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

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

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 (&lt;50) or a low number of non-survivors (5%). 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. Septic adults with or without shock. Gas6 and sAxl were prospectively measured by ELISA at day 0, 3, 7, and then weekly until discharge or death. 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%. 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

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

MOLECULAR BASES OF THE MODULATION OF COAGULATION FACTORS LEVELS: IN VITRO AND IN VIVO STUDY.

Hemostasis is a nely tuned equilibrium status between procoagulant and anticoagulant activities. Despite the presence of several regulatory factors, hemostatic balance is nevertheless sensitive to perturbation. In this thesis, we have analyzed the effects of some acquired and inherited components that could modulate specific coagulation factors. The study of coagulation factor level variations could be instrumental to discover new parameters for the evaluation of the risk of thrombotic and hemorrhagic events. In the first study we evaluated the effects of a whole-diet therapy on a wide panel of hemostatic and in ammatory parameters in a population of overweighted premenopausal women with moderate CVD risk. We observed specific changes on proteins important in coagulation initiation (factor VII, [FVII]) and amplication phases (factor VIII, [FVIII]). Levels of Tumor Necrosis Factor-alfa, a cytokine over-produced by adipocytes and macrophages of adipose tissue in the obese state, were correlated with those of FVIII, thus suggesting new relations between coagulation and cellular components of in ammation. Also temporal parameters of thrombin generation (lag time and time to peak) were significantly prolonged, perhaps re ecting changes related with initial stages of coagulation cascade. In the second study, we evaluated the effect of exercise training on end-stage renal disease (ESRD) patients on hemodialysis (HD), a population characterized by high CVD risk and a seriously impaired physical function. The results we have obtained suggest the presence of factors capable of modulating the coagulation phenotype in both the two groups of patients, albeit independently of physical activity. The lack of association between physical exercise and the coagulation phenotype, cannot exclude that exercise alone is not able to modulate blood coagulation in the ESRD population. We have found an extremely significant reduction in the levels of FVIII, FVII and FX levels, that are of potential clinical importance, even though they proved to be independent from lifestyle change. For a better understanding of these results, we will need the expertise of clinicians, and particularly of nephrologists. Finally, we have investigated the molecular mechanisms induced by a new mutation (R1698W) of coagulation factor V (FV), in the modulation of its protein levels. Our results indicate that the R1698W mutation has a pleiotropic effect on FV by reducing its cellular secretion, impairing its binding affinity to FXa, reducing the prothrombinase catalytic efficiency and deeply increasing its instability after activation. These data also provide an interpretation of the cross-reacting material negative (CRM -) phenotype shown in R1698W carriers. This study gives new structural information at the A3-A2 domains interface, a region presenting several differences between the two available FV models. The replacement of the arginine with a relatively large and hydrophobic residue (tryptophan or alanine) at 1698 is not tolerated since it probably abolishes an important inter-domains interaction. The next step of our research will be focused at understanding the potentially unique role of 1698R in FVa stability by the creation of new FV mutants

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

Reduced FVII and FVIII levels and shortened thrombin-generation times during a healthy diet in middle-aged women with mild to moderate CVD risk.

No experimental study has investigated the effect of whole-diet therapies on a wide panel of haemostatic parameters, and their relationship with metabolic and inflammatory markers. These information were sought in middle-aged women with moderate CVD risk subjected to an integrated healthy diet. Research design: Forty-nine pre-menopausal women were screened for C-reactive protein levels >/=1 mg/L and at least one additional cardiovascular disease risk factor. Sixteen women (age: 43-54 years) were selected and received a 12-week diet (four phases) integrating National Cholesterol Education Program-Adult Treatment Panel-III recommendations with components of Mediterranean-style diet. Results: We observed a reduction in body mass index (P=0.001), waist circumference (P=0.005), total (P=0.011) and LDL cholesterol levels (P=0.035). Antigen levels of coagulation factor VII (P=0.003) and VIII (P=0.005) were clearly reduced by dietary intervention, which also appeared to decrease circulating tissue factor but not fibrinogen and von Willebrand factor antigen levels. Levels of factor VIII and tumour necrosis factor-alpha, among the inflammation markers, showed the best correlation, particularly before the intervention (r=0.55, P=0.032). Only this cytokine affected FVIII variation over time, thus highlighting new relations between coagulation and cellular components of inflammation. The functional effect of diet on coagulation was indicated by markedly prolonged thrombin generation initiation and propagation times (lag time, P=0.002; time to peak, P=0.005). Conclusions: The changes observed in coagulation initiation and amplification phases, body composition and lipid profile would translate into a remarkable decreased risk for cardiovascular disease. Our observations suggest novel relationships between coagulation and inflammatory components

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

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

Upon activation, platelets release plasma-membrane derived microparticles (PMPs) exposing phosphatidylserine (PS) on their surface. The function and clearance mechanism of these MPs 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 PS-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 MPs in the presence of protein S and Gas6 in human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) was monitored by flow cytometry, western blotting and confocal/electron microscopy. We found that both endothelial cell types can phagocytose PMPs, and using TAM-blocking antibodies or siRNA knock-down of individual TAMs we show that the uptake is mediated by endothelial Axl and Gas6. As circulating PMPs-levels were not altered in Gas6-/- mice compared to Gas6+/+ mice, we hypothesize that the Gas6-mediated uptake is not a means to clear the bulk of circulating PMPs but may serve to phagocytose PMPs locally generated at sites of platelet activation and as a way to affect endothelial responses

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