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International audience; IntroductionCerebral venous sinus thrombosis (CVST) is a rare disease with highly variable clinical presentation and outcomes. Clinical studies suggest a role of inflammation and coagulation in CVST outcomes. The aim of this study was to investigate the association of inflammation and hypercoagulability biomarkers with CVST clinical manifestations and prognosis.MethodsThis prospective multicenter study was conducted from July 2011 to September 2016. Consecutive patients referred to 21 French stroke units and who had a diagnosis of symptomatic CVST were included. High-sensitivity C-reactive protein (hs-CRP), neutrophil-to-lymphocyte ratio (NLR), D-dimer, and thrombin generation using calibrated automated thrombogram system were measured at different time points until 1 month after anticoagulant therapy discontinuation.ResultsTwo hundred thirty-one patients were included. Eight patients died, of whom 5 during hospitalization. The day 0 hs-CRP levels, NLR, and D-dimer were higher in patients with initial consciousness disturbance than in those without (hs-CRP: 10.2 mg/L [3.6-25.5] vs 23.7 mg/L [4.8-60.0], respectively; NLR: 3.51 [2.15-5.88] vs 4.78 [3.10-9.59], respectively; D-dimer: 950 μg/L [520-2075] vs 1220 μg/L [950-2445], respectively). Patients with ischemic parenchymal lesions (n = 31) had a higher endogenous thrombin potential5pM than those with hemorrhagic parenchymal lesions (n = 31): 2025 nM min (1646-2441) vs 1629 nM min (1371-2090), respectively (P = .0082). Using unadjusted logistic regression with values >75th percentile, day 0 hs-CRP levels of >29.7 mg/L (odds ratio, 10.76 [1.55-140.4]; P = .037) and day 5 D-dimer levels of >1060 mg/L (odds ratio, 14.63 [2.28-179.9]; P = .010) were associated with death occurrence.ConclusionTwo widely available biomarkers measured upon admission, especially hs-CRP, could help predict bad prognosis in CVST in addition to patient characteristics. These results need to be validated in other cohorts.

Background Double-filtration plasmapheresis (DFPP), a selective therapeutic apheresis, can deplete pathogenic antibodies/substances, but also important coagulation factors. Aim To determine if the use of a separator filter with different characteristics (CascadefloEC-50 W) as compared to the reference filter (PlasmafloOP-08 W) is as efficient in terms of immunoglobulin loss, but can reduce coagulation factor losses and have similar tolerability. Patients/methods This is a single-center prospective study including 14 patients divided into two groups (7 each): that is, group1 = CascadefloEC-50 W and group2 = PlasmafloOP-08 W. We measured immunoglobulins, lipid profiles, blood-cell counts, hemostasis (prothrombin time, activated partial thromboplastin time), coagulation factors, and natural anticoagulants at before and after the first DFPP-session. Results In group 1, the loss of coagulation factors was significantly reduced as compared to group 2 for proteins with a molecular weight of >150 kDa: there was, respectively, an average decrease of 70% vs 31% for fibrinogen (P = 0.004), 66% vs 21% for factor V (P = 2.16e-07), 60% vs 32% for factor XI (P = 6.96e-06), 75% vs 17% for XIII-antigen (P = 0.0002), and 47% vs 0% for VWF-antigen(P = 0.02). The decrease in post-session IgG was, on average, 45% in group 1 and 50% in group 2 (P = 0.13). Those results remained significant even when adjusted to the treated-plasma volume and the pre-DFPP factor values. Conclusion DFPP, using a CascadefloEC-50W as a first-filter, reduces efficiently IgGs similarly to PlasmafloOP-08W but spares clotting factors.

Gla-domainless factor Xa (GD-FXa) was proposed as a trap to the endogenous anticoagulant Tissue Factor Pathway Inhibitor (TFPI) to restore thrombin generation in hemophilia. Using computational chemistry and experimental approaches, we previously showed that S195A GD-FXa also binds TFPI and restores ex vivo coagulation in hemophilia plasmas.To design a GD-FXa variant with improved anti-TFPI activity and identify suitable sites for mutagenesis, we performed molecular dynamics simulations. The calculations identified residues R150FXa and K96FXa as cold-spots of interaction between GD-FXa and the K2 domain of TFPI. In the three-dimensional model, both residues are facing TFPI hydrophobic residues and are thus potential candidates for mutagenesis into hydrophobic residues to favor an improved protein-protein interaction.Catalytically inactive GD-FXa variants containing the S195A mutation and additional mutations as K96Y, R150I, R150G and R150F were produced to experimentally confirm these computational hypotheses. Among these mutants, the R150FFXA showed increased affinity for TFPI as theoretically predicted, and was also more effective than S195A GD-FXa in restoring coagulation in FVIII deficient plasmas. Moreover, the R150 mutants lost interaction with antithrombin, which is favorable to extend their half-life.

BACKGROUND Fibrinogen reconstitution after therapeutic apheresis has been poorly studied. Apheresis modalities, for example, plasma exchange (PE), double-filtration plasmapheresis (DFPP), or selective immunoadsorption (IA), may have different impacts. METHODS We retrospectively investigated therapeutic apheresis sessions performed at our center across four modalities (PE, DFPP, and IA with or without plasma filtration). Fibrinogen levels were assessed at the beginning and end of each apheresis session, and immediately before the subsequent session. We adjusted measurements on hematocrit values to account for hemoconcentration. RESULTS Between January 10, 2016 and March 2, 2020, we included 90 patients for a total of 754 apheresis sessions (PE: 35; DFPP: 351; IA only: 109; IA + plasma filtration: 259). Each patient received a median of five sessions (1Q 3; 3Q 9); median plasma volume treated was 5.5 L (1Q 4.3 L; 3Q 7.0 L). Within a session, DFPP and PE induced a significantly greater depletion of fibrinogen than both IA modalities, even after adjustment for the treated plasma volume. Median fibrinogen reconstitution was 0.8 (0.4-1.2) g/L (median time between sessions: 38 hours). In multivariate analysis, fibrinogen reconstitution was significantly associated with intersession time (+0.66 g/L/log-hour P

Coronavirus disease 2019 (COVID-19) is associated with a high risk of thrombotic events, particularly in critically ill patients. Anticoagulants at higher than standard thromboprophylaxis doses have been advocated by our group [...].

BACKGROUND Plasmapheresis can deplete pathogenic antibodies and allow ABO- and/or HLA-incompatible transplantation. AIM To determine the impacts of three modalities of plasmapheresis (centrifugal plasmapheresis [cTPE], single-filtration plasmapheresis [mTPE], double-filtration plasmapheresis [DFPP]) on hemostasis parameters and thrombin generation. MATERIALS/METHODS Prospective, comparative study on 21 patients that received three modalities of plasmapheresis (7 patients/group). Hemostasis (prothrombin time [PT], activated partial thromboplastin time [aPTT], procoagulant factors and natural anticoagulants) were measured before and after the first plasmapheresis session. Thrombin generation was also assessed in platelet-poor plasma using an STA-Genesia (Stago) analyzer and Thromboscreen reagents (Stago) in 4-5 patients from each group. RESULTS Both cTPE and mTPE resulted in high decreases in proteins, whatever their molecular weights. Median post/pre ratios were 0.27 to 0.55 for cTPE for most proteins (except FVIII [0.64] and VWF [0.57]). Median post/pre-ratios of mTPE were 0.28 to 0.56 for all proteins. DFPP decreased high-molecular-weight proteins (fibrinogen, FV, FVIII, FXI, VWF) and proteins strongly bound to large molecules (protein SandTFPI). Median post/pre ratios with cTPE and mTPE were similar to DFPP for fibrinogen and FXIII. Regarding thrombin generation, cTPE and mTPE did not significantly modify endogenous thrombin potential (ETP) and DFPP induced a slight decrease in ETP (median post/pre ratio at 0.73) in the absence of thrombomodulin. ETP inhibition by thrombomodulin was decreased for all procedures. CONCLUSIONS DFPP depleted high molecular-weight proteins in contrast to cTPE and mTPE, which significantly decreased all proteins. Regarding thrombin generation, depletion of procoagulant factors was counterbalanced by a decrease in some natural anticoagulants whatever plasmapheresis method used; with all methods, fibrinogen and FXIII were highly depleted.

INTRODUCTION ABO- or HLA-incompatible kidney transplantation is possible thanks to pretransplant antibody-depletion achieved by extracorporeal-treatment modalities. These methods induce depletion of some plasma proteins and may also impact on proteins involved in hemostasis. METHODS To determine the impact of one session of immunoadsorption (IA) alone or combined with membrane filtration (MF) on clotting factors and natural anticoagulants, we performed a prospective, observational study on 13 patients waiting for HLA-/ABO-incompatible kidney transplants. Plasma hemostasis parameters were measured before and immediately after a first session of IA alone in six patients and of IA + MF in seven patients. RESULTS IA alone induced depletion of fibrinogen and factor XIII (FXIII) whereas IA + MF caused greater depletion of all high-molecular-weight hemostatic proteins (fibrinogen, FV, FVIII, FXI, FXIII, von-Willebrand factor [VWF]). After an IA session, median reductions were 30% for fibrinogen and 43% for FXIII compared to baseline values. After a session of IA + MF, median decreases were 70% for fibrinogen, 54% for FV, 56% for FVIII, 37% for FXI, 78% for FXIII, and 62% for VWF. Noticeably, levels of low-molecular-weight factors (

Introduction: Cerebral venous thrombosis (CVT) is a rare disease with highly variable clinical presentation and outcome. Etiological assessment may be negative. The clinical and radiological presentation and evolution can be highly variable. The mechanisms involved in this variability remain unknown.Objective: The aim of this multicenter French study registered on ClinicalTrials.gov (NCT02013635) was therefore to prospectively recruit a cohort of patients with cerebral venous thrombosis (FPCCVT) in order to study thrombin generation and clot degradation, and to evaluate their influence on clinical radiological characteristics. The first part of the study was to compare our cohort with a reference cohort.Methods: This prospective, multicenter, French study was conducted from July 2011 to September 2016. Consecutive patients (aged >15 years) referred to the stroke units of 21 French centers and who had a diagnosis of symptomatic CVT were included. All patients gave their written informed consent. The diagnosis of CVT had to be confirmed by imaging. Clinical, radiological, biological, and etiological characteristics were recorded at baseline, at acute phase, at 3 months and at last follow-up visit. Thrombophilia screening and the choice of treatment were performed by the attending physician. All data were compared with data from the International Study on CVT published by Ferro et al.Results: Two hundred thirty-one patients were included: 117 (50.6%) had isolated intracranial hypertension, 96 (41.5%) had focal syndrome. During hospitalization, 229 (99.1%) patients received anticoagulant treatment. Median length of hospital stay was 10 days. Five patients died during hospitalization (2.2%). At 3 months, 216 patients (97.0%) had follow-up with neurological data based on an outpatient visit. The mean duration of antithrombotic treatment was 9 months, and the mean time to last follow-up was 10.5 months. At the end of follow-up, eight patients had died, and 26 patients were lost to follow-up. At least one risk factor was identified in 200 patients.Conclusions: We demonstrated that the FPCCVT cohort had radiological, biological, and etiological characteristics similar to the historical ISCVT cohort. Nevertheless, the initial clinical presentation was less severe in our study probably due to an improvement in diagnostic methods between the two studies.

Laboratory Method to Detect VITT The rare vaccine-induced immune thrombotic thrombocytopenia that may follow adenovirus-based Covid-19 vaccination resembles heparin-induced thrombocytopenia, but ra...

Nearly 18% of patients on a waiting list for kidney transplantation (KT) are highly sensitized, which make access to KT more difficult. We assessed the efficacy and tolerance of different techniques (plasma exchanges [PE], double-filtration plasmapheresis [DFPP], and immunoadsorption [IA]) to remove donor specific antibodies (DSA) in the setting of HLA-incompatible (HLAi) KT. All patients that underwent apheresis for HLAi KT within a single center were included. Intra-session and inter-session Mean Fluorescence Intensity (MFI) decrease in DSA, clinical and biological tolerances were assessed. A total of 881 sessions were performed for 45 patients: 107 DFPP, 54 PE, 720 IA. The procedures led to HLAi KT in 39 patients (87%) after 29 (15–51) days. A higher volume of treated plasma was associated with a greater decrease of inter-session class I and II DSA (p = 0.04, p = 0.02). IA, PE, and a lower maximal DSA MFI were associated with a greater decrease in intra-session class II DSA (p &lt, 0.01). Safety was good: severe adverse events occurred in 17 sessions (1.9%), more frequently with DFPP (6.5%) p &lt, 0.01. Hypotension occurred in 154 sessions (17.5%), more frequently with DFPP (p &lt, 0.01). Apheresis is well tolerated (IA and PE &gt, DFPP) and effective at removing HLA antibodies and allows HLAi KT for sensitized patients.

L’impact d’un traitement Intercept sur la capacite hemostatique plaquettaire reste debattu. L’etude par viscoelastometrie (VE) quantifie les proprietes mecaniques plaquettaires dans un caillot de sang total. Objectifs Etude fonctionnelle plaquettaire par VE en condition thrombocytopenique creee in vitro puis application a l’etude ex vivo de concentres plaquettaires (EFFIPAP : plaquettes traitees Intercept vs. non traitees ; transfusion pour thrombocytopenie d’une hemopathie maligne – Garban, 2018). Methodes Avec VE-ROTEM, la composante plaquettaire est obtenue par la difference EXTEM – FIBTEM des elasticites maximales (MCE) derivees du parametre MCF (Solomon, 2015). Du sang de 20 donneurs sains consentants est fractionne pour reconstitution autologue : culot erythrocytaire avec plasma et plaquettes lavees afin d’obtenir des numerations plaquettaires (NP) normales et basses, jusqu’a 20 G/L. Des prelevements sont effectues avant et apres transfusion : etude ancillaire EFFIPAP-bio (Dijon, Grenoble, Lille). Resultats (1) Avec la relation etablie entre MCE et NP un intervalle de reference est defini sur l’etendue des NP ( Fig. 1 ). L’incubation avec aspirine a un effet faible mais statistiquement significatif : l’approche est sensible a un defaut fonctionnel mineur sur l’etendue des NP. (2) Aucune difference des MCE entre les types de concentres n’est trouvee, en prenant en compte la NP ( Figure 1 , Figure 2 ). Les rendements transfusionnels sont en accord avec ceux de EFFIPAP. Conclusion L’etude fonctionnelle plaquettaire par VE (resultat exprime en MCE) est faisable en condition thrombocytopenique. Les resultats d’EFFIPAP-bio n’indiquent pas une alteration fonctionnelle due a Intercept.

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We have previously developed a fibrin structural assay dedicated to purified fibrinogen-thrombin system. Here, we extend the pertinence of this test, called Fibrinography, to tissue factor-triggered plasma coagulation. We show that Fibrinography determines quantitatively the structure of fibrin fibers in plasma with an excellent reproducibility. We compare this assay with the commonly used single wavelength turbidity method, showing that the latter is not a proper structural assay, but determines essentially the fibrinogen content in plasma. In addition, we also show, in model plasmas, that Fibrinography is able to discriminate normal and hypocoagulant plasmas, and even between hypercoagulant plasmas. Therefore, Fibrinography, by measuring the final step of the coagulation cascade, may be used to evaluate patients’ plasma in hypo- or hypercoagulant diseases.

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Ticagrelor is a direct and reversible competitive antagonist of the P2Y12 receptor and inhibits platelet activation. Although adverse bleeding is common, fatal intoxication has never been documented.A 47-year-old man died from a severe cerebral hemorrhage secondary to a fall and cranial trauma 4 d after the massive intake of ticagrelor. Iterative platelet transfusions did not improve his condition. Toxicological analyses by liquid chromatography tandem mass spectrometry (LC-MS/MS) revealed high plasma concentrations of ticagrelor (3343 µg/L) and its active metabolite AR-C124910XX (656 µg/L) 10 h after intake. The approximate ingested dose was extrapolated to 1677 mg. Assessment of ADP-induced platelet aggregation and platelet Vasodilator Stimulated Phosphoprotein phosphorylation (VASP), 2 and 3 d after admission, respectively, showed the persistence of platelet inhibition.To the best of our knowledge, no prior fatal cases have been reported and documented with both ticagrelor and AR-C124910XX concentrations. Our findings highlight the need for a specific antidote to manage such complications resulting from ticagrelor overdose.

Donor-specific alloantibodies (DSAs) cause kidney-allograft loss in chronic antibody-mediated rejection (CAMR). Treatment relies on blocking antibody-producing cells and removing DSAs by apheresis: e.g., double-filtration plasmapheresis (DFPP).To determine the impact of DFPP (6 or 8 sessions/patient) on clotting factors and natural anticoagulants, and on thrombin generation, we performed a prospective and observational study in five CAMR kidney-transplant patients who received DFPP plus rituximab therapy. Thrombin generation was performed in poor platelet plasma (PPP) with 5 pM tissue factor without and with 2 nM recombinant human thrombomodulin.After the first DFPP session, median levels of high molecular-weight proteins (fibrinogen, FV, FVIII, FXI, FXIII, von Willebrand factors and α2-MG) decreased significantly to50% of baseline values, whereas levels of low molecular-weight factors (100 kDa) were not significantly modified, except for protein S and TFPI. Of note, binding-protein (BP) S, i.e., C4BP, was significantly decreased. Over the course of successive DFPP sessions, both high and lower molecular-weight proteins (100 kDa) with longer half-lives (2 days, prothrombin and factor XII) were significantly decreased. DFPP also highly affected thrombin generation in the absence of thrombomodulin but not significantly in the presence of thrombomodulin. After the first DFPP session, mean endogenous thrombin potential (ETP) and peak thrombin (PH) significantly decreased when the thrombin generation assay was performed without thrombomodulin (respectively, 1084 nM·min for ETP and 210 nM for PH after the first DFPP session compared to 1616 nM·min and 264 nM at baseline). In the presence of thrombomodulin, there was only a slight decrease in ETP and PH (respectively 748 nM·min, and 172 nM after the first DFPP session compared to 822 nM·min and 179 nM at baseline). After the last session, median ETP and PH decreased respectively to 646 nM·min and 143 nM without thrombomodulin, and, to 490 nM·min and 117 nM with thrombomodulin.DFPP significantly removed high molecular-weight proteins from the haemostatic system and profoundly decreased levels of protein S and TFPI. Overall thrombin-generation balance was only moderately affected in the presence of thrombomodulin. Nevertheless, high depletion of fibrinogen, FXIII and Von Willebrand Factor may expose patients to an increased risk of bleeding.

Introduction Rotational Thromboelastometry (ROTEM) is a point of care method used to monitor coagulation during surgery and to guide transfusion strategies in patients presenting with severe bleeding. The aim of our study was to determine the impact of four direct oral anticoagulants (DOACs) on 3 commonly used ROTEM tests. Methods Whole blood samples from 20 healthy donors were spiked in vitro with apixaban, edoxaban, rivaroxaban or dabigatran at 5 different plasma concentrations (0-1000 ng/mL). EXTEM, INTEM and FIBTEM tests were systematically performed. Results There was a linear relationship between the increase in clotting time (CT) and plasma DOAC concentrations in both the EXTEM and INTEM tests. We found that the DOAC concentration required to double EXTEM CT was 1042 ± 225 ng/mL for apixaban, 134 ± 38 ng/mL for edoxaban, 176 ± 26 ng/mL for rivaroxaban and 284 ± 73 ng/mL for dabigatran. INTEM CT was less sensitive than EXTEM CT whatever the anticoagulant. EXTEM CT was above the normal range for 5 of 5 spiked samples when the plasma concentrations were ~1000 ng/mL for apixaban, ~100 ng/mL for edoxaban, ~200 ng/mL for rivaroxaban and ~200 ng/mL for dabigatran. Maximum Clot Firmness in EXTEM, INTEM and FIBTEM tests was not affected whatever the DOAC or its concentration. Conclusion This study found a DOAC dose-dependent increase in ROTEM CTs. ROTEM tests were only poorly impacted by low levels of edoxaban, rivaroxaban or dabigatran. Apixaban had only a low effect even at high concentrations.

Background Calibrated Automated Thrombography (CAT) has been widely used to assess in vitro thrombin generation as an informative intermediary phenotype of coagulation. Interlaboratory exercises have documented a worrisome poor reproducibility. There are some data on the normalisation with an appropriate external reference plasma (RP). This multicentre study of the French-speaking CAT Club aimed at providing further evidence for the usefulness of such a normalisation. Materials and Methods Lyophilised aliquots of a RP along with 3 plasmas (P1 = normal; P2 = hypo-; P3 = hypercoagulable) were sent to 34 laboratories (corresponding to 38 instruments). CAT was studied using 1 and 5 pM tissue factor and other dedicated reagents. Normalisation with the local RP in use in the laboratory could also be performed. Interlaboratory CVs were calculated for each plasma before and after normalisation. Results Regarding endogenous thrombin potential, a good discrimination between the 3 plasmas was achieved in all laboratories but there was no overlap after normalisation only. CVs were generally not reduced with the use of local RP but were generally improved with normalisation using the external RP, often becoming lower than 10%. Regarding P2 however, the benefit of normalisation was poor, and there were analytical difficulties as well, some laboratories being unable to get a useable signal. Conclusions We confirm that normalisation of CAT results with a suitable external RP is useful in “real life” practice as it often permits an acceptable level of interlaboratory variability. In case of frank hypocoagulability, further improvements are required to get reliable, potentially clinically relevant results.

Background/Aims: Antibody-mediated rejection (AMR) is related to circulating donor-specific anti-human leukocyte antigen alloantibodies (DSAs). DSAs can be removed by apheresis, for example, double-filtration plasmapheresis (DFPP). However, DFPP removes some clotting factors (fibrinogen and factor XIII [FXIII]). Methods: This was a prospective trial including 6 DSA-mediated AMR kidney transplant recipients. Patients received 2 cycles of 3–4 consecutive DFPP sessions followed by 1 injection of rituximab (break of 4–5 days between the 2 cycles). We monitored fibrinogen and FXIII levels before and after each session of DFPP. Results: Overall, fibrinogen and FXIII levels were significantly decreased after each session, and were significantly reduced between the very first and very last sessions. In addition, we established a model that predicted fibrinogen and FXIII values after each session and after 2 cycles. Conclusion: We established a model in order to predict fibrinogen and FXIII depletion after DFPP sessions; it may help clinicians supplement fibrinogen and/or FXIII when appropriate.

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Objective Ticagrelor is a direct and reversible competitive antagonist of the P2Y12 receptor and inhibits platelet activation. From 2013 to 2016, 1027 ticagrelor-affiliated deaths were reported from 59 countries to the Food and Drug Administration and the trend is on the rise [1] . In order to highlight ticagrelor-related risk of hemorrhage, we report herein a documented lethal intoxication and a disproportionality analysis using World Heath Organization (WHO) VigiBase®. Case history A 47-year-old man died from a severe cerebral hemorrhage secondary to a fall and cranial trauma 4 days after an assumed massive ticagrelor intake. Iterative platelet transfusions did not improve his condition. Methods Ticagrelor and its active metabolite AR-C124910XX plasma levels were determined by LC-MS/MS during patient hospitalization, using an Sciex API 3200 QTRAP mass spectrometer in negative electro-spray ionization mode [2] . Pharmacokinetics parameters were calculated using PK Solver® 2.0. We performed a disproportionality analysis using VigiBase®, the WHO pharmacovigilance database. Up to December 2018, more than 18 million Individual Case Safety Reports (ICSRs) have been collected from 131 countries. We extracted all the ICSRs with the MedDRA (version 21.1) preferred terms “cerebral hemorrhage” associated with antiplatelet agents, recorded from December 9, 1970 to December 16, 2018. Results LC-MS/MS toxicological analyses revealed high plasma concentrations of ticagrelor (3343 μg/L) and AR-C124910XX (656 μg/L) 10 h after the intake and remained quantifiable until patient death. Ticagrelor and AR-C124910XX elimination half-lives were 12.5 h and 32.8 h, respectively (normally 7.3 and 8.6 h). The ingested dose was therefore extrapolated to 1677 mg, which was a massive overdose (recommended dosage: 90 mg bid). In addition, the relationship between the use of ticagrelor and the occurrence of cerebral hemorrhage was assessed by calculating the reporting odds ratio (ROR) associated with its 95% confidence interval (CI) in a case-non-case model compared to other drugs. ROR value was significant (95% CI: lower boundary > 1 and ≥ 3 cases): ticagrelor (n = 229, ROR = 8,01, 95% CI: 7,03–9,13), confirming that ticagrelor treatment is associated with intracerebral hemorrhage. Conclusion Further studies are needed to better understand ticagrelor pharmacokinetics in cases of massive intoxication. These findings emphasized the need for a specific antidote to manage such complications resulting from ticagrelor overdose, but also the contribution of LC-MS/MS analysis to confirm a ticagrelor intoxication.

Objective— Because of the widespread clinical use of heparins, their effects on the enzymatic cascade are very well known. In contrast, little is known about the direct effect of heparins on the nanostructure of fibrin fibers, even though this nanostructure plays a major role in the mechanical strength and lysis of clots. This lack of reliable data can be correlated with the lack of a nonintrusive, quantitative method to determine this structure. We recently developed such a method that allows the simultaneous determination of the average fiber radius and the protein content using spectrometric data. In this study, we assessed the nanostructure of fibrin in a system composed of human thrombin and fibrinogen. Methods and Results— We provide quantitative evidence showing that both unfractionated heparin and low molecular weight heparin directly alter the nanostructure of fibrin fibers independent of their other actions on the coagulation cascade; as expected, the pentasaccharide fondaparinux has no effect. Conclusion— Our results show that in addition to the effect of heparin on the coagulation cascade, modifications of the fibrin nanostructure may also contribute to improved fibrinolysis.

SummaryThe new anticoagulants dabigatran and rivaroxaban can be responsible for haemorrhagic complications. As for any anticoagulant, bleeding management is challenging. We aimed to test the effect of all putative haemostatic agents on the anticoagulant activity of these new drugs using thrombin generation tests. In an ex vivo study, 10 healthy white male subjects were randomised to receive rivaroxaban (20 mg) or dabigatran (150 mg) in one oral administration. After a two weeks washout period, they received the other anticoagulant. Venous blood samples were collected just before drug administration (H0) and 2 hours thereafter. Reversal of anticoagulation was tested in vitro using prothrombin complex concentrate (PCC), rFVIIa or FEIBA® at various concentrations. Rivaroxaban affects quantitative and kinetic parameters, including the endogenous thrombin potential (ETP-AUC and more pronouncedly the thrombin peak), the lag-time and time to peak. PCC strongly corrected ETP-AUC, whereas rFVIIa only modified the kinetic parameters. FEIBA corrected all parameters. Dabigatran specially affects the kinetics of thrombin generation with prolonged lag-time and time to peak. Although PCC increased ETP-AUC, only rFVIIa and FEIBA corrected the altered lag-time. For both anticoagulants, lower doses of FEIBA, corresponding to a quarter to half the dose usually used, have potential reversal profile of interest. In conclusion, some non-specific reversal agents appear to be able to reverse the anticoagulant activity of rivaroxaban or dabigatran. However, clinical evaluation is needed regarding haemorrhagic situations, and a meticulous risk-benefit evaluation regarding their use in this context is required.

International audience; Immunotherapeutic approaches, based on the generation of tumor-specific cytotoxic T-lymphocytes (CTL), are currently emerging as promising strategies of anti-tumor therapy. The potential use of attenuated bacteria as engineered vectors for vaccine development offers several advantages, including the stimulation of innate immunity. We developed an attenuated live bacterial vector using the type III secretion system (TTSS) of Pseudomonas aeruginosa to deliver in vivo tumor antigens. Using an inducible and rapid expression plasmid, vaccination with several antigens of different length and epitope composition, including TRp-2, gp100 and MUC18, was evaluated against glioma tumor cells. We observed similar CTL immunity and T-cell receptor (TCR) repertoire diversity with the vaccines, TRP2(125-243), TRP2L(125-376) and TRP2S(291-376). However, only immunization with TRP2L(125-376) induced significant anti-tumor immunity. Taken together, our data indicate the importance of the epitopes composition and/or peptide length of these peptides for inducing cytotoxic T-lymphocyte (CTL) mediated immunity. Characteristics that consistently improved anti-tumor immunity include: long peptides with immunodominant and cryptic CD8(+) epitopes, and strong CD4(+) Th epitopes. Our bacterial vector is versatile, easy-to-use and quick to produce. This vector is suitable for rapid screening and evaluation of antigens of varying length and epitope composition.

During the last few years, the use of type III secretion system-based bacterial vectors for immunotherapy purposes has been assessed in various applications. We showed that a type III secretion-based Pseudomonas aeruginosa vector delivering the ovalbumin (OVA) antigen induced an efficient specific CD8 + T-lymphocyte immune response against OVA-expressing cells. Because of the intrinsic toxicity of the vector, further virulence attenuation was needed. Therefore, we explored the effects of the deletion of quorum-sensing genes and the aroA gene toward toxicity and efficiency of the vector strain. The aroA mutation in our strain (making the strain auxotrophic for aromatic amino acids) conferred a strikingly reduced toxicity, with the bacterial lethal dose being more than 100 times higher than that of the parental strain. The quorum-sensing gene mutation alone was associated with a slightly reduced toxicity. In a prophylactic OVA-expressing melanoma mouse model, an OVA-delivering aroA -deficient mutant was the most efficient at a low dose (10 5 ), but dose enhancement was not associated with a greater immune response. The quorum-sensing-deficient strain was the most efficient at a mild dose (10 6 ), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at an elevated dose (10 7 ), in agreement with the known negative effects of quorum-sensing molecules upon T-cell activation. In conclusion, we have obtained a promising immunotherapy vector regarding toxicity and efficiency for further developments in both antitumor and anti-infectious strategies.

Background: Hemophilia is caused by deficiencies in coagulation factor VIII or IX, resulting in direct blockade of the intrinsic tenase complex and indirect blockade of the extrinsic tenase complex which is rapidly inhibited upon binding of factor Xa to tissue factor pathway inhibitor (TFPI). We evaluated the ability of Gla-domainless factor Xa (GDXa), a truncated form of factor Xa devoid of procoagulant properties, to bind to TFPI and to alleviate the physiological inhibition of the extrinsic tenase. Design and Methods: In the first part of this work, we evaluated the ability of GDXa to restore coagulation in plasmas from hemophilia A and B patients without and with inhibitors, using a thrombin generation assay triggered by a low concentration of tissue factor. We then compared its efficacy to generate thrombin to depletion of antithrombin or TFPI by specific antibodies. Finally, we compared the kinetics of neutralization of factor Xa and GDXa by antithrombin and TFPI. In the second part of this work, we realized an in silico study of the interactions between factor Xa heavy chain and TFPI. The aim was to detect unfavorable interactions and to identify amino-acid candidates for mutagenesis in order to increase affinity for TFPI. Taking into account the results of this in silico study, we produced by genic engienering different molecular variants of GDXa (R138F, R138G, R138I) where Arg138 was substituted by site directed mutagenesis. Proteins were produced in HEK293E cells. We tested dialyzed cell culture supernatants containing each variant to restore thrombin generation in plasmas from severe hemophilia patients. Results: In the first part of this work, we showed that GDXa was able to restore thrombin generation in plasma samples from hemophiliacs. This effect was observed for plasma from hemophilia A patients without or with inhibitors and for plasma from hemophilia B patients. GDXa had a lower affinity than factor Xa for TFPI whereas the affinities of both proteins for antithrombin were similar. Finally, despite a short half-life in plasma, the effect of GDXa on thrombin generation was sustained for at least one hour. In the second part of this work, we produced the different variants R138F, R138G et R138I in HEK293E cells and showed that cell culture supernatants were able to restore thrombin generation in a more efficient way than GDXa. Conclusions: As GDXa was able to restore thrombin generation in plasma from hemophilia patients, our results suggest that it may be an effective alternative to current treatments for hemophilia with or without inhibitors. Results sustained the hypothesis that GDXa coagulant activity is through TFPI binding and competition with factor Xa to bind TFPI resulting in limiting factor Xa-TFPI formation, which is essential for inhibition of extrinsic tenase complex. Furthermore, rational design of GDXa variants based on an in silico study lead to production of proteins whose coagulant activity is increased compared to GDXa."; Introduction : L'hémophilie est une maladie génétique de la coagulation due à un déficit en facteur VIII ou en facteur IX. Ces déficits sont responsables d'un déficit du complexe ténase intrinsèque (VIIIa-IXa). De plus, le complexe ténase extrinsèque (facteur tissulaire - VIIa) est physiologiquement rapidement inhibé par le TFPI lié au facteur Xa. Nous avons évalué la capacité d'une forme tronquée du facteur Xa (GDXa), dépourvue de domaine Gla à se lier au TFPI et à soulager l'inhibition physiologique du complexe ténase extrinsèque. Matériel et Méthodes : Dans une première partie, nous avons évalué la capacité du GDXa à restaurer la génération de thrombine de plasmas d'hémophiles A et B sévères sans et avec inhibiteurs. Nous avons également comparé les profils de génération de thrombine obtenus après addition du GDXa à ceux obtenus en présence d'anticorps neutralisants anti-TFPI ou anti-antithrombine. Enfin, nous avons comparé les cinétiques enzymatiques de neutralisation du facteur Xa et du GDXa par le TFPI et l'antithrombine. Dans une seconde partie, nous avons étudié in silico les interactions entre la chaîne lourde du facteur Xa et le TFPI pour détecter les zones d'interaction défavorables. Cette étude a identifié des acides aminés du facteur Xa qui pourraient être substitués pour optimiser l'interaction avec le TFPI. Les résultats in silico ont orienté nos choix de mutagenèse dirigée pour concevoir différents variants moléculaires du GDXa (R138F, R138G, R138I) où l'arginine 138 est substituée. Ces variants protéiques ont été produits de façon recombinante dans des cellules HEK293E. La capacité des différents variants à restaurer la génération de thrombine de plasmas d'hémophiles a été testée avec les surnageants de culture cellulaires correspondants. Résultats : Dans la première partie, nous avons montré que le GDXa est capable de restaurer la génération de thrombine de plasmas d'hémophiles A et B sans et avec inhibiteurs. Comparativement au facteur Xa, le GDXa montre une affinité moindre pour le TFPI tandis que les affinités du GDXa et du facteur Xa pour l'antithrombine sont identiques. Enfin, malgré une demi-vie courte, l'effet du GDXa sur la génération de thrombine est maintenu pendant au moins une heure. Dans la seconde partie, nous avons produit les différents variants R138F, R138G et R138I en cellules HEK293E et montré que les surnageants de culture cellulaire étaient capables de restaurer la génération de thrombine de plasmas d'hémophiles de façon plus efficace que le GDXa. Conclusion : Comme le GDXa est capable de restaurer la génération de thrombine de plasmas d'hémophiles, nos résultats suggèrent que le GDXa pourrait être une alternative efficace aux thérapeutiques hémostatiques court-circuitantes actuelles chez les hémophiles sans ou avec inhibiteurs. Les résultats obtenus renforcent l'hypothèse que l'activité pro-coagulante du GDXa serait liée à la formation d'un complexe GDXa-TFPI limitant la formation du complexe Xa-TFPI nécessaire à l'inhibition physiologique du complexe ténase extrinsèque. De plus, notre approche rationnelle basée sur une étude in silico visant à augmenter l'affinité du TFPI pour le GDXa a permis de produire différents variants moléculaires du GDXa dont l'activité procoagulante in vitro est augmentée par rapport au GDXa.

International audience; BACKGROUND: Hemophilia is caused by deficiencies in coagulation factor VIII or IX, resulting in direct blockade of the intrinsic tenase complex and indirect blockade of the extrinsic tenase complex which is rapidly inhibited upon binding of factor Xa to tissue factor pathway inhibitor. We evaluated the ability of Gla-domainless factor Xa, a truncated form of factor Xa devoid of procoagulant properties, to bind to tissue factor pathway inhibitor and to alleviate the physiological inhibition of the extrinsic tenase. DESIGN AND METHODS: Using a thrombin generation assay triggered by a low concentration of tissue factor, we evaluated the ability of Gla-domainless factor Xa to restore blood coagulation in plasma from hemophilia A and B patients without and with inhibitors. We then compared its efficacy to generate thrombin to depletion of antithrombin or tissue factor pathway inhibitor by specific antibodies. Finally, we compared the kinetics of neutralization of factor Xa and Gla-domainless factor Xa by antithrombin and tissue factor pathway inhibitor. RESULTS: Gla-domainless factor Xa was able to restore thrombin generation in plasma samples from hemophiliacs. This effect was observed for plasma from hemophilia A patients without or with inhibitors and for plasma from hemophilia B patients. Gla-domainless factor Xa had a lower affinity than factor Xa for tissue factor pathway inhibitor whereas the affinities of both proteins for antithrombin were similar. Finally, despite a short half-life in plasma, the effect of Gla-domainless factor Xa on thrombin generation was sustained for at least 1 hour. CONCLUSIONS: As Gla-domainless factor Xa was able to restore thrombin generation in plasma from hemophilia patients, our results suggest that it may be an effective alternative to current treatments for hemophilia with or without an inhibitor.

The Escherichia coli system is the system of choice for recombinant protein production because it is possible to obtain a high protein yield in inexpensive media. The accumulation of protein in an insoluble form in inclusion bodies remains a major disadvantage. Use of the Pseudomonas aeruginosa type III secretion system can avoid this problem, allowing the production of soluble secreted proteins.

International audience

Abstract 1155 Introduction: Although a great deal is known about the properties and functions of fibrin(ogen), very little is known about the lateral nanostructure of the fibrin fibers composing clots. This structure is important as it controls both the Young and Poisson moduli of the fibers, i.e. the overall mechanical properties of the clot. However, a quantitative characterization of the lateral nanostructure of fibrin fibers is still lacking, as well as its dependence on environmental parameters and anticoagulant molecules. This nanostructure was investigated with the help of Small Angle X-Ray Spectra (SAXS) obtained at the European Synchrotron Radiation Facility, and a newly developped light spectrometry technique (Yeromonahos et al. BioPhys. J., to appear). As we used purified fibrinogen essentially devoid of any coagulation factors, or antithrombin, only the effect of molecular interactions between Heparins, Thrombin and Fibrin(ogen) and their effects on the fibrin fibers nanostructure were observed. Materials: Experiments were performed with purified human thrombin and fibrinogen, purchased respectively from Cryopep and LFB, France. Clots were prepared from mixtures that contained 0.1–1mg/ml fibrinogen and 0.1–1.25 IU/ml thrombin. Polymerization was carried out during 90 minutes in HEPES buffer at 300 mOsm. Results and discussions: At low Fg concentrations ([Fg]=0.1mg/ml), results show that the nanostructure of the clot is quasi cristalline. At [IIa]=1.25 IU/ml, when increasing [Fg] up to 1mg/ml, the average radius increases significantly (52 n to 83 nm) while the number of protofibrils stays constant, in perfect agreement with existing small angle light scattering and dynamic light scattering data. This means that the density of the fibers decreases for increasing [Fg], the fibers looking more and more hollow. In standard conditions ([Fg]=1mg/ml, [IIa]=1.25 IU) the fibers contain half as much protofibrils as would crystalline ones, their inner lateral nanostructure being a fractal as suggested by SAXS spectra (df=1.5). On the other hand, at [Fg]=1mg/ml, decreasing [IIa] from 1.25 to 0.1 UI/ml leads to an increase of the average radius (25%) and density (35%) of the fibers. The effect on the fibrin clot nanostructure of unfractionated heparin (UFH), enoxaparine (LMWH), and pentasaccharide was studied using the light spectrometry method. No significant effect of pentasaccharide was found, indicating that this molecule acts mainly on the enzymatic cascade and not on IIa or on fibrin(ogen). As expected, the UFH has a strong structural effect. At low concentrations ([UHF]>0.1 UI/ml, however, there is a brutal drop in the protein quantity inside the fibers, the clot no longer forming at around 1UI/ml. These effects can be interpreted as the consequences of the formation of thrombin-fibrin monomers-heparin and thrombin-fibrin oligomers-heparin complexes. The effect of LMWH is more progressive and subtle. At low concentrations ([LMWH] In conclusion, we have shown how the reaction parameters ([Fg] and [IIa]) control the nanostructure of the fibrin clot. The addition of UFH, in absence of factor Xa and antithrombin, has a clear structural effect through the inhibition of IIa, probably due to the formation of heparin complexes. Enoxaparin, unlike heparin, has a progressive structural effect that cannot be reduced to an anti-IIa effect. Indeed, it produces clots that are more and more hollow, which should decrease their strength and help for their lysis. Understanding the way these molecules acts may open the way to new molecules altering the structure and mechanical properties of the clot, but not its generation. Disclosures: No relevant conflicts of interest to declare.

La TIH est une complication iatrogène pro-thrombotique d’origine immunologique potentiellement grave compliquant les traitements hépariniques, associant une thrombopénie et parfois une/des thrombose(s) veineuse et/ou artérielle. Il s’agit d’une pathologie complexe où le diagnostic est encore aujourd’hui difficile. Les nombreux tests immunologiques et fonctionnels disponibles ont des performances diagnostiques ainsi que des avantages et inconvénients variables. Les tests fonctionnels requièrent généralement une grande expertise technique. L’objectif dans les années à venir serait d’évoluer vers des tests fonctionnels plus rapides. Le test fonctionnel (HIT Confirm®) que nous avons évalué en cytométrie de flux présente ces avantages mais les résultats obtenus sur un petit effectif de patients ont abouti à plusieurs résultats douteux et des résultats dépendants des plaquettes des témoins testés. Une évaluation à plus large échelle en sélectionnant les plaquettes de donneurs paraît nécessaire. L’étude rétrospective réalisée au cours de cette thèse sur 395 patients suspects de TIH et attachée à décrire les résultats des tests diagnostiques ainsi que la prise en charge thérapeutique anticoagulante illustre la complexité de cette pathologie notamment concernant le choix et la posologie du traitement anticoagulant alternatif et de son monitoring biologique. Les récentes recommandations des sociétés savantes pour le diagnostic et la prise en charge des TIH permettront sans doute l’uniformisation de la prise en charge de cette pathologie.