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Background Antibodies binding to domain I of β2-glycoprotein I (aDI) and activated protein C (APC) resistance are associated with an increased risk of thrombosis in cross-sectional studies. The objective of this study was to assess their predictive value for future thromboembolic events in patients with antiphospholipid antibodies (aPL) or antiphospholipid syndrome. Methods This prospective multicenter cohort study included consecutive patients with aPL or systemic lupus erythematosus. We followed 137 patients (43.5 ± 15.4 year old; 107 women) for a mean duration of 43.1 ± 20.7 months. Results We detected aDI IgG antibodies by ELISA in 21 patients. An APC sensitivity ratio (APCsr) was determined using a thrombin generation–based test. The APCsr was higher in patients with anti–domain I antibodies demonstrating APC resistance (0.75 ± 0.13 vs 0.48 ± 0.20, P 95th percentile; HR, 6.07 [95% CI, 1.69–21.87]; P = 0.006). A sensitivity analysis showed an increased risk of higher aDI IgG levels up to HR 5.61 (95% CI, 1.93–16.31; P = 0.01). In multivariate analysis, aDI IgG (HR, 3.90 [95% CI, 1.33–11.46]; P = 0.01) and APC resistance (HR, 4.98 [95% CI, 1.36–18.28]; P = 0.02) remained significant predictors of thrombosis over time. Conclusions Our study shows that novel tests for antibodies recognizing domain I of β2-glycoprotein I and functional tests identifying APC resistance are significant predictors of thrombosis over time and may be useful for risk stratification.

Background Anticardiolipin (aCL) and anti-β2 glycoprotein I (aβ2GPI) immunoglobulin A (IgA) antiphospholipid antibodies (aPL) have shown to associate with thrombosis and pregnancy morbidity. However, inclusion of IgA aPL in the classification criteria of the antiphospholipid syndrome (APS) has been debated. We investigated the value of aCL and aβ2GPI IgA aPL in the detection of thrombosis and pregnancy morbidity in addition to the current aPL panel for APS. Methods We included 1,068 patients from eight European medical centers: 259 thrombotic APS patients, 122 obstetric APS patients, 204 non-APS thrombosis patients, 33 non-APS obstetric patients, 60 APS patients with unspecified clinical manifestations, 196 patients with autoimmune diseases, and 194 controls. aCL and aβ2GPI IgG/M/A were detected with four commercial assays and lupus anticoagulant was determined by the local center. Results Positivity for IgA aPL was found in 17 to 26% of the patients with clinical manifestations of APS and in 6 to 13% of the control population. Both aCL and aβ2GPI IgA were significantly associated with thrombosis and pregnancy morbidity. Isolated IgA positivity was rare in patients with clinical manifestations of APS (0.3–5%) and not associated with thrombosis and/or pregnancy morbidity. Addition of IgA to the current criterion panel did not increase odds ratios for thrombosis nor pregnancy morbidity. Conclusion aCL and aβ2GPI IgA are associated with clinical manifestations of APS. However, isolated IgA positivity was rare and not associated with thrombosis or pregnancy morbidity. These data do not support testing for aCL and aβ2GPI IgA subsequent to conventional aPL assays in identifying patients with thrombosis or pregnancy morbidity.

Background Fluorogenic thrombin generation (TG) assays are commonly used to determine global coagulation phenotype in plasma. Whole blood (WB)-TG assays reach one step closer to physiology by involving the intrinsic blood cells, but erythrocytes cause variable quenching of the fluorescence signals, hampering its routine application.Objective To develop a new assay for continuous WB-TG measurement.Methods In the new WB-TG assay, the erythrocyte-caused distortion of signal was solved by continuously mixing the sample during the measurement. The assay was validated by evaluating the reproducibility and comparing with the paper-based WB-TG assay. Reconstituted human blood and WB from 119 healthy donors was tested to explore the influences of hematocrit and platelet count on TG.Results This novel WB-TG assay showed good reproducibility while being less affected by contact activation compared with the previous paper-based assay. Reconstitution experiments showed that the lag time of TG was shortened by the addition of platelets but not erythrocytes. Increasing hematocrit strongly augmented the peak thrombin, even in the presence of high platelet counts. The lag time and peak of WB-TG of 119 healthy donors were positively related to erythrocyte count after adjusting for age, sex, and oral contraceptive use with multiple linear regression analyses. The reference range and interindividual variation of WB-TG were determined in the healthy cohort.Conclusions A novel WB-TG assay was developed, which is a straightforward tool to measure the involvement of platelets and erythrocytes in TG and may assist the research of blood cell-associated coagulation disorders.

Background Classification of the antiphospholipid syndrome (APS) relies predominantly on detecting antiphospholipid antibodies (aPLs). Antibodies against a domain I (DI) epitope of anti-beta 2glycoprotein I (beta 2GPI) proved to be pathogenic, but are not included in the current classification criteria. Objectives Investigate the clinical value of detecting anti-DI IgG in APS. Patients/Methods From eight European centers 1005 patients were enrolled. Anti-cardiolipin (CL) and anti-beta 2GPI were detected by four commercially available solid phase assays; anti-DI IgG by the QUANTA Flash (R) beta 2GPI domain I assay. Results Odds ratios (ORs) of anti-DI IgG for thrombosis and pregnancy morbidity proved to be higher than those of the conventional assays. Upon restriction to patients positive for anti-beta 2GPI IgG, anti-DI IgG positivity still resulted in significant ORs. When anti-DI IgG was added to the criteria aPLs or used as a substitute for anti-beta 2GPI IgG/anti-CL IgG, ORs for clinical symptoms hardly improved. Upon removing anti-DI positive patients, lupus anticoagulant remained significantly correlated with clinical complications. Anti-DI IgG are mainly present in high-risk triple positive patients, showing higher levels. Combined anti-DI and triple positivity confers a higher risk for clinical symptoms compared to only triple positivity. Conclusions Detection of anti-DI IgG resulted in higher ORs for clinical manifestations than the current APS classification criteria. Regardless of the platform used to detect anti-beta 2GPI/anti-CL, addition of anti-DI IgG measured by QUANTA Flash (R) did not improve the clinical associations, possibly due to reduced exposure of the pathogenic epitope of DI. Our results demonstrate that anti-DI IgG potentially helps in identifying high-risk patients.

Background The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of lupus anticoagulant (LAC), anti-cardiolipin (aCL) and/or anti-beta 2glycoprotein I (a beta 2GPI) antibodies of the immunoglobulin G/immunoglobulin M (IgG/IgM) isotype. However, the role of aCL and a beta 2GPI IgM as a serologic marker in APS is debated. Objectives We aimed to assess the diagnostic and clinical value of IgM antiphospholipid antibodies (aPL) in APS within the classification criteria. Patients/Methods Our multicenter study comprised 1008 patients, including APS patients and controls. Anti-CL and a beta 2GPI IgG and IgM antibodies were detected with four commercially available solid phase assays. Results Positivity for aCL and/or a beta 2GPI antibodies was significantly correlated with thrombosis and pregnancy morbidity, independent of the isotype and solid phase assay. Higher odds ratios were obtained for IgG compared to IgM positivity. Isolated IgM was rare in thrombotic APS, but more frequent in obstetric APS, ranging from 3.5% to 5.4% and 5.7% to 12.3%, respectively, dependent on the solid phase assay. In a multivariate logistic regression analysis of aPL, IgM positivity was found to be associated with pregnancy morbidity. However, detection of IgM was not independently associated with thrombosis. Combined positivity for LAC, IgG, and IgM was highly associated with thrombosis and pregnancy morbidity. Conclusions Our data support testing for aCL and a beta 2GPI IgM in women suspected of obstetric APS. However, no added value was found for testing IgM in patients suspected of thrombotic APS. Still, IgM aPL might be useful as a second-line test to improve thrombotic risk stratification.

Background: Anticardiolipin (aCL) and anti-β2 glycoprotein I (aβ2GPI) immunoglobulin (Ig) G/IgM antibodies are 2 of the 3 laboratory criteria for classification of antiphospholipid syndrome (APS). The threshold for clinically relevant levels of antiphospholipid antibodies (aPL) for the diagnosis of APS remains a matter of debate. The aim of this study was to evaluate the variation in cutoffs as determined in different clinical laboratories based on the results of a questionnaire as well as to determine the optimal method for cutoff establishment based on a clinical approach.Methods: The study included samples from 114 patients with thrombotic APS, 138 patients with non-APS thrombosis, 138 patients with autoimmune disease, and 183 healthy controls. aCL and aβ2GPI IgG/IgM antibodies were measured at 1 laboratory using 4 commercial assays. Assay-specific cutoff values for aPL were obtained by determining 95th and 99th percentiles of 120 compared to 200 normal controls by different statistical methods.Results: Normal reference value data showed a nonparametric distribution. Higher cutoff values were found when calculated as 99th rather than 95th percentiles. These values also showed a stronger association with thrombosis. The use of 99th percentile cutoffs reduced the chance of false positivity but at the same time reduced sensitivity. The decrease in sensitivity was higher than the gain in specificity when 99th percentiles were calculated by methods wherein no outliers were eliminated.Conclusions: We present cutoff values for aPL determined by different statistical methods. The 99th percentile cutoff value seemed more specific. However, our findings indicate the need for standardized statistical criteria to calculate 99th percentile cutoff reference values.

Background The anti-phospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with persistent presence of anti-phospholipid antibodies (aPL). Laboratory criteria include aPL detection by coagulation tests for lupus anticoagulant (LAC) or solid phase assays measuring anti-β2 glycoprotein I (aβ2GPI) or anti-cardiolipin (aCL) immunoglobulin (Ig) G/IgM antibodies. External quality control programs illustrate that commercially available aPL assays produce variable results. Objective We aimed to investigate the agreement and diagnostic accuracy of solid phase assays. Materials and Methods In this multi-centre study, 1,168 patient samples were tested on one site for aCL and aβ2GPI IgG/IgM antibodies by four solid phase test systems. Samples included APS patients, controls and monoclonal antibodies (MoAB) against different epitopes of β2GPI. LAC was determined by the local centre. Results aCL IgM assays resulted in the most discrepancies (60%), while aCL IgG and aβ2GPI IgM assays resulted in lower discrepancies (36%), suggesting better agreement. Discrepant samples displayed lower median aPL titers. Dependent on the solid phase test system, odds ratios (ORs) for thrombosis and pregnancy morbidity ranged from 1.98 to 2.56 and 3.42 to 4.78, respectively. Three platforms showed lower sensitivity for MoAB directed against the glycine (Gly) 40-arginine (Arg) 43 epitope of domain I of β2GPI. Conclusion Poor agreement was observed between different commercially available aCL and aβ2GPI IgG/IgM assays, hampering uniformity in the identification of aPL-positive patients. Clinical association was globally concordant between solid phase test systems considering results of the four aPL together. An assay sensitive in detecting the MoAB against Gly40-Arg43 of domain I of β2GPI reached the highest OR for thrombosis.

As the clinical symptoms of the antiphospholipid syndrome (APS) frequently occur irrespective of the syndrome, diagnosis predominantly depends on the laboratory assays measuring the level or function of antiphospholipid antibodies (aPLs). beta 2-glycoprotein I (beta 2GPI) is increasingly accepted as the most important target of aPLs. Anti-beta 2GPI antibodies constitute a heterogeneous population, but current in vivo and in vitro evidence show that especially the first domain (DI) of beta 2GPI contains an important pathogenic epitope. This epitope containing Glycine40-Arginine43 (G40-R43) has proven to be cryptic and only exposed when beta 2GPI is in its open conformation. A previous study demonstrated a highly variable exposure of the cryptic epitope in commercial anti-beta 2GPI assays, with implications on correct patient classification. Unexpectedly, recent unpublished data revealed impaired exposure of the pathogenic epitope in the commercially available anti-DI chemiluminescence immunoassay (CIA) assay detecting specific antibodies directed to DI. In this review we summarize the laboratory and clinical performance characteristics of the different anti-DI assays in published data and conclude with inconsistent results for both the correlation of anti-DI antibodies with clinical symptoms and the added value of anti-DI antibodies in the classification criteria of APS. Additionally, we hypothesize on possible explanations for the observed discrepancies. Finally, we highly advise manufacturers to use normal pooled plasma spiked with the monoclonal anti-DI antibodies to verify correct exposure of the cryptic epitope.

International audience; Azathioprine (AZA), an oral immunosuppressant, is safe during pregnancy. Some reports suggested different impairments in the offspring of mothers with autoimmune diseases (AI) exposed in utero to AZA. These observations are available from retrospective studies or case reports. However, data with respect to the long-term safety in the antenatally exposed child are still lacking. The aim of this study is to summarize the current knowledge in this field and to focus on the need for a prospective study on this population. We performed a PubMed search using several search terms. The actual data show that although the risk of congenital anomalies in offspring, as well as the infertility risk, are similar to those found in general population, there is a higher incidence of prematurity, of lower weight at birth and an intra-uterine delay of development. There is also an increased risk of materno- fetal infections, especially cytomegalovirus infection. Some authors raise the interrogations about neurocognitive impairment. Even though the adverse outcomes might well be a consequence of maternal illness and disease activity, interest has been raised about a contribution of this drug. However, the interferences between the external agent (in utero exposure to AZA), with the host (child genetic susceptibility, immune system anomalies, emotional status), environment (public health, social context, availability of health care), economic, social, and behavioral conditions, cultural patterns, are complex and represent confounding factors. In conclusion, it is necessary to perform studies on the medium and long-term outcome of children born by mothers with autoimmune diseases, treated with AZA, in order to show the safety of AZA exposure. Only large-scale population studies with long-term follow-up will allow to formally conclude in this field

The antiphospholipid syndrome (APS) is characterized by vascular thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPLs). Progress is being made in understanding the pathogenesis of the syndrome, but difficulties persist in the identification of patients at risk for thrombosis and/or pregnancy morbidity. Beta-2 glycoprotein I (β2GPI), a plasma protein consisting of five sushi domains, is thought to be the main antigenic target of aPLs. Antibodies recognizing domain I of β2GPI are predominantly present in patients with an elevated risk of thrombosis, whereas antidomain IV/V antibodies are found in nonthrombotic autoimmune diseases. Indeed, domain I antibodies proved to be pathogenic in multiple studies. Retrospective studies have provided evidence for an added clinical value of antidomain I antibodies in the risk stratification of patients with APS. Still, wide ranges of odds ratio exist between studies, probably due to differences in the study and control population, and detection methods used. Despite the proven pathogenicity of antidomain I antibodies and their correlations with clinical manifestations of APS, heterogeneity of the current studies has prohibited their acceptance in the official diagnostic criteria. Well-designed large longitudinal prospective studies with available and new, preferentially functional, assays for the risk stratification of patients with APS are required.

Antiphospholipid syndrome (APS) is a condition in which the presence of antibodies against phospholipid-binding proteins is associated with thrombophilia and/or pregnancy morbidity. Although antiphospholipid antibodies have anticoagulant characteristics in vitro, they are associated with thromboembolic complications. Thrombin generation (TG) is a sensitive global test of coagulation, and elevated TG is associated with thrombosis. Increased TG can be caused by increased prothrombin conversion, decreased thrombin inactivation, or a combination of both. In this study, we measured TG in APS patients and healthy controls with and without vitamin K antagonist (VKA) treatment at 1 and 5 pM tissue factor and with thrombomodulin. Prothrombin conversion and thrombin inactivation were determined by thrombin dynamics analysis. The TG peak was increased in nontreated APS patients at 1 pM TF compared with nontreated controls. Prothrombin conversion was significantly increased in nontreated APS patients. In contrast, prothrombin conversion did not differ in controls and patients that were on VKA therapy. Thrombin inactivation was comparable between controls and APS patients in the presence and absence of VKAs. Both TG (peak and ETP) and prothrombin conversion were significantly higher in APS patients with prior thrombosis compared with patients without a history of thrombosis. In this study, we demonstrate that in APS, the hemostatic balance shifts toward a more prothrombotic phenotype due to elevated prothrombin conversion but unchanged thrombin inactivation rates. Within the group of APS patients, increased TG and prothrombin conversion are associated with a history of thrombosis.

Accurate thrombin generation determination by calibrated automated thrombinography can be sustained when reducing the plasma and reagent volumes up to half, but not for higher reductions or plasma dilutions.

Background Measuring thrombin generation (TG) in plasma increasingly gained attention as a diagnostic tool in the field of thrombosis and hemostasis. To include the contribution of all blood cells, recently, the whole blood TG method was developed. Methods We changed the calculation method of the standard calibrated automated thrombography (CAT) to a method only taking into account the data until the peak of TG, thereby considerably reducing the time from blood draw to result. By redesigning the method, the blood volume per test was reduced to 15 μL. Results For all TG parameters, the interassay variation proved to be below 15%. The interindividual variation of all parameters was comparable to the CAT method. Thirty-three patients undergoing cardiothoracic surgery were included to investigate whether our assay correlates with postoperative blood loss. On dividing patients into severe and mild bleeders, significant differences between both groups were found for the peak endogenous thrombin potential (peakETP) and peak values determined by our near-patient device. Importantly, patients with a peakETP below the median experienced significantly more blood loss compared to those with a peakETP above the median. A similar division based on the peak as well as the body mass index of the patient yielded similar significant differences. A combination of the peakETP, the body mass index, and the lag time even resulted in a better predictor of blood loss compared to each parameter separately. Conclusions Our adapted whole blood TG assay can be used near patients and is indicative for the amount of blood loss post cardiothoracic surgery.

BackgroundInteraction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF Al domain, enabling platelet glycoprotein Iba binding. Quantification of this "active" conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.MethodsWe developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF Al domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.ResultsIntra- and inter-assay CVs were between 2.4-7.2% and 4.1-9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103 +/- 3%. The assay was linear in the range of 90.1-424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6-154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF: GP1bM binding.ConclusionsWe developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.

SummaryWhile experimental data state that protamine exerts intrinsic anticoagulation effects, protamine is still frequently overdosed for heparin neutralisation during cardiac surgery with cardiopulmonary bypass (CPB). Since comparative studies are lacking, we assessed the influence of two protamine-to-heparin dosing ratios on perioperative haemostasis and bleeding, and hypothesised that protamine overdosing impairs the coagulation status following cardiac surgery. In this open-label, multicentre, single-blinded, randomised controlled trial, patients undergoing on-pump coronary artery bypass graft surgery were assigned to a low (0.8; n=49) or high (1.3; n=47) protamine-to-heparin dosing group. The primary outcome was 24-hour blood loss. Patient haemostasis was monitored using rotational thromboelastometry and a thrombin generation assay. The low protamine-to-heparin dosing ratio group received less protamine (329 ± 95 vs 539 ± 117 mg; p

Essentials The clinical value of IgM antibodies in thrombotic antiphospholipid syndrome (APS) is debated. By review of literature, we reconsidered the clinical value of IgM antibodies in thrombotic APS. More significant correlations with thrombosis were found for the IgG compared to IgM isotype. Unavailability of paired IgG/IgM results hampers evaluating the added value of IgM positivity. Click to hear Dr de Groot's perspective on antiphospholipid syndrome SummaryBackground Despite the update of the classification criteria for the antiphospholipid syndrome (APS), difficulties persist in the identification of patients at risk for thrombosis. Current guidelines include assays detecting IgG/IgM anti-β2-glycoprotein I and anti-cardiolipin antibodies, although the relevance of IgM antibodies has been debated. Objectives Through a review of the literature from 2001 to 2014, we aimed to formally establish the thrombotic risk stratification potential of IgM as compared with IgG anti-phospholipid antibodies (aPLs). Patients/methods One thousand two hundred and twenty-eight articles were selected by a computer-assisted search of the literature. Of the 177 studies that met our inclusion criteria, the clinical value of IgG/IgM aPLs was established through analysis of odds ratios for thrombosis or percentage of positives in the thrombotic population. Results/conclusions We clearly found more significant correlations with thrombosis for the IgG than for the IgM isotype. Nonetheless, in a minority of studies, significant associations with thrombosis were found for IgM but not IgG antibodies. The unavailability of paired results of IgG and IgM for each separate patient hampers evaluation of the added value of isolated IgM positivity. To fully take advantage of results obtained by future studies, we strongly encourage scientists to provide all studied information per patient. We planned a large multicenter study to investigate clinical associations of isolated/combined positivity for criteria/non-criteria aPLs. Importantly, because of the presence of non-pathogenic aPLs, quantitative assays are characterized by a high false-positivity rate. Optimization of functional assays, such as thrombin generation measuring the whole scheme of coagulation, may help to reduce APS-related morbidity and mortality.

SummaryAssays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a ‘cone and base principle’-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

Background and objectivesThe effect of oral contraceptive (OC) usage on coagulation has been studied worldwide. However, no such studies have been conducted in Saudi Arabia on Saudi women using OCs. The aim of this study was to investigate the effects of OC-induced changes of thrombin generation (TG) in the absence and presence of activated protein C (APC) or thrombomodulin (TM) in Saudi women.MethodsA total of 115 adult women, 47 on oral contraception (OC users) and 68 controls (not using OCs) were recruited from the obstetrics-gynecology outpatient clinic in Saudi Arabia. OCs that were used in this study include the following: Marvelon, Gynera, Cerrazetem, Yasmine, Microlut, Gracial and Diane. The plasma calibrated automated thrombinography (CAT) was used to determine TG which was expressed as endogenous thrombin potential (ETP; nM/ min), lag time (min), peak (nM) and time-to-peak (ttpeak; min). In the presence of TM or APC, TG parameters were expressed relative to the parameters in the absence of TM or APC.Results and conclusionAs in other populations, our study demonstrated that OC usage induced prothrombotic changes in plasma of Saudi women, including resistance to the inhibitory actions of TM and APC. More specifically, OC usage in our population predominantly influenced TG and APC/ TM sensitivity in overweight and obese individuals, a finding that needs confirmation in large cohort studies. The effects of APC and TM on TG parameters showed a positive association, and the correlation coefficients were higher in OC users for both ETP and peak values.

Essentials Triple-positivity is associated with a high risk for a first thrombotic event and recurrence. Identification of triple-positives is dependent on the solid phase assay used. In triple-positivity, IgM only adds value in thrombotic risk stratification together with IgG. Thrombotic risk in triple-positive patients with IgM only, depends on the platform. Abstract Background The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPL). Triple-positivity (i.e. positivity for lupus anticoagulant [LAC], anti-cardiolipin [aCL] and anti-β2glycoprotein I [aβ2GPI] antibodies) is associated with a high thrombotic risk. Objectives We investigated the variability in triple-positivity detection by measuring the same samples with four commercially available solid phase assays. In addition, the added clinical value of aPL in LAC-positive patients was investigated, as well as the association of IgM triple-positivity and thrombosis. Patients/Methods We included 851 patients from seven European medical centers. Anti-CL and aβ2GPI IgG/IgM antibodies were determined by four platforms: BioPlex® 2200, ImmunoCap® EliA, ACL AcuStar® and QUANTA Lite ELISA® . Results Triple-positivity detection by solid phase assays varied, ranging from 89 up to 118 in thrombotic APS patients (n = 258), of which 86 were detected independent of the platform. Lupus anticoagulant positivity resulted in an odds ratio (OR) for thrombosis of 3.4; triple-positivity (irrespective of the isotype) increased the OR from 4.3 up to 5.2, dependent on the platform. Triple-positivity solely for the IgM isotype did not increase the OR for thrombosis compared with LAC positivity. The highest OR for thrombosis was reached for positivity for IgG and IgM aβ2GPI and aCL (8.6 up to 28.9). Conclusions Triple-positivity proved to be highly associated with thrombosis, but identification is assay dependent. Within triple-positivity, IgM antibodies only have an added clinical value in patients positive for IgG antibodies.

Exercise is protective against cardiovascular disease, but can also provoke sudden cardiac death, a phenomenon referred to as “the exercise paradox.” Extreme exertion is known to induce a rebalanced hemostatic state by causing hypercoagulability and concomitantly enhanced fibrinolysis. Over the past decade, novel techniques for quantifying hemostasis have been introduced, which may provide new insights into this process. This review summarizes recent literature on the effect of extreme exertion of both short and long duration on coagulation, fibrinolysis, and recovery of hemostatic balance. Extreme exertion introduces a temporary hypercoagulable state, mainly through upregulation of the contact pathway by increased FVIII levels. Additionally, von Willebrand factor levels and platelet activation are increased. Simultaneously, increased fibrinolysis results from increased tissue-type plasminogen activator levels, suggesting a rebalanced hemostasis. The vascular endothelium appears to play a pivotal role in both processes. Data on the effect of exercise on endogenous anticoagulants are scarce. Hypercoagulability persists for hours to a day after prolonged extreme exertion, while fibrinolytic parameters appear to return to baseline levels more quickly. Hence, the balance in the rebalanced hemostatic state may be lost during recovery. Training induces lower amplitude of the hypercoagulable response, and quicker recuperation toward baseline. Repetitive exercise exhausts the endothelium, attenuating procoagulant changes. Additional research is needed to identify if the hemostatic balance is lost during recovery, and if so, when the shift toward thrombosis appears. Moreover, differences between sexes need to be addressed, since women are known to have a different pathophysiological mechanism behind cardiovascular events, but are underrepresented in recent literature.

Introduction Platelet function testing with flow cytometry has additional value to existing platelet function testing for diagnosing bleeding disorders, monitoring anti-platelet therapy, transfusion medicine and prediction of thrombosis. The major challenge is to use this technique as a diagnostic test. The aim of this study is to standardize preparation, optimization and validation of the test kit and to determine reference values in a population of 129 healthy individuals. Methods Platelet function tests with 3 agonists and antibodies against P-selectin, activated alpha IIb beta 3 and glycoprotein Ib (GPIb), were prepared and stored at -20 degrees C until used. Diluted whole blood was added and platelet activation was quantified by the density of activation markers, using flow cytometry. Anti-mouse Ig. particles were included to validate stability of the test and to standardize results. Reference intervals were determined. Results Blood stored at room temperature (RT) for up to 4h after blood donation and preheated/tested at 37 degrees C resulted in stable results (%CV

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.

SummaryThe Calibrated Automated Thrombogram (CAT) assay that measures thrombin generation (TG) in platelet-poor and -rich plasma, is increasingly being recognised as a more sensitive tool to determine the overall function of the haemostatic system. We developed a method enabling the measurement of TG in a small aliquot of blood. The objective was to validate this assay in mouse blood and to examine the rate and extent of TG in a mouse model of premature aging. TG was assayed in blood from 20– to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. Bmal1-KO mice are known to display symptoms of premature aging. TG was initiated by adding calcium, tissue factor and a thrombin specific substrate. After TG, the samples were prepared for scanning electron microscopy (SEM). The intra-assay variations (%) in mouse blood of the endogenous thrombin potential (ETP), peak height, lag time, time-to-peak and velocity index were 10% or less (n=24). We found that Bmal1-KO mice have a significantly (p

Introduction: The antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPL). The current aPL panel consists of detection of lupus anticoagulant (LAC), anti-cardiolipin (aCL) IgG/IgM and anti-β2glycoprotein I (aβ2GPI) IgG/IgM antibodies. Despite the fact that these aPL associate differently with the clinical symptoms of APS, all of them have the same diagnostic value in the Sydney criteria. Combined positivity has been shown to significantly increase the risk of thrombosis and pregnancy morbidity. Nonetheless, risk stratification of APS patients remains challenging. Objective: We aimed to assess the thrombotic and obstetric association of the different aPL profiles. Methods: In this multicenter study, 1068 patients from eight European medical centers were enrolled within a timespan of one year with patient samples stored less than 5 years. We included 259 thrombotic APS patients, 122 obstetric APS patients, 204 diseased controls for thrombosis (non-APS thrombosis), 33 diseased controls for obstetric complications (non-APS obstetric), 196 patients with an autoimmune disease other than APS without thrombotic complications (AID controls) and 194 patients that were referred for aPL testing for other reasons than the clinical criteria of APS (e.g. subfertility and prolonged activated partial thromboplastin time; controls). Four platforms, ACL AcuStar®, Bioplex 2200®, ImmunoCap®EliA and QUANTA Lite ELISA®, were selected based on frequency of use and the willingness of manufacturers to provide their assays. All samples with all assays were tested at one location by a single technician. Lupus anticoagulant positivity was determined by the local center, according to the ISTH guidelines. The study was approved by the local ethical committees. Results: Isolated LAC significantly correlated with thrombosis, while isolated IgG and IgM did not (Figure 1). Looking at pregnancy morbidity, significant correlations were found for both isolated LAC and isolated IgG, but not for isolated IgM (Figure 2). Based on these findings, we included both LAC and IgG testing in the first steps of risk stratification. Compared to isolated LAC positivity, with an odds ratio (OR) for thrombosis of 3.4, combined positivity for anti-β2GPI and/or anti-CL IgG antibodies increased the OR up to 5.9 (95% confidence interval [95% CI], 3.4-10.5). Positivity for LAC, IgG and IgM antibodies resulted in the highest OR for thrombosis, ranging from 5.5 (95% CI, 2.6-11.8) up to 9.6 (95% CI, 3.4-27.1), depending on the solid phase assay used (Figure 1). Other profiles including IgM showed no increased risk (insignificant OR) for thrombosis or failed to result in a higher OR compared to other profiles without IgM (Figure 1). In line with thrombosis, the highest OR for pregnancy morbidity was achieved in patients positive for LAC, IgG and IgM antibodies (Figure 2). Interestingly, a positive IgM result in the presence of a negative IgG result was not associated with pregnancy morbidity, independent of the LAC status (Figure 2). Although also other profiles were associated with pregnancy morbidity, the computed ORs were similar to isolated LAC positivity (Figure 2). Conclusions: Positivity for LAC in combination with aCL and/or aβ2GPI IgG or in combination with aCL IgM and/or aβ2GPI IgM antibodies is highly associated with thrombosis and pregnancy morbidity. Other tested aPL profiles showed equal or less correlation than isolated LAC or showed no association with thrombosis or pregnancy morbidity. Based on these findings, depending on the outcome of LAC and IgG positivity, further testing of IgM testing may not be advised as this won't provide additional information regarding risk stratification of APS patients (indicated in the red lines of the flow chart). This flowchart should be validated in additional patient cohorts and may potentially lead to an improved risk stratification of APS patients. Disclosures No relevant conflicts of interest to declare.

SummaryPhysical exercise is recommended for a healthy lifestyle. Strenuous exercise, however, may trigger the haemostatic system, increasing the risk of vascular thrombotic events and the incidence of primary cardiac arrest. Our goal was to study the effects of strenuous exercise on risk factors of cardiovascular disease. Blood was collected from 92 healthy volunteers who participated in the amateur version of the protour Amstel Gold cycling race, before and directly after the race. Thrombin generation showed a shortening of the lag time and time to peak and an increase of the velocity index. Interestingly, the endogenous thrombin potential measured in plasma decreased due to reduced prothrombin conversion. Platelet reactivity increased and this effect was stronger in men than in women. Lower fibrinogen and higher D-dimer levels after exercise indicated higher fibrin formation. On the other hand, fibrinolysis was also elevated as indicated by a shortening of the clot lysis time. Exercise activated the endothelium (von Willebrand factor (VWF) and active VWF levels were elevated) and the immune system (concentrations IL-6, IL-8, MCP-1, RANTES and PDGF increased). Additionally, an increased cardiac troponin T level was measured post-exercise. Strenuous exercise induces a temporary hyperreactive state in the body with enhanced proand anticoagulant responses. As strenuous exercise has a more pronounced effect on platelet function in male subjects, this gives a possible explanation for the higher incidence of sudden cardiac death during exercise compared to women. This trial is registered at www.clinicaltrials.gov as NCT02048462.

To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis.

The absence of IFN-γ signaling leads to an increased inflammatory response in many murine models of autoimmune diseases induced by a CFA-assisted immunization schedule. We investigated the role of endogenous IFN-γ in arthritis induced by immunization with glucose-6-phosphate isomerase (G6PI) in CFA in DBA/1 mice. Surprisingly, and in contrast to our previous findings in collagen-induced arthritis (CIA), G6PI-induced arthritis was found to be reduced in IFN-γ receptor-deficient (IFN-γR KO) mice, demonstrating a proinflammatory role for IFN-γ in this model. Milder disease in IFN-γR KO mice was associated with less vigorous innate and adaptive immune responses early (day 9) after immunization: less proliferation of myeloid cells in the spleen, less osteoclast formation, less G6PI-reactive Th cells (as measured by ex vivo stimulation and flow cytometry and by in vivo skin reactivity to G6PI) and lower G6PI-specific immunoglobulin serum levels. Surprisingly, on day 21, despite continued milder disease in IFN-γR KO mice, their Th cell responses were no longer diminished but augmented as compared to wild-type mice, and their numbers of immature myeloid splenocytes were also more increased. These data reveal that IFN-γ signaling is critical for the induction of the early immune responses which trigger G6PI-induced arthritis. The strikingly different clinical consequences of absent IFN-γ signaling in G6PI-induced arthritis compared with the very similarly induced CIA emphasize that the role of a single cytokine in experimentally induced arthritis depends critically on the very nature of the inciting (auto)antigen and in particular on the kinetics of the disease manifestation elicited by the antigen.

Objective. To investigate the role of interleukin-22 (IL-22) in collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. Methods. C57BL/6 mice were immunized with type II collagen (CII) in Freund's incomplete adjuvant with added Mycobacterium tuberculosis, and levels of IL-22 and its specific receptor, IL-22 receptor type I (IL-22RI), were measured in sera and tissue by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction analysis. Clinical and histologic signs of arthritis were recorded and compared with those in C57BL/6 mice deficient in the IL-22 gene (IL-22(-/-)). Humoral and cellular immune responses against CII were analyzed. In vitro osteoclastogenesis assays were performed on splenocytes. Results. Upon immunization with CII in Freund's incomplete adjuvant plus heat-killed Mycobacterium tuberculosis, sera from C57BL/6 mice were found to contain high levels of IL-22, and the specific IL-22RI was expressed in lymphoid tissue, including splenocytes. IL-22(-/-) mice were less susceptible to CIA than were wild-type mice, as evidenced by their decreased incidence of arthritis and decreased pannus formation. Remarkably, the less severe form of arthritis in IL-22(-/-) mice was associated with increased production of CII-specific and total IgG antibodies, whereas cellular CII responses were unchanged. In vitro, IL-22 was found to promote osteoclastogenesis, a process that might contribute to its proinflammatory activity in CIA. Conclusion. Endogenous IL-22 plays a proinflammatory role in CIA in C57BL/6 mice. Our data also indicate that IL-22 promotes osteoclastogenesis and regulates antibody production.

Interferon-gamma (IFN-gamma) is regarded traditionally as a proinflammatory factor and as the signature cytokine of Th1-dominated autoimmune processes. Early evidence indicative of an opposite, protective role has recently received further attention from reports revealing an increasing number of pathways by which IFN-gamma can counteract harmful inflammation in Th1-associated autoimmune diseases. Here, we review evidence for IFN-gamma's anti-inflammatory effects primarily from the perspective of one experimental model, collagen-induced arthritis (CIA), and question the classic proinflammatory role of IFN-gamma and also the Th1-Th2 paradigm as a basis for explaining the regulation of autoimmune diseases. We conclude that endogenous production of IFN-gamma during inflammatory and autoimmune diseases should be considered as a process with bidirectional immunoregulatory consequences, often resulting in moderation of pathology.

Objectives:Patients with rheumatoid arthritis (RA) have defective CD4+CD25+ regulatory T (Treg) cells and increased osteoclastogenesis. A similar situation has been described in collagen-induced arthritis (CIA). In this study, it was investigated whether a single transfer of polyclonally activated Treg cells inhibits CIA and osteoclastogenesis.Methods:Purified Treg cells were expanded in vitro with anti-CD3 and anti-CD28 antibody-coated beads and injected into DBA/1 mice. Mice were immunised with collagen type II (CII) in complete Freund adjuvant (CFA) and scores of arthritis were recorded. In vitro osteoclastogenesis assays were performed on splenocytes by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)κB ligand (RANKL). Levels of anti-CII antibody and cytokines were determined in the supernatant using ELISA and Bio-Plex protein array system.Results:It was found that 106 activated Treg cells significantly counteracted the development of CIA, which was accompanied by decreased serum levels of TNFα and IL6, but not by inhibition of autoimmune antibody responses. The differentiation of osteoclasts in splenocyte cultures was significantly reduced in the presence of prestimulated Treg cells. Expression of cytokines that are described to inhibit osteoclastogenesis, including granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)γ, interleukin (IL)5 and IL10, were dramatically increased upon addition of Treg cells. Furthermore, splenocytes from mice that had been treated with Treg cells displayed an impaired capacity to develop into mature osteoclasts, suggesting that Treg cells abrogated osteoclastogenesis in vivo.Conclusions:Activated CD4+CD25+ Treg cells improve clinical symptoms of CIA, regulate cytokine production and inhibit osteoclastogenesis in vitro and in vivo.

Mice with a disrupted IFN-γ system are remarkably susceptible to experimental autoimmune diseases, such as collagen-induced arthritis (CIA), which rely on the use of CFA. The inflammatory lesions of these IFN-γ knockout (KO) mice are characterized by an excessive proportion of neutrophils. Here, we show that the increased severity of CIA in IFN-γR KO as compared with wild-type mice is accompanied by increased levels of the CXC chemokine granulocyte chemotactic protein-2 (GCP-2), a major neutrophil-attracting chemokine in mice. We demonstrated that the heat-killed mycobacteria present in CFA elicited production of GCP-2 in mouse embryo fibroblast cultures and that this production was inhibited by IFN-γ. Inhibition of GCP-2 production by IFN-γ was STAT-1-dependent. IFN-γ receptor KO mice treated with neutralizing anti-GCP-2 antibodies were protected from CIA, indicating the in vivo importance of GCP-2 in the pathogenesis of CIA. Our data support the notion that one of the mechanisms whereby endogenous IFN-γ mitigates the manifestations of CIA consists of inhibiting production of GCP-2, thereby limiting mobilization and infiltration of neutrophils, which are important actors in joint inflammation. These results may also be applicable to other experimental models of autoimmunity that rely on the use of CFA.

International audience; Objective. To investigate the validity of the global APS score (GAPSS) to predict thrombosis in patients with autoimmune diseases.Methods. This prospective cohort study included consecutive patients with aPL or SLE. aPL, aPS-PT and GAPSS were determined. A Cox proportional hazards model assessed the validity of GAPSS and identified other potential independent predictors of thrombosis.Results. One hundred and thirty-seven patients [43.5 (s.d. 15.4) years old; 107 women] were followed up for a mean duration of 43.1 (s.d. 20.7) months. Mean GAPSS was significantly higher in patients who experienced a thrombotic event compared with those without [10.88 (s.d. 5.06) vs 8.15 (s.d. 5.31), respectively, P = 0.038]. In univariate analysis, age [hazard ratio (HR) = 1.04 (95% CI 1.01, 1.08)] and GAPSS above 16 [HR = 6.86 (95% CI 1.90, 24.77)] were each significantly associated with thrombosis during follow-up, while history of arterial thrombosis [HR = 2.61 (95% CI 0.87, 7.82)] failed to reach significance. Among aPL assays, IgG aPS/PT—a component of the GAPSS—was significantly associated with thrombosis [HR = 2.95 (95% CI 1.02, 8.51)]. In multivariate analysis, GAPSS above 16 remained the only significant predictor of thrombosis [HR = 6.17 (95% CI 1.70, 22.40)].Conclusion. This first external validation study confirmed that GAPSS can predict thrombosis in patients with aPL and associated autoimmune diseases.

Introduction Infants and children have a lower incidence of thrombosis compared with adults. Yet, the mechanism of blood clot formation and structure in infants and children, as the end product of coagulation, has not been studied. This study aimed to establish differences in the mechanism of thrombin generation, fibrin clot formation and response to thrombolysis in infants and children compared with adults. Materials and methods We studied thrombin generation, fibrin clot formation, structure and fibrinolysis in healthy infants, children and adults. Results Younger populations had a decreased potential to generate thrombin, at a slower velocity compared with adults, correlating positively with age. Clot formation at venous shear rate was decreased in infants and children compared with adults, with increased time for fibrin formation, decreased fibrin formation velocity, resulting in decreased tendency for fibrin formation in younger populations. These differences were less pronounced at arterial shear rate. Studies of the fibrin clot structure in paediatric age groups showed a significantly larger pore size compared with adults, suggestive of a clot that is less resistant to fibrinolysis. The presence of tissue plasminogen activator (tPA) resulted in a significant decrease in the pore size of infants and children, but not in adults. Conclusions This is the first study to suggest that the mechanism of blood clot formation and nanostructure, as well as response to thrombolytic therapy is different in infants and children compared with adults.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immunological disorder that is characterized by systemic inflammation, widespread organ damage, and hypercytokinemia. Primary HLH is caused by mutations in granule-mediated cytotoxicity, whereas secondary HLH occurs, without a known genetic background, in a context of infections, malignancies, or autoimmune and autoinflammatory disorders. Clinical manifestations of both HLH subtypes are often precipitated by a viral infection, predominantly with Herpesviridae. Exploiting this knowledge, we established an animal model of virus-associated secondary HLH by infecting immunocompetent wild-type mice with the β-herpesvirus murine CMV. C57BL/6 mice developed a mild inflammatory phenotype, whereas BALB/c mice displayed the clinicopathologic features of HLH, as set forth in the Histiocyte Society diagnostic guidelines: fever, cytopenia, hemophagocytosis, hyperferritinemia, and elevated serum levels of soluble CD25. BALB/c mice also developed lymphadenopathy, liver dysfunction, and decreased NK cell numbers. Lymphoid and myeloid cells were in a hyperactivated state. Nonetheless, depletion of CD8+ T cells could not inhibit or cure the HLH-like syndrome, highlighting a first dissimilarity from mouse models of primary HLH. Immune cell hyperactivation in BALB/c mice was accompanied by a cytokine storm. Notably, plasma levels of IFN-γ, a key pathogenic cytokine in models of primary HLH, were the highest. Nevertheless, murine CMV–infected IFN-γ–deficient mice still developed the aforementioned HLH-like symptoms. In fact, IFN-γ–deficient mice displayed a more complete spectrum of HLH, including splenomegaly, coagulopathy, and decreased NK cell cytotoxicity, indicating a regulatory role for IFN-γ in the pathogenesis of virus-associated secondary HLH as opposed to its central pathogenic role in primary HLH.

Thrombin generation in the presence of platelets is sensitive to the activation status of von Willebrand factor

Current practice in the prevention of venous thromboembolism (VTE) is either controlled dosage of vitamin K antagonists (VKA) or standard dosage of a low molecular weight heparin (LMWH) or an orally administrated direct inhibitor of thrombin or factor Xa (DOACs). This is justified by clinical trials showing the non-inferiority of standard dosage to VKA treatment - disregarding that the latter could be improved upon. Whether standard dosage is optimal for the individual patient depends upon how well (s)he is represented by the "average" patient that meets the inclusion criteria of the trial. Here we show that in a set of normal plasmas the individual thrombin generating power (the endogenous thrombin potential: ETP), after spiking with a fixed concentration (~ IC50) of different anticoagulants show such a wide variation that, even when plasma levels would be identical, a considerable percentage of patients could be over- or under-anticoagulated. Consequently adapted doses must be considered if better results than those with VKA are our aim. We recall that the ETP is close to constant in the individual person but varies in the population with a broad log-normal distribution (CV 16%). The ETP is highly correlated to the risk of thrombosis: The relative risk of VTE in the upper quartile being 5 - 7 times higher than that in the lower one (Winckers K., et al., poster PO617, ISTH 2015, Toronto). Reducing the ETP is the common feature of all antithrombotic therapy. The desired range of reduction is not exactly known. In congenital bleeding disorders bleeding risks increase sharply at ETP below 33%. In monitoring VKA treatment an INR below 2, which corresponds to an ETP > 66% of normal, is generally considered inadequate. We therefore arbitrarily choose the range 33 - 66% of normal, as a target range (TR). Experimental: Individual plasmas from 60 normal healthy volunteers were spiked with a fixed amount of either unfractionated heparin (UFH), low molecular weight heparin (LMWH), antithrombin binding pentasaccharide (penta), DOAC acting on thrombin or DOAC acting on factor Xa. We found the residual ETP to be highly variable (table column 3), obviously because the variation in susceptibility to the inhibitor superimposes upon the natural variation. Using these CVs we calculated what % falls either above or below the target range. The total variance of the ETP under treatment is the combined effect of natural variance, the pharmacokinetic- and the pharmacodynamic -variance. Here we determined pharmacodynamic effects only and calculated the % of ETP values that would fall outside the target range A: if there would be no pharmacokinetic variance (table column 4 & 5) and B: If the pharmacokinetic variance would equal the pharmacodynamic variance (table column 6 & 7). It is seen that in any case over 15% of the population will be outside the target range and that in the more likely case that pharmacokinetic variation counts as much as pharmacodynamic variation does around half of all patients will be outside the safe range. This could be avoided by measuring the effect of a standard dose of anticoagulant on the ETP once and increasing the dose in those with an ETP > 66% and decreasing it in those with ETP < 33%. The present work is meant to provide the rationale for starting clinical studies on the actual variation of the ETP attained under standard dosage of different anticoagulants and the effects thereon of personalised dose adjustment. Table Table. Disclosures Hemker: Diagnostica Stago: Consultancy.

Background: The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies directed mainly against prothrombin and β2-glycoprotein I. The syndrome is associated with an increased risk of thrombosis. The global hemostatic state in a patient can be tested by measuring thrombin generation (TG). Recently, we developed a method to study the main pro- and anticoagulant processes at the basis of TG, called the thrombin dynamics method. Aim: In this study we investigated the dynamics of thrombin generation in healthy subjects and APS patients. Materials and methods: Healthy subjects (n=129) and antiphospholipid syndrome (APS) patients (n=31) were included in the study. Sixty-eight percent of the APS patients were lupus anticoagulant positive, anti-cardiolipin antibodies were detected in 84% of the patients, and 52% presented with anti-β2-glycoprotein I antibodies. Patients on anticoagulant therapy were excluded from the study. Thrombin generation was measured at 1 pM tissue factor (TF) and activated protein C (APC) system sensitivity was tested by measuring TG in the presence and absence of 20 nM thrombomodulin (TM). Results: Thrombin generation was measured in platelet poor plasma at 1 pM tissue factor. The lag time and time-to-peak were significantly prolonged in APS patients compared to healthy subjects (lag time: 3.30 ± 0.59 vs. 6.69 ± 4.26 min, p The pro- and anticoagulant processes underlying thrombin generation were studied separately. The total amount of prothrombin converted during thrombin generation (PCtot) did not differ between healthy subjects and patients (1234 vs. 1165 nM). However, the maximum rate of prothrombin conversion (PCmax) was significantly elevated in APS patients (291 vs 425 nM/min; p Thrombomodulin reduced the ETP by 45% in healthy subjects, but had significantly less effect in APS patients (10%). The addition of TM decreased total prothrombin conversion by 40% and the maximum prothrombin conversion rate by 50% in healthy subjects. In patients, TM only slightly reduced total prothrombin conversion (8%) and the maximum prothrombin conversion rate (7%). Discussion: The thrombin generation results indicate a predisposition to thrombosis in APS patients, as the TG parameters peak height and the velocity index are increased. Examination of the underlying pro- and anticoagulant processes of prothrombin conversion and thrombin inactivation revealed that although the same amount of prothrombin is converted in patients, the maximum activity of the prothrombinase complex is higher, indicating that patients can generate thrombin faster. In addition, APS patients have a dysfunctional APC system, as prothrombin conversion and thrombin generation could be only slightly inhibited by the addition of thrombomodulin. Disclosures No relevant conflicts of interest to declare.

Introduction: Thrombin generation (TG) assesses the hemostatic capacity of an individual and is indicative of a person's bleeding and thrombosis risk. TG dependson the balance between the pro- and anticoagulant processes in clotting plasma. Recently, we developed a method to study the main pro- and anticoagulant processes at the basis of TG, called the thrombin dynamics method. Vitamin K antagonist (VKA) treatment is known to reduce TG and that the reduction of TG correlates with the international normalized ratio (INR). In this study, we aim to investigate the effect of VKA therapy on the dynamics of TG by examining prothrombin conversion and thrombin inactivation. Materials and methods: TG was measured in platelet poor plasma at 1 pM tissue factor in 129 healthy subjects and 129 patients treated with VKA. The patients were classified according to the INR value: INR4 (n=19). Thrombin dynamics was calculated from TG curves and plasma antithrombin (AT), α2-macroglobulin (α2M) and fibrinogen levels. Three thrombin dynamics parameters were determined: the total amount of prothrombin conversion (PCtot), the maximum rate of prothrombin conversion (PCmax) and the thrombin decay capacity (TDC) of each plasma sample. Results: Vitamin K antagonist treatment reduced the ETP and peak height significantly at all INR values (p As previously reported, the thrombin decay capacity is mainly dependent on the plasma antithrombin and fibrinogen level. Plasma antithrombin levels were comparable between healthy subjects and patients on VKA (figure 2A) and showed a clear positive correlation with the thrombin decay constant in the patient group (R2=0.72, p Conclusion: Thrombin dynamics analysis shows that prothrombin conversion is reduced in patients treated with VKA and that thrombin inactivation is significantly attenuated. We demonstrate that the latter effect can be attributed to the elevated fibrinogen levels that accompany VKA treatment. Figure 1 Thrombin dynamics in healthy subjects and VKA patients. (A) Prothrombin conversion curves. (B) PCtot: total amount of prothrombin converted; (C) PCmax: maximum rate of prothrombin conversion; (D) TDC: thrombin decay capacity. *p Disclosures Hemker: Diagnostica Stago: Consultancy.

The antiphospholipid syndrome (APS) is diagnosed by the combination of vascular thrombosis and/or pregnancy morbidity and the detection of antiphospholipid antibodies (aPLs) in plasma. In the last few years, a great effort has been made to unravel the mechanism by which aPLs cause thrombosis and a vast amount of mechanisms have been proposed. aPLs were proposed to induce a prothrombotic state by influencing the cellular blood compartment, the plasma compartment, the vascular wall and even metabolic pathways beyond the hemostatic system. However, due to the diversity in the mechanisms and the differences in the methodology, the focus of the mechanistical studies in this field seems to be largely diffused. It is hard to imagine that aPLs can exert such a diversity of effects, resulting in either thrombosis and/or pregnancy morbidity and the relationship between aPLs and the clinical manifestations remains to be a mysterious "black box". In an attempt to get insight in what takes place inside the black box, we have analyzed 126 mechanistical studies on aPLs and discussed differences in the type of antibodies that were used, the involvement of beta2-glycoprotein I (beta(2)GPI), and the criteria used to diagnose APS patients.

A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-beta(2)glycoprotein I (beta(2)GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces beta(2)GPI opens up, thereby exposing G40-R43. Objectives: To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays. Methods: Two patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes beta(2)GPI irrespective of its conformation. These antibodies were tested in commercial anti-beta(2)GPI assays (A-E). Results: In assay A, both antibodies showed equal reactivity towards beta(2)GPI, indicating that all the beta(2)GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43. Conclusions: Exposure of epitope G40-R43 on beta(2)GPI is highly variable between commercial anti-beta(2)GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43.

beta 2glycoprotein I (beta 2GPI) is the major antigen in the antiphospholipid syndrome. It has been shown that beta 2GPI can adapt to different conformations, a circular, a S-shaped and a J-shaped conformation. In literature anticoagulant properties of beta 2GPI have been indicated, though there is no consensus on how beta 2GPI exerts a certain action. This article will first review existing data on the conformation of beta 2GPI. In addition, we will investigate whether the conformation of beta 2GPI plays a role in in the proposed anticoagulant activity of beta 2GPI. We investigated the effect of native beta 2GPI and phospholipid-bound beta 2GPI on thrombin generation (TG). Native beta 2GPI was found to have no significant effect on the TG regardless of the concentration of tissue factor. On the contrary, beta 2GPI preincubated with phospholipids significantly inhibited TG triggered with low TF concentration, suggesting an effect on the intrinsic pathway. This indicates that native beta 2GPI in circulation obtains its anticoagulant activity in the presence of anionic phospholipids such as activated blood cells thereby serving as an inhibitory modulator in hemostasis.