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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Summary Background We observed that minute amounts of thrombin or the enzyme Russell's viper venom activating factor V (RVV-V) added to plasma strongly diminish the potential of that plasma to generate thrombin after being triggered by tissue factor. Objective To find the mechanism behind this phenomenon. Methods and Results Thrombin generation (TG) initiated by tissue factor (TF) is strongly and dose-dependently inhibited by addition of activated factor V (FVa) or by addition of a factor V activator (thrombin or RVV-V). No inhibition is seen when TG is triggered via the intrinsic pathway or by direct activation of factor X. The effect is independent of proteins C and S and tissue factor pathway inhibitor (TFPI). In factor VII-deficient plasma the effect is seen when it is spiked with recombinant factor VII (FVII) and to a much lesser extent when spiked with recombinant FVIIa. In a purified system, FVa also dose-dependently inhibits the activation of FX by TF/FVII(a). The inhibitory effect is neutralized by antibodies against the light chain of FVa but not by antibodies against the heavy chain. Conclusions Our observations can be explained by assuming that FVa, via its light chain, binds to the complex TF/FVII(a) and prevents it from activating FX. We assume that this mechanism reduces the possibility that thrombin and factor Xa escaping from a wound area into the circulation, together with blood-borne tissue factor, would trigger intravascular coagulation.

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 that VWF crosslinks to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). The aim of our study was to investigate the mechanism of VWF incorporation into a fibrin network and thereby characterize the role of VWF in arterial thrombus growth. We monitored the interactions of von Willebrand factor with fibrin(ogen) using different techniques and enzymes in purified test system at high shear stress. Applying surface techniques, ellipsometry and surface plasmon resonance, we real-time observed that VWF does not bind to either a fibrinogen monolayer or to a polymerized fibrin layer formed on the solid surface. However, 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. Using either Arvin (cleaves FpA but not FpB) or protease III from snake venom Crotalus atrox (cleaves 42 amino acids, where FpB is located) we were able to show that VWF interacts with fibrin monomers. Furthermore, using transmittance measurements we observed that in the presence of VWF the density of the fibrin clot increases suggesting that VWF incorporates into fibrin network in solution. These findings indicate that incorporation of VWF into a fibrin network occurs via the E domain of fibrinogen where FpA and/or FpB were situated. Using a domain deletion mutant deltaC1C2-VWF we demonstrated the involvement of the C1C2 domain of VWF in the binding to fibrin monomers. Our results did not show any interaction of deltaC1C2-VWF with fibrin monolayer in the presence of thrombin. Application of the inhibitor K9-DON against factor XIIIa in the presence of calcium and thrombin slightly decreased the amount of VWF adsorbed on fibrin monolayer but not completely abolished it, 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. Additionally, under high shear conditions, we were able to show that platelets adhere to fibrin only if VWF had been incorporated. Addition of a GPIb blocking antibody almost completely abolished the adhesion of platelets to the fibrin monomers surface with bound VWF. These data provided evidence that the binding of platelets to a fibrin monomer layer under high shear rate is completely dependent on the VWF-GPIb interaction. All experiments were performed in the presence of abciximab suggesting that the GPIIb/IIIa receptor is not involved in this binding. In conclusion, our experiments show that the C1C2 domain 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. Additionally it may help to explain the observed phenotype in types I and III of von Willebrand Disease and the Bernard-Soulier Syndrome. Low amount of (functional) VWF or deficiency in GPIb-IX-V, here lead to disruption in thrombus formation and wound healing. Disclosures No relevant conflicts of interest to declare.

Introduction Recently, the whole blood calibrated automated thrombogram (CAT) was miniaturized enabling the measurement of thrombin generation (TG) in a limited volume (5 µl) of whole blood. Consequently, this approach may be used to determine TG in small lab animals. Zebrafish are readily available test animals for genetic research. However, their small size has been a hurdle in thrombosis and hemostasis research since most assays require large amounts of plasma. In this study we verified the possibility to measure TG in zebrafish using our newly developed miniaturized whole blood assay. Methods For TG, 5 µl of whole blood was mixed with 5 µl of buffer containing a rhodamin-based thrombin-sensitive P2Rho substrate (final concentration (fc) 300 µM). 5 µl of this mixture was put on a paper disk and covered with mineral oil to prevent evaporation. Calibration was done as described previously (Ninivaggi et al. Clin Chem 2012) by adding 5 µl of whole blood to 5 µl of a mixture containing P2Rho (fc 300 µM), a2M-thrombin calibrator (fc 100 nM) and citrate (fc 9,8 mM). Fluorescence was detected with a fluorometer (485/538 nm). Results Due to their limited blood volume, it is impossible to perform both a TG and calibrator measurement on the same fish. Since calibrator measurements performed on blood from different fish demonstrated acceptable variation (CV calibrator slopes < 15%), the average calibrator slope was used for calculations. The calculated TG parameters from 2 independent experiments are depicted in Table 1. TG measured in individual fish showed the same amount of inter-individual variation as in humans. Striking differences with human TG parameters were the short lag time, high peak and high velocity index. Moreover a further analysis of the fibrin network of the clot, by means of scanning electron microscopy (SEM), showed a much denser network composed of thinner fibers compared to humans. Table 1. Thrombin generation parameters and their coefficient of variance (CV) in zebrafish in two independent experiments. Experiment 1(n=4) Experiment 2(n=9) Mean CV Mean CV Peak ETP (nM.min) 575.68 19.69 689.71 24.91 Peak (nM) 1573.16 29.58 1668.05 14.46 Lag time (min) 0.27 0.00 0.49 10.92 Time to peak (min) 1.08 5.30 1.37 11.49 Velocity (nM/min) 4002.33 38.21 3826.35 17.04 Conclusion These results demonstrate the feasibility of measuring TG in whole blood collected from zebrafish. Consequently, zebrafish may be used as a in vivo model to test the effect of (novel) anticoagulant therapeutics on thrombin generation and serve as a model organism for mechanistical research in thrombosis and haemostasis. Disclosures No relevant conflicts of interest to declare.

Background. Thrombin is the key enzyme converting fibrinogen into fibrin and assays based on the formation of thrombin and fibrin are currently often used separately both in research and clinical settings. Recently it was shown that thrombin generation and fibrin formation do not always go hand in hand as factor XIIa independently influences the crosslinking of fibrin and thereby the stability of the clot. Objective. The development and clinical validation of an assay that is able to simultaneously measure the generation of thrombin and formation of fibrin in whole blood. Methods and results. We have redesigned an air-bearing rheometer, based on the plate and cone viscositor, rendering it sensitive enough to measure fibrin formation based on the change in viscosity. A confocal fluorescent camera is placed into the rheometer, allowing the measurement of thrombin generation based on the conversion of a thrombin-sensitive substrate as in the original Calibrated Automated Thrombinography (CAT) method. Furthermore, the method allows the application of laminar flow conditions within the range of 100 s-1 until 1200 s-1. For all variables related to fibrin formation and thrombin generation, the intra- and inter-assay variation were below 10% in platelet-poor plasma (PPP). For both parameters endogenous thrombin potential (ETP) and peak we demonstrated an inverse relationship with increasing flow rates (100-1200 s-1) in PPP. This was in line with fibrin formation as maximum clot strength (based on viscosity) was inversely related to an increase in flow rate. Furthermore, as expected, the addition of increasing concentrations of fibrinogen to defibrinated plasma was accompanied with an increase in ETP, peak and maximum clot strength. Our method proved to be sensitive enough to detect an increase of 0.1 mg/ml fibrinogen. Furthermore, in the presence of tissue plasminogen activator, it allows to study fibrinolysis. The system was clinically validated in 70 patients undergoing cardiothoracic surgery. Blood samples were taken pre-bypass before heparinisation. Besides thrombin generation and fibrin formation measurements, clinical parameters together with the blood loss and the amount of transfusion products were recorded until 24 hours after surgery. Thrombin generation parameters obtained with the specially designed rheometer correlated well with the traditional whole blood CAT, whereas fibrin formation parameters correlated well with the fibrinogen content and the rotational thromboelastometry (ROTEM) parameters. Importantly, when patients were divided into two groups based on the median clot strength determined with the rheometer, a significant difference in perioperative and total blood loss was established. Conclusion. We have technically and clinically validated a new method capable of simultaneously measuring thrombin generation and fibrin formation in plasma and whole blood under continuous flow. Our method proved to be sensitive enough to detect differences in fibrinogen content and revealed an effect of different flow rates on both thrombin generation and clot strength. We hereby have not only combined thrombin generation and fibrin formation in whole blood, but by introducing continuous flow, our method is also one step closer to physiology.Importantly,in contrast to the traditionally used methods, our test applied pre-operatively was found to be predictive for the amount of blood loss during and after cardiothoracic surgery. Disclosures No relevant conflicts of interest to declare.

Introduction The occurrence of venous thromboembolism is strongly age-dependent and has a substantial morbidity and mortality. While the incidence is Objectives By modifying the classic plasma Calibrated Automated Thrombogram (CAT) assay, we were able of measuring TG in whole blood in mice. The objective was to validate this assay in mice blood and to examine the rate and extent TG in a mouse model of premature aging. Methods TG was assayed in 20- to 28-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout, KO) mice and wild-type (WT) littermates. The Bmal1-KO mice have an impaired circadian behavior and demonstrate loss of rhythmicity in the expression of their target genes. They are known to have a reduced lifespan and display symptoms of premature aging. Mice blood samples were taken from the orbital sinus using a capillary anticoagulated with citrate (3.8%). Coagulation and therefore TG was initiated with a solution containing calcium, tissue factor (TF) and a thrombin specific substrate. Varying the TF concentration revealed an optimal of 0.5 pM. At the end of the TG assay, the samples were fixated, prepared for and analysed by scanning electron microscopy (SEM). Results The intra-assay variations (CV%) in mice blood of the endogenous thrombin potential (ETP), peak height, lagtime, time-to-peak and velocity index were 10% or less (n=24). The whole blood TG test showed that Bmal1-KO mice have a significantly (p Conclusions The whole blood TG assay in mice blood revealed to be reproducible and we demonstrated that aging Bmal1-KO mice have a hypercoagulable state when compared with WT mice indicating that a higher thrombotic risk in elderly is partially due to a more procoagulation state. Disclosures: No relevant conflicts of interest to declare.

Abstract 632 Introduction. Diagnosis of the antiphospholipid syndrome (APS) depends on the detection of autoantibodies against phospholipid-bound β2-glycoprotein I (β2GPI), the most prominent antigen in APS. One of the main problems faced is the high variability observed between different commercially available anti-β2GPI assays. Anti-β2GPI antibodies constitute a heterogeneous population, but predominantly antibodies reacting to a cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I of β2GPI are associated with a strong risk to develop thrombosis. b2GPI is present in blood in a native conformation (either circular or S-shaped). After interaction with anionic surfaces it opens up (J-shaped conformation), resulting in exposure of the epitope G40-R43 in domain I. It is therefore important that in diagnostic assays b2GPI is presented in the open conformation enabling antibodies to react with the G40-R43 epitope. We hypothesize that the high variability between different commercial anti-b2GPI ELISA assays arise from variation in exposure of the G40-R43 epitope of b2GPI. Methods. Two patient-derived monoclonal antibodies P2-6 and P1-117 were tested for their reactivity towards β2GPI in different conformations. Using both antibodies, we compared exposure of epitope G40-R43 on β2GPI in five different commercial anti-β2GPI IgG assays. 10 patient samples selected for their low to high positivity towards epitope G40-R43, were tested in two anti-β2GPI IgG assays. Results. Using neutral versus anionic ELISA plates, we have shown that antibody P1-117 specifically reacts with epitope G40-R43, exposed only in the open conformation, while antibody P2-6 recognizes β2GPI irrespective of its conformation (Fig. 1). In one of the tested anti-β2GPI assays, both antibodies showed equal reactivity towards β2GPI, indicating that all the coated β2GPI exposes epitope G40-R43. In this assay, all ten anti-domain I positive patients tested positive. In other assays P1-117 displayed lower reactivity towards β2GPI than P2-6, demonstrating a reduced exposure of G40-R43. Only 3 out of the 10 patients tested positive in such assay, illustrating that a significant number of patients can be falsely assigned negative in assays characterized by a reduced exposure of epitope G40-R43. Conclusions. The exposure of epitope G40-R43 on β2GPI is highly variable in commercial anti-β2GPI assays, and influences the diagnosis of APS. Our results have major implications for the diagnosis of APS, as it provides suitable controls to ensure sufficient exposure of the G40-R43 epitope or suggests the development of alternative assays coating only domain I of β2GPI. Disclosures: No relevant conflicts of interest to declare.

Interleukin (IL)-17 is a pro-inflammatory cytokine in rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). Since interferon (IFN)-gamma inhibits Th17 cell development, IFN-gamma receptor knockout (IFN-gammaR KO) mice develop CIA more readily. We took advantage of this model to analyse the mechanisms of action of IL-17 in arthritis. The role of IFN-gamma on the effector mechanisms of IL-17 in an in vitro system was also investigated.IFN-gammaR KO mice induced for CIA were treated with anti-IL-17 or control antibody. The collagen type II (CII)-specific humoral and cellular autoimmune responses, myelopoiesis, osteoclastogenesis, and systemic cytokine production were determined. Mouse embryo fibroblasts (MEF) were stimulated with IL-17, tumor necrosis factor (TNF)-alpha and the expression of cytokines and chemokines were determined.A preventive anti-IL-17 antibody treatment inhibited CIA in IFNgammaR KO mice. In the joints of anti-IL-17-treated mice, neutrophil influx and bone destruction were absent. Treatment reduced the cellular autoimmune response as well as the splenic expansion of CD11b+ cells, and production of myelopoietic cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-6. IL-17 and TNF-alpha synergistically induced granulocyte chemotactic protein-2 (GCP-2), IL-6 and receptor activator of NFkappaB ligand (RANKL) in MEF. This induction was profoundly inhibited by IFN-gamma in a STAT-1 (signal transducer and activator of transcription-1)-dependent way.In the absence of IFN-gamma, IL-17 mediates its pro-inflammatory effects mainly through stimulatory effects on granulopoiesis, neutrophil infiltration and bone destruction. In vitro IFN-gamma profoundly inhibits the effector function of IL-17. Thus, aside from the well-known inhibition of the development of Th17 cells by IFN-gamma, this may be an additional mechanism through which IFN-gamma attenuates autoimmune diseases.

CXCL12 (stromal cell-derived factor 1) is a unique biological ligand for the chemokine receptor CXCR4. We previously reported that treatment with a specific CXCR4 antagonist, AMD3100, exerts a beneficial effect on the development of collagen-induced arthritis (CIA) in the highly susceptible IFN-γ receptor-deficient (IFN-γR KO) mouse. We concluded that CXCL12 plays a central role in the pathogenesis of CIA in IFN-γR KO mice by promoting delayed type hypersensitivity against the auto-antigen and by interfering with chemotaxis of CXCR4+ cells to the inflamed joints. Here, we investigated whether AMD3100 can likewise inhibit CIA in wild-type mice and analysed the underlying mechanism. Parenteral treatment with the drug at the time of onset of arthritis reduced disease incidence and modestly inhibited severity in affected mice. This beneficial effect was associated with reduced serum concentrations of IL-6. AMD3100 did not affect anti-collagen type II antibodies and, in contrast with its action in IFN-γR KO mice, did not inhibit the delayed type hypersensitivity response against collagen type II, suggesting that the beneficial effect cannot be explained by inhibition of humoral or cellular autoimmune responses. AMD3100 inhibited the in vitro chemotactic effect of CXCL12 on splenocytes, as well as in vivo leukocyte infiltration in CXCL12-containing subcutaneous air pouches. We also demonstrate that, in addition to its effect on cell infiltration, CXCL12 potentiates receptor activator of NF-κB ligand-induced osteoclast differentiation from splenocytes and increases the calcium phosphate-resorbing capacity of these osteoclasts, both processes being potently counteracted by AMD3100. Our observations indicate that CXCL12 acts as a pro-inflammatory factor in the pathogenesis of autoimmune arthritis by attracting inflammatory cells to joints and by stimulating the differentiation and activation of osteoclasts.

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4+CD25+ Treg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of Treg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4+CD25+ T cells in the central and peripheral lymphoid tissues. In addition, CD4+CD25+ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4+CD25+ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for Treg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both Treg cells and accessory cells. Our results demonstrate that the decrease in Treg cell activity in CIA is counter-regulated by endogenous IFN-γ.

Introduction The goal of this study is to analyze the potential immunosuppressive properties of mesenchymal stem cells (MSC) on T cell proliferation and in collagen-induced arthritis (CIA). An additional aim is to investigate the role of interferon-γ (IFN-γ) in these processes. Methods MSC were isolated from bone marrow of DBA/1 wild type and IFN-γ receptor knock-out (IFN-γR KO) mice and expanded in vitro. Proliferation of anti-CD3-stimulated CD4+ T cells in the presence or absence of MSC was evaluated by thymidine incorporation. CIA was induced in DBA/1 mice and animals were treated with MSC by intravenous or intraperitoneal injections of wild type or IFN-γR KO MSC. Results Purity of enriched MSC cultures was evaluated by flow cytometry and their ability to differentiate into osteoblasts and adipocytes. In vitro, wild type MSC dose-dependently suppressed anti-CD3-induced T cell proliferation whereas IFN-γR KO MSC had a significantly lower inhibitory potential. A role for inducible nitric oxide (iNOS), programmed death ligand-1 (PD-L1) and prostaglandin E2 (PGE2), but not indoleamine 2,3-dioxigenase (IDO), in the T cell inhibition was demonstrated. In vivo, neither wild type nor IFN-γR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II. Conclusions Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-γ, MSC do not influence in vivo T cell proliferation nor the disease course of CIA. Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.