2% increase in sensitivity with a 2.7% decrease in specificity regarding thrombotic events or obstetric complications. CONCLUSIONS: We provide refined cutoff values for the detection of anticardiolipin and anti-beta2 glycoprotein-1 antibodies with the HemosIL AcuStar Antiphospholipid assay that should be preferred for routine use.

Novel products for haemostasis.

The primary major issue in haemophilia treatment remains the development of inhibitors. Recently two novel bypassing products have been developed. First, a humanized bispecific antibody against FIXa and FX, termed hBS23, was produced utilizing these two molecules placed into a spatially appropriate position to mimic FVIIIa, and recently this mimetic activity and the pharmacokinetics of the original antibody were improved by engineering the charge properties of the variable region within the immunoglobulin. Using the new antibody, termed ACE910, a phase 1 study in 64 Japanese and Caucasian healthy adults was performed and data from this trial suggested that the product had medically acceptable safety and tolerability profiles. The other new bypassing agent is named MC710, and consists of a mixture of plasma-derived FVIIa and FX. Preclinical studies using in vitro and in vivo haemophilia B inhibitor monkey models indicated that the haemostatic effects of FVIIa and FX were enhanced by simultaneous administration. Results from phase I and II clinical studies suggested that MC710 had equal or greater pharmacokinetic (PK), pharmacodynamic (PD), efficacy and safety profiles than conventional bypassing agents in the treatment of joint bleeding in haemophilia patients with inhibitors. Another significant current issue in this context is the increased medical cost of conventional treatment due to the higher consumption of concentrates. Biosimilar products may offer advantages in these circumstances and may offer a less expensive alternative. Regulatory issues, however, together with acceptability of biosimilar materials and reimbursement policies as well as supply and demand incentives remain to be considered. Rare bleeding disorders (RBDs) have attracted less attention from the pharmaceutical industry than haemophilia or von Willebrand disease due to the limited number of patients involved. Many cases of this type have been treated, therefore, using fresh frozen plasma (FFP) or prothrombin complex concentrates (PCCs) which carry serious risks of infections, allergic reactions and fluid overload. Several specific plasma-derived or recombinant products including fibrinogen, FVIIa, FXI and FXIII have now become available, however, and a phase III clinical study of recombinant FXIIIa has recently been completed demonstrating safety and efficacy of substances of this nature.

NF-κB is activated from endosomal compartments in antiphospholipid antibodies-treated human monocytes.

BACKGROUND: The antiphospholipid antibody syndrome (APS) is an autoimmune disease associated with arterial or venous thrombosis and/or recurrent fetal loss and is caused by pathogenic antiphospholipid antibodies (aPLA). We recently demonstrated that Toll-like receptor 2 (TLR2) and CD14 contribute to monocyte activation of aPLA. OBJECTIVE: To study the mechanisms of cell activation by aPLA, leading to pro-coagulant and pro-inflammatory responses. METHODS AND RESULTS: For this study, we used purified antibodies from the plasmas of 10 different patients with APS and healthy donors. We demonstrate that aPLA, but not control IgG, co-localizes with TLR2 and TLR1 or TLR6 on human monocytes. Blocking antibodies to TLR2, TLR1 or TLR6, but not to TLR4, decreased TNF and tissue factor (TF) responses to aPLA. Pharmacological and siRNA approaches revealed the importance of the clathrin/dynamin-dependent endocytic pathway in cell activation by aPLA. In addition, soluble aPLA induced NF-κB activation, while bead-immobilized aPLA beads, which cannot be internalized, were unable to activate NF-κB. Internalization of aPLA in monocytes and NF-κB activation were dependent on the presence of CD14. CONCLUSION: We show that TLR2 and its co-receptors, TLR1 and TLR6, contribute to the pathogenicity of aPLA, that aPLA are internalized via clathrin- and CD14-dependent endocytosis and that endocytosis is required for NF-κB activation. Our results contribute to a better understanding of the APS and provide a possible therapeutic approach.