Search

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.

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

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 (

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...

International audience

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.

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/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.

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; 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.

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.