Raphaël Marlu

Effect of non-specific reversal agents on anticoagulant activity of dabigatran and rivaroxaban: a randomised crossover ex vivo study in healthy volunteers.

The 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.

Optimization of a Type III Secretion System-Based Pseudomonas aeruginosa Live Vector for Antigen Delivery

ABSTRACT 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 (105), but dose enhancement was not associated with a greater immune response. The quorum-sensing-deficient strain was the most efficient at a mild dose (106), but this dose was close to the toxic dose. Combination of both mutations conferred the highest efficiency at an elevated dose (107), 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.

High-Yield Production of Secreted Active Proteins by the Pseudomonas aeruginosa Type III Secretion System

ABSTRACT 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.

Antithrombin-Independent Effects of Heparins on Fibrin Clot Nanostructure

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.

Optimal epitope composition after antigen screening using a live bacterial delivery vector: application to TRP-2.

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, TRP2125-243, TRP2L125-376 and TRP2S291-376. However, only immunization with TRP2L125-376 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 rapide screening and evaluation of antigens of varying length and epitope composition.

Evaluation of dabigatran, rivaroxaban and apixaban target-specific assays in a multicenter French study

Dabigatran etexilate, rivaroxaban and apixaban (DOACs) are widely used and measurement of their concentration is desirable in certain clinical situations. Target-specific assays are available but limited information exists on their performance especially in their ability to accurately measure low and high concentrations. AIMS To define, in a multicenter study, the precision and accuracy of DOAC measurements in daily practice. METHODS 15 plasma samples (kindly provided by Hyphen-Biomed) spiked with 5 blinded concentrations of dabigatran, rivaroxaban or apixaban (targeted 0-40-100-250-500ng/mL, actual concentrations measured by HPLC-MS/MS), were sent to 30 haemostasis laboratories. DOAC concentration, PT and aPTT were measured once in each sample using local reagents. Interlaboratory precision was determined by its coefficient of variation (CV) and accuracy by its bias. RESULTS 464 DOAC measurements were performed in the 30 laboratories using 4 dabigatran and 5 rivaroxaban/apixaban calibrated assays on 3 analysers. Inter-laboratory CVs were below 18% for concentrations ≥100ng/mL, and higher for concentrations ~40ng/mL; biases were below 8% for all drugs and concentrations. In DOAC-free samples, concentrations were all below the lower limit of quantification except for one value (dabigatran: 35ng/mL). Depending on the concentrations, significant differences were found between reagents in rivaroxaban and apixaban concentration values. PT and aPTT ratios displayed a low sensitivity to apixaban. CONCLUSION Our results suggest that calibrated DOAC assays allow the reliable measurement of a wide range of drug concentrations, even though improvement of their performances is necessary, especially for measuring low concentrations.

Letter by Marlu et al Regarding Article, “Reversal of Rivaroxaban and Dabigatran by Prothrombin Complex Concentrate: A Randomized, Placebo-Controlled, Crossover Study in Healthy Subjects”

To the Editor: Eerenberg et al1 recently published a very interesting article on the reversal of rivaroxaban and dabigatran anticoagulation by prothrombin complex concentrate (PCC). In their study, the authors reported that a single bolus of a high dose of PCC (50 IU/kg) was responsible for the normalization of both rivaroxaban-induced prothrombin time (PT) and endogenous thrombin potential (ETP) alteration and therefore for a complete reversion of the anticoagulation effect. These results concerning the reversion efficacy based on correction of hemostatic …

Gla-domainless factor Xa: molecular bait to bypass a blocked tenase complex

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.

Reducing Fibrinogen and Factor XIII Using Double-Filtration Plasmapheresis for Antibody-Mediated Rejection: Predictive Models

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.