Publications by authors named "Nathalie Hézard"

26 Publications

  • Page 1 of 1

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

Authors:
Elisabetta Castoldi Nathalie Hézard Guillaume Mourey Kanin Wichapong Marjorie Poggi Manal Ibrahim-Kosta M Christella L G D Thomassen Alexandra Fournel Catherine P M Hayward Marie-Christine Alessi Tilman M Hackeng Jan Rosing Pierre-Emmanuel Morange

J Thromb Haemost 2021 Feb 19. Epub 2021 Feb 19.

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
February 2021

Laboratory Techniques Used to Diagnose Constitutional Platelet Dysfunction.

Authors:
Manal Ibrahim-Kosta Marie-Christine Alessi Nathalie Hezard

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

Receptor for Advanced Glycation End Products is Involved in Platelet Hyperactivation and Arterial Thrombosis during Chronic Kidney Disease.

Authors:
Jérémy Ortillon Nathalie Hézard Karim Belmokhtar Charlotte Kawecki Christine Terryn Guenter Fritz Alexandre Kauskot Ann Marie Schmidt Philippe Rieu Philippe Nguyen Pascal Maurice Fatouma Touré

Thromb Haemost 2020 Sep 29;120(9):1300-1312. Epub 2020 Jul 29.

UMR CNRS 7369 Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), Team 2 "Matrix Aging and Vascular Remodelling," Université de Reims Champagne Ardenne, Reims, France.

Background:  Chronic kidney disease (CKD) is associated with a high cardiovascular mortality due to increased rates of vascular lesions and thrombotic events, as well as serum accumulation of uremic toxins. A subgroup of these toxins (advanced glycation end products [AGEs] and S100 proteins) can interact with the receptor for AGEs (RAGE). In this study, we analyzed the impact of CKD on platelet function and arterial thrombosis, and the potential role of RAGE in this process.

Methods:  Twelve weeks after induction of CKD in mice, platelet function and time to complete carotid artery occlusion were analyzed in four groups of animals (sham-operated, CKD, apolipoprotein E [Apoe], and Apoe/Ager mice).

Results:  Analysis of platelet function from whole blood and platelet-rich plasma showed hyperactivation of platelets only in CKD Apoe mice. There was no difference when experiments were done on washed platelets. However, preincubation of such platelets with AGEs or S100 proteins induced RAGE-mediated platelet hyperactivation. In vivo, CKD significantly reduced carotid occlusion times of Apoe mice (9.2 ± 1.1 vs. 11.1 ± 0.6 minutes for sham,  < 0.01). In contrast, CKD had no effect on occlusion times in Apoe/Ager mice. Moreover, carotid occlusion in Apoe CKD mice occurred significantly faster than in Apoe/Ager CKD mice ( < 0.0001).

Conclusion:  Our results show that CKD induces platelet hyperactivation, accelerates thrombus formation in a murine model of arterial thrombosis, and that RAGE deletion has a protective role. We propose that RAGE ligands binding to RAGE is involved in CKD-induced arterial thrombosis.
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http://dx.doi.org/10.1055/s-0040-1714101DOI Listing
September 2020

Emicizumab treatment: Impact on coagulation tests and biological monitoring of haemostasis according to clinical situations (BIMHO group proposals).

Authors:
Christophe Nougier Emmanuelle Jeanpierre Catherine Ternisien Valérie Proulle Nathalie Hezard Claire Pouplard Dominique Lasne

Eur J Haematol 2020 Dec 16;105(6):675-681. Epub 2020 Sep 16.

Laboratoire d'hématologie générale, Hôpital Necker, AP-HP, Paris, France.

Emicizumab, a bispecific humanised monoclonal antibody restoring to some extent the function of activated FVIII deficient in haemophilia A, represents a major therapeutic advance in the management of haemophilia A patients. No dosage adjustment is required, which leads to a major change for patients used to regular biological monitoring which is particularly burdensome in the case of substitution therapy. In some circumstances, such as before an invasive procedure or in case of bleeding, biological monitoring will be necessary and emicizumab's interference with haemostasis tests, particularly those based on an activated partial thromboplastin times (aPTT), must be known to best interpret the tests and to select the most appropriate methods to guide therapy. The normalisation of aPTT in patients treated with emicizumab is not sufficient to consider haemostasis as normalised. In the event of administration of FVIII to a patient receiving emicizumab, the determination of FVIII should use a chromogenic method using non-human reagents. Coagulation global tests have been proposed to evaluate the biological response when using bypassing agents in patients treated with emicizumab, but the usefulness must be confirmed. The French group BIMHO presents proposals for biological monitoring of a patient treated with emicizumab according to clinical situations.
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http://dx.doi.org/10.1111/ejh.13490DOI Listing
December 2020

Multicentre evaluation of CK Prest for assaying plasma levels of factor IX fused with albumin (Idelvion ).

Authors:
Claire Pouplard Hubert Galinat Catherine Ternisien Florence Blanc Jouvan Emmanuel De Maistre Jérôme Duchemin Claire Flaujac Nathalie Hézard François Grand Véronique Le Cam-Duchez Raphael Marlu Guillaume Mourey Fabienne Nedelec Fabienne Pineau-Vincent Yohann Repesse Alain Stépanian Jean Szymezak Sophie Voisin Anne Lise Voyer Emmanuelle Jeanpierre Dominique Lasne

Haemophilia 2019 Sep 11;25(5):e327-e330. Epub 2019 Jul 11.

Department of biologic Haematology, AP-HP, Hôpital Necker Paris, INSERM, UMR-S1176, University Paris Sud, University Paris-Saclay, Le Kremlin Bicêtre, France.

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http://dx.doi.org/10.1111/hae.13812DOI Listing
September 2019

Elastin-derived peptides are new regulators of thrombosis.

Authors:
Charlotte Kawecki Nathalie Hézard Olivier Bocquet Gaël Poitevin Fanja Rabenoelina Alexandre Kauskot Laurent Duca Sébastien Blaise Béatrice Romier Laurent Martiny Philippe Nguyen Laurent Debelle Pascal Maurice

Arterioscler Thromb Vasc Biol 2014 Dec 23;34(12):2570-8. Epub 2014 Oct 23.

From the URCA, CNRS UMR 7369 (Matrice Extracellulaire et Dynamique Cellulaire, MEDyC), laboratoire SiRMa, UFR Sciences Exactes et Naturelles, Reims, France (C.K., O.B., F.R., L. Ducca, S.B., B.R., L.M., L. Debelle, P.M.); EA3801, Hémostase et remodelage vasculaire post-ischémique (HERVI), UFR de Médecine, Reims, France (N.H., G.P., P.N.); CHU Reims, Hôpital Robert Debré, Laboratoire d'Hématologie, Reims, France (N.H., P.N.); INSERM UMRS 1140, Université Paris Descartes, Sorbonne Paris Cité, France (A.K.); and INSERM U770, Le Kremlin Bicêtre, Université Paris-Sud, Le Kremlin Bicêtre, France (A.K.).

Objective: Elastin is the major structural extracellular matrix component of the arterial wall that provides the elastic recoil properties and resilience essential for proper vascular function. Elastin-derived peptides (EDP) originating from elastin fragmentation during vascular remodeling have been shown to play an important role in cell physiology and development of cardiovascular diseases. However, their involvement in thrombosis has been unexplored to date. In this study, we investigated the effects of EDP on (1) platelet aggregation and related signaling and (2) thrombus formation. We also characterized the mechanism by which EDP regulate thrombosis.

Approach And Results: We show that EDP, derived from organo-alkaline hydrolysate of bovine insoluble elastin (kappa-elastin), decrease human platelet aggregation in whole blood induced by weak and strong agonists, such as ADP, epinephrine, arachidonic acid, collagen, TRAP, and U46619. In a mouse whole blood perfusion assay over a collagen matrix, kappa-elastin and VGVAPG, the canonical peptide recognizing the elastin receptor complex, significantly decrease thrombus formation under arterial shear conditions. We confirmed these results in vivo by demonstrating that both kappa-elastin and VGVAPG significantly prolonged the time for complete arteriole occlusion in a mouse model of thrombosis and increased tail bleeding times. Finally, we demonstrate that the regulatory role of EDP on thrombosis relies on platelets that express a functional elastin receptor complex and on the ability of EDP to disrupt plasma von Willebrand factor interaction with collagen.

Conclusions: These results highlight the complex nature of the mechanisms governing thrombus formation and reveal an unsuspected regulatory role for circulating EDP in thrombosis.
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http://dx.doi.org/10.1161/ATVBAHA.114.304432DOI Listing
December 2014

Performance of two new automated assays for measuring von Willebrand activity: HemosIL AcuStar and Innovance.

Authors:
Emmanuel de Maistre Fabienne Volot Guillaume Mourey Ludwig S Aho Catherine Ternisien Marie-Elisabeth Briquel Marie-Anne Bertrand Brigitte Tardy Birgit Frotscher Philippe Nguyen Laure Dumont David Vandroux Nathalie Hézard Marc Trossaërt

Thromb Haemost 2014 Oct 7;112(4):825-30. Epub 2014 Aug 7.

Dr. Marc Trossaërt, Centre Régional de Traitement de l'Hémophilie, 1 Place Alexis RICORDEAU, Centre Hospitalier Universitaire, 44093 Nantes Cedex 1, France, Tel.: +33 2 40 08 74 68, Fax: +33 2 40 08 42 59, E-mail:

The ristocetin cofactor activity assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity but remains difficult to perform, and the coefficient of variation of the method is high (about 20-30%). This study evaluated and compared the performance for measuring the VWF activity of two newly commercialised assays [VWF:Ac Innovance (VWF:Ac) and VWF:RCo Acustar (VWF:RCo Acu)] with the reference VWF:RCo aggregation in 123 pathological plasma samples. The correlation and concordance between both new tests (VWF:RCo-Acu and VWF:Ac) and the reference VWF:RCo were good. The results of the VWF activity to VWF antigen ratio were also comparable whatever the method for the classification of VWF deficiency in all patients. Our results showed that both new tests could replace the "gold standard" VWF:RCo in aggregometry with several benefits: they are fully automated, easier and faster to perform, better adapted to emergency situations if necessary.
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http://dx.doi.org/10.1160/TH14-02-0108DOI Listing
October 2014

Spectrum of the mutations in Bernard-Soulier syndrome.

Authors:
Anna Savoia Shinji Kunishima Daniela De Rocco Barbara Zieger Margaret L Rand Nuria Pujol-Moix Umran Caliskan Huseyin Tokgoz Alessandro Pecci Patrizia Noris Alok Srivastava Christopher Ward Marie-Christine Morel-Kopp Marie-Christine Alessi Sylvia Bellucci Philippe Beurrier Emmanuel de Maistre Rémi Favier Nathalie Hézard Marie-Françoise Hurtaud-Roux Véronique Latger-Cannard Cécile Lavenu-Bombled Valérie Proulle Sandrine Meunier Claude Négrier Alan Nurden Hanitra Randrianaivo Fabrizio Fabris Helen Platokouki Nurit Rosenberg Basma HadjKacem Paula G Heller Mehran Karimi Carlo L Balduini Annalisa Pastore Francois Lanza

Hum Mutat 2014 Sep 15;35(9):1033-45. Epub 2014 Jul 15.

Institute for Maternal and Child Health - IRCCS "Burlo Garofolo", Trieste, Italy; Department of Medical Sciences, University of Trieste, Trieste, Italy.

Bernard-Soulier syndrome (BSS) is a rare autosomal recessive bleeding disorder characterized by defects of the GPIb-IX-V complex, a platelet receptor for von Willebrand factor (VWF). Most of the mutations identified in the genes encoding for the GP1BA (GPIbα), GP1BB (GPIbβ), and GP9 (GPIX) subunits prevent expression of the complex at the platelet membrane or more rarely its interaction with VWF. As a consequence, platelets are unable to adhere to the vascular subendothelium and agglutinate in response to ristocetin. In order to collect information on BSS patients, we established an International Consortium for the study of BSS, allowing us to enrol and genotype 132 families (56 previously unreported). With 79 additional families for which molecular data were gleaned from the literature, the 211 families characterized so far have mutations in the GP1BA (28%), GP1BB (28%), or GP9 (44%) genes. There is a wide spectrum of mutations with 112 different variants, including 22 novel alterations. Consistent with the rarity of the disease, 85% of the probands carry homozygous mutations with evidence of founder effects in some geographical areas. This overview provides the first global picture of the molecular basis of BSS and will lead to improve patient diagnosis and management.
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http://dx.doi.org/10.1002/humu.22607DOI Listing
September 2014

Prospective evaluation of a rapid nanoparticle-based lateral flow immunoassay (STic Expert(®) HIT) for the diagnosis of heparin-induced thrombocytopenia.

Authors:
Dorothée Leroux Nathalie Hezard Aurélien Lebreton Anne Bauters Pierre Suchon Emmanuel de Maistre Christine Biron Marie-Genevieve Huisse Catherine Ternisien Sophie Voisin Yves Gruel Claire Pouplard

Br J Haematol 2014 Sep 12;166(5):774-82. Epub 2014 May 12.

Haemostasis Laboratory and UMR CNRS 7292, Hôpital Trousseau and Université François Rabelais Tours, Tours, France.

A rapid lateral flow immunoassay (LFIA) (STic Expert(®) HIT), recently developed for the diagnosis of heparin-induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme-linked immunosorbent assay (Asserachrom(®) HPIA IgG) and serotonin release assay. The inter-reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4-PaGIA(®) ) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert(®) HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT.
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http://dx.doi.org/10.1111/bjh.12939DOI Listing
September 2014

Anticoagulant properties of the anti-inflammatory cytokine IL-10 in a factor Xa-activated human monocyte model.

Authors:
Sonia Ben-Hadj-Khalifa Philippe Nguyen Touhami Mahjoub Nathalie Hézard

Eur Cytokine Netw 2012 Jul-Sep;23(3):87-92

Unité de recherche des maladies hématologiques et auto-immunes, faculté de pharmacie, 5000 Monastir, Tunisie.

Background: Monocytes and factor Xa (FXa) are procoagulant agents implicated in the physiopathological processes of atherosclerosis and thrombosis.

Objective: we evaluated the anticoagulant effect of the anti-inflammatory cytokine IL-10 on an FXa-activated human monocyte (Hu-monocyte) procoagulant phenotype.

Methods: Hu-monocytes were purified by elutriation and activated by FXa. The effect of IL-10 was assessed by means of a 2 h pre-incubation step with recombined human IL-10 (0.5 and 1 ng/mL). Real-time RT-PCR and Western blotting were used to evaluate the effect of IL-10 on tissue factor (TF) mRNA and protein synthesis. A thrombin generation (TG) assay was used as a functional test to assess the effect of IL-10 on TF-dependent TG.

Results: we showed that IL-10 inhibited both TFmRNA and TF protein expression in a dose-dependant manner.We showed, as a functional consequence, that IL-10 inhibited Hu-monocyte-triggered TG and that this inhibition was concentration-dependant, and significant for all TG phases. The rate index of the propagation phase (rate index) was the most sensitive parameter while the endpoint of TG decay (S-tail) and the endogenous thrombin potential (ETP) were the least sensitive (inhibition of 80, 40 and 30% respectively). The IL-10 pattern of TG inhibition was similar to TF-Ab-induced inhibition: IC(50) were not reached by ETP and S-tail, and the lowest IC(50) values were reached by the rate index (0.61 ± 0.12 ng/mL and 1.87 ± 0.35 μg/mL respectively).

Conclusion: the anticoagulant effect of the anti-inflammatory cytokine IL-10 in an FXa-activated Hu-monocyte model is an additional illustration of the cross-talk between inflammation and coagulation, opening new approaches in the field of arteriosclerosis and thrombosis.
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http://dx.doi.org/10.1684/ecn.2012.0315DOI Listing
July 2013

A new feature of the MYH9-related syndrome: chronic transaminase elevation.

Authors:
Rémi Favier Analisa DiFeo Nathalie Hezard Monique Fabre Pierre Bedossa John A Martignetti

Hepatology 2013 Mar 27;57(3):1288-9. Epub 2012 Nov 27.

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http://dx.doi.org/10.1002/hep.25913DOI Listing
March 2013

IL-10 modulates fondaparinux inhibition of monocyte-induced thrombin generation.

Authors:
Sonia Ben-Hadj-Khalifa Nathalie Hézard Wassim Y Almawi Souad Lakbakbi Catherine Macé Pascale Cornillet-Lefebvre Touhami Mahjoub Philippe Nguyen

J Thromb Thrombolysis 2011 Oct;32(3):311-7

Faculté de Pharmacie, Unité de Recherche des Maladies Hématologiques et Auto-Immunes, Monastir, Tunisia.

In addition to its established immuno-regulating capacity, the anti-inflammatory cytokine interleukin (IL)-10 exerts direct effects on coagulation. IL-10 down regulates the expression of tissue factor (TF) and thrombin generation (TG). Thus, we hypothesised that IL-10 could enhance the effect of anticoagulants. To evaluate in vitro the potential additive effect of IL-10 on fondaparinux-induced anticoagulation. Human monocytes were purified by elutriation, and were activated by factor Xa (FXa). Real-time RT-PCR and Western blotting were used to evaluate FXa-induced TF synthesis. TG test was used as a functional test to assess TF-dependent monocyte procoagulation, and to evaluate the effects of IL-10 (200 and 500 pg/ml) and fondaparinux (0.0, 0.1, 0.4, 0.7 and 1.2 μg/ml), separately and in combination. We confirmed that FXa induced TF mRNA and protein synthesis by monocyte in a concentration dependent manner. We showed that FXa-activated monocytes triggered TG via TF expression. We reported that IL-10 inhibited TG with a marginal effect seen at 200 pg/ml. Results with fondaparinux showed a concentration-dependent TG inhibition. The combination of IL-10 and fondaparinux effects demonstrated that IL-10: (i) potentiates the inhibitory effect of fondaparinux on TG by 10-30%, and (ii) dramatically modifies fondaparinux IC50 for each TG parameter. IL-10 enhances in vitro the extent of anticoagulation induced by fondaparinux.
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http://dx.doi.org/10.1007/s11239-011-0618-1DOI Listing
October 2011

Differential coagulation inhibitory effect of fondaparinux, enoxaparin and unfractionated heparin in cell models of thrombin generation.

Authors:
Sonia Ben-Hadj-Khalifa Nathalie Hézard Wassim Y Almawi Marie G Remy Bernadette Florent Touhami Mahjoub Philippe Nguyen

Blood Coagul Fibrinolysis 2011 Jul;22(5):369-73

Unité de Recherche des Maladies Hématologiques et Auto-Immunes, Monastir, Tunisia.

Anticoagulants, including unfractionated heparin (UFH), enoxaparin and fondaparinux, are approved drugs in acute coronary syndrome (ACS). Monocytes and monocyte-derived microparticles (MMPs) play an important procoagulant role in ACS by expressing high tissue factor (TF) levels, which in turn triggers thrombin generation. The objective of our study is to compare the in-vitro inhibitory effect of UFH, enoxaparin and fondaparinux in monocytes and MMP models. Human-elutriated monocytes were activated for 5 and 18 h by lipopolysaccharide to obtain activated monocytes (ac-M) or MMPs, respectively. Thrombin generation inhibition was assessed using ac-M or MMPs mixed with platelet-poor plasma containing increased concentrations of anticoagulants. Thrombin generation inhibition was dose-dependent with a differential effect according to the drug: the highest for UFH, the lowest for fondaparinux. Rate index was the most sensitive parameter. For fondaparinux, its IC50 values (anti-Xa IU/ml) were 0.59±0.05 for ac-M and 0.17±0.03 for MMPs. For enoxaparin, rate index IC50 values were 0.27±0.03 for ac-M and 0.19±0.02 for MMPs. Our data support the notion that cell-induced thrombin generation assay may be a reliable alternative to anti-Xa assessment in determining patient anticoagulation level.
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http://dx.doi.org/10.1097/MBC.0b013e328344f7d0DOI Listing
July 2011

Differential inhibitory effect of fondaparinux on the procoagulant potential of intact monocytes and monocyte-derived microparticles.

Authors:
Sonia Ben-Hadj-Khalifa-Kechiche Nathalie Hezard Stephane Poitevin Marie-Geneviève Remy Bernadette Florent Touhami Mahjoub Philippe Nguyen

J Thromb Thrombolysis 2010 Nov;30(4):412-8

Laboratoire d'Hématologie, CHU Robert Debré, 51092 Reims Cedex, France.

Monocytes and monocyte-derived microparticles (MMPs) play a major role in acute coronary syndrome (ASC). Activated monocytes (ac-M) and MMPs support thrombin generation via tissue factor (TF). The aim of this study was to evaluate the inhibitory effect of fondaparinux, a selective Xa inhibitor, on thrombin generation supported by activated monocytes and MMPs. Monocytes were purified by elutriation. They were activated by LPS, allowing to obtain both ac-M and MMPs. Thrombin generation was performed using Fluoroscan(®) in these two cell models, in comparison with a cell-free model (TF 5 pM final). Two concentrations of ac-M (0.2 × 10⁶ and 1 × 10⁶/well) and four concentrations of MMPs (40,000; 80,000; 120,000 and 160,000/well) were tested. TGT was evaluated for increasing fondaparinux concentrations (0, 0.1, 0.4, 0.7 and 1.2 μg/ml). Without fondaparinux, 0.2 × 10⁶ ac-M and 160,000 MMPs induced comparable results. Fondaparinux inhibited thrombin generation in the three models. Inhibition was fondaparinux concentration dependent. Rate index was the most sensitive parameter, compared to lag-time, peak and endogenous thrombin potential. The rate index IC(50) were 0.69 ± 0.03 μg/ml for ac-M, 0.20 ± 0.03 μg/ml for MMPs, and 0.22 ± 0.02 μg/ml for cell-free model. Fondaparinux exerted an inhibitory effect at all concentrations, including the lowest (0.1 μg/ml). The extend of inhibition was similar between MMPs and cell-free models, and stronger than ac-M model. We assume that the efficacy of fondaparinux 2.5 mg once daily in ACS patients may be in part attributed to its inhibitory effect on MMPs.
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http://dx.doi.org/10.1007/s11239-010-0490-4DOI Listing
November 2010

AlphaIIbbeta3 integrin: new allelic variants in Glanzmann thrombasthenia, effects on ITGA2B and ITGB3 mRNA splicing, expression, and structure-function.

Authors:
Vincent Jallu Mathilde Dusseaux Simon Panzer Marie-Françoise Torchet Nathalie Hezard Jenny Goudemand Alexandre G de Brevern Cécile Kaplan

Hum Mutat 2010 Mar;31(3):237-46

Platelet Immunology Laboratory, INTS, Paris, France.

Glanzmann thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation due to defects in integrin alphaIIbbeta3 (ITGA2B, ITGB3), a fibrinogen receptor. Mutations from 24 GT patients and two carriers of various origins, Caucasian, North-African and Asian were characterized. Promoter and exon sequences of alphaIIb and beta3 genes were amplified and directly sequenced. Among 29 identified mutations, 17 new allelic variants resulting from nonsense, missense and deletion/insertion mutations were described. RNA alterations were evaluated by using Web servers. The alphaIIb p.S926L, p.V903F, and beta3 p.C38Y, p.M118R, p.G221D substitutions prevented complex expression at the surface of COS-7 cells by altering the alphaIIb or the beta3 subunit structure. As shown by free energy analyses applied on the resolved structure of alphaIIbbeta3 and structural modeling of the mutant, the p.K253M substitution of beta3 helped to define a key role of the K253 in the interaction of the alphaIIb beta-propeller and the beta3 beta-I domains. finally, the alphaIIb p.Q595H substitution allowed cell surface expression of the complex but its corresponding c.2800G>T mutation is predicted to alter normal RNA splicing. In conclusion, our study yielded the discovery of 17 new GT allelic variants, revealed the key role of K253 of alphaIIb for the alphaIIbbeta3 complex formation and provides an additional example of an apparently missense mutation causing a splicing defect.
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http://dx.doi.org/10.1002/humu.21179DOI Listing
March 2010

Is flow cytometry accurate enough to screen platelet autoantibodies?

Authors:
Nathalie Hézard Gérard Simon Catherine Macé Vincent Jallu Cécile Kaplan Philippe Nguyen

Transfusion 2008 Mar 7;48(3):513-8. Epub 2007 Dec 7.

Laboratoire d'Hématologie, CHU Robert Debré, Reims, France.

Background: The diagnosis of immune thrombocytopenic purpura (ITP) is a diagnosis of exclusion, as stated by international guidelines. Nevertheless, the assessment of platelet (PLT) antibodies has been reported as helpful for the diagnosis and the follow-up of ITP patients. PLT antibodies are detected by highly specialized assays, such as monoclonal antibody-specific immobilization of PLT antigen (MAIPA) test. Flow cytometry for PLT-associated immunoglobulin G (PAIgG) detection has been described more recently. This study was meant to evaluate the utility of flow cytometry to screen accurately patients needing further MAIPA testing.

Study Design And Methods: PAIgG, PAIgM, and PAIgA were determined in 107 consecutive patients and in 147 healthy controls in parallel. MAIPA testing was performed in all patients. The accuracy of flow cytometry was assessed with a receiver operating characteristics (ROC) curve analysis versus MAIPA.

Results: MAIPA assay found PLT-specific IgG in 27 patients (25%). The ROC curve analysis showed that no false-negative result in flow cytometry was obtained for a mean fluorescence intensity (MFI) cutoff of 0.2. With this cutoff, PAIgG were positive in 61 patients (57%). In this series, MAIPA was unnecessary in 42 percent of patients (corresponding to true-negative results). When MAIPA was positive, PAIgM values ranged from 0.1 to 1.0, and PAIgA from 0.1 to 2.

Conclusion: Flow cytometry for PAIgG assessment may be used to accurately decide whether or not MAIPA must be subsequently performed. In this series, MAIPA was unnecessary in 42 percent of patients. Moreover, PAIgM results suggested that its determination combined with PAIgG may be of interest in ITP investigation.
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http://dx.doi.org/10.1111/j.1537-2995.2007.01556.xDOI Listing
March 2008

A case control study of deep venous thrombosis in relation to factor V G1691A (Leiden) and A4070G (HR2 Haplotype) polymorphisms.

Authors:
Lobna Bouaziz-Borgi Philipe Nguyen Nathalie Hezard Umayya Musharrafieh Wassim Y Almawi Touhami Mahjoub

Exp Mol Pathol 2007 Dec 6;83(3):480-3. Epub 2007 May 6.

Faculty of Pharmacy, University of Monastir, Monastir, Tunisia.

Activated protein C resistance (APCR) is a significant risk factor for venous thromboembolism (VTE), with the factor V (FV) G1691A (Leiden) mutation accounting for the majority of inherited APCR cases. An additional FV polymorphism, A4074G (FV-HR2), reportedly increased VTE risk by some, but not all groups. We determined the prevalence of FV-Leiden and FV-HR2 SNPs in 126 patients with deep venous thrombosis (DVT), and 197 control subjects. Frequencies of FV-Leiden A and HR2 G alleles, together with FV-Leiden G/A and A/A (but not HR2 A/G) genotypes were significantly higher among patients. While no significant linkage disequilibrium was noted between FV 1691A and 4070G or A alleles, significantly higher prevalence of single-mutant 1691G/4070G and 1691A/4070A haplotypes were seen in patients. FV Leiden and FV HR2 haplotype are independent risk factors for DVT, and their coinheritance does not seem to increase significantly DVT risk imparted by either.
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http://dx.doi.org/10.1016/j.yexmp.2007.04.006DOI Listing
December 2007

Protein C deficiency screening using a thrombin-generation assay.

Authors:
Nathalie Hézard Lobna Bouaziz-Borgi Marie-Geneviève Remy Bernadette Florent Philippe Nguyen

Thromb Haemost 2007 Jan;97(1):165-6

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January 2007

Reliability of thrombin generation assay on frozen-thawed platelet-rich plasma: a reply.

Authors:
Nathalie Hézard Marie-Geneviève Remy Bernadette Florent Philippe Nguyen

Clin Chem 2006 Nov;52(11):2127-8

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http://dx.doi.org/10.1373/clinchem.2006.074567DOI Listing
November 2006

Distinct association of factor V-Leiden and prothrombin G20210A mutations with deep venous thrombosis in Tunisia and Lebanon.

Authors:
Lobna Bouaziz-Borgi Wassim Y Almawi Nabil Mtiraoui Brahim Nsiri Sose H Keleshian Raghid Kreidy Bessem Louzir Nathalie Hezard Touhami Mahjoub

Am J Hematol 2006 Aug;81(8):641-3

Faculty of Pharmacy, University of Monastir, Tunisia.

Factor V G1691A (FV-Leiden) and prothrombin (PRT) G20210A single nucleotide polymorphisms (SNPs) were associated with venous thrombosis among Caucasians. We assessed the contribution of both SNPs to the genetic susceptibility of deep venous thrombosis (DVT) among Lebanese and Tunisian patients. Subjects comprised 198 DVT patients and 540 healthy controls from Lebanon and 126 Tunisian DVT patients and 197 control subjects; FV-Leiden (MnlI) and PRT G20210A (HindIII) genotyping was done by PCR-RFLP. While the prevalence of FV-Leiden mutant A allele and the G/A and A/A genotypes were significantly higher among DVT patients from Lebanon and Tunisia, the association of PRT G20210A with DVT was pronounced among Lebanese but not Tunisian patients. The prevalence of PRT G20210A mutant A allele (P < 0.001 vs. P = 181) and G/A genotype (P < 0.001 vs. P = 0.994) was significantly higher among Lebanese but not Tunisians, respectively. While FV-Leiden was a common genetic risk factor for DVT in both communities, the contribution of PRT G20210A to the genetic susceptibility of DVT differed among Lebanese and Tunisians, which underscores the need to determine prothrombotic gene polymorphisms associated with DVT among Arab and Mediterranean basin communities.
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http://dx.doi.org/10.1002/ajh.20582DOI Listing
August 2006

Interleukin-10 promoter polymorphism and venous thrombosis: a case-control study.

Authors:
Eva Cochery-Nouvellon Fabien Vitry Pascale Cornillet-Lefebvre Nathalie Hezard Lucile Gillot Philippe Nguyen

Thromb Haemost 2006 Jul;96(1):24-8

Laboratoire Central d'hématologie, CHU Robert Debré, Université de Reims Champagne-Ardenne, France.

The human interleukin-10 promoter gene is highly polymorphic. IL-10 polymorphisms have been associated with various autoimmune and lymphoproliferative disorders. Although IL-10 has been shown to modulate thrombin generation in several experimental models, it is not known whether IL-10 polymorphisms could be a risk factor for venous thrombosis. We therefore conducted a case-control study comparing 74 consecutive patients who experienced at least one episode of documented venous thromboembolic event and 100 healthy controls. All subjects were Caucasians. Five polymorphisms of the IL-10 promoter gene were studied: two highly polymorphic dinucleotide repeats, IL-10 R and IL-10G, and three single nucleotide polymorphisms at position -1082, -819 and -592. Factor VG1691C Leiden mutation was systematically determined. Multivariate logistic regression analysis showed that IL-10 G13 and G10 alleles are independent risk factors for venous thrombosis (Odds ratio:OR = 3.33, p = 0.003 and OR = 2.83, p = 0.03, respectively). Furthermore, IL-10 G10 allele is more frequent in recurrent disease.
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http://dx.doi.org/10.1160/TH06-01-0027DOI Listing
July 2006

Utility of thrombin-generation assay in the screening of factor V G1691A (Leiden) and prothrombin G20210A mutations and protein S deficiency.

Authors:
Nathalie Hézard Lobna Bouaziz-Borgi Marie-Geneviève Remy Philippe Nguyen

Clin Chem 2006 Apr 9;52(4):665-70. Epub 2006 Feb 9.

Laboratoire d'Hématologie, Centre Hospitalaire et Régional, CHU, Robert Debré, Reims, France.

Background: The thrombin-generation assay has a variety of clinical uses, including diagnosis of thromboembolism-related disease, and particular profiles are associated with thrombophilic risk factors. The aim of this study was to evaluate the use of this assay in screening and identifying patients who require specific thrombophilic testing.

Methods: We used a 2-step approach to perform specific thrombophilic testing and thrombin-generation assays on 169 consecutive patients. The first step was to identify particular profiles of thrombin generation corresponding to each type of thrombophilic risk factor and to determine the pertinent variables related to thrombin generation. We then performed ROC curve analysis for each predefined variable to determine the relevant cutoffs for identification of patients in need of further testing (negative predictive value, 100%).

Results: Suggestive profiles were seen in factor V Leiden (n = 49) and prothrombin (n = 12) mutations and in protein S deficiency (n = 12). ROC curves showed that factor V Leiden may be excluded when the difference between lag times obtained in the absence and presence of activated protein C (APC) is >1.5 min and that prothrombin G20210A may also be excluded when the peak thrombin concentration is 63%.

Conclusion: The thrombin-generation assay represents a promising tool for screening thrombophilic risk factors, particularly in patients who are carriers of factor V Leiden or prothrombin G20210A mutations and patients with protein S deficiency.
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http://dx.doi.org/10.1373/clinchem.2005.063339DOI Listing
April 2006

Platelet VASP phosphorylation assessment in clopidogrel-treated patients: lack of agreement between Western blot and flow cytometry.

Authors:
Nathalie Hezard Damien Metz Roselyne Garnotel Gerard Simon Catherine Mace Pascale Koebel Philippe Nguyen

Platelets 2005 Dec;16(8):474-81

Laboratory of Haematology, CHU Rober Debré, Reims, France.

Vasodilator-stimulated phosphoprotein (VASP) 239 phosphorylation flow cytometric assessment has been reported as a tool to evaluate the responsiveness to clopidogrel in coronary heart disease (CHD) patients. We report for the first time the comparison between flow cytometry and two challenger assays, aggregometry and Western blot. We studied 21clopidogrel-treated CHD patients, and 28 healthy volunteers. Aggregometry showed platelet function inhibition inpatients. VASP 239 phosphorylation was assessed using flow cytometry and Western blot. ADP receptor response index (RI) were calculated using the formula (PGE1) - (PGE1 + ADP)/(PGE1) x 100. Flow cytometry was not able to detect clopidogrel intake, as RI were 99 +/- 10% [68-130] in healthy volunteers, and 91 +/- 17% [66-127] in treated patients (ns). On the contrary, RI mean in Western blot was 91 + 8% [76-127] in healthy volunteers, and 37 i 25% [4-80] in patients (p<0.05). The extreme values in Western blot revealed inter-individual variability in response to treatment. The comparison between both tests showed a total lack of agreement. Flow cytometric VASP 239 phosphorylation assay lacks sensitivity to detect clopidogrel intake, contrary to Western blot and aggregometry. Caution is required before classifying patients as 'low-responders' to thienopyridines using such method.
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http://dx.doi.org/10.1080/09537100500212551DOI Listing
December 2005

Allelic frequency of the factor V Leiden mutation and of the pro-thrombin gene 20210A mutation in healthy Tunisian population.

Authors:
Lobna Bouaziz Nathalie Hézard Mahjoub Touhami Gérard Potron Brahim N'siri Philippe Nguyen

Thromb Haemost 2004 Apr;91(4):824-5

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April 2004

Unexpected persistence of platelet hyporeactivity beyond the neonatal period: a flow cytometric study in neonates, infants and older children.

Authors:
Nathalie Hézard Gérard Potron Nicole Schlegel Catherine Amory Bernard Leroux Philippe Nguyen

Thromb Haemost 2003 Jul;90(1):116-23

Laboratoire Central d'Hématologie, CHU Robert Debré, Reims, France.

Previous studies, using flow cytometry, have reported a lower platelet reactivity in neonates compared to adults. Only few studies were carried out in older children, and results were controversial in terms of age to reach adult platelet function. We studied a total of 125 healthy neonates, infants and older children, and 15 adults. alphaIIbbeta3 expression on resting and activated platelets was lower in all children, with an impaired capability of alphaIIbbeta3 activation (PAC1 and bound fibrinogen). This defect was observed until the age of fifteen with a gradual recovery with age. In neonates, we observed a defect of GPIalpha internalization, and demonstrated that this defect persisted in older children as well. In contrast with alphaIIbbeta3 integrin activation, we did not observe a gradual age-dependent recovery. These unexpected results point out the need for reference values in childhood.
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July 2003

PFA-100 and flow cytometry: can they challenge aggregometry to assess antiplatelet agents, other than GPIIbIIIa blockers, in coronary angioplasty?

Authors:
Nathalie Hézard Damien Metz Pierre Nazeyrollas Chantal Droullé Gérard Potron Philippe Nguyen

Thromb Res 2002 Oct;108(1):43-7

Laboratoire Central d'Hématologie, CHU Robert Debré, 51092 Reims Cédex, France.

Introduction: Platelet response to inhibitors varies widely, leading to a higher risk of abrupt closure events in insufficiently treated-coronary heart disease patients. The aim of this study was to compare, in patients under various antiplatelet regimens, three platelet function assays: aggregometry, PFA-100 and flow cytometry. These assays stand for available tests, as "ready-to-use" device (PFA-100) and sophisticated assay (cytometry). We chose the setting of percutaneous coronary intervention as a standardized procedure to determine which test was appropriate to detect the effect of (1) an aspirin bolus in patients under long-term aspirin treatment, and (2) ticlopidin in case of stent implantation.

Methods: Fifty patients under oral aspirin treatment were randomized to receive a bolus of 500 mg aspirin before angioplasty (n=25). Ticlopidin was given at a 500 mg loading dose in the case of stent implantation (n=38). Platelet function was assessed before, at 2 and 24 h after angioplasty.

Results: Considering aspirin antiplatelet effect, the following was observed: (1) a lack of further inhibition after the bolus whatever assay was used and (2) a disagreement between aggregometry and PFA-100 to classify patients as being poor or good aspirin responders (kappa were 0.11 and 0.28 between ADP 4 or 6 microM aggregation, respectively, and PFA-100). Another finding was the good performance of flow cytometry, which evaluated GPIIbIIIa activation, and aggregometry, to detect ticlopidin the day after the loading dose. In contrast, PFA-100 was insensitive to ticlopidin.

Conclusion: Current assays are not interchangeable to monitor antiplatelet treatment in daily practice.
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http://dx.doi.org/10.1016/s0049-3848(02)00391-2DOI Listing
October 2002