ATG5 promotes eosinopoiesis but inhibits eosinophil effector functions.

Eosinophils are white blood cells that contribute to the regulation of immunity and are involved in the pathogenesis of numerous inflammatory diseases. In contrast to other cells of the immune system, no information is available regarding the role of autophagy in eosinophil differentiation and functions. To study the autophagic pathway in eosinophils, we generated conditional knockout mice in which Atg5 is deleted within the eosinophil lineage only (designated Atg5eoΔ mice). Eosinophilia was provoked by crossbreeding Atg5eoΔ mice with Il5 (IL-5) overexpressing transgenic mice (designated Atg5eoΔIl5tg mice). Deletion of Atg5 in eosinophils resulted in a dramatic reduction in the number of mature eosinophils in blood and an increase of immature eosinophils in the bone marrow. Atg5-knockout eosinophil precursors exhibited reduced proliferation under both in vitro and in vivo conditions but no increased cell death. Moreover, reduced differentiation of eosinophils in the absence of Atg5 was also observed in mouse and human models of chronic eosinophilic leukemia. Atg5-knockout blood eosinophils exhibited augmented levels of degranulation and bacterial killing in vitro. Moreover, in an experimental in vivo model, we observed that Atg5eoΔ mice achieve better clearance of the local and systemic bacterial infection with Citrobacter rodentium. Evidence for increased degranulation of ATG5low-expressing human eosinophils was also obtained in both tissues and blood. Taken together, mouse and human eosinophil hematopoiesis and effector functions are regulated by ATG5, which controls the amplitude of overall antibacterial eosinophil immune responses.

Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus.

Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre- mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

Targeting anticoagulant protein S to improve hemostasis in hemophilia.

Improved treatments are needed for hemophilia A and B, bleeding disorders affecting 400 000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by rebalancing coagulation. Protein S (PS) is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting PS rebalances coagulation in hemophilia. PS gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking PS in plasma of hemophilia patients normalized in vitro thrombin generation. Both PS and TFPIα were detected in hemophilic mice joints. PS and TFPI expression was stronger in the joints of hemophilia A patients than in those of hemophilia B patients when receiving on-demand therapy, for example, during a bleeding episode. In contrast, PS and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, comparable to osteoarthritis patients. These results establish PS inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine PS silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.

The Gas6-Axl Protein Interaction Mediates Endothelial Uptake of Platelet Microparticles.

Upon activation, platelets release plasma membrane-derived microparticles (PMPs) exposing phosphatidylserine on their surface. The functions and clearance mechanism of these microparticles are incompletely understood. As they are pro-coagulant and potentially pro-inflammatory, rapid clearance from the circulation is essential for prevention of thrombotic diseases. The tyrosine kinase receptors Tyro3, Axl, and Mer (TAMs) and their ligands protein S and Gas6 are involved in the uptake of phosphatidylserine-exposing apoptotic cells in macrophages and dendritic cells. Both TAMs and their ligands are expressed in the vasculature, the functional significance of which is poorly understood. In this study, we investigated how vascular TAMs and their ligands may mediate endothelial uptake of PMPs. PMPs, generated from purified human platelets, were isolated by ultracentrifugation and labeled with biotin or PKH67. The uptake of labeled microparticles in the presence of protein S and Gas6 in human aortic endothelial cells and human umbilical vein endothelial cells was monitored by flow cytometry, Western blotting, and confocal/electron microscopy. We found that both endothelial cell types can phagocytose PMPs, and by using TAM-blocking antibodies or siRNA knockdown of individual TAMs, we show that the uptake is mediated by endothelial Axl and Gas6. As circulating PMP levels were not altered in Gas6(-/-) mice compared with Gas6(+/+) mice, we hypothesize that the Gas6-mediated uptake is not a means to clear the bulk of circulating PMPs but may serve to locally phagocytose PMPs generated at sites of platelet activation and as a way to effect endothelial responses.

Thrombin Generation Is Associated with Venous Thromboembolism Recurrence, but Not with Major Bleeding and Death in the Elderly: A Prospective Multicenter Cohort Study.

It is currently unknown whether thrombin generation is associated with venous thromboembolism (VTE) recurrence, major bleeding, or mortality in the elderly. Therefore, our aim was to prospectively study the association between thrombin generation and VTE recurrence, major bleeding, and mortality in elderly patients with acute VTE. Consecutive patients aged ≥65 years with acute VTE were followed for 2 years, starting from 1 year after the index VTE. Primary outcomes were VTE recurrence, major bleeding, and mortality. Thrombin generation was assessed in 551 patients 1 year after the index VTE. At this time, 59% of the patients were still anticoagulated. Thrombin generation was discriminatory for VTE recurrence, but not for major bleeding and mortality in non-anticoagulated patients. Moreover, peak ratio (adjusted subhazard ratio 4.09, 95% CI, 1.12-14.92) and normalized peak ratio (adjusted subhazard ratio 2.18, 95% CI, 1.28-3.73) in the presence/absence of thrombomodulin were associated with VTE recurrence, but not with major bleeding and mortality after adjustment for potential confounding factors. In elderly patients, thrombin generation was associated with VTE recurrence, but not with major bleeding and/or mortality. Therefore, our study suggests the potential usefulness of thrombin generation measurement after anticoagulation completion for VTE to help identify among elderly patients those at higher risk of VTE recurrence.

Resistance to thrombomodulin correlates with liver stiffness in chronic liver disease a prospective single-center cohort study.

INTRODUCTION: Chronic liver disease (CLD) is characterized by changes in haemostasis, embracing both hypo- and hypercoagulability. Global hemostatic tests such as thrombin generation assays evaluate the hemostatic balance, to better assess bleeding and thrombotic risks. In addition, procoagulant state in patients with CLD has been demonstrated using modified thrombin generation assays with thrombomodulin, a cofactor for protein C activation. In this study, we prospectively determined thrombin generation and thrombomodulin resistance in patients with CLD staged with liver stiffness measurement (LSM), using both the fully automated analyzer ST Genesia® Thrombin Generation System (STG) and the calibrated automated thrombogram assay (CAT). MATERIALS AND METHODS: Demographic, clinical and laboratory characteristics, and blood samples were collected from 65 patients with CLD. Liver stiffness was measured by transient elastography, and thrombin generation and thrombomodulin resistance, by STG and CAT. RESULTS: Patients were separated based on LSM of <21 and ≥21 kilopascals (kPa). The propagation rate of thrombin generation was higher in patients with LSM ≥21 kPa and the thrombin generation rate increased as LSM increased. In addition, thrombomodulin resistance assessed by STG and CAT was higher in patients with LSM ≥21 kPa. However, ETP inhibition by activated protein C was comparable in patients with LSM <21 and ≥21 kPa. Finally, LSM correlated with most thrombin generation parameters. CONCLUSION: The STG automated system may have value in the assessment of patients with chronic liver disease in the routine coagulation laboratory. LSM ≥21 kPa identify a procoagulant phenotype in these patients, including thrombomodulin resistance.

Thrombin generation measurement using the ST Genesia Thrombin Generation System in a cohort of healthy adults: Normal values and variability.

BACKGROUND: Thrombin generation (TG) assays evaluate the balance between pro- and anticoagulant forces, to better assess bleeding and thrombotic risks. Although TG readouts obtained with the calibrated automated TG have been investigated in multiple clinical conditions, TG still needs standardization and clinical validation. The automated TG instrument ST Genesia® (STG, Stago, Asnières-sur-Seine, France) provides a normalization of TG parameters based on a reference plasma aiming to reduce the interlaboratory variability and the variability between different measurement runs. OBJECTIVES: To evaluate STG in a group of healthy adults. METHODS: Reference intervals in healthy adults and variability of the new standardized reagents for bleeding (BleedScreen) and thrombophilic (ThromboScreen) conditions were determined using STG. RESULTS: TG was measured in platelet-free plasma (PFP) samples of 123 healthy adults. Reference intervals were determined for TG parameters. Intra- and interassay coefficients of variation were calculated on quality controls and PFP samples from healthy adults. Oral contraception (OC) possibly influenced TG parameters, resulting in a higher median and a broader reference interval for peak height and endogenous thrombin potential (ETP) in women aged 20 to 49 years than in all other sex and age categories. Therefore, we propose the following reference interval categories: men, women aged <50 years not using OC, women aged <50 years using OC, and women aged ≥50 years. Normalization was effective to reduce the interassay variability of quality controls for ETP (BleedScreen assay), and peak height and ETP (ThromboScreen assay without thrombomodulin), but had little impact on PFP sample variability. CONCLUSION: STG appears suitable for accurate measurement of TG in healthy adults.

A high Gas6 level in plasma predicts venous thromboembolism recurrence, major bleeding and mortality in the elderly: a prospective multicenter cohort study.

Essentials Predictive ability of pro-hemostatic Gas6 for recurrent venous thromboembolism (VTE) is unknown. We measured Gas6 levels in 864 patients with VTE over 3 years. High Gas6 (> 157%) at diagnosis is associated with VTE recurrence, major bleeding and mortality. Gas6 plasma levels measured 12 months after the index VTE are discriminatory for VTE recurrence. SUMMARY: Background Growth arrest-specific gene 6 (Gas6) is a prohemostatic protein with an unknown predictive ability for recurrent venous thromboembolism (VTE). In the elderly, VTE results in higher mortality but does not have a higher rate of recurrence than in younger patients. Consequently, anticoagulation management in the elderly is challenging. Objective To prospectively investigate the performance of Gas6 in predicting VTE recurrence, major bleeding and mortality in the elderly. Methods Consecutive patients aged ≥ 65 years with acute VTE were followed for a period of 3 years. Primary outcomes were symptomatic VTE recurrence, major bleeding, and mortality. Plasma Gas6 was measured with ELISA. Results Gas6 levels were measured in 864 patients at the time of the index VTE (T1) and, in 70% of them, also 12 months later (T2). The Gas6 level at T1 was discriminatory for VTE recurrence (C-statistic, 0.56; 95% confidence interval [CI] 0.51-0.62), major bleeding (0.60, 95% CI 0.55-0.65) and mortality (0.69, 95% CI 0.65-0.73) up to 36 months. VTE recurrence up to 24 months after T2 was discriminated by the Gas6 level at T2 (0.62, 95% CI 0.54-0.71). High Gas6 levels (> 157%) and continuous Gas6 levels at T1 were associated with VTE recurrence up to 6 months and 12 months, respectively. Conclusions In elderly patients, a high Gas6 level is associated with higher risks of VTE recurrence, major bleeding, and death. These findings support further studies to assess the performance of Gas6 in adjusting the length of anticoagulation.

Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles.

Receptors for extracellular nucleotides are the focus of increasing attention for their ability to cause release of plasma membrane vesicles (microparticles, MPs). Here, we show that monocyte-derived human dendritic cells (DCs) stimulated with a P2X7 receptor (P2X7R) agonist undergo a large release of MPs endowed with procoagulant activity. Functional and Western blot studies revealed that MPs contain the membrane-bound form of tissue factor (TF), a glycoprotein acting as essential cofactor of activated factor VII and triggering blood coagulation. Quiescent DCs express the membrane-bound (full length), as well as truncated alternatively spliced TF forms. DC reactivity to anti-TF Abs disappeared almost completely on stimulation with ATP or benzoyl ATP (BzATP), as shown by immunoblot and confocal microscopy analysis. Concurrently, TF reactivity and activity appeared in the vesicular fraction, indicating that MPs are important carriers for the dissemination of full-length TF form. Activity of MP-bound TF, comparable to that of relipidated recombinant TF, was dose dependently inhibited by the addition of a specific anti-human TF antibody. We infer that a large fraction of this protein, and its procoagulant potential, are "deliverable" after physiological or pathological stimuli. These findings might have implications for triggering and propagating coagulation in healthy and atherosclerotic vessels.

Peptide Macrocycle Inhibitor of Coagulation Factor XII with Subnanomolar Affinity and High Target Selectivity.

Factor XII (FXII) is a plasma protease that has emerged in recent years as a potential target to treat or prevent pathological thrombosis, to inhibit contact activation in extracorporeal circulation, and to treat the swelling disorder hereditary angioedema. While several protein based inhibitors with high affinity for activated FXII (FXIIa) were developed, the generation of small molecule inhibitors has been challenging. In this work, we have generated a potent and selective FXIIa inhibitor by optimizing a peptide macrocycle that was recently evolved by phage display (Ki = 0.84 ± 0.03 nM). A fluorine atom introduced in the para-position of phenylalanine enhanced the binding affinity as much as 10-fold. Furthermore, we improved the proteolytic stability by substituting the N-terminal arginine by norarginine. The resulting inhibitor combines high inhibitory affinity and selectivity with a good stability in plasma (Ki = 1.63 ± 0.18 nM, >27 000-fold selectivity, t1/2(plasma) =16 ± 4 h). The inhibitor efficiently blocked activation of the intrinsic coagulation pathway in human blood ex vivo.

Study of Early Elevated Gas6 Plasma Level as a Predictor of Mortality in a Prospective Cohort of Patients with Sepsis.

BACKGROUND: Growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein interacting with anionic phospholipids and TAM tyrosine kinase receptors, is elevated in plasma of septic patients. Previous studies did not find different levels between survivors and non-survivors at admission because either they included a low number of patients (<50) or a low number of non-survivors (5%). OBJECTIVES: To determine, in a larger cohort of septic patients comprising an expected number of non-survivors, the performance of the plasma level of Gas6 and its soluble receptor Axl (sAxl) within 24 hours of admission to predict in-ICU mortality. PATIENTS: Septic adults with or without shock. METHODS: Gas6 and sAxl were prospectively measured by ELISA at day 0, 3, 7, and then weekly until discharge or death. RESULTS: We evaluated 129 septic patients, including 82 with and 47 without shock, with in-ICU mortality rate of 19.4% and in-hospital mortality rate of 26%. Gas6 level was higher in non-survivors than in survivors (238 vs. 167%, P = 0.003); this difference remained constant during the ICU stay. The area under the ROC curve for Gas6 (0.695 [95% CI: 0.58-0.81]) was higher than for sAxl, procalcitonin, CRP, IL-1beta, IL-6 and-alpha, and slightly higher than for IL-8, IL-10, SOFA and APACHEII scores in predicting in-ICU mortality. Considering 249% as a cut-off value, Gas6 measurement had a negative predictive value for mortality of 87%. CONCLUSION: It seems that Gas6 plasma level within 24 hours of ICU admission may predicts in-ICU mortality in patients with sepsis. If our result are confirmed in external validation, Gas6 plasma level measurement could contribute to the identification of patients who may benefit most from more aggressive management.

A Synthetic Factor XIIa Inhibitor Blocks Selectively Intrinsic Coagulation Initiation.

Coagulation factor XII (FXII) inhibitors are of interest for the study of the protease in the intrinsic coagulation pathway, for the suppression of contact activation in blood coagulation assays, and they have potential application in antithrombotic therapy. However, synthetic FXII inhibitors developed to date have weak binding affinity and/or poor selectivity. Herein, we developed a peptide macrocycle that inhibits activated FXII (FXIIa) with an inhibitory constant Ki of 22 nM and a selectivity of >2000-fold over other proteases. Sequence and structure analysis revealed that one of the two macrocyclic rings of the in vitro evolved peptide mimics the combining loop of corn trypsin inhibitor, a natural protein-based inhibitor of FXIIa. The synthetic inhibitor blocked intrinsic coagulation initiation without affecting extrinsic coagulation. Furthermore, the peptide macrocycle efficiently suppressed plasma coagulation triggered by contact of blood with sample tubes and allowed specific investigation of tissue factor initiated coagulation.

Development of a selective peptide macrocycle inhibitor of coagulation factor XII toward the generation of a safe antithrombotic therapy.

Inhibition of coagulation factor XII (FXII) activity represents an attractive approach for the treatment and prevention of thrombotic diseases. The few existing FXII inhibitors suffer from low selectivity. Using phage display combined to rational design, we developed a potent inhibitor of FXII with more than 100-fold selectivity over related proteases. The highly selective peptide macrocycle is a promising candidate for the control of FXII activity in antithrombotic therapy and a valuable tool in hematology research.

Molecular basis of coagulation factor V deficiency caused by the R1698W inter-domain mutation.

Coagulation factor V (FV) deficiency is characterised by variable bleeding phenotypes and heterogeneous mutations. To add new insights into the FV genotype-phenotype relationship, we characterised the R1698W change in the A3 domain, at the poorly investigated interface with the A2 domain. The FV R1698W mutation was responsible for a markedly reduced expression level (10% of FV-WT) and specific activity in thrombin generation (0.39). Interestingly, the FVa1698W showed rapid activity decay upon activation due to increased dissociation rate between the heavy and light chains. The importance of the size and charge of the residue at position 1698 was investigated by three additional recombinant mutants, FVR1698A, FVR1698Q, and FVR1698E. FVR1698A and FVR1698Q expression (30 and 45% of FV-WT), specific activity (both 0.57) and stability were all reduced. Noticeably, FVR1698E showed normal activity and stability despite poor expression (10% of FV-WT). These data indicate the essential role of R1698 for normal biosynthetic process and support local flexibility for positively or negatively charged residues to produce stable and functional A3-A2 domain interactions. Their experimental alteration produces a gradient of FV defects, which help to interpret the wide spectrum of phenotypes in FV-deficient patients.