Bernard F. Le Bonniec

Ticks are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Hard tick feeding is a slow process—taking up to several days—and necessitates extended control over the host response. The success of the feeding process depends upon injection of tick saliva, which not only controls host hemostasis and wound healing, but also subverts the host immune response to avoid tick rejection that creates a favorable niche for the survival and propagation of diverse tick-borne pathogens. Here, we report on the molecular and biochemical features and functions of an Ixodes ricinus serine protease inhibitor (IrSPI). We characterize IrSPI as a Kunitz elastase inhibitor that is overexpressed in several tick organs—especially salivary glands—during blood-feeding. We also demonstrated that when IrSPI is injected into the host through saliva, it had no impact on tissue factor pathway-induced coagulation, fibrinolysis, endothelial cell angiogenesis or apoptosis, but the protein exhibits immunomodulatory activity. In particular, IrSPI represses proliferation of CD4⁺ T lymphocytes and proinflammatory cytokine secretion from both splenocytes and macrophages. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in I. ricinus feeding.

Increasing number of patients are treated with direct oral anticoagulants (DOAC). An antidote for dabigatran inhibiting thrombin (idarucizumab) is available but no antidote is yet approved for the factor Xa (FXa) inhibitors (xabans). We hypothesized that a complex between Gla-domainless FXa and α₂-macroglobulin (GDFXa-α₂M) may neutralize the xabans without interfering with normal blood coagulation. Purified α₂M was incubated with GDFXa to form GDFXa-α₂M. Affinity of apixaban and rivaroxaban for GDFXa-α₂M was only slightly decreased compared to FXa. Efficacy and harmlessness of GDFXa-α₂M were tested in vitro and in vivo. Stoichiometric excess of GDFXa-α₂M neutralized rivaroxaban and apixaban as attested by clot waveform assay and rotational thromboelastometry, whereas GDFXa-α₂M alone had no effect on these assays. Efficacy and pro-thrombotic potential of GDFXa-α₂M were also assessed in vivo. Half-life of GDFXa-α₂M in C57BL6 mice was 4.9 ± 1.1 minutes, but a 0.5 mg/mouse dose resulted in uptake saturation such that 50% persistence was still observed after 170 minutes. Single administration of GDFXa-α₂M significantly decreased the rivaroxaban-induced bleeding time (p < 0.001) and blood loss (p < 0.01). GDFXa-α₂M did not increase D-dimer or thrombin–antithrombin complex formation, suggesting a lack of pro-thrombotic potential. GDFXa-α₂M is therefore an attractive candidate for xaban neutralization neither pro- nor anticoagulant in vitro as well as in vivo. B.L.B. conceived the study and together with G.J. designed and performed research, analysed data and wrote the manuscript; I.G.-T., V.S., S.G. and P.G. critically discussed the data, revised the manuscript and gave final approval.

Post-thrombotic syndrome, a frequent complication of deep venous thrombosis, can be reduced with early vein recanalization. Pulsed cavitational therapy (PCT) using ultrasound is a recent non-invasive approach. We propose to test the efficacy and safety of high-frequency focused PCT for drug-free thrombolysis (thrombotripsy) in a realistic in vitro model of venous thrombosis. To reproduce venous thrombosis conditions, human whole blood was allowed to clot by stasis in silicone tubes (6 mm internal diameter) at a 30 cm H ₂ O pressure, maintained during the whole experiment. We engineered an ultrasound device composed of dual 2.25 MHz transducers centered around a 6 MHz imaging probe. A therapeutic focus was generated at a 3.2 cm depth from the probe. Thrombotripsy was performed by longitudinally scanning the thrombus at three different speeds: 1 mm s ⁻¹ ( n = 6); 2 mm s ⁻¹ ( n = 6); 3 mm s ⁻¹ ( n = 12). Restored outflow was measured every three passages. Filters were placed to evaluate the debris size. Twenty-four occlusive thrombi, of 2.5 cm mean length and 4.4 kPa mean stiffness, were studied. Flow restoration was systematically obtained by nine subsequent passages (4.5 min maximum). By varying the device’s speed, we found an optimal speed of 1 mm s ⁻¹ to be efficient for effective recanalization with 90 s (three passages). Within 90 s, flow restoration was of 80, 62 and 74% at respectively 1, 2 and 3 mm s ⁻¹ . For all groups, cavitation cloud drilled a 1.7 mm mean diameter channel throughout the clot. Debris analysis showed 92% of debris 200 µ m.

Preterm birth is associated with altered angiogenesis and with increased risk of cardiovascular dysfunction and hypertension at adulthood. We previously demonstrated that in preterm newborns circulating cord blood endothelial progenitor cells (ECFC), responsible for angio/vasculogenesis, are reduced in number and display altered angiogenic properties. Altered angiogenic function was associated with a decreased expression of pro-angiogenic genes, among which the AMOT gene which is a strong positive regulator of angiogenesis. Such dysregulation may be related to epigenetic factors. In this study we analyse the methylation profiling of the AMOT gene during development, through a comparative analysis of the cord blood ECFC of preterm newborns and their term counterpart. We used both cloning-sequencing and pyrosequencing experiments to perform a comparative analysis of the DNA methylation profile of the promoter CpG island of AMOT gene in the cord blood ECFC of 16 preterm newborns (28–35 weeks gestational age-GA) and 15 term newborns (>37 weeks GA). Twenty nine clones (obtained from 2 term newborns) and forty clones (obtained from 3 preterm newborns) were sequenced. The AMOT gene methylation rate was significantly higher in preterm compared to term newborns (4.5% versus 2.5% respectively: χ2 = 3.84; P = 1.8 10⁻⁰²). Bisulfite pyrosequencing identified four CpG dinucleotides with significantly higher methylation levels in preterm newborns. This CpG-targeted methylation significantly decreased with increasing gestational age. These findings highlight importance of pro-angiogenic AMOT gene methylation in ECFC, suggesting that epigenetic mechanisms may control the regulation of angiogenesis during development. Therefore they pave the way to specific short term and long term complications of preterm birth by altered angiogenesis.

Removed.

Aim: Management of ticagrelor-induced bleeding is challenging as platelet transfusion is ineffective. An effective strategy is needed. This study aimed to investigatein vitrothe efficacy of four haemostatic drugs (HDs), namely recombinant activated factor VII (rFVIIa), fibrinogen concentrate (Fib), tranexamic acid (TXA) and factor XIII concentrate (FXIII) to improve the haemostatic capacity in the presence of ticagrelor.Methods: Blood was spiked with ticagrelor then supplemented by either HD or control. Several assays were performed: ADP-induced platelet aggregation measured by impedance aggregometry, light transmission and two global assays, thrombolastography with the platelet mapping device (TEG-PM) and a platelet-dependent thrombin generation assay (TGA).Results: Ticagrelor inhibited ADP-induced platelet aggregation and decreased the clot strength maximum amplitude (MA) in TEG-PM_ADP. None of the HDs corrected these parameters. However, rFVIIa shortened the coagulation time R using TEG-PM_thrombinand the time to peak prolonged by ticagrelor in TGA. Fib increased MA_thrombinand FXIII decreased LY30. TXA had no effects.Conclusions: Whereas none of the HDs corrected ticagrelor-induced platelet inhibition, rFVIIa shortened coagulation times, Fib increased clot firmness and FXIII decreased fibrinolysis. Consequently, they may bypass ticagrelor effects by acting on fibrin formation or fibrinolysis. Further studies are needed to confirm these datain vivo.

Rivaroxaban and apixaban are selective direct inhibitors of free and prothrombinase-bound factor Xa (FXa). Surprisingly prothrombin time (PT) is little sensitive to clinically relevant changes in drug concentration, especially with apixaban. To investigate this pharmacodynamic discrepancy we have compared the kinetics of FXa inhibition in strictly identical conditions (pH 7.48, 37 °C, 0.15 M). K_I values of 0.74 ± 0.03 and 0.47 ± 0.02 nM and k_on values of 7.3 ± 1.6 10⁶ and 2.9 ± 0.6 10⁷ M^-1 s^-1 were obtained for apixaban and rivaroxaban, respectively. To investigate if these constants rationalise the inhibitor pharmacodynamics, we used numerical integration to evaluate impact of FXa inhibition on thrombin generation assay (TGA) and PT. Simulation predicted that in TGA triggered with 20 pM tissue factor, 100 ng/ml apixaban or rivaroxaban increased 1.8– or 3.0-fold the lag time and 1.4– or 2.0-fold the time to peak, whilst decreasing 1.2– or 3.1-fold the maximum thrombin and 1.7– or 3.5-fold the endogenous thrombin potential. These numbers were consistent with those obtained through the corresponding TGA triggered in plasma spiked with apixaban or rivaroxaban. Simulated PT ratios were also consistent with the corresponding plasma PT: markedly less sensitive to apixaban than to rivaroxaban. Analogous differences in TGA and PT were obtained irrespective of the drug amount added. We concluded that kon values for FXa of apixaban and rivaroxaban rationalise the unexpected lower sensitivity of PT and TGA to the former.