Publications by authors named "Pascale Gaussem"

115 Publications

Lupus anticoagulant single positivity at acute phase is not associated with venous thromboembolism or in-hospital mortality in COVID-19.

Arthritis Rheumatol 2021 Apr 21. Epub 2021 Apr 21.

Université de Paris, Innovative Therapies in Haemostasis, INSERM, F-75006 Paris, France, Respiratory Medicine Department and Biosurgical Research Lab (Carpentier Foundation), Assistance Publique - Hôpitaux de Paris-Centre (APHP-CUP), F-75015, Paris, France.

Introduction: Antiphospholipid antibodies (APA) clinical relevance in COVID-19 is controversial. We aimed to investigate the prevalence and prognostic value of conventional and non-conventional APA in COVID-19 patients.

Methods: This study was a multi-centric, prospective observational French cohort of patients hospitalized for COVID-19 suspicion.

Results: 249 patients were hospitalized for suspected COVID-19, including 154 with confirmed COVID-19 and 95 not confirmed. We found a significant increase in lupus anticoagulant (LA) positivity among COVID-19 positive patients (60.9% versus 23.7% in non-COVID19 patients, p<0.001), while prevalence of conventional (anti-cardiolipin and anti-beta-2-GP1, IgG and IgM isotypes) and non-conventional APA (IgA, anti-phosphatidylserine/prothrombin and anti-prothrombin IgG and IgM) were low in both groups. COVID-19 patients with LA positivity had higher levels of fibrinogen (6.0 IQR 5.0-7.0 versus 5.3 g/L IQR 4.3-6.4, p=0.028) and C-reactive protein (CRP, 115.5 IQR 66.0-204.8 versus 91.8 mg/L IQR 27.0-155.1, p=0.019). Univariate analysis did not show any association between LA positivity and higher risk of venous thromboembolism (VTE, OR 1.02, 95% CI 0.44-2.43, p=0.95) or in-hospital mortality (OR 1.80, 95% CI 0.70-5.05, p=0.24). Unadjusted and adjusted (to CRP, age and sex) Kaplan-Meier survival curves according to LA positivity confirmed the absence of association with VTE or in-hospital mortality (unadjusted: p=0.64 and p=0.26, respectively; adjusted: hazard ratio = 1.13 95% CI 0.48-2.60 and 1.80 95% CI 0.67-5.01).

Conclusions: COVID-19 patients have an increased prevalence of LA positivity associated with biological inflammation markers. However, positive LA at admission is not associated with VTE risk and/or in-hospital mortality.
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http://dx.doi.org/10.1002/art.41777DOI Listing
April 2021

Role of membrane lipid rafts in MRP4 (ABCC4)-dependent regulation of the cAMP pathway in blood platelets.

Thromb Haemost 2021 Apr 13. Epub 2021 Apr 13.

Université de Paris, Innovative Therapies in Haemostasis, INSERM U1140, Paris, France.

Background: Platelet cytosolic cAMP levels are balanced by synthesis, degradation, and efflux. Efflux can occur via MRP4 (multidrug resistant protein-4, ABCC4) present on dense granule and/or plasma membranes. As lipid rafts have been shown to interfere on cAMP homeostasis, we evaluated the relationships between the distribution and activity of MRP4 in lipid rafts and cAMP efflux.

Methods: Platelet activation and cAMP homeostasis were analyzed in human and wild-type or MRP4-deleted mouse platelets in the presence of methyl-β-cyclodextrin (MβCD) to disrupt lipid rafts, and of activators of the cAMP signalling pathways. Human platelet MRP4 and effector proteins of the cAMP pathway were analyzed by immunoblots in lipid rafts isolated by differential centrifugation.

Results: MβCD dose-dependently inhibited human and mouse platelet aggregation without affecting per se cAMP levels. An additive inhibitory effect existed between the adenylate cyclase (AC) activator forskolin and MβCD that was accompanied by an overincrease of cAMP, and which was significantly enhanced upon MRP4 deletion. Finally, an efflux of cAMP out of resting platelets incubated with PGE1 was observed that was partly dependent on MRP4. Lipid rafts contained a small fraction (≈ 15%) of MRP4 and most of the inhibitory G-protein Gi, whereas Gs protein, AC3, and phosphodiesterases PDE2 and PDE3A were all present as only trace amounts.

Conclusion: Our results are in favor of part of MRP4 present at the platelet surface, including in lipid rafts. Lipid raft integrity is necessary for cAMP signalling regulation, although MRP4 and most players of cAMP homeostasis are essentially located outside rafts.
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http://dx.doi.org/10.1055/a-1481-2663DOI Listing
April 2021

D-dimer at hospital admission for COVID-19 are associated with in-hospital mortality, independent of venous thromboembolism: Insights from a French multicenter cohort study.

Arch Cardiovasc Dis 2021 Mar 9. Epub 2021 Mar 9.

Université de Paris, PARCC, INSERM, 75015 Paris, France.

Background: Coronavirus disease 2019 (COVID-19) has been associated with coagulation disorders, in particular high concentrations of D-dimer, and increased frequency of venous thromboembolism.

Aim: To explore the association between D-dimer at admission and in-hospital mortality in patients hospitalised for COVID-19, with or without symptomatic venous thromboembolism.

Methods: From 26 February to 20 April 2020, D-dimer concentration at admission and outcomes (in-hospital mortality and venous thromboembolism) of patients hospitalised for COVID-19 in medical wards were retrospectively analysed in a multicenter study in 24 French hospitals.

Results: Among 2878 patients enrolled in the study, 1154 (40.1%) patients had D-dimer measurement at admission. Receiver operating characteristic curve analysis identified a D-dimer concentration>1128ng/mL as the best cut-off value for in-hospital mortality (area under the curve 64.9%, 95% confidence interval [CI] 60-69), with a sensitivity of 71.1% (95% CI 62-78) and a specificity of 55.6% (95% CI 52-58), which did not differ in the subgroup of patients with venous thromboembolism during hospitalisation. Among 545 (47.2%) patients with D-dimer concentration>1128ng/mL at admission, 86 (15.8%) deaths occurred during hospitalisation. After adjustment, in Cox proportional hazards and logistic regression models, D-dimer concentration>1128ng/mL at admission was also associated with a worse prognosis, with an odds ratio of 3.07 (95% CI 2.05-4.69; P<0.001) and an adjusted hazard ratio of 2.11 (95% CI 1.31-3.4; P<0.01).

Conclusions: D-dimer concentration>1128ng/mL is a relevant predictive factor for in-hospital mortality in patients hospitalised for COVID-19 in a medical ward, regardless of the occurrence of venous thromboembolism during hospitalisation.
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http://dx.doi.org/10.1016/j.acvd.2021.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7942155PMC
March 2021

Placental growth factor level in plasma predicts COVID-19 severity and in-hospital mortality.

J Thromb Haemost 2021 Apr 8. Epub 2021 Apr 8.

Université de Paris, PARCC, INSERM, F-75015, Paris, France.

Background: Coronavirus disease 2019 (COVID-19) is a respiratory disease associated with vascular inflammation and endothelial injury.

Objectives: Correlate circulating angiogenic markers VEGF-A, PlGF and FGF-2 to in-hospital mortality in COVID-19 adult patients.

Methods: Consecutive ambulatory and hospitalized patients with COVID-19 infection were enrolled. VEGF-A, PlGF and FGF-2 were measured in each patient ≤48 h following admission.

Results: Study enrolled 237 patients with suspected COVID-19: 208 patients had a positive diagnostic for COVID-19 of whom 23 were mild outpatients and 185 patients hospitalized after admission. Levels of VEGF-A, PlGF and FGF-2 significantly increase with the severity of the disease (p<0.001). Using a logistic regression model we found a significant association between the increase of FGF-2 or PlGF and mortality (OR 1.11, 95% CI [1.07-1.16], p<0.001 for FGF-2 and OR 1.07 95% CI [1.04-1.10], p<0.001 for PlGF) while no association were found for VEGF-A levels. ROC curve analysis was performed and we identified PlGF above 30 pg/mL as the best predictor of in-hospital mortality in COVID-19 patients. Survival analysis for PlGF confirmed its interest for in-hospital mortality prediction, by using a Kaplan-Meier survival curves (p=0.001) and a Cox proportional hazard model adjusted to age, body mass index, D-dimer and CRP (3.23 95% CI [1.29-8.11], p=0.001).

Conclusion: Angiogenic factor PlGF is a relevant predictive factor for in-hospital mortality in COVID-19 patients. More than a biomarker, we hypothesize that PlGF blocking strategies could be a new interesting therapeutic approach in COVID-19.
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http://dx.doi.org/10.1111/jth.15339DOI Listing
April 2021

Anticoagulation Before Hospitalization Is a Potential Protective Factor for COVID-19: Insight From a French Multicenter Cohort Study.

J Am Heart Assoc 2021 04 8;10(8):e018624. Epub 2021 Feb 8.

PARCC INSERM Université de Paris France.

Background Coronavirus disease 2019 (COVID-19) is a respiratory disease associated with thrombotic outcomes with coagulation and endothelial disorders. Based on that, several anticoagulation guidelines have been proposed. We aimed to determine whether anticoagulation therapy modifies the risk of developing severe COVID-19. Methods and Results Patients with COVID-19 initially admitted in medical wards of 24 French hospitals were included prospectively from February 26 to April 20, 2020. We used a Poisson regression model, Cox proportional hazard model, and matched propensity score to assess the effect of anticoagulation on outcomes (intensive care unit admission or in-hospital mortality). The study enrolled 2878 patients with COVID-19, among whom 382 (13.2%) were treated with oral anticoagulation therapy before hospitalization. After adjustment, anticoagulation therapy before hospitalization was associated with a better prognosis with an adjusted hazard ratio of 0.70 (95% CI, 0.55-0.88). Analyses performed using propensity score matching confirmed that anticoagulation therapy before hospitalization was associated with a better prognosis, with an adjusted hazard ratio of 0.43 (95% CI, 0.29-0.63) for intensive care unit admission and adjusted hazard ratio of 0.76 (95% CI, 0.61-0.98) for composite criteria intensive care unit admission or death. In contrast, therapeutic or prophylactic low- or high-dose anticoagulation started during hospitalization were not associated with any of the outcomes. Conclusions Anticoagulation therapy used before hospitalization in medical wards was associated with a better prognosis in contrast with anticoagulation initiated during hospitalization. Anticoagulation therapy introduced in early disease could better prevent COVID-19-associated coagulopathy and endotheliopathy, and lead to a better prognosis.
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http://dx.doi.org/10.1161/JAHA.120.018624DOI Listing
April 2021

Role of oculocerebrorenal syndrome of Lowe (OCRL) protein in megakaryocyte maturation, platelet production and functions: a study in patients with Lowe syndrome.

Br J Haematol 2021 Mar 2;192(5):909-921. Epub 2021 Feb 2.

Université de Paris, Innovations Thérapeutiques en Hémostase, Paris, INSERM U1140, France.

Lowe syndrome (LS) is an oculocerebrorenal syndrome of Lowe (OCRL1) genetic disorder resulting in a defect of the OCRL protein, a phosphatidylinositol-4,5-bisphosphate 5-phosphatase containing various domains including a Rho GTPase-activating protein (RhoGAP) homology domain catalytically inactive. We previously reported surgery-associated bleeding in patients with LS, suggestive of platelet dysfunction, accompanied with a mild thrombocytopenia in several patients. To decipher the role of OCRL in platelet functions and in megakaryocyte (MK) maturation, we conducted a case-control study on 15 patients with LS (NCT01314560). While all had a drastically reduced expression of OCRL, this deficiency did not affect platelet aggregability, but resulted in delayed thrombus formation on collagen under flow conditions, defective platelet spreading on fibrinogen and impaired clot retraction. We evidenced alterations of the myosin light chain phosphorylation (P-MLC), with defective Rac1 activity and, inversely, elevated active RhoA. Altered cytoskeleton dynamics was also observed in cultured patient MKs showing deficient proplatelet extension with increased P-MLC that was confirmed using control MKs transfected with OCRL-specific small interfering(si)RNA (siOCRL). Patients with LS also had an increased proportion of circulating barbell-shaped proplatelets. Our present study establishes that a deficiency of the OCRL protein results in a defective actomyosin cytoskeleton reorganisation in both MKs and platelets, altering both thrombopoiesis and some platelet responses to activation necessary to ensure haemostasis.
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http://dx.doi.org/10.1111/bjh.17346DOI Listing
March 2021

Circulating Von Willebrand factor and high molecular weight multimers as markers of endothelial injury predict COVID-19 in-hospital mortality.

Angiogenesis 2021 Jan 15. Epub 2021 Jan 15.

Université de Paris, Institut Cochin, INSERM, 75014 Paris, France, Hematology Department Assistance Publique Hôpitaux de Paris. Centre-Université de Paris (APHP-CUP), Cochin Hospital, 75014, Paris, France.

Background: Coronavirus disease 2019 (COVID-19) is a respiratory disease associated with endotheliitis and microthrombosis.

Objectives: To correlate endothelial dysfunction to in-hospital mortality in a bi-centric cohort of COVID-19 adult patients.

Methods: Consecutive ambulatory and hospitalized patients with laboratory-confirmed COVID-19 were enrolled. A panel of endothelial biomarkers and von Willebrand factor (VWF) multimers were measured in each patient ≤ 48 h following admission.

Results: Study enrolled 208 COVID-19 patients of whom 23 were mild outpatients and 189 patients hospitalized after admission. Most of endothelial biomarkers tested were found increased in the 89 critical patients transferred to intensive care unit. However, only von Willebrand factor antigen (VWF:Ag) scaled according to clinical severity, with levels significantly higher in critical patients (median 507%, IQR 428-596) compared to non-critical patients (288%, 230-350, p < 0.0001) or COVID-19 outpatients (144%, 133-198, p = 0.007). Moreover, VWF high molecular weight multimers (HMWM) were significantly higher in critical patients (median ratio 1.18, IQR 0.86-1.09) compared to non-critical patients (0.96, 1.04-1.39, p < 0.001). Among all endothelial biomarkers measured, ROC curve analysis identified a VWF:Ag cut-off of 423% as the best predictor for in-hospital mortality. The accuracy of VWF:Ag was further confirmed in a Kaplan-Meier estimator analysis and a Cox proportional Hazard model adjusted on age, BMI, C-reactive protein and D-dimer levels.

Conclusion: VWF:Ag is a relevant predictive factor for in-hospital mortality in COVID-19 patients. More than a biomarker, we hypothesize that VWF, including excess of HMWM forms, drives microthrombosis in COVID-19.
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http://dx.doi.org/10.1007/s10456-020-09762-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809553PMC
January 2021

Dabigatran Level Before Reversal Can Predict Hemostatic Effectiveness of Idarucizumab in a Real-World Setting.

Front Med (Lausanne) 2020 16;7:599626. Epub 2020 Dec 16.

Université de Paris, Innovative Therapies in Haemostasis, INSERM, Paris, France.

Idarucizumab has been included in guidelines for the management of bleeding or surgical procedure in dabigatran-treated patients without need for biological monitoring. The aim of the study was to assess the prognostic value of dabigatran plasma level before reversal to test the hemostatic efficacy of idarucizumab. The secondary objectives were (i) to analyze plasma dabigatran level according to the risk of rebound and (ii) to evaluate the incidence of post-reversal non-favorable clinical outcomes (including thromboembolism, bleeding, antithrombotic, and death) and antithrombotic resumption. This was an observational multicentric cohort study, which included all French patients who required idarucizumab for dabigatran reversal. Between May 2016 and April 2019, 87 patients from 21 French centers were enrolled. Patients received idarucizumab for overt bleeding ( = 61), urgent procedures ( = 24), or overdose without bleeding ( = 2). Among patients with major bleeding ( = 57), treatment with idarucizumab was considered effective in 44 (77.2%) of them. Patients who did not achieve effective hemostasis after reversal had a significantly higher mean level of plasma dabigatran at baseline (524.5 ± 386 vs. 252.8 ng/mL ± 235, = 0.033). Furthermore, patients who did not achieve effective hemostasis after reversal had less favorable outcomes during follow-up (46.2 vs. 81.8%, = 0.027). ROC curve identified a cutoff of 264 ng/mL for dabigatran level at admission to be predictive of ineffective hemostasis. No plasma dabigatran rebound was observed after reversal in patients with dabigatran plasma level < 264 ng/mL at baseline. This retrospective study shows that dabigatran level before reversal could predict hemostatic effectiveness and dabigatran plasma rebound after idarucizumab injection.
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http://dx.doi.org/10.3389/fmed.2020.599626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772865PMC
December 2020

No impact of cancer and plague-relevant polymorphisms on COVID-19.

Oncoimmunology 2020 12 8;9(1):1857112. Epub 2020 Dec 8.

Equipe Labellisée Par La Ligue Contre Le Cancer, Université De Paris, Sorbonne Université, INSERM U1138, Centre De Recherche Des Cordeliers, Paris, France.

Formyl peptide receptor 1 (FPR1) is a pattern-recognition receptor that detects bacterial as well as endogenous danger-associated molecular patterns to trigger innate immune responses by myeloid cells. A single nucleotide polymorphism, rs867228 (allelic frequency 19-20%), in the gene coding for FPR1 accelerates the manifestation of multiple carcinomas, likely due to reduced anticancer immunosurveillance secondary to a defect in antigen presentation by dendritic cells. Another polymorphism in , rs5030880 (allelic frequency 12-13%), has been involved in the resistance to plague, correlating with the fact that FPR1 is the receptor for . Driven by the reported preclinical effects of FPR1 on lung inflammation and fibrosis, we investigated whether rs867228 or rs5030880 would affect the severity of coronavirus disease-19 (COVID-19). Data obtained on patients from two different hospitals in Paris refute the hypothesis that rs867228 or rs5030880 would affect the severity of COVID-19.
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http://dx.doi.org/10.1080/2162402X.2020.1857112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734042PMC
December 2020

Predictive Factor for COVID-19 Worsening: Insights for High-Sensitivity Troponin and D-Dimer and Correlation With Right Ventricular Afterload.

Front Med (Lausanne) 2020 12;7:586307. Epub 2020 Nov 12.

Innovative Therapies in Haemostasis, INSERM, Université de Paris, Paris, France.

Coronavirus disease 2019 (COVID-19) has been associated with cardiovascular complications and coagulation disorders. To explore clinical and biological parameters of COVID-19 patients with hospitalization criteria that could predict referral to intensive care unit (ICU). Analyzing the clinical and biological profiles of COVID-19 patients at admission. Among 99 consecutive patients that fulfilled criteria for hospitalization, 48 were hospitalized in the medicine department, 21 were first admitted to the medicine ward department and referred later to ICU, and 30 were directly admitted to ICU from the emergency department. At admission, patients requiring ICU were more likely to have lymphopenia, decreased SpO, a D-dimer level above 1,000 ng/mL, and a higher high-sensitivity cardiac troponin (Hs-cTnI) level. A receiver operating characteristic curve analysis identified Hs-cTnI above 9.75 pg/mL as the best predictive criteria for ICU referral [area under the curve (AUC), 86.4; 95% CI, 76.6-96.2]. This cutoff for Hs-cTnI was confirmed in univariate [odds ratio (OR), 22.8; 95% CI, 6.0-116.2] and multivariate analysis after adjustment for D-dimer level (adjusted OR, 20.85; 95% CI, 4.76-128.4). Transthoracic echocardiography parameters subsequently measured in 72 patients showed an increased right ventricular (RV) afterload correlated with Hs-cTnI ( = 0.42, = 0.010) and D-dimer ( = 0.18, = 0.047). Hs-cTnI appears to be the best relevant predictive factor for referring COVID-19 patients to ICU. This result associated with the correlation of D-dimer with RV dilatation probably reflects a myocardial injury due to an increased RV wall tension. This reinforces the hypothesis of a COVID-19-associated microvascular thrombosis inducing a higher RV afterload.
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http://dx.doi.org/10.3389/fmed.2020.586307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689153PMC
November 2020

Multidimensional Proteomic Approach of Endothelial Progenitors Demonstrate Expression of KDR Restricted to CD19 Cells.

Stem Cell Rev Rep 2021 04 17;17(2):639-651. Epub 2020 Nov 17.

Innovative Therapies in Haemostasis, INSERM, Université de Paris, F-75006, Paris, France.

Endothelial progenitor cells (EPCs) are involved in vasculogenesis and cardiovascular diseases. However, the phenotype of circulating EPCs remains elusive but they are more often described as CD34KDR. The aim of the study was to extensively characterize circulating potential vasculogenic stem cell candidates in two populations of patients with cardiovascular disease by powerful multidimensional single cell complementary cytometric approaches (mass, imaging and flow). We identified cellular candidates in one patient before and after bioprosthetic total artificial heart implantation and results were confirmed in healthy peripheral and cord blood by mass cytometry. We also quantified cellular candidates in 10 patients with different COVID-19 severity. Both C-TAH implantation and COVID-19 at critical stage induce a redistribution of circulating CD34 and CD19 sub-populations in peripheral blood. After C-TAH implantation, circulating CD34 progenitor cells expressed c-Kit stem marker while specific subsets CD34CD133CD45c-KitKDR were mobilized. KDR was only expressed by CD19 B-lymphocytes and CD14 monocytes subpopulations in circulation. We confirmed by mass cytometry this KDR expression on CD19 in healthy peripheral and cord blood, also with a VE-cadherin expression, confirming absence of endothelial lineage marker on CD34 subtypes. In COVID-19, a significant mobilization of CD34c-KitKDR cells was observed between moderate and critical COVID-19 patients regardless CD133 or CD45 expression. In order to better evaluate EPC phenotype, we performed imaging flow cytometry measurements of immature CD34KDR cells in cord blood and showed that, after elimination of non-circular events, those cells were all CD19. During COVID-19, a significant mobilization of CD19KDR per million of CD45 cells was observed between moderate and critical COVID-19 patients regardless of CD34 expression. CD34c-Kit cells are mobilized in both cardiovascular disease described here. KDR cells in peripheral blood are CD19 positive cells and are not classic vasculogenic stem and/or progenitor cells. A better evaluation of c-Kit and KDR expressing cells will lead to the redefinition of circulating endothelial progenitors.Graphical abstract Central illustration figure. Multidimensional proteomic approach of endothelial progenitors demonstrate expression of KDR restricted to CD19 cells. Endothelial progenitor cells (EPCs) are involved in cardiovascular diseases, however their phenotype remains elusive. We elucidated here EPCs phenotype by a deep characterization by multidimensional single cell complementary cytometric approaches after Bioprosthetic total artificial heart implantation and during COVID-19. We showed a redistribution of circulating CD34 and CD19 sub-populations in both situations. None of the immature cell population expresses KDR. Mobilized CD34 expressed c-Kit. Imaging flow cytometry demonstrated that CD34KDR cells, after elimination of non-circular events, are all CD19. Our results suggest a new definition of circulating EPCs and emphasize involvement of CD19 cells in cardiovascular disease.
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http://dx.doi.org/10.1007/s12015-020-10062-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7670993PMC
April 2021

Interleukin-8 Receptors CXCR1 and CXCR2 Are Not Expressed by Endothelial Colony-forming Cells.

Stem Cell Rev Rep 2021 Apr 13;17(2):628-638. Epub 2020 Nov 13.

Innovative Therapies in Haemostasis, INSERM, Université de Paris, F-75006 , Paris, France.

Endothelial colony-forming cells (ECFCs) are human vasculogenic cells described as potential cell therapy product and good candidates for being a vascular liquid biopsy. Since interleukin-8 (IL-8) is a main actor in senescence, its ability to interact with ECFCs has been explored. However, expression of CXCR1 and CXCR2, the two cellular receptors for IL-8, by ECFCs remain controversial as several teams published contradictory reports. Using complementary technical approaches, we have investigated the presence of these receptors on ECFCs isolated from cord blood. First, CXCR1 and CXCR2 were not detected on several clones of cord blood- endothelial colony-forming cell using different antibodies available, in contrast to well-known positive cells. We then compared the RT-PCR primers used in different papers to search for the presence of CXCR1 and CXCR2 mRNA and found that several primer pairs used could lead to non-specific DNA amplification. Last, we confirmed those results by RNA sequencing. CXCR1 and CXCR2 were not detected in ECFCs in contrary to human-induced pluripotent stem cell-derived endothelial cells (h-iECs). In conclusion, using three different approaches, we confirmed that CXCR1 and CXCR2 were not expressed at mRNA or protein level by ECFCs. Thus, IL-8 secretion by ECFCs, its effects in angiogenesis and their involvement in senescent process need to be reanalyzed according to this absence of CXCR-1 and - 2 in ECFCs.Graphical Abstract.
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http://dx.doi.org/10.1007/s12015-020-10081-yDOI Listing
April 2021

Human Aortic Valve Interstitial Cells Display Proangiogenic Properties During Calcific Aortic Valve Disease.

Arterioscler Thromb Vasc Biol 2021 01 5;41(1):415-429. Epub 2020 Nov 5.

Université de Paris, Innovative Therapies in Haemostasis, INSERM, France (N.G., A.B., E.R., S.I., S.L., A. Cras, N.N., J.R., P.G., D.M.S.).

Objective: The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results: VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VIC) mesenchymal-like phenotype was confirmed by CD90/CD73/CD44 expression and multipotent-like differentiation ability. When VIC were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VIC and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A was observed at the protein level in VIC-conditioned media and confirmed at the mRNA level in VIC compared with control VIC. Conditioned media from VIC induced in vitro a significant increase in endothelial colony-forming cell proliferation, migration, and sprouting compared with conditioned media from control VIC. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VIC involvement in angiogenesis by a VEGF-A dependent mechanism.

Conclusions: We provide here the first proof of an angiogenic potential of human VICs isolated from patients with calcific aortic valve disease. These results point to a novel function of VIC in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.
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http://dx.doi.org/10.1161/ATVBAHA.120.314287DOI Listing
January 2021

Platelet Functions During Extracorporeal Membrane Oxygenation. Platelet-Leukocyte Aggregates Analyzed by Flow Cytometry as a Promising Tool to Monitor Platelet Activation.

J Clin Med 2020 Jul 23;9(8). Epub 2020 Jul 23.

Rennes University Hospital, INSERM-CIC 1414, F-35000 Rennes, France.

Extracorporeal membrane oxygenation (ECMO) is an extracorporeal circulation used to manage patients with severe circulatory or respiratory failure. It is associated with both high bleeding and thrombosis risks, mainly as a result of biomaterial/blood interface phenomena, high shear stress, and complex inflammatory response involving the activation of coagulation and complement systems, endothelial cells, leukocytes, and platelets. Besides their critical role in hemostasis, platelets are important players in inflammatory reactions, especially due to their ability to bind and activate leukocytes. Hence, we reviewed studies on platelet function of ECMO patients. Moreover, we addressed the issue of platelet-leukocyte aggregates (PLAs), which is a key step in both platelet and leukocyte activation, and deserves to be investigated in these patients. A reduced expression of GPIb and GPVI was found under ECMO therapy, due to the shedding processes. However, defective platelet aggregation is inconsistently reported and is still not clearly defined. Due to the high susceptibility of PLAs to pre-analytical conditions, defining and strictly adhering to a rigorous laboratory methodology is essential for reliable and reproducible results, especially in the setting of complex inflammatory situations like ECMO. We provide results on sample preparation and flow cytometric whole blood evaluation of circulating PLAs.
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http://dx.doi.org/10.3390/jcm9082361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464627PMC
July 2020

Autoregulation of Pulsatile Bioprosthetic Total Artificial Heart is Involved in Endothelial Homeostasis Preservation.

Thromb Haemost 2020 Sep 20;120(9):1313-1322. Epub 2020 Jul 20.

Department of Cardiovascular Surgery, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.

Pulsatile Carmat bioprosthetic total artificial heart (C-TAH) is designed to be implanted in patients with biventricular end-stage heart failure. Since flow variation might contribute to endothelial dysfunction, we explored circulating endothelial biomarkers after C-TAH implantation in seven patients and compared the manual and autoregulated mode. Markers of endothelial dysfunction and regeneration were compared before and during a 6- to 9-month follow-up after implantation. The follow-up was divided into three periods (< 3, 3-6, and > 6 months) and used to estimate the temporal trends during the study period. A linear mixed model was used to analyze repeated measures and association between tested parameters according to the mode of C-TAH and the time. Relevance of soluble endoglin (sEndoglin) level increase has been tested on differentiation and migration potential of human vasculogenic progenitor cells (endothelial colony forming cells [ECFCs]). Normal sEndoglin and soluble endothelial protein C receptor (sEPCR) levels were found in patients after implantation with autoregulated C-TAH, whereas they significantly increased in the manual mode, as compared with pretransplant values ( = 0.005 and 0.001, respectively). In the autoregulated mode, a significant increase in the mobilization of cytokine stromal cell-derived factor 1 was found ( = 0.03). After adjustment on the mode of C-TAH, creatinine or C-reactive protein level, sEndoglin, and sEPCR, were found significantly associated with plasma total protein levels. Moreover, a significant decrease in pseudotubes formation and migration ability was observed in vitro in ECFCs receiving sEndoglin activation. Our combined analysis of endothelial biomarkers confirms the favorable impact of blood flow variation achieved with autoregulation in patients implanted with the bioprosthetic total artificial heart.
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http://dx.doi.org/10.1055/s-0040-1713751DOI Listing
September 2020

[Usefulness of immature platelet fraction measurement for diagnosis and monitoring of iron deficiency associated thrombocytopenia: about two cases].

Ann Biol Clin (Paris) 2020 08;78(4):433-437

Université de Paris, Hôpital européen Georges Pompidou, AP-HP, Service d'hématologie biologique, Paris, France, Université de Paris, Innovative Therapies in Haemostasis, Inserm 1140, Paris, France.

Iron deficiency anemia is frequently associated with thrombocytosis. However, in some rare cases of very severe iron deficiency, a thrombocytopenia may occur. This condition may lead to a misdiagnosis of immune thrombocytopenic purpura and thus to unnecessary tests in this context. Here we report two patients who presented with iron deficiency associated thrombocytopenia rapidly corrected after martial supplementation. We then discuss the value of measuring immature platelet fraction (IPF), which represents the population of newly formed platelets containing a greater amount of residual RNA. For both cases, low IPF values at admission indicated a central origin of thrombocytopenia with decreased platelet production, which is the pathophysiological mechanism of iron deficiency associated thrombocytopenia.
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http://dx.doi.org/10.1684/abc.2020.1564DOI Listing
August 2020

Curative anticoagulation prevents endothelial lesion in COVID-19 patients.

J Thromb Haemost 2020 09 30;18(9):2391-2399. Epub 2020 Jul 30.

Innovative Therapies in Haemostasis, INSERM, Université de Paris, Paris, France.

Background: Coronavirus disease-2019 (COVID-19) has been associated with cardiovascular complications and coagulation disorders.

Objectives: To explore the coagulopathy and endothelial dysfunction in COVID-19 patients.

Methods: The study analyzed clinical and biological profiles of patients with suspected COVID-19 infection at admission, including hemostasis tests and quantification of circulating endothelial cells (CECs).

Results: Among 96 consecutive COVID-19-suspected patients fulfilling criteria for hospitalization, 66 were tested positive for SARS-CoV-2. COVID-19-positive patients were more likely to present with fever (P = .02), cough (P = .03), and pneumonia at computed tomography (CT) scan (P = .002) at admission. Prevalence of D-dimer >500 ng/mL was higher in COVID-19-positive patients (74.2% versus 43.3%; P = .007). No sign of disseminated intravascular coagulation were identified. Adding D-dimers >500 ng/mL to gender and pneumonia at CT scan in receiver operating characteristic curve analysis significantly increased area under the curve for COVID-19 diagnosis. COVID-19-positive patients had significantly more CECs at admission (P = .008) than COVID-19-negative ones. COVID-19-positive patients treated with curative anticoagulant prior to admission had fewer CECs (P = .02) than those without. Interestingly, patients treated with curative anticoagulation and angiotensin-converting-enzyme inhibitors or angiotensin receptor blockers had even fewer CECs (P = .007).

Conclusion: Curative anticoagulation could prevent COVID-19-associated coagulopathy and endothelial lesion.
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http://dx.doi.org/10.1111/jth.14968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323356PMC
September 2020

Angiopoietin-2 as a marker of endothelial activation is a good predictor factor for intensive care unit admission of COVID-19 patients.

Angiogenesis 2020 11 27;23(4):611-620. Epub 2020 May 27.

Université de Paris, Innovative Therapies in Haemostasis, INSERM, 75006, Paris, France.

Background: Coronavirus disease-2019 (COVID-19), a respiratory disease has been associated with ischemic complications, coagulation disorders, and an endotheliitis.

Objectives: To explore endothelial damage and activation-related biomarkers in COVID-19 patients with criteria of hospitalization for referral to intensive care unit (ICU) and/or respiratory worsening.

Methods: Analysis of endothelial and angiogenic soluble markers in plasma from patients at admission.

Results: Study enrolled 40 consecutive COVID-19 patients admitted to emergency department that fulfilled criteria for hospitalization. Half of them were admitted in conventional wards without any ICU transfer during hospitalization; whereas the 20 others were directly transferred to ICU. Patients transferred in ICU were more likely to have lymphopenia, decreased SpO2 and increased D-dimer, CRP and creatinine levels. In those patients, soluble E-selectin and angiopoietin-2 were significantly increased (p value at 0.009 and 0.003, respectively). Increase in SELE gene expression (gene coding for E-selectin protein) was confirmed in an independent cohort of 32 patients using a whole blood gene expression profile analysis. In plasma, we found a strong association between angiopoetin-2 and CRP, creatinine and D-dimers (with p value at 0.001, 0.001 and 0.003, respectively). ROC curve analysis identified an Angiopoietin-2 cut-off of 5000 pg/mL as the best predictor for ICU outcome (Se = 80.1%, Sp = 70%, PPV = 72.7%, NPV = 77%), further confirmed in multivariate analysis after adjustment for creatinine, CRP or D-dimers.

Conclusion: Angiopoietin-2 is a relevant predictive factor for ICU direct admission in COVID-19 patients. This result showing an endothelial activation reinforces the hypothesis of a COVID-19-associated microvascular dysfunction.
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http://dx.doi.org/10.1007/s10456-020-09730-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250589PMC
November 2020

Comparative Study of Various α-Adrenoreceptor Agonist Drugs for Ticagrelor Reversal.

J Clin Med 2020 Mar 16;9(3). Epub 2020 Mar 16.

Université de Paris, Innovations Thérapeutiques en Hémostase, INSERM 1140, 75006 Paris, France.

Ticagrelor, an antiplatelet adenosine diphosphate (ADP)-P2Y receptor antagonist, increases the risk of bleeding. Its management is challenging because platelet transfusion is ineffective and no specific antidote is currently available. Epinephrine, a vasopressor catecholamine prescribed during shock, restores platelet functions inhibited by ticagrelor through stimulation of α-adrenoreceptors. It subsequently inhibits cyclic adenosine monophosphate (cAMP) pathway and PI3K signaling. However, since epinephrine may expose a patient to deleterious hemodynamic effects, we hypothesized that other α-adrenoreceptor agonist drugs used in clinical practice with fewer side effects could reverse the antiplatelet effects of ticagrelor. We compared the efficacy of clonidine, dexmedetomidine, brimonidine, and norepinephrine with epinephrine to restore ADP- and PAR-1-AP-induced washed platelet aggregation inhibited by ticagrelor, as well as resulting platelet cAMP levels. In ticagrelor-free samples, none of the α-adrenoreceptor agonists induced aggregation by itself but all of them potentiated ADP-induced aggregation. Compared with epinephrine, norepinephrine, and brimonidine partially restored ADP- and fully restored PAR-1-AP-induced aggregation inhibited by ticagrelor while clonidine and dexmedetomidine were ineffective. Indeed, this lack of effect resulted from a lower decrease in cAMP concentration elicited by these partial α-adrenoreceptor agonists, clonidine, and dexmedetomidine, compared with full α-agonists. Our results support the development of specific full and systemic α-adrenoreceptor agonists for ticagrelor reversal.
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http://dx.doi.org/10.3390/jcm9030809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141185PMC
March 2020

P2Y Inhibition beyond Thrombosis: Effects on Inflammation.

Int J Mol Sci 2020 Feb 19;21(4). Epub 2020 Feb 19.

INSERM, CIC 1414 (Centre d'Investigation Clinique de Rennes), Université de Rennes, CHU de Rennes, F-35000 Rennes, France.

The P2Y receptor is a key player in platelet activation and a major target for antithrombotic drugs. The beneficial effects of P2Y receptor antagonists might, however, not be restricted to the primary and secondary prevention of arterial thrombosis. Indeed, it has been established that platelet activation also has an essential role in inflammation. Additionally, nonplatelet P2Y receptors present in immune cells and vascular smooth muscle cells might be effective players in the inflammatory response. This review will investigate the biological and clinical impact of P2Y receptor inhibition beyond its platelet-driven antithrombotic effects, focusing on its anti-inflammatory role. We will discuss the potential molecular and cellular mechanisms of P2Y-mediated inflammation, including cytokine release, platelet-leukocyte interactions and neutrophil extracellular trap formation. Then we will summarize the current evidence on the beneficial effects of P2Y antagonists during various clinical inflammatory diseases, especially during sepsis, acute lung injury, asthma, atherosclerosis, and cancer.
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http://dx.doi.org/10.3390/ijms21041391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073040PMC
February 2020

Epinephrine restores platelet functions inhibited by ticagrelor: A mechanistic approach.

Eur J Pharmacol 2020 Jan 15;866:172798. Epub 2019 Nov 15.

Université de Paris, Innovations Thérapeutiques en Hémostase, INSERM, F-75006 Paris, France.

Ticagrelor, an antagonist of the platelet adenosine diphosphate (ADP)-P2Y receptor is recommended for patients with acute coronary syndromes. However, ticagrelor exposes to a risk of bleeding, the management of which is challenging because platelet transfusion is ineffective, and no antidote is yet available. We hypothesized that the vasopressor drug epinephrine could counter the antiplatelet effects of ticagrelor and restore platelet functions. We assessed in vitro the efficiency of epinephrine in restoring platelet aggregation inhibited by ticagrelor and investigated the underlying mechanisms. Washed platelet aggregation and secretion were measured upon stimulation by epinephrine alone or in combination with ADP, in the presence or absence of ticagrelor. Mechanistic investigations used P2Y and phosphoinositide 3-kinase (PI3K) inhibitors and included vasodilator-stimulated phosphoprotein (VASP) and Akt phosphorylation assays as well as measurement of Ca mobilisation. We found that epinephrine restored ADP-induced platelet aggregation, but not dense granule release. Epinephrine alone failed to induce aggregation whereas it fully induced VASP dephosphorylation and Akt phosphorylation regardless of the presence of ticagrelor. In the presence of ticagrelor, blockage of the P2Y receptor prevented restoration of platelet aggregation by the combination of epinephrine and ADP, as well as intracellular Ca mobilisation. In combination with ADP, epinephrine induced platelet aggregation of ticagrelor-treated platelets through inhibition of the cAMP pathway and activation of the PI3K pathway, thus enabling the P2Y receptor signalling and subsequent Ca mobilisation. This proof-of-concept study needs to be challenged in vivo for the management of bleeding in ticagrelor-treated patients.
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http://dx.doi.org/10.1016/j.ejphar.2019.172798DOI Listing
January 2020

Potential usefulness of activated charcoal (DOAC remove®) for dRVVT testing in patients receiving Direct Oral AntiCoagulants.

Thromb Res 2019 Dec 5;184:86-91. Epub 2019 Nov 5.

Service d'Hématologie Biologique, CHU Pontchaillou, Rennes, France; University of Rennes 1, CIC-Inserm1414, Rennes, France.

Introduction: Lupus Anticoagulant testing using dilute Russell Viper Venom Time (dRVVT) is challenging in patients receiving Direct Oral AntiCoagulants (DOAC) due to potential false positive results. In a multicenter study, we evaluated the in vitro removal of DOAC by activated charcoal (DOAC remove®), allowing reliable dRVVT testing.

Materials And Methods: Patient samples were analyzed before and after treatment with DOAC remove®: 49 apixaban, 48 rivaroxaban, 24 dabigatran and 30 none. DOAC plasma concentrations were measured using anti-Xa or diluted thrombin time assays. In a subset of 28 samples, DOAC concentrations were also measured using HPLC-MS/MS following treatment with DOAC remove®. DRVVT was performed using STA-Staclot dRVVT Screen®/Confirm® (Stago) or LAC-Screening®/Confirmation® (Siemens).

Results: Baseline median [min-max] concentrations were 94 [<20-479] for apixaban, 107 [<20-501] for rivaroxaban and 135 ng/mL [<20-792] for dabigatran; dRVVT screen ratio/confirm ratio was positive in 47, 90 and 42% of apixaban, rivaroxaban and dabigatran samples. Treatment with DOAC remove® did not affect dRVVT results in non-DOAC patients while it resulted in DOAC concentrations <20 ng/mL in 82, 98 and 100% of samples, respectively. Concentrations were <5 ng/mL with HPLC-MS/MS in 5 out of 10, 8 out of 10 and 7 out of 8 samples, respectively. DOAC remove® corrected DOAC interference with dRVVT assays in 76, 85 and 95% of the patients, respectively.

Conclusion: For dRVVT testing in DOAC patients, we suggest the use of DOAC remove® for every rivaroxaban sample, whereas it might only be used in positive apixaban and dabigatran samples. A residual DOAC interference cannot be ruled out in case of persisting dRVVT positive results after treatment with DOAC remove®.
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http://dx.doi.org/10.1016/j.thromres.2019.11.001DOI Listing
December 2019

DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.

FASEB J 2019 12 4;33(12):14270-14280. Epub 2019 Nov 4.

Innovative Therapies in Haemostasis, INSERM Unité Mixte de Recherche (UMR)_S 1140, Université de Paris, Paris, France.

Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin-NETs matrices by seeding and activating neutrophils onto a fibrin surface and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE-DNA complexes is protected from inhibition by plasma antiproteases and sustains its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains (K, k) and the serine protease (SP) region (K-SP). We further demonstrate that patients with septic shock with disseminated intravascular coagulation have circulating HNE-DNA complexes, HNE-derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE-DNA complexes reduce plasminogen into fragments, thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin, and localized plasmin formation.-Barbosa da Cruz, D., Helms, J., Aquino, L. R., Stiel, L., Cougourdan, L., Broussard, C., Chafey, P., Riès-Kautt, M., Meziani, F., Toti, F., Gaussem, P., Anglés-Cano, E. DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.
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http://dx.doi.org/10.1096/fj.201901363RRRDOI Listing
December 2019

Effect of rivaroxaban and dabigatran on platelet functions: in vitro study.

Thromb Res 2019 Nov 22;183:159-162. Epub 2019 Oct 22.

Université de Paris, U1140 Innovative Therapies in Haemostasis, INSERM, F-75006 Paris, France; Service d'Hematologie Biologique, AP-HP, Georges Pompidou European Hospital, F-75015 Paris, France.

Introduction: Clinical benefit-risk balance of direct oral anticoagulants (DOAC) in atherothrombosis prevention differs between anti-Xa and anti-IIa drugs and their specific effect on platelet functions remains controversial. We hence investigated rivaroxaban and dabigatran effect on platelets in identical experimental conditions.

Materials And Methods: Blood of fifteen healthy volunteers was spiked with DOAC which plasma concentrations were measured by specific anti-Xa or anti-IIa assays. Light transmission aggregometry measured in platelet-rich plasma used low doses of agonists: 0.5 mM arachidonic acid, 2.5 μM ADP, 0.5 μM epinephrine, 0.8 μg/ml collagen, 7.5 μM TRAP-6 and 0.5 pM tissue factor in the presence of H-Gly-Pro-Arg-Pro-OH to prevent fibrin polymerization. Platelet adhesion on collagen fibres was evaluated in whole blood under flow. Same experiments were reproduced in the presence of aspirin.

Results: Median [95% CI] plasma concentrations were of 28 [23-36], 128 [119-144] and 321 [293-361] ng/ml for rivaroxaban and 39 [34-45], 171 [166-193] and 353 [349-382] ng/ml for dabigatran. DOAC did not modify platelet aggregation or adhesion on collagen fibres at any tested concentrations. However, they delayed platelet aggregation secondary to coagulation activation with a more potent effect with dabigatran (p < 0.001). Aspirin did not modify DOAC effect.

Conclusion: Efficacy of combining DOAC and aspirin in atherothrombosis prevention would not stem from a direct antiplatelet effect of the formers but to their additive inhibitory effect on platelet aggregation secondary to coagulation activation. This effect differs according to DOAC molecules and may also result from the pleiotropic roles of the different coagulation factors targeted by DOAC.
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http://dx.doi.org/10.1016/j.thromres.2019.10.007DOI Listing
November 2019

Pain assessment and factors influencing pain during bone marrow aspiration: A prospective study.

PLoS One 2019 29;14(8):e0221534. Epub 2019 Aug 29.

AP-HP, Hôpital Européen Georges Pompidou, Service d'Hématologie Biologique, Paris, France.

Although bone marrow aspiration (BMA) is still considered a painful procedure, pain level remains poorly documented. We therefore conducted a prospective study intended to evaluate pain level in adult patients undergoing BMA at the sternal or iliac crest site to identify factors associated with pain. We enrolled a total of 448 patients who underwent 461 BMA and asked those patients to score their pain intensity after BMA using numerical pain rating scale (NPRS). The following factors: level of anxiety, quality of the information given to the patient, operator's experience, and bone texture were recorded using a standardized questionnaire. The median NPRS score was 3.5 (IQR [2.0; 5.0]) the sternal site (n = 405) was associated with an increased median NPRS score (3.5 [2.0; 5.0]) compared to the iliac crest (n = 56, 2.5 [1.0; 4.0]; p<0.0001). For those patients who underwent sternal BMA, the median NPRS score was significantly lower when using lidocaine infiltration (p = 0.0159) as compared with no anesthetic use. Additionally there was no significant effect of anesthetic cream found. After multivariate analysis, the model of NPRS score at the sternal site included patient anxiety (p<0.0001) and the use of lidocaine infiltration (0.0378). This study underlines the usefulness of a comprehensive management including pain relief and efforts to reduce anxiety including appropriate information given to the patient during BMA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221534PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715342PMC
March 2020

Evaluation of commonly used tests to measure the effect of single-dose aspirin on mouse hemostasis.

Prostaglandins Leukot Essent Fatty Acids 2019 10 8;149:46-51. Epub 2019 Aug 8.

Université de Paris, Innovative Therapies in Haemostasis, INSERM, F-75006 Paris, France.

Discrepancies in preclinical studies of aspirin (ASA) antiplatelet activity in mouse models of bleeding and arterial thrombosis led us to evaluate commonly reported methods in order to propose a procedure for reliably measuring the effects of single dose ASA on mouse hemostasis. FVB and C57Bl6 mice received 100 mg/kg of ASA or vehicle orally 30 min or 3 h prior to investigate either hemostasis using the tail bleeding assay or carotid thrombosis induced by FeCl, or to blood sampling for isolated platelet aggregation and TXB generation. Expected inhibition of COX1 by ASA was ascertained by a strong decrease in TXB production, and its effect on platelet function and hemostasis, by decreased collagen-induced aggregation and increased bleeding time, respectively. Strikingly, we determined that anti-hemostatic effects of ASA were more predictable 30 min after administration than 3 h later. Conversely, ASA did not alter time to arterial occlusion of the carotid upon FeCl-induced thrombosis, suggesting ASA not to be used as reference inhibitor drug in this model of arterial thrombosis.
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http://dx.doi.org/10.1016/j.plefa.2019.08.002DOI Listing
October 2019

Successful Use of Recombinant Activated Factor VII to Reverse Ticagrelor-Induced Bleeding Risk: A Case Report.

TH Open 2018 Jul 27;2(3):e346-e349. Epub 2018 Sep 27.

Sorbonne Paris Cité, Faculté de Pharmacie, Université Paris Descartes, Paris, France.

Management of ticagrelor-associated bleeding is challenging, especially in neurosurgery. Platelet transfusion is inefficient and no antidote is currently available. We report here the first case of recombinant activated factor VII (rFVIIa) use to bypass ticagrelor-induced platelet inhibition. A woman treated with ticagrelor and requiring emergent neurosurgery for an intracranial hematoma received preoperative high-dose platelet transfusion and 60 μg/kg rFVIIa. Laboratory monitoring demonstrated that platelet transfusion failed to reverse ticagrelor-induced platelet inhibition while rFVIIa improved hemostasis by shortening the thromboelastometric clotting time. Neurosurgery occurred without any bleeding event but the patient presented with a postoperative pulmonary embolism. In conclusion, rFVIIa may decrease ticagrelor-induced bleeding risk but careful assessment of the benefit-risk balance is warranted before using rFVIIa to reverse ticagrelor effects.
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http://dx.doi.org/10.1055/s-0038-1672211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524896PMC
July 2018

Rivaroxaban pharmacodynamics in healthy volunteers evaluated with thrombin generation and the active protein C system: Modeling and assessing interindividual variability.

J Thromb Haemost 2019 10 9;17(10):1670-1682. Epub 2019 Jul 9.

Laboratoire d'Hématologie Biologique, Centre Hospitalier Universitaire Pontchaillou, Rennes, France.

Background: Rivaroxaban is a direct factor Xa inhibitor with substantial inter-individual pharmacokinetic (PK) variability. Pharmacodynamic (PD) variability, especially assessed with thrombin generation (TG), has been less documented.

Objectives: (i) To assess TG parameter time profiles in healthy volunteers, with TG being studied under different conditions and (ii) to model the relationship between rivaroxaban concentrations and TG parameters and subsequently estimate interindividual variability.

Methods: Sixty healthy male volunteers (DRIVING-NCT01627665) received a single 40-mg rivaroxaban dose. Blood sampling was performed at baseline and 10 predefined time points over 24 h. The TG was investigated with the fully automated ST-Genesia system (Stago), using two tissue-factor (TF) concentrations, in the absence (-), or presence (+) of thrombomodulin (TM) for the lowest one. The PD models were built to characterize the relationships between plasma rivaroxaban concentrations and endogenous thrombin potential (ETP) or peak height induced by the lowest TF concentration.

Results: Thrombin generation parameter time profiles with the lowest TF concentration showed a good sensitivity to rivaroxaban, especially +TM (active protein C negative feedback). The relationship between rivaroxaban concentrations and TG parameters was modeled with a sigmoidal relation. Mean rivaroxaban concentrations halving the baseline value of ETP and peak height (-TM) (C ) were of 284 and 33.2 ng/mL, respectively: +TM, C declined to 19.4 and 13.8 ng/mL, reflecting a powerful inhibitory effect. The estimated C population coefficients of variation were of 12.2% (-TM) and 31.3% (+TM) with the peak height models, 34.8% (+TM) with the ETP model.

Conclusions: This low-rivaroxaban to moderate-rivaroxaban PD variability in healthy volunteers contrasts with the substantial PK variability and deserves to be studied in different patient settings.
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http://dx.doi.org/10.1111/jth.14541DOI Listing
October 2019

Murine platelet production is suppressed by S1P release in the hematopoietic niche, not facilitated by blood S1P sensing.

Blood Adv 2019 06;3(11):1702-1713

INSERM U970, Paris Cardiovascular Research Centre, Paris, France.

The bioactive lipid mediator sphingosine 1-phosphate (S1P) was recently assigned critical roles in platelet biology: whereas S1P receptor-mediated S1P gradient sensing was reported to be essential for directing proplatelet extensions from megakaryocytes (MKs) toward bone marrow sinusoids, MK sphingosine kinase 2 (Sphk2)-derived S1P was reported to further promote platelet shedding through receptor-independent intracellular actions, and platelet aggregation through S1P Yet clinical use of S1P pathway modulators including fingolimod has not been associated with risk of bleeding or thrombosis. We therefore revisited the role of S1P in platelet biology in mice. Surprisingly, no reduction in platelet counts was observed when the vascular S1P gradient was ablated by impairing S1P provision to plasma or S1P degradation in interstitial fluids, nor when gradient sensing was impaired by deletion selectively in MKs. Moreover, S1P expression and signaling were both undetectable in mature MKs in situ, and MK deletion did not affect platelet aggregation or spreading. When deletion was induced in hematopoietic progenitor cells, platelet counts were instead significantly elevated. Isolated global Sphk2 deficiency was associated with thrombocytopenia, but this was not replicated by MK-restricted deletion and was reversed by compound deletion of either or , suggesting that this phenotype arises from increased S1P export and S1P activation secondary to redistribution of sphingosine to Sphk1. Consistent with clinical observations, we thus observe no essential role for S1P in facilitating platelet production or activation. Instead, S1P restricts megakaryopoiesis through S1P, and can further suppress thrombopoiesis through S1P when aberrantly secreted in the hematopoietic niche.
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http://dx.doi.org/10.1182/bloodadvances.2019031948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560353PMC
June 2019

Human Endothelial Colony Forming Cells Express Intracellular CD133 that Modulates their Vasculogenic Properties.

Stem Cell Rev Rep 2019 08;15(4):590-600

Sorbonne Paris Cité, Université Paris Descartes, Paris, France.

Stem cells at the origin of endothelial progenitor cells and in particular endothelial colony forming cells (ECFCs) subtype have been largely supposed to be positive for the CD133 antigen, even though no clear correlation has been established between its expression and function in ECFCs. We postulated that CD133 in ECFCs might be expressed intracellularly, and could participate to vasculogenic properties. ECFCs extracted from cord blood were used either fresh (n = 4) or frozen (n = 4), at culture days <30, to investigate the intracellular presence of CD133 by flow cytometry and confocal analysis. Comparison with HUVEC and HAEC mature endothelial cells was carried out. Then, CD133 was silenced in ECFCs using specific siRNA (siCD133-ECFCs) or scramble siRNA (siCtrl-ECFCs). siCD133-ECFCs (n = 12), siCtrl-ECFCs (n = 12) or PBS (n = 12) were injected in a hind-limb ischemia nude mouse model and vascularization was quantified at day 14 with H&E staining and immunohistochemistry for CD31. Results of flow cytometry and confocal microscopy evidenced the positivity of CD133 in ECFCs after permeabilization compared with not permeabilized ECFCs (p < 0.001) and mature endothelial cells (p < 0.03). In the model of mouse hind-limb ischemia, silencing of CD133 in ECFCs significantly abolished post-ischemic revascularization induced by siCtrl-ECFCs; indeed, a significant reduction in cutaneous blood flows (p = 0.03), capillary density (CD31) (p = 0.01) and myofiber regeneration (p = 0.04) was observed. Also, a significant necrosis (p = 0.02) was observed in mice receiving siCD133-ECFCs compared to those treated with siCtrl-ECFCs. In conclusion, our work describes for the first time the intracellular expression of the stemness marker CD133 in ECFCs. This feature could resume the discrepancies found in the literature concerning CD133 positivity and ontogeny in endothelial progenitors.
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http://dx.doi.org/10.1007/s12015-019-09881-8DOI Listing
August 2019