Publications by authors named "Marie-Christine Alessi"

177 Publications

The ISTH bleeding assessment tool as predictor of bleeding events in inherited platelet disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

J Thromb Haemost 2021 May;19(5):1364-1371

Department of Internal Medicine, IRCCS Policlinico S. Matteo Foundation, University of Pavia, Pavia, Italy.

Background: The ISTH Bleeding Assessment Tool (ISTH-BAT) has been validated for clinical screening of suspected von Willebrand disease (VWD) and for bleeding prediction. Recently it has been validated for subjects with inherited platelet disorders (IPD) (BAT-VAL study).

Objectives: To determine whether the ISTH-BAT bleeding score (BS) predicts subsequent bleeding events requiring treatment in IPD patients.

Methods: Patients with IPD, type 1 VWD (VWD-1) and age- and sex-matched healthy controls enrolled in the BAT-VAL study were prospectively followed-up for 2 years and bleeding episodes requiring treatment were recorded.

Results: Of the 1098 subjects initially enrolled, 955 were followed-up and 124 suffered hemorrhages during follow-up, 60% of whom had inherited platelet function disorders (IPFD). Total number of events was significantly higher in IPFD (n = 235) than VWD-1 (n = 52) or inherited thrombocytopenia (IT; n = 20). Events requiring transfusions were 66% in IPFD, 5.7% in VWD-1, and 3% in IT. Baseline BS was significantly higher in IPFD patients with a bleeding event at follow-up than in those without (p < .01) and the percentage of subjects suffering a bleeding event increased proportionally to baseline BS quartile. A significant association between the BS and the chance of suffering severe bleeding was found in the overall, IPFD, and VWD-1 populations. Similar results were obtained for the pediatric population.

Conclusions: Inherited platelet function disorder patients with high BS at enrollment are more likely to suffer from bleeding events requiring treatment at follow-up. Moreover, the higher the baseline BS quartile the greater the incidence of subsequent events, suggesting that independently from diagnosis a high BS is associated with a greater risk of subsequent hemorrhage.
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http://dx.doi.org/10.1111/jth.15263DOI Listing
May 2021

Severe thrombophilia in a factor V-deficient patient homozygous for the Ala2086Asp mutation (FV Besançon).

J Thromb Haemost 2021 May 8;19(5):1186-1199. Epub 2021 Apr 8.

C2VN, INSERM, INRA, Aix Marseille University, Marseille, France.

Background: Coagulation factor V (FV), present in plasma and platelets, has both pro- and anticoagulant functions.

Objective: We investigated an FV-deficient patient (FV:C 3%, FV:Ag 4%) paradoxically presenting with recurrent venous thrombosis (11 events) instead of bleeding.

Methods/results: Thrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet-poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet-rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC-cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FV ) that favors a "closed conformation" of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FV was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVa has slightly (≤1.5-fold) unfavorable kinetic parameters (K , V ) of prothrombin activation, but also a lower rate of APC-catalyzed inactivation in the presence of protein S.

Conclusions: FV induces a hypercoagulable state via quantitative (markedly decreased FV level) and qualitative (phospholipid-binding defect) effects that affect anticoagulant pathways (anticoagulant activities of FV, FVa inactivation, tissue factor pathway inhibitor α level) more strongly than the prothrombinase activity of FVa. A possible specific role of platelet FV cannot be excluded.
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http://dx.doi.org/10.1111/jth.15274DOI Listing
May 2021

Impaired adhesion of neutrophils expressing Slc44a2/HNA-3b to VWF protects against NETosis under venous shear rates.

Blood 2021 Apr;137(16):2256-2266

Aix-Marseille University, INSERM, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Center for CardioVascular and Nutrition Research (C2VN), Marseille, France.

Genome-wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for von Willebrand factor (VWF). To explain the link between Slc44a2 and VT, we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a- or HNA-3b-coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100 s-1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100 s-1. This adhesion could occur independently of β2 integrin and was enhanced when neutrophils were preactivated with lipopolysaccharide. Moreover, specific shear conditions with high neutrophil concentration could act as a "second hit," inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. The fact that neutrophils expressing Slc44a2/HNA-3b have a different response on VWF in the conditions tested could thus explain the association between HNA-3b and a reduced risk for VT in humans.
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http://dx.doi.org/10.1182/blood.2020008345DOI Listing
April 2021

A rare coding mutation in the MAST2 gene causes venous thrombosis in a French family with unexplained thrombophilia: The Breizh MAST2 Arg89Gln variant.

PLoS Genet 2021 01 19;17(1):e1009284. Epub 2021 Jan 19.

EA3878-GETBO, Univ Brest, Department of internal medicine and chest diseases, FCRIN_INNOVTE, CHU Brest, Brest, France.

Rare variants outside the classical coagulation cascade might cause inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (Arg89Gln) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2 Gln89 variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type. This study identifies a novel thrombophilia-causing Arg89Gln variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells.
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http://dx.doi.org/10.1371/journal.pgen.1009284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846112PMC
January 2021

On-Ticagrelor Platelet Reactivity and Clinical Outcome in Patients Undergoing Percutaneous Coronary Intervention for Acute Coronary Syndrome.

Thromb Haemost 2020 Dec 1. Epub 2020 Dec 1.

Intensive care Unit, Department of Cardiology, Assistance publique des hopitaux de Marseille, Hôpital Nord, Aix Marseille University, Mediterranean Association for Research and Studies in Cardiology (MARS Cardio), Marseille, France.

Background:  A strong association between on-thienopyridine platelet reactivity (PR) and the risk of both thrombotic and bleeding events in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) has been demonstrated. However, no study has analyzed the relationship between on-ticagrelor PR and clinical outcome in this clinical setting.

Objectives:  We aimed to investigate the relationship between on-ticagrelor PR, assessed by the vasodilator-stimulated phosphoprotein (VASP) index, and clinical outcome in patients with ACS undergoing PCI.

Methods:  We performed a prospective, multicenter, observational study of patients undergoing PCI for ACS. PR was measured using the VASP index following ticagrelor loading dose. The primary study endpoint was the rate of Bleeding Academic Research Consortium (BARC) type ≥2 at 1 year. The key secondary endpoint was the rate of major adverse cardiovascular events (MACE) defined as the composite of cardiovascular death, myocardial infarction, stroke, and urgent revascularization.

Results:  We included 570 ACS patients, among whom 33.9% had ST-elevation myocardial infarction. BARC type ≥2 bleeding occurred in 10.9% and MACE in 13.8%. PR was not associated with BARC ≥2 or with MACE ( = 0.12 and  = 0.56, respectively). No relationship between PR and outcomes was observed, neither when PR was analyzed quantitatively nor when it was analyzed qualitatively (low on-treatment PR [LTPR] vs. no LTPR).

Conclusion:  On-ticagrelor PR measured by the VASP was not associated with bleeding or thrombotic events in ACS patients undergoing PCI. PR measured by the VASP should not be used as a surrogate endpoint in studies on ticagrelor.
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http://dx.doi.org/10.1055/a-1326-5110DOI Listing
December 2020

A Combination of Single Nucleotide Polymorphisms is Associated with the Interindividual Variability of Cholesterol Bioavailability in Healthy Adult Males.

Mol Nutr Food Res 2020 11 15;64(22):e2000480. Epub 2020 Oct 15.

Aix-Marseille University, INRAE, INSERM, C2VN, 13005, Marseille, France.

Scope: Cholesterol bioavailability displays a high interindividual variability, partly due to genetic factors. Existing studies have focused on single nucleotide polymorphisms (SNPs) analyzed individually, which only explained a minor fraction of the variability of this complex phenotype. The aim is to identify a combination of SNPs associated with a significant part of the variability in cholesterol bioavailability.

Methods And Results: Thirty-nine healthy adult males are given a standard test snack containing 80 mg heptadeuterated (D7) cholesterol. The plasma D7-cholesterol concentration is measured at equilibrium 40 h after test snack intake. The D7-cholesterol response (D7-cholesterol/total cholesterol concentration) exhibits a relatively high interindividual variability (CV = 32%). The association of exonic SNPs in candidate genes (188 genes involved in or related to cholesterol metabolism) with the plasma D7-cholesterol response is assessed by univariate statistics followed by partial least squares regression. A significant model (p-value after cross-validation ANOVA = 1.64 × 10 ) that includes 8 SNPs (SOAT2-rs9658625, DNAH11-rs11768670, LIPC-rs690, MVK-rs2287218, GPAM-rs10787428, APOE-rs7412, CBS-rs234706, and WRN-rs1801196) explains 59.7% of the variance in cholesterol bioavailability (adjusted R²).

Conclusion: Here a combination of SNPs is significantly associated with the variability in dietary cholesterol bioavailability in healthy adult males.
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http://dx.doi.org/10.1002/mnfr.202000480DOI Listing
November 2020

Laboratory Techniques Used to Diagnose Constitutional Platelet Dysfunction.

Hamostaseologie 2020 Nov 15;40(4):444-459. Epub 2020 Sep 15.

Laboratory of Hematology, CHU Timone, Marseille Cedex 05, France.

Platelets play a major role in primary hemostasis, where activated platelets form plugs to stop hemorrhaging in response to vessel injuries. Defects in any step of the platelet activation process can cause a variety of platelet dysfunction conditions associated with bleeding. To make an accurate diagnosis, constitutional platelet dysfunction (CPDF) should be considered once von Willebrand disease and drug intake are ruled out. CPDF may be associated with thrombocytopenia or a genetic syndrome. CPDF diagnosis is complex, as no single test enables the analysis of all aspects of platelet function. Furthermore, the available tests lack standardization, and repeat tests must be performed in specialized laboratories especially for mild and moderate forms of the disease. In this review, we provide an overview of the laboratory tests used to diagnose CPDF, with a focus on light transmission platelet aggregation (LTA), flow cytometry (FC), and granules assessment. Global tests, mainly represented by LTA, are often initially performed to investigate the consequences of platelet activation on platelet aggregation in a single step. Global test results should be confirmed by additional analytical tests. FC represents an accurate, simple, and reliable test to analyze abnormalities in platelet receptors, and granule content and release. This technique may also be used to investigate platelet function by comparing resting- and activated-state platelet populations. Assessment of granule content and release also requires additional specialized analytical tests. High-throughput sequencing has become increasingly useful to diagnose CPDF. Advanced tests or external research laboratory techniques may also be beneficial in some cases.
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http://dx.doi.org/10.1055/a-1223-3306DOI Listing
November 2020

Perioperative Open-lung Approach, Regional Ventilation, and Lung Injury in Cardiac Surgery.

Anesthesiology 2020 11;133(5):1029-1045

Background: In the Protective Ventilation in Cardiac Surgery (PROVECS) randomized, controlled trial, an open-lung ventilation strategy did not improve postoperative respiratory outcomes after on-pump cardiac surgery. In this prespecified subanalysis, the authors aimed to assess the regional distribution of ventilation and plasma biomarkers of lung epithelial and endothelial injury produced by that strategy.

Methods: Perioperative open-lung ventilation consisted of recruitment maneuvers, positive end-expiratory pressure (PEEP) = 8 cm H2O, and low-tidal volume ventilation including during cardiopulmonary bypass. Control ventilation strategy was a low-PEEP (2 cm H2O) low-tidal volume approach. Electrical impedance tomography was used serially throughout the perioperative period (n = 56) to compute the dorsal fraction of ventilation (defined as the ratio of dorsal tidal impedance variation to global tidal impedance variation). Lung injury was assessed serially using biomarkers of epithelial (soluble form of the receptor for advanced glycation end-products, sRAGE) and endothelial (angiopoietin-2) lung injury (n = 30).

Results: Eighty-six patients (age = 64 ± 12 yr; EuroSCORE II = 1.65 ± 1.57%) undergoing elective on-pump cardiac surgery were studied. Induction of general anesthesia was associated with ventral redistribution of tidal volumes and higher dorsal fraction of ventilation in the open-lung than the control strategy (0.38 ± 0.07 vs. 0.30 ± 0.10; P = 0.004). No effect of the open-lung strategy on the dorsal fraction of ventilation was noted at the end of surgery after median sternotomy closure (open-lung = 0.37 ± 0.09 vs. control = 0.34 ± 0.11; P = 0.743) or in extubated patients at postoperative day 2 (open-lung = 0.63 ± 0.18 vs. control = 0.59 ± 0.11; P > 0.999). Open-lung ventilation was associated with increased intraoperative plasma sRAGE (7,677 ± 3,097 pg/ml vs. 6,125 ± 1,400 pg/ml; P = 0.037) and had no effect on angiopoietin-2 (P > 0.999).

Conclusions: In cardiac surgery patients, open-lung ventilation provided larger dorsal lung ventilation early during surgery without a maintained benefit as compared with controls at the end of surgery and postoperative day 2 and was associated with higher intraoperative plasma concentration of sRAGE suggesting lung overdistension.

Editor’s Perspective:
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http://dx.doi.org/10.1097/ALN.0000000000003539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907969PMC
November 2020

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Blood 2020 10;136(17):1956-1967

Service d'Hématologie Biologique, Hospices Civils de Lyon, Lyon, France.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.
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http://dx.doi.org/10.1182/blood.2019004776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582559PMC
October 2020

Strengths and Weaknesses of Light Transmission Aggregometry in Diagnosing Hereditary Platelet Function Disorders.

J Clin Med 2020 Mar 12;9(3). Epub 2020 Mar 12.

Inserm U1048, I2MC et Université Paul Sabatier, 31024 Toulouse Cedex, France.

Hereditary defects in platelet function are responsible for sometimes severe mucocutaneous hemorrhages. They are a heterogeneous group of abnormalities whose first-line diagnosis typically involves interpreting the results of in vitro light transmission aggregometry (LTA) traces. Interpretation of LTA is challenging. LTA is usually performed in specialized laboratories with expertise in platelet pathophysiology. This review updates knowledge on LTA, describing the various platelet aggregation profiles typical of hereditary platelet disorders to guide the physician in the diagnosis of functional platelet disorders.
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http://dx.doi.org/10.3390/jcm9030763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141357PMC
March 2020

Contribution of exome sequencing to the identification of genes involved in the response to clopidogrel in cardiovascular patients.

J Thromb Haemost 2020 06 20;18(6):1425-1434. Epub 2020 Mar 20.

Geneva Platelet Group, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

Background: On-clopidogrel platelet reactivity (PR) is associated with the risk of thrombotic or bleeding event in selected populations of high-risk patients. PR is a highly heritable phenotype and a few variants of cytochrome genes, essentially CYP2C19, are associated with PR but only explain 5% to 12% of the variability.

Objective: The aim of this study is to delineate genetic determinants of on-clopidogrel PR using high-throughput sequencing.

Methods: We performed a whole exome sequencing of 96 low- and matched high-PR patients in a discovery cohort. Exomes from genes with variants significantly associated with PR were sequenced in 96 low- and matched high-PR patients from an independent replication cohort.

Results: We identified 585 variants in 417 genes with an adjusted P value < .05. In the replication cohort, all top variants including CYP2C8, CYP2C18, and CYP2C19 from the discovery population were found again. An original network analysis identified several candidate genes of potential interest such as a regulator of PI3K, a key actor in the downstream signaling pathway of the P2Y receptor.

Conclusion: This study emphasizes the role of CYP-related genes as major regulators of clopidogrel response, including the poorly investigated CYP2C8 and CYP2C18.
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http://dx.doi.org/10.1111/jth.14776DOI Listing
June 2020

RasGRP2 Structure, Function and Genetic Variants in Platelet Pathophysiology.

Int J Mol Sci 2020 Feb 6;21(3). Epub 2020 Feb 6.

Aix Marseille University, INSERM, INRAE, C2VN, 13005 Marseille, France.

RasGRP2 is calcium and diacylglycerol-regulated guanine nucleotide exchange factor I that activates Rap1, which is an essential signaling-knot in "inside-out" αIIbβ3 integrin activation in platelets. Inherited platelet function disorder caused by variants of RASGRP2 represents a new congenital bleeding disorder referred to as platelet-type bleeding disorder-18 (BDPLT18). We review here the structure of RasGRP2 and its functions in the pathophysiology of platelets and of the other cellular types that express it. We will also examine the different pathogenic variants reported so far as well as strategies for the diagnosis and management of patients with BDPLT18.
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http://dx.doi.org/10.3390/ijms21031075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037602PMC
February 2020

An integrated approach to inherited platelet disorders: results from a research collaborative, the Sydney Platelet Group.

Pathology 2020 Feb 10;52(2):243-255. Epub 2020 Jan 10.

Northern Blood Research Centre, Kolling Institute of Medical Research, University of Sydney, Sydney, NSW, Australia; Department of Haematology and Transfusion Medicine, Royal North Shore Hospital, Sydney, NSW, Australia.

Inherited disorders of platelet function (IPFD) and/or number (IPND) are heterogeneous conditions that result in variable mucocutaneous bleeding symptoms as a result of deranged primary haemostasis caused by platelet dysfunction or thrombocytopenia. Diagnosis is important to guide post-operative bleeding prophylactic strategies, to avoid treatment with inappropriate medications, and inform prognosis. Achieving an accurate diagnosis has traditionally been hampered by the requirement of multiple, often complex, laboratory tests that are not always available at single centres. To improve the diagnosis of these disorders a research collaborative was established, the Sydney Platelet Group, that explored an integrated approach combining traditional and contemporary platelet phenotypic and genetic diagnostic platforms available at four Sydney tertiary hospitals. Herein we report the outcomes of the first 50 patients evaluated using this approach. The cohort included 22 individuals with suspected IPFD and 28 with thrombocytopenia. Bleeding scores were higher in individuals with IPFD (mean 5.75; SD 4.83) than those with IPNDs (mean 2.14; SD 2.45). In cases with suspected IPFD, diagnosis to the level of the defective pathway was achieved in 71% and four individuals were found not to have a definitive platelet function defect. Dense granule secretion disorders were the most common platelet pathway abnormality detected (n=5). Mean bleeding scores in these individuals were not significantly different to individuals with defects in other commonly detected platelet pathways (dense granules, signal transduction and 'undetermined'). A molecular diagnosis was achieved in 52% of individuals with IPNDs and 5% with IPFD. Likely pathogenic and pathogenic variants detected included variants associated with extra-haematological complications (DIAPH1, MYH9) and potential for malignancy (ANKRD26 and RUNX1). The level of platelet investigation undertaken by this initiative is currently not available elsewhere in Australia and initial results confirm the utility of this integrated phenotypic-genetic approach.
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http://dx.doi.org/10.1016/j.pathol.2019.10.005DOI Listing
February 2020

High prevalence of mutations in perilipin 1 in patients with precocious acute coronary syndrome.

Atherosclerosis 2020 01 12;293:86-91. Epub 2019 Dec 12.

MARS Cardio, Mediterranean Association for Research and Studies in Cardiology, Intensive Care Unit, Hospital Nord, Marseille, France; Aix Marseille Univ, INSERM, INRA, C2VN, Marseille, France. Electronic address:

Background And Aims: Genetic partial lipodystrophies are rare heterogeneous disorders characterized by abnormalities of fat distribution and associated metabolic complications including a predisposition for atherosclerotic cardiovascular disease. We hypothesized that the milder forms of these diseases might be underdiagnosed and might result in early acute coronary syndrome (ACS) as the first sign of the pathology.

Methods: We performed targeted sequencing on a panel of 8 genes involved in genetic lipodystrophy for 62 patients with premature ACS, and selected heterozygous missense variations with low frequency. To confirm those results, we analyzed a second independent group of 60 additional patients through Sanger sequencing, and compared to a control group of 120 healthy patients.

Results: In the first cohort, only PLIN1 exhibited variants in more than 1 patient. In PLIN1, 3 different variants were found in 6 patients. We then analyzed PLIN1 sequence in the second cohort with premature ACS and found 2 other patients. Altogether, 8 patients were carriers of 4 different mutations in PLIN1. The variant frequencies in the total cohort of 122 patients were compared to frequencies observed in a local control cohort and in 2 different public databases showing a significant difference between patient vs control group frequencies for two mutations out of 4 (c.245C > T p = 10; c.839G > A p = 0.014).

Discussion: This is the first study that identifies a high frequency of potential pathogenic mutations in PLIN1 related to early onset ACS. These findings could contribute to the prevention and care of precocious ACS in families carrying those mutations.
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http://dx.doi.org/10.1016/j.atherosclerosis.2019.12.002DOI Listing
January 2020

Validation of the ISTH/SSC bleeding assessment tool for inherited platelet disorders: A communication from the Platelet Physiology SSC.

J Thromb Haemost 2020 03 16;18(3):732-739. Epub 2019 Dec 16.

Department of Internal Medicine, IRCCS Policlinico S. Matteo Foundation, University of Pavia, Pavia, Italy.

Background: Careful assessment of bleeding history is the first step in the evaluation of patients with mild/moderate bleeding disorders, and the use of a bleeding assessment tool (BAT) is strongly encouraged. Although a few studies have assessed the utility of the ISTH-BAT in patients with inherited platelet function disorders (IPFD) none of them was sufficiently large to draw conclusions and/or included appropriate control groups.

Objectives: The aim of the present study was to test the utility of the ISTH-BAT in a large cohort of patients with a well-defined diagnosis of inherited platelets disorder in comparison with two parallel cohorts, one of patients with type-1 von Willebrand disease (VWD-1) and one of healthy controls (HC).

Patients/methods: We enrolled 1098 subjects, 482 of whom had inherited platelet disorders (196 IPFD and 286 inherited platelet number disorders [IT]) from 17 countries.

Results: IPFD patients had significantly higher bleeding score (BS; median 9) than VWD-1 patients (median 5), a higher number of hemorrhagic symptoms (4 versus 3), and higher percentage of patients with clinically relevant symptoms (score > 2). The ISTH-BAT showed excellent discrimination power between IPFD and HC (0.9 < area under the curve [AUC] < 1), moderate (0.7 < AUC < 0.9) between IPFD and VWD-1 and between IPFD and inherited thrombocytopenia (IT), while it was inaccurate (AUC ≤ 0.7) in discriminating IT from HC.

Conclusions: The ISTH-BAT allows to efficiently discriminate IPFD from HC, while it has lower accuracy in distinguishing IPFD from VWD-1. Therefore, the ISTH-BAT appears useful for identifying subjects requiring laboratory evaluation for a suspected IPFD once VWD is preliminarily excluded.
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http://dx.doi.org/10.1111/jth.14683DOI Listing
March 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

Platelet CD40 ligand and bleeding during P2Y12 inhibitor treatment in acute coronary syndrome.

Res Pract Thromb Haemost 2019 Oct 26;3(4):684-694. Epub 2019 Jul 26.

Aix Marseille University INSERM, INRA, C2VN Marseille France.

Antiplatelet therapy through inhibition of the adenosine diphosphate (ADP)/P2Y12 pathway is commonly used in the treatment of acute coronary syndrome (ACS). Although efficient in preventing platelet activation and thrombus formation, it increases the risk of bleeding complications. In patients with ACS receiving platelet aggregation inhibitors, that is, P2Y12 blockers (n = 923), we investigated the relationship between plasma and platelet-associated CD40L levels and bleeding events (n = 71). Treatment with P2Y12 inhibitors in patients with ACS did not affect plasma-soluble CD40L levels, but decreased platelet CD40L surface expression (pCD40L) and platelet-released CD40L (rCD40L) levels in response to stimulation as compared to healthy controls. In vitro inhibition of the ADP pathway in healthy control platelets reduced both pCD40L and rCD40L levels. In a multivariable analysis, the reduced pCD40L level observed in ACS patients was significantly associated with the risk of bleeding occurrence (adjusted odds ratio = 0.15; 95% confidence interval = 0.034-0.67). P2Y12 inhibitor-treated (ticagrelor) mice exhibited a 2.5-fold increase in tail bleeding duration compared with controls. A significant reduction in bleeding duration was observed on CD40L but not CD40L platelet infusion. In addition, CD40L blockade in P2Y12 inhibitor-treated blood samples from a healthy human reduced thrombus growth over immobilized collagen under arterial flow. In conclusion, measurement of pCD40L may offer a novel approach to assessing bleeding risk in patients with ACS who are being treated with P2Y12 inhibitors.
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http://dx.doi.org/10.1002/rth2.12244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781928PMC
October 2019

Platelet reactivity inhibition following ticagrelor loading dose in patients undergoing percutaneous coronary intervention for acute coronary syndrome.

J Thromb Haemost 2019 12 27;17(12):2188-2195. Epub 2019 Jul 27.

Intensive Care Unit, Department of Cardiology, Assistance Publique-Hôpitaux de Marseille, Hôpital Nord, Aix-Marseille University, Marseille, France.

Background: Ticagrelor induces more potent platelet reactivity (PR) inhibition with reduced interindividual variability compared to clopidogrel. Although on-clopidogrel PR was shown to correlate with ischemia and bleeding events, no study has investigated the relationship between on-ticagrelor PR and outcome.

Objectives: We aimed to evaluate the relationship between on-ticagrelor PR, assessed by the vasodilator-stimulated phosphoprotein index (VASP), and thrombotic and bleeding events in patients with acute coronary syndrome (ACS) treated by percutaneous coronary intervention (PCI).

Methods: We performed a prospective, multicenter observational study on patients treated with PCI for ACS. The VASP index was used to assess PR after ticagrelor loading dose (LD). The primary endpoint was the link between major adverse cardiovascular events (MACE) and PR.

Results: Among the 530 patients with ACS included, 183 (34.5%) were admitted for ST elevation myocardial infarction. We observed high potency and limited interindividual variability after the ticagrelor LD (VASP 19.1% ± 16.6%). At 1 month, 21 (3.8%) MACE and 29 (5.5%) bleedings ≥ 2 according to the Bleedings Academic Research Consortium (BARC) scale were recorded. Neither MACE nor bleeding was associated with PR (P = .34 and P = .78, respectively). However, there was a strong association between PR and the occurrence of definite acute stent thrombosis (P = .03). Platelet reactivity was the only factor associated with acute definite stent thrombosis.

Conclusion: In patients receiving a ticagrelor LD while undergoing PCI for ACS, PR using the VASP did not predict MACE or bleeding, but it was significantly associated with the occurrence of definite acute stent thrombosis.
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http://dx.doi.org/10.1111/jth.14592DOI Listing
December 2019

A Novel Rapid Method of Red Blood Cell and Platelet Permeabilization and Staining for Flow Cytometry Analysis.

Cytometry B Clin Cytom 2019 09 13;96(5):426-435. Epub 2019 Jul 13.

Aix-Marseille Univ, INSERM, INRA, C2VN, Marseille, France.

Background: Flow cytometry essentially focuses on surface-expressed proteins, with few protocols being devoted to intracellular components. We evaluated a two-step procedure using new formaldehyde-free permeabilization and staining reagents that allow the staining of platelets and red blood cells (RBCs) from whole blood.

Methods: Citrated blood was treated with the new staining protocol (NSP) or control reagent (phosphate-buffered solution bovine serum albumin) and stained with antibodies against surface or intracellular markers. The effects of the NSP on cell integrity, morphology, and content were evaluated.

Results: The NSP slightly reduced the cell count (~20%) and changed the RBC morphology with a 42% mean diameter reduction. Conversely, the NSP did not affect platelet discoid morphology and led to a minor size decrease (11%). These morphological changes neither impelled a gating strategy modification nor interfered with the discrimination among populations based on surface markers. The NSP provided intracellular access to all the tested antigens: CD62P, FXIII, and CD63 in platelets and glycated and fetal hemoglobin (HbA1c and HbF) and nucleic acid in RBCs. The NSP gave excellent intra-assay precision with minimal impact on cell morphology and fluorescence labelling over time (up to 24 h).

Conclusions: With the ability to detect surface and intracellular antigens through a rapid preparation protocol without washing steps or toxic formaldehyde treatment, this NSP designed for research offers a marked improvement in the analysis of platelets and RBCs isolated directly from whole blood. Consequently, the NSP opens new avenues to investigate platelet degranulation and erythrocyte subpopulations. © 2019 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21839DOI Listing
September 2019

Increased levels of the megakaryocyte and platelet expressed cysteine proteases stefin A and cystatin A prevent thrombosis.

Sci Rep 2019 07 3;9(1):9631. Epub 2019 Jul 3.

Aix Marseille Univ, INSERM, INRA, C2VN, Marseille, 13385, France.

Increased platelet activity occurs in type 2 diabetes mellitus (T2DM) and such platelet dysregulation likely originates from altered megakaryopoiesis. We initiated identification of dysregulated pathways in megakaryocytes in the setting of T2DM. We evaluated through transcriptomic analysis, differential gene expressions in megakaryocytes from leptin receptor-deficient mice (db/db), exhibiting features of human T2DM, and control mice (db/+). Functional gene analysis revealed an upregulation of transcripts related to calcium signaling, coagulation cascade and platelet receptors in diabetic mouse megakaryocytes. We also evidenced an upregulation (7- to 9.7-fold) of genes encoding stefin A (StfA), the human ortholog of Cystatin A (CSTA), inhibitor of cathepsin B, H and L. StfA/CSTA was present in megakaryocytes and platelets and its expression increased during obesity and diabetes in rats and humans. StfA/CSTA was primarily localized at platelet membranes and granules and was released upon agonist stimulation and clot formation through a metalloprotease-dependent mechanism. StfA/CSTA did not affect platelet aggregation, but reduced platelet accumulation on immobilized collagen from flowing whole blood (1200 s). In-vivo, upon laser-induced vascular injury, platelet recruitment and thrombus formation were markedly reduced in StfA1-overexpressing mice without affecting bleeding time. The presence of CA-074Me, a cathepsin B specific inhibitor significantly reduced thrombus formation in-vitro and in-vivo in human and mouse, respectively. Our study identifies StfA/CSTA as a key contributor of platelet-dependent thrombus formation in both rodents and humans.
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http://dx.doi.org/10.1038/s41598-019-45805-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610149PMC
July 2019

Common Risk Factors Add to Inherited Thrombophilia to Predict Venous Thromboembolism Risk in Families.

TH Open 2019 Jan 28;3(1):e28-e35. Epub 2019 Jan 28.

Laboratory of Haematology, La Timone Hospital, Marseille, France.

The clinical venous thromboembolism (VTE) pattern often shows wide heterogeneity within relatives of a VTE-affected family, although they carry the same thrombophilia defect. It is then mandatory to develop additional tools for assessing VTE risk in families with thrombophilia. This study aims to assess whether common environmental and genetic risk factors for VTE contribute to explain this heterogeneity. A total of 2,214 relatives from 651 families with known inherited thrombophilia were recruited at the referral center for thrombophilia in Marseilles, France, from 1986 to 2013. A thrombophilia screening was systematically performed in all included relatives. According to the severity of the thrombophilia defect, individuals were split into three groups: no familial defect, mild thrombophilia, and severe thrombophilia. In addition, common genetic factors (ABO blood group and 11 polymorphisms selected on the basis of their association with VTE in the general population) were genotyped. Furthermore, body mass index and smoking were collected. VTE incidence was 1.74, 3.64, and 6.40 per 1,000 person-years in individuals with no familial defect, mild thrombophilia, and severe thrombophilia, respectively. Five common risk factors were associated with VTE in this population: obesity, smoking, ABO blood group, and _rs2036914 and _rs2066865 polymorphisms. These common factors were then included into a three-level risk score. The score was highly efficient for assessing VTE risk in mild thrombophilia patients by identifying two groups with different VTE risk; individuals with low score had the same risk as individuals with no familial defect whereas individuals with high score had the same risk as individuals with severe thrombophilia. An overall score including the five items plus the thrombophilia status was built and displayed an area under the receiver operating characteristic curve of 0.702 for discriminating VTE and non-VTE relatives. In conclusion, integrating common environmental and genetic risk factors improved VTE risk assessment in relatives from families with thrombophilia.
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http://dx.doi.org/10.1055/s-0039-1677807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524901PMC
January 2019

Novel ACTN1 variants in cases of thrombocytopenia.

Hum Mutat 2019 12 6;40(12):2258-2269. Epub 2019 Nov 6.

Aix-Marseille Univ, INSERM, INRA, C2VN, Marseille, France.

The ACTN1 gene has been implicated in inherited macrothrombocytopenia. To decipher the spectrum of variants and phenotype of ACTN1-related thrombocytopenia, we sequenced the ACTN1 gene in 272 cases of unexplained chronic or familial thrombocytopenia. We identified 15 rare, monoallelic, nonsynonymous and likely pathogenic ACTN1 variants in 20 index cases from 20 unrelated families. Thirty-one family members exhibited thrombocytopenia. Targeted sequencing was carried out on 12 affected relatives, which confirmed presence of the variant. Twenty-eight of 32 cases with monoallelic ACTN1 variants had mild to no bleeding complications. Eleven cases harbored 11 different unreported ACTN1 variants that were monoallelic and likely pathogenic. Nine variants were located in the α-actinin-1 (ACTN1) rod domain and were predicted to hinder dimer formation. These variants displayed a smaller increase in platelet size compared with variants located outside the rod domain. In vitro expression of the new ACTN1 variants induced actin network disorganization and led to increased thickness of actin fibers. These findings expand the repertoire of ACTN1 variants associated with thrombocytopenia and highlight the high frequency of ACTN1-related thrombocytopenia cases. The rod domain, like other ACTN1 functional domains, may be mutated resulting in actin disorganization in vitro and thrombocytopenia with normal platelet size in most cases.
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http://dx.doi.org/10.1002/humu.23840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900141PMC
December 2019

Binding of Coagulation Factor XIII Zymogen to Activated Platelet Subpopulations: Roles of Integrin αIIbβ3 and Fibrinogen.

Thromb Haemost 2019 Jun 1;119(6):906-915. Epub 2019 Apr 1.

Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russia.

Factor XIIIa (fXIIIa) is a transglutaminase that plays a crucial role in fibrin clot stabilization and regulation of fibrinolysis. It is known to bind to procoagulant platelets. In contrast, the zymogen fXIII interaction with platelets is not well characterized. We investigated the interaction of zymogen fXIII with activated platelet subpopulations. Confocal microscopy and flow cytometry using fluorescently labelled factors and antibodies. Phosphatidylserine (PS)-positive activated platelets bound 700 to 800 molecules/cell of fXIII at 100 nM, while both PS-negative activated platelets and resting platelets bound 200 to 400 molecules/cell. The binding was reversible, calcium-independent and linear within the fXIII concentration range of up to 1,000 nM. fXIII predominantly bound to the caps of procoagulant platelets and co-localized with fibrinogen. Exogenous fibrinogen promoted fXIII binding by activated PS-negative platelets; this effect was abolished by the integrin αIIbβ3 antagonist monafram. The fXIII binding was 1.5- to 3-fold decreased for platelets from four patients with grey platelet syndrome, and was variable for platelets from six patients with Glanzmann's thrombasthenia. Strong platelet stimulation, fibrinogen and αIIbβ3 play essential roles in fXIII binding, without any of them fXIII does not bind to platelets. The preferential binding in the cap-like structures might be important for increasing local fXIII concentration in platelet thrombi.
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http://dx.doi.org/10.1055/s-0039-1683912DOI Listing
June 2019

Lipodystrophy-like features after total body irradiation among survivors of childhood acute leukemia.

Endocr Connect 2019 Apr;8(4):349-359

Aix Marseille University, INSERM, INRA, C2VN, Marseille, France.

Background/objective: The number of long-term survivors of childhood acute leukemia (AL) is substantially growing. These patients are at high risk for metabolic syndrome (MS), especially those who received total body irradiation (TBI). The consequences of children's irradiation on adipose tissue (AT) development in adulthood are currently unknown. The objective of this study is to assess the impact of TBI on AT of childhood AL survivors.

Design: We compared the morphological and functional characteristics of AT among survivors of childhood AL who developed MS and received (n = 12) or not received (n = 12) TBI.

Subjects/methods: Body fat distribution and ectopic fat stores (abdominal visceral and liver fat) were evaluated by DEXA, MRI and 1H-spectroscopy. Functional characteristics of subcutaneous AT were investigated by studying gene expression and pre-adipocyte differentiation in culture.

Results: Patients who have received TBI exhibited a lower BMI (minus 5 kg/m2) and a lower waist circumference (minus 14 cm), especially irradiated women. Despite the lower quantity of intra-abdominal AT, irradiated patient displayed a nearly two-fold greater content of liver fat when compared to non-irradiated patient (17 vs 9%, P = 0.008). These lipodystrophic-like features are supplemented by molecular abnormalities in subcutaneous AT of irradiated patients: decrease of gene expression of SREBP1 (minus 39%, P = 0.01) and CIDEA (minus 36%, P = 0.004) and a clear alteration of pre-adipocyte differentiation.

Conclusions: These results strongly support the direct effect of irradiation on AT, especially in women, leading to specific nonalcoholic fatty liver disease, despite lower BMI. A long-term appropriate follow-up is necessary for these patients.
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http://dx.doi.org/10.1530/EC-18-0497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6454302PMC
April 2019

Thromboxane-prostaglandin receptor antagonist, terutroban, prevents neurovascular events after subarachnoid haemorrhage: a nanoSPECT study in rats.

Crit Care 2019 Feb 11;23(1):42. Epub 2019 Feb 11.

Department of Anaesthesiology and Critical Care Medicine, INT (Institut de Neurosciences de la Timone), University Hospital Timone, Aix Marseille University, Marseille, France.

Background: Several lipid metabolites in cerebrospinal fluid are correlated with poor outcomes in aneurysmal subarachnoid haemorrhage. Most of these metabolites bind to ubiquitous thromboxane-prostaglandin (TP) receptors, causing vasoconstriction and inflammation. Here, we evaluated terutroban (TBN), a specific TP receptor antagonist, for the prevention of post-haemorrhage blood-brain barrier disruption, neuronal apoptosis and delayed cerebral hypoperfusion.

Methods: The rat double subarachnoid haemorrhage model was produced by twice injecting (days 1 and 2) autologous blood into the cisterna magna. Seventy-eight male Sprague-Dawley rats were assigned to experimental groups. Rats exposed to subarachnoid haemorrhage were allocated to no treatment (SAH group) or TBN treatment by gastric gavage during the first 5 days after haemorrhage (SAH+TBN group). Control rats received artificial cerebrospinal fluid injections (CSF group). Sham-operated rats with or without TBN administration were also studied. Body weight and Garcia neurological scores were assessed on day 2 and day 5. We used nanoscale single-photon emission computed tomography (nanoSPECT) to measure brain uptake of three radiolabelled agents: Technetium-diethylenetriaminepentacetate (Tc-DTPA), which indicated blood-brain barrier permeability on day 3, Technetium-annexin V-128 (Tc-Anx-V128), which indicated apoptosis on day 4, and Technetium-hexamethylpropyleneamineoxime (Tc-HMPAO), which indicated cerebral perfusion on day 5. Basilar artery narrowing was verified histologically, and cerebral TP receptor agonists were quantified.

Results: Tc-DTPA uptake unveiled blood-brain barrier disruption in the SAH group. TBN mitigated this disruption in the brainstem area. Tc-Anx-V128 uptake was increased in the SAH group and TBN diminished this effect in the cerebellum. Tc-HMPAO uptake revealed a global decreased perfusion on day 5 in the SAH group that was significantly counteracted by TBN. TBN also mitigated basilar artery vasoconstriction, neurological deficits (on day 2), body weight loss (on day 5) and cerebral production of vasoconstrictors such as Thromboxane B2 and Prostaglandin F2α.

Conclusions: Based on in vivo nanoscale imaging, we demonstrated that TBN protected against blood-brain barrier disruption, exerted an anti-apoptotic effect and improved cerebral perfusion. Thus, TP receptor antagonists showed promising results in treating post-haemorrhage neurovascular events.
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http://dx.doi.org/10.1186/s13054-019-2338-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6371436PMC
February 2019

Phenotype analysis and clinical management in a large family with a novel truncating mutation in , the CalDAG-GEFI encoding gene.

Res Pract Thromb Haemost 2017 Jul 20;1(1):128-133. Epub 2017 Jun 20.

IHU-LIRYC Plateforme Technologique D'Innovation Biomédicale Hopital Xavier Arnozan Pessac France.

Background: Genetic variants in the gene encoding calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) represent a new inherited bleeding disorder linked to major defects of platelet aggregation and activation of αIIbβ3 integrin. They are of major interest as CalDAG-GEFI is receiving attention as a potential target for antiplatelet therapy for prevention and treatment of cardiovascular disorders including arterial thrombosis and atherosclerosis.

Objectives: To better understand the phenotypical and clinical profiles of patients with CalDAG-GEFI deficiency.

Patients: We report a five-generation family with a novel truncating CalDAG-GEFI mutation detailing clinical management and phenotypic variability.

Results: Patients IV.6 & IV.4 manifested with episodes of serious mucocutanous bleeding or bleeding after surgery not responding to platelet transfusion but responding well to recombinant Factor VIIa infusions. Their blood counts and coagulation parameters were normal but platelet aggregation to ADP and collagen was defective. Further work-up confirmed normal levels of αIIb and β3 in their platelets but decreased αIIbβ3 function. DNA analysis by whole exome sequencing within the BRIDGE-BPD consortium (Cambridge, UK), allowed us to highlight a homozygous c.1490delT predicted to give rise to a p.F497Sfs*22 truncating mutation near to the C-terminal domain of CalDAG-GEFI. Sanger sequencing confirmed that both patients were homozygous for the c.1490delT and 3 out of 4 close family members were heterozygous.

Conclusions: A long-term prospective study is warranted for full clinical exploration of CalDAG-GEFI to understand the bleeding phenotyes and their management.
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http://dx.doi.org/10.1002/rth2.12019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974916PMC
July 2017

From Naproxen Repurposing to Naproxen Analogues and Their Antiviral Activity against Influenza A Virus.

J Med Chem 2018 08 3;61(16):7202-7217. Epub 2018 Aug 3.

Gustave Roussy Institute, Paris Saclay University, UMR8200 CNRS , 94805 Villejuif , France.

The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00557DOI Listing
August 2018

The Annexin A1 Receptor FPR2 Regulates the Endosomal Export of Influenza Virus.

Int J Mol Sci 2018 May 8;19(5). Epub 2018 May 8.

C2VN Aix Marseille Univ, INSERM, INRA, C2VN, 13005 Marseille, France.

The Formyl Peptide Receptor 2 (FPR2) is a novel promising target for the treatment of influenza. During viral infection, FPR2 is activated by annexinA1, which is present in the envelope of influenza viruses; this activation promotes virus replication. Here, we investigated whether blockage of FPR2 would affect the genome trafficking of influenza virus. We found that, upon infection and cell treatment with the specific FPR2 antagonist WRW4 or the anti-FPR2 monoclonal antibody, FN-1D6-AI, influenza viruses were blocked into endosomes. This effect was independent on the strain and was observed for H1N1 and H3N2 viruses. In addition, blocking FPR2signaling in alveolar lung A549 epithelial cells with the monoclonal anti-FPR2 antibody significantly inhibited virus replication. Altogether, these results show that FPR2signaling interferes with the endosomal trafficking of influenza viruses and provides, for the first time, the proof of concept that monoclonal antibodies directed against FPR2 inhibit virus replication. Antibodies-based therapeutics have emerged as attractive reagents in infectious diseases. Thus, this study suggests that the use of anti-FPR2 antibodies against influenza hold great promise for the future.
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http://dx.doi.org/10.3390/ijms19051400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983815PMC
May 2018

Anti-α β immunization in Glanzmann thrombasthenia: review of literature and treatment recommendations.

Br J Haematol 2018 04 2;181(2):173-182. Epub 2018 Apr 2.

Laboratory of Haematology, University Hospital of La Timone, French Reference Centre for Rare Platelet Disorders, Marseille, France.

Glanzmann thrombasthenia (GT) is caused by inherited defects of the α β platelet glycoprotein. This bleeding disorder can be treated with platelet transfusion therapy, but some patients will be immunized and begin to form anti-human leucocyte antigen (HLA) and/or anti-α β antibodies. These antibodies can bind and interfere with the function of the transfused platelets, rendering treatment ineffective. However, platelet transfusion refractoriness attributable to HLA antibodies may be managed by the selection of compatible donors, although they are not always readily available, particularly in an emergency. Thus, anti-α β antibodies represent one of the most severe complications in GT. Both genetic and environmental factors may contribute to the risk of anti-α β development, but the underlying pathogenic mechanisms are still unknown. This review will summarize the current knowledge of the risk factors for development of anti-α β antibodies in patients with GT and discuss how these findings may influence the clinical management of patients.
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http://dx.doi.org/10.1111/bjh.15087DOI Listing
April 2018