Dominique Lasne

Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations

The glycoprotein VI (GPVI)/FcRgamma complex is a key receptor for platelet activation by collagen. We describe, for the first time, 2 genetic abnormalities in one patient. This 10-year-old girl presented ecchymoses since infancy, a prolonged bleeding time despite a normal platelet count and no antiplatelet antibodies. Collagen-induced platelet activation was null, whereas GPVI quantification by flow cytometry evidenced an incomplete deficiency. Immunoblotting showed an abnormal migration of residual GPVI, and no FcRgamma defect. GPVI DNA sequencing revealed (1) an R38C mutation in exon 3 of one allele and (2) an insertion of 5 nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA. Introduction of the R38C mutation into recombinant GPVI-Fc resulted in abnormal protein migration and a loss of collagen binding. Thus, this composite genetic GPVI deficiency and dysfunction cause absence of platelet responses to collagen and a mild bleeding phenotype.

Vitamin K antagonists in children with heart disease: height and VKORC1 genotype are the main determinants of the warfarin dose requirement.

Managing vitamin K antagonist (VKA) therapy is challenging in children because of a narrow therapeutic range and wide inter- and intra-individual variability in dose response. Only a few small studies have investigated the effect of nongenetic and genetic factors on the dose response to VKAs in children. In a cohort study including 118 children (median age 9 years; range, 3 months-18 years) mostly with cardiac disease, we evaluated by multivariate analysis the relative contribution of nongenetic factors and VKORC1/CYP2C9/CYP4F2 genotypes on warfarin (n = 83) or fluindione (n = 35) maintenance dose and the influence of these factors on the time spent within/above/below the range. The results showed that height, target international normalized ratio and VKORC1 and CYP2C9 genotypes were the main determinants of warfarin dose requirement, accounting for 48.1%, 4.4%, 18.2%, and 2.0% of variability, respectively, and explaining 69.7% of the variability. Our model predicted the warfarin dose within 7 mg/wk in 86.7% of patients. None of the covariates was associated with the time spent above or below the international normalized ratio range. Whether this model predicts accurately the effective maintenance dose is currently being investigated.

Acquired protein S deficiency leads to obliterative portal venopathy and to compensatory nodular regenerative hyperplasia in HIV-infected patients.

Objective:To identify the mechanism of nodular regenerative hyperplasia in HIV-infected patients. Design:Case–control study. Setting:The hepatology and the infectious disease units of two tertiary care centers in France. Patients:We compared 13 consecutive HIV-positive patients with unexplained nodular regenerative hyperplasia to 16 consecutive HIV-positive patients without nodular regenerative hyperplasia, to eight HIV-negative patients with nodular regenerative hyperplasia from an identified cause and to 10 anonymous healthy blood donors. Main outcome measure:Patients and controls were screened for diminished protein S activity and antiprotein S immunoglobulin G (IgG) antibodies. The antiprotein S activity of purified IgG from patients and controls was assessed in a functional test of activation of protein C in which protein S serves as a cofactor. A full liver CT portography was realized on the liver explant of a case patient. Results:The CT portography disclosed diffuse obliterative portal venopathy. Levels of protein S activity were lower among patients with HIV-associated nodular regenerative hyperplasia when compared with HIV-positive patients without nodular regenerative hyperplasia and when compared with HIV-negative patients with nodular regenerative hyperplasia (P < 0.005 for all comparisons). HIV-positive patients with nodular regenerative hyperplasia had significantly higher levels of antiprotein S IgG than HIV-positive patients without nodular regenerative hyperplasia and healthy controls. Purified IgG from patients with HIV-associated nodular regenerative hyperplasia specifically inhibited the protein S-dependent protein C activation. Conclusion:Acquired autoimmune protein S paucity and secondary thrombophilia appear to be causes of obliterative portal venopathy and compensatory nodular regenerative hyperplasia in HIV-positive patients.

From normal to pathological hemostasis.

PurposeTo review the evolution of knowledge on physiological hemostasis and the main abnormalities that may interfere with hemostasis in the perioperative period.MethodsNarrative review of the literature, including relevant papers published in English. Principal findings: Physiological hemostasis controls blood fluidity and rapidly induces hemostatic plug formation in order to stop or limit bleeding. The three distinct phases of the hemostatic process, primary hemostasis, coagulation and fibrinolysis are closely linked to each other and precisely regulated in order to efficiently close vessel wounds, promote vascular healing and maintain vessel patency. Primary hemostasis is the result of complex interactions between the vascular wall, platelets and adhesive proteins. Initiation of the coagulation pathway in vivo is secondary to the exposure of tissue factor (TF) and the formation of TF/VIIa complex which can activate both FIX and FX. This initiation phase is followed by a propagation phase with amplification of thrombin generation. Several control mechanisms exist for localizing fibrin formation to the site of injury including tissue factor pathway inhibitor, protein C system, antithrombin, and glycosaminoglycans on the vessel wall. Fibrinolysis is also a highly regulated system that controls fibrin dissolution. Both constitutive and acquired hemostasic defects exist. The consequences of these abnormalities are highly variable according to the type of defect, and to the genetic and environmental background.ConclusionHemostasis is one of the most complex physiological self-defence systems, not only involved in control of blood fluidity but also interfering in major physiopathological processes. The evolution of our knowledge of the physiology of hemostasis has numerous implications for therapy.RésuméObjectifRevoir ľévolution des connaissances sur ľhémostase physiologique et les principales anomalies qui peuvent nuire à ľhémostase périopératoire.MéthodeUne revue documentaire traditionnelle, dont les articles pertinents publiés en anglais.Constatations principalesĽhémostase physiologique contrôle la fluidité du sang et induit rapidement la formation ďun caillot hémostatique pour stopper ou limiter ľhémorragie. Les trois phases distinctes du processus hémostatique, ľhémostase primaire, la coagulation et la fibrinolyse, sont intimement liées entre elles et précisément réglées pour fermer efficacement les blessures vasculaires, activer la cicatrisation et maintenir la perméabilité vasculaire. Ľhémostase primaire est le résultat ďinteractions complexes entre la paroi vasculaire, les plaquettes et les protéines adhésives. Le déclenchement du processus de coagulation in vivo est secondaire à ľexposition du facteur tissulaire (FT) et à la formation du complexe FT/VIIa qui peut activer les facteurs IX et X. Cette phase initiale est suivie ďune phase de propagation avec amplification de la génération de thrombine. Des mécanismes de contrôle existent pour localiser la formation de fibrine au site de la lésion (ľinhibiteur du mécanisme du facteur tissulaire, le système de protéine C, ľantithrombine et les glycosaminoglycanes sur la paroi vasculaire). La fibrinolyse est aussi un système très réglé qui contrôle la dissolution de la fibrine. Il existe des anomalies constitutives et acquises de ľhémostase. Leurs conséquences sont hautement variables selon le type ďanomalie et le bagage génétique et environnemental.ConclusionĽhémostase est ľun des systèmes physiologiques ďautodéfense le plus complexe, qui non seulement participe au contrôle de la fluidité du sang, mais aussi intervient dans les principaux processus physiopathologiques. Ľévolution de nos connaissances sur la physiologie de ľhémostase a de nombreuses implications pour le traitement.Objectif Revoir ľevolution des connaissances sur ľhemostase physiologique et les principales anomalies qui peuvent nuire a ľhemostase perioperatoire.

Minimizing perioperative blood loss and transfusions in children.

PurposeTo summarize the physiology and pathophysiology relevant to perioperative blood loss in children. Strategies to reduce blood losses are reviewed.MethodsThe literature was reviewed using the electronic library PUBMED and the Cochrane Database of Systematic Reviews. Relevant studies published in English or French with an English abstract are included. The following keywords were used: children, blood transfusion, surgical blood loss, erythropoietin, autologous blood, red blood cell saver, normovolemic hemodilution, desmopressin, aminocaproic acid, tranexamic acid, aprotinin, cardiac surgery, liver transplantation and scoliosis surgery.Main findingsFor patients with idiopathic scoliosis, predonation with or without the addition of erythropoietin is a safe and effective way to avoid the use of allogenic blood products. For open heart procedures: whole blood of less than 48 hr is helpful for children of less than two years of age undergoing complex procedures; tranexamic acid may be helpful for cyanotic heart disease and, to a lesser degree, for reoperations; while antikallikrein blood levels of aprotinin may both reduce the need for allogenic blood transfusions and improve postoperative oxygenation in infants.ConclusionReducing perioperative allogenic blood transfusions is possible in pediatric patients provided that prophylactic measures are adapted to age, disease and type of surgery.ObjectifRevoir la littérature pertinente à la prise en charge des pertes sanguines péri-opératoires de ľenfant ainsi que les stratégies ďépargne sanguine.MéthodeLa littérature a été revue à ľaide de la banque de données électronique PUBMED et du Cochrane Database of Systematic Reviews. Les études pertinentes publiées en langue française ou anglaise avec résumé en langue anglaise ont été revues. Les mots-clés suivants ont été utilisés: enfant, transfusion sanguine, pertes sanguines chirurgicales, érythropoïétine, transfusion autologue préprogrammée, récupérateur de globules rouges, hémodilution isovolémique, desmopressine, acide aminocaproïque, acide tranexamique, aprotinine, chirurgie cardiaque, transplantation hépatique et arthrodèse vertébrale.Constatations principalesPour les scolioses idiopathiques, la transfusion autologue programmée avec ou sans ľajout ďérythropoïétine réduit ľadministration de sang homologue. Pour les chirurgies à cœur ouvert, le sang complet de moins de 48 h est utile pour les corrections de cardiopathies complexes avant ľâge de deux ans, ľacide tranexamique est utile pour les corrections de cardiopathies cyanogènes et à un degré moindre pour les réinterventions alors que ľaprotinine à dose anti-kallikréïne diminue les besoins transfusionnels et améliore ľoxygénation postopératoire en dessous de ľâge de un an.ConclusionIl est possible de réduire les transfusions allogènes chez ľenfant si des mesures adaptées à ľâge, à la pathologie et au type de chirurgie sont appliquées.Purpose To summarize the physiology and pathophysiology relevant to perioperative blood loss in children. Strategies to reduce blood losses are reviewed.

Polymorphonuclear leukocyte and monocyte activation induced by plasma from patients with heparin-induced thrombocytopenia in whole blood

Heparin-induced thrombocytopenia (HIT), a severe complication of heparin therapy, results from platelet activation by heparin-dependent antibodies. Previously, we have shown that plasma from patients with HIT (HIT plasma) induces leukocyteplatelet aggregation in blood. In this report, we examined leukocyte activation by HIT plasma and the contribution of heparin and platelets to this activation, in whole blood. Degranulation of leukocytes from HIT patients was evaluated as a leukocyte activation marker. We showed that polymorphonuclear leukocytes (PMN) and monocytes were the leukocyte subpopulations involved in platelet-leukocyte aggregation induced by HIT plasma in healthy donor blood. PMN and monocyte activation, reflected by increased surface expression of the CD11b adhesion molecule, was induced by HIT plasma in a heparin-dependent manner. The CD11b increase induced by HIT plasma was observed on PMN only when they were associated with platelets. Moreover, the increased CD11b expression on monocytes and PMN correlated strongly with the degree of platelet adhesion to these cells. Degranulation of leukocytes from HIT patients and control subjects (non-HIT heparin-treated patients and healthy subjects) was evaluated in vivo by measuring the plasma myeloperoxidase concentration. HIT plasma contained higher myeloperoxidase concentrations than control plasma, suggesting leukocyte degranulation during HIT. In conclusion, this study provides the first evidence that PMN activation is induced by HIT plasma. HIT plasma induced PMN and monocyte activation in a heparin-dependent manner. In whole blood, platelet association with monocytes and PMN, and the activation of these leukocytes by HIT plasma were interrelated. Finally, leukocyte degranulation could be involved in HIT physiopathology.

Bleeding disorders in Lowe syndrome patients: evidence for a link between OCRL mutations and primary haemostasis disorders

Lowe syndrome (LS) is a rare X‐linked disorder caused by mutations in the oculocerebrorenal gene (OCRL), encoding OCRL, a phosphatidylinositol 5–phosphatase with a RhoGAP domain. An abnormal rate of haemorrhagic events was found in a retrospective clinical survey. Herein, we report the results of exploration of haemostasis in six LS patients. All patients had normal coagulation tests but prolonged closure times (CTs) in the PFA–100 system. Healthy donors’ blood samples incubated with a RhoA kinase inhibitor had prolonged CTs. This suggests that an aberrant RhoA pathway in platelets contributes to CT prolongation and primary haemostasis disorders in LS.

Type I interferon mediated autoinflammation due to DNase II deficiency

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.Nucleic acid sensing is important to ensure that an innate immune response is only mounted against microbial nucleic acid. Here, the authors identify loss-of-function mutations in the DNASE2 gene that cause type I interferon-mediated autoinflammation due to enhanced systemic interferon signaling.

Diagnosis and management of heparin-induced thrombocytopenia.

Objectif Revoir ľevolution de la pathogenese, des caracteristiques cliniques, des tests de laboratoire et du traitement de la thrombopenie induite par ľheparine (TIH).PurposeTo review recent developments in the pathogenesis, clinical features, laboratory testing and treatment of heparininduced thrombocytopenia (HIT).MethodsNarrative review of the literature, including relevant papers published in English or French.Principal findingsAlthough the prevalence of HIT has decreased with the widespread use of low molecular weight heparin in the past ten years, HIT remains a life-threatening prothrombotic state. This immune adverse event due to heparin-dependent antibodies that bind to chemokines (such as platelet factor 4) induces platelet activation and hypercoagulability. Heparin-induced thrombocytopenia can be complicated by thrombosis even after withdrawing heparin, explaining why substituting heparin with an alternative anticoagulant (danaparoid, lepirudin, argatroban) is always necessary. However, management of these alternative treatments is difficult, and in some patients there is the risk of withdrawing heparin without taking the time to diagnose HIT properly on the basis of clinical and laboratory findings (evolution of platelet count, laboratory testing such as antigen assays and platelet activation tests).ConclusionsManagement of HIT has become easier in recent years with the development of more specific and sensitive laboratory tests and new antithrombotic drugs. However, the diagnosis of HIT is often difficult, and it remains very important to investigate this adverse reaction systematically in every patient treated with heparin who develops thrombocytopenia.RésuméObjectifRevoir ľévolution de la pathogenèse, des caractéristiques cliniques, des tests de laboratoire et du traitement de la thrombopénie induite par ľhéparine (TIH).MéthodeUne revue descriptive de la littérature, incluant les articles pertinents publiés en anglais ou en français.Constatations principalesQuoique la prévalence de TIH ait diminué avec ľusage répandu de ľhéparine de bas poids moléculaire au cours des dix dernières années, la TIH demeure un état prothrombotique grave. Cet événement immunitaire indésirable, causé par des anticorps reliés à ľhéparine qui se lient aux chimiokines (comme le facteur plaquettaire 4), induit ľactivation des plaquettes et ľhypercoagulabilité. La TIH peut être compliquée par une thrombose même après le retrait de ľhéparine, ce qui explique pourquoi la substitution de ľhéparine par un autre anticoagulant (danaparoïde, lépirudine, argatroban) est toujours nécessaire. Cependant, ľutilisation de ces traitements de remplacement est difficile, et chez certains patients, il y a le risque de retirer ľhéparine sans prendre le temps de diagnostiquer correctement la TIH ďaprès les signes cliniques et les résultats de laboratoire (évolution de la numération plaquettaire, tests immunologiques et fonctionnels).ConclusionLe traitement de la TIH est maintenant plus facile avec le développement de tests de laboratoire plus spécifiques et plus sensibles et de nouveaux médicaments antithrombotiques. Mais le diagnostic de TIH est souvent difficile à poser et il demeure très important de rechercher cette réaction indésirable de façon systématique chez chaque patient traité avec de ľhéparine chez qui se développe une thrombopénie.

Extracorporeal adsorption of anti-factor VIII allo-antibodies on randomly functionalized polystyrene resins.

The occurrence of anti-factor VIII (FVIII) allo-antibodies is a severe complication of the treatment of haemophilia A patients, leading to the inhibition of transfused FVIII activity. The effective elimination of these inhibitory antibodies plays a decisive role in the management of affected patients. To achieve this, immunoadsorption devices employing synthetic adsorbers, which selectively eliminate inhibitors, are of interest in the treatment strategy of haemophilia A patients with inhibitors. Adsorbers consisting of polystyrene-based beads substituted with sulphonate and L-tyrosyl methylester groups, which mimic part of epitope of FVIII molecule recognized by inhibitors, exhibit selective binding capacities towards anti-FVIII antibodies. The adsorption of FVIII inhibitors was investigated by simulating an extracorporeal circulation of haemophilic plasma over these functionalized resins. These innovative adsorbers are able to remove around 25% of anti-FVIII antibodies in 15 minutes depending on the plasma tested. Furthermore, they do not modify the amount of essential plasmatic proteins or residual immunoglobulins G. Experiments which were carried out using different plasmas with various inhibitor titres demonstrate a good reproducibility regarding the adsorption capacity of the synthetic resin. The characteristics of adsorption are similar on either native or regenerated resins. Both the purely synthetic nature of the resin and its easy processability demonstrate the real advantages over currently available protocols. This synthetic adsorber is a major technological advance in selective removal of FVIII inhibitory antibodies.