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International audience; BackgroundThe optimal dose of tranexamic acid to inhibit hyperfibrinolysis in postpartum haemorrhage is unclear. Tranexamic Acid to Reduce Blood Loss in Hemorrhagic Cesarean Delivery (TRACES) was a double-blind, placebo-controlled, randomised, multicentre dose-ranging study to determine the dose–effect relationship for two regimens of intravenous tranexamic acid vs placebo.MethodsWomen experiencing postpartum haemorrhage during Caesarean delivery were randomised to receive placebo (n=60), tranexamic acid 0.5 g (n=57), or tranexamic acid 1 g i.v. (n=58). Biomarkers of fibrinolytic activation were assayed at five time points, with inhibition of hyperfibrinolysis defined as reductions in the increase over baseline in D-dimer and plasmin–antiplasmin levels and in the plasmin peak time.ResultsIn the placebo group, hyperfibrinolysis was evidenced by a mean increase over baseline [95% confidence interval] of 93% [68–118] for D-dimer level at 120 min and 56% [25–87] for the plasmin–antiplasmin level at 30 min. A dose of tranexamic acid 1 g was associated with smaller increases over baseline (D-dimers: 38% [13–63] [P=0.003 vs placebo]; plasmin–antiplasmin: –2% [–32 to 28] [P=0.009 vs placebo]). A dose of tranexamic acid 0.5 g was less potent, with non-significant reductions (D-dimers: 58% [32–84] [P=0.06 vs placebo]; plasmin–antiplasmin: 13% [18–43] [P=0.051]). Although both tranexamic acid doses reduced the plasmin peak, reduction in plasmin peak time was significant only for the 1 g dose of tranexamic acid.ConclusionsFibrinolytic activation was significantly inhibited by a dose of intravenous tranexamic acid 1 g but not 0.5 g. Pharmacokinetic–pharmacodynamic modelling of these data might identify the best pharmacodynamic monitoring criteria and the optimal tranexamic acid dosing regimen for treatment of postpartum haemorrhage.

Rapid assessment of hemostasis during postpartum hemorrhage (PPH) is essential to allow characterization of coagulopathy, to estimate bleeding severity, and to improve outcome. Point of care (POC) coagulation monitors could be of great interest for early diagnosis and treatment of coagulation disorders in PPH.Women with ongoing PPH500 mL who clinically required an assessment of coagulation with thromboelastography (TEG) were included. The primary aim of this retrospective observational cohort study was to assess the predictive accuracy of TEG parameters for the diagnosis of coagulation disorders (hypofibrinogenemia ≤2 g/L, thrombocytopenia ≤80,000/mm, prothrombin ratio ≤50%, or activated partial thromboplastin time ratio ≥1.5) during PPH. The analyzed TEG parameters were Kaolin-maximum amplitude (K-MA), Kaolin-maximum rate of thrombus generation using G (K-MRTGG), functional fibrinogen-maximum amplitude (FF-MA), and functional fibrinogen-maximum rate of thrombus generation using G (FF-MRTGG). Secondary aims of this study were (1) comparison of the time delay between classical parameters and velocity curve-derived parameters (K-MA versus K-MRTGG and FF-MA versus FF-MRTGG) and (2) evaluation of the accuracy of TEG parameters to predict severe hemorrhage estimated by calculated blood losses.Ninety-eight patients were included with 98 simultaneous TEG analyses and laboratory assays. All parameters had an excellent predictive performance. For the Kaolin assay, no significant difference was evidenced between K-MA and K-MRTGG for the predictive performance for hypofibrinogenemia ≤2 g/L and/or thrombocytopenia ≤80,000/mm (respective area under the curve [AUC], 0.970 vs 0.981). For the functional fibrinogen assay, no significant difference was evidenced between FF-MA and FF-MRTGG for the predictive performance for hypofibrinogenemia ≤2 g/L (respective AUC, 0.988 vs 0.974). For both assays, the time to obtain results was shorter for the velocity parameters (K-MRTGG: 7.7 minutes [2.4 minutes] versus K-MA: 24.7 minutes [4.2 minutes], P.001; FF-MRTGG: 2.7 minutes [2.7 minutes] versus FF-MA: 14.0 minutes [4.3 minutes], P.001). All TEG parameters derived from the Kaolin and functional fibrinogen assays and Clauss fibrinogen were significantly predictive of severe PPH2500 mL.During PPH, when coagulation assessment is indicated, TEG provides a rapid and reliable detection of hypofibrinogenemia ≤2 g/L and/or thrombocytopenia ≤80,000/mm. No difference in performance was evidenced between the velocity-derived parameters (K-MRTGG and FF-MRTGG) and the classical parameters (K-MA and FF-MA). However, velocity-derived parameters offer the advantage of a shorter time to obtain results: FF-MRTGG parameter is available within ≤5 minutes. POC assessment of hemostasis during PPH management may help physicians to diagnose clotting disorders and to provide appropriate hemostatic support.

MYH9-related disease (MYH9-RD) is a rare, autosomal dominant disorder caused by mutations in MYH9, the gene encoding the actin-activated motor protein non-muscle myosin IIA (NMIIA). MYH9-RD patients suffer from bleeding syndromes, progressive kidney disease, deafness, and/or cataracts, but the impact of MYH9 mutations on other NMIIA-expressing tissues remains unknown. In human red blood cells (RBCs), NMIIA assembles into bipolar filaments and binds to actin filaments (F-actin) in the spectrin-F-actin membrane skeleton to control RBC biconcave disk shape and deformability. Here, we tested the effects of MYH9 mutations in different NMIIA domains (motor, coiled-coil rod, or non-helical tail) on RBC NMIIA function. We found that MYH9-RD does not cause clinically significant anemia and that patient RBCs have normal osmotic deformability as well as normal membrane skeleton composition and micron-scale distribution. However, analysis of complete blood count data and peripheral blood smears revealed reduced hemoglobin content and elongated shapes, respectively, of MYH9-RD RBCs. Patients with mutations in the NMIIA motor domain had the highest numbers of elongated RBCs. Patients with mutations in the motor domain also had elevated association of NMIIA with F-actin at the RBC membrane. Our findings support a central role for motor domain activity in NMIIA regulation of RBC shape and define a new sub-clinical phenotype of MYH9-RD.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a severe inherited thrombocytopenia due to loss-of-function mutations affecting the thrombopoietin (TPO) receptor, MPL. Here, we report a new homozygous MPL variant responsible for CAMT in 1 consanguineous family. The propositus and her sister presented with severe thrombocytopenia associated with mild anemia. Next-generation sequencing revealed the presence of a homozygous MPLR464G mutation resulting in a weak cell-surface expression of the receptor in platelets. In cell lines, we observed a defect in MPLR464G maturation associated with its retention in the endoplasmic reticulum. The low cell-surface expression of MPLR464G induced very limited signaling with TPO stimulation, leading to survival and reduced proliferation of cells. Overexpression of a myeloproliferative neoplasm–associated calreticulin (CALR) mutant did not rescue trafficking of MPLR464G to the cell surface and did not induce constitutive signaling. However, it unexpectedly restored a normal response to eltrombopag (ELT), but not to TPO. This effect was only partially mimicked by the purified recombinant CALR mutant protein. Finally, the endogenous CALR mutant was able to restore the megakaryocyte differentiation of patient CD34+ cells carrying MPLR464G in response to ELT.

MYH9-related disease (MYH9-RD) is an inherited rare autosomal dominant macrothrombocytopenia. Patients with MYH9-RD have giant platelets and leukocyte inclusion bodies caused by mutations in the MYH9 gene encoding the non-muscle myosin heavy chain II-A. Before identification of the causative gene, patients were diagnosed as Epstein or Fechtner or Sebastian syndromes or May-Hegglin anomaly. As with other inherited thrombocytopenias, the risk of increased bleeding during perioperative period or delivery is a major concern. We report here the first successful cesarean delivery of a woman with MYH9-RD treated with eltrombopag during the last month of pregnancy.

Filamin A (FLNa) links the cell membrane with the cytoskeleton and is central in several cellular processes. Heterozygous mutations in the X-linked FLNA gene are associated with a large spectrum of conditions, including macrothrombocytopenia, called filaminopathies. Using an isogenic pluripotent stem cell model derived from patients, we show that the absence of the FLNa protein in megakaryocytes (MKs) leads to their incomplete maturation, particularly the inability to produce proplatelets. Reduction in proplatelet formation potential is associated with a defect in actomyosin contractility, which results from inappropriate RhoA activation. This dysregulated RhoA activation was observed when MKs were plated on fibrinogen but not on other matrices (fibronectin, vitronectin, collagen 1, and von Willebrand factor), strongly suggesting a role for FLNa/αIIbβ3 interaction in the downregulation of RhoA activity. This was confirmed by experiments based on the overexpression of FLNa mutants deleted in the αIIbβ3-binding domain and the RhoA-interacting domain, respectively. Finally, pharmacological inhibition of the RhoA-associated kinase ROCK1/2 restored a normal phenotype and proplatelet formation. Overall, this work suggests a new etiology for macrothrombocytopenia, in which increased RhoA activity is associated with disrupted FLNa/αIIbβ3 interaction.

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbβ3 activation, and granule secretion induced by G-protein–coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbβ3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet–platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin–mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.

The number of genes involved in the identification of macrothrombocytopenia (MTP) is growing but the clinical consequences for the affected patients are not well determined. Here, we report the management of the bleeding risk for a patient with the newly reported and rare DIAPH1-related disease during surgery for infertility and then during her subsequent pregnancy. The R1213* DIAPH1 variant responsible for a mild bleeding syndrome in six families was considered a potential risk factor for our patient. Preliminary laparoscopic surgery was followed by neosalpingostomy to open the obstructed fallopian tube that was followed by an ectopic pregnancy requiring further surgery, tranexamic acid was used on each occasion and no bleeding complications were observed. A second pregnancy proceeded to term; the mother's platelet count was controlled throughout the gestation period and remained close to her basal values. No bleeding occurred at delivery or during the postpartum period. In conclusion, with strict repeated assessments of blood parameters and maintenance of the platelet count, the bleeding risk in pregnancy in DIAPH1-related disease can be successfully controlled.

The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first

Fil: Antony Debre, I.. Albert Einstein College of Medicine; Estados Unidos. Universite de Paris XI; Francia

The most common diagnosis for pediatric thrombocytopenia is immune thrombocytopenia. Nevertheless, in atypical cases, the hypothesis of an inherited thrombocytopenia has to be investigated. We report a series of cases of a newly described entity, genetic thrombocytopenia with mutation in the ankyrine 26 gene, diagnosed from the exploration of five pediatric cases of thrombocytopenia. This entity is characterized by a moderate thrombocytopenia with normal mean platelet volume, and poorly bleeding. Its transmission is autosomal dominant. Final diagnosis is made by sequencing of a short DNA region of ANKRD26 gene. This pathology can be considered as an hematological malignancy predisposition syndrome.We report the first cohort of pediatric patients diagnosed with thrombocytopenia with mutation in the ankyrine 26. The aim is to underline the specificities of this entity in children and bring it to the knowledge of pediatricians who may be in first place to manage these patients.• Genetic thrombocytopenia with mutation in the ankyrine 26 gene is a recently described entity, which seems to be considered as a predisposition for hematologic malignancies. • The first cohort has been reported in 2011, by Noris et al., in 78 Italian adult patients. What is New: • We describe clinical and biological features of the first pediatric cohort diagnosed with genetic thrombocytopenia with mutation in the ankyrine 26 gene. • It seemed important to consider the pediatric specificities of this entity to enable pediatricians to investigate, diagnose, and manage pediatric patients and their families.

The most common cause of thrombocytopenia in children is immune thrombocytopenia. Nevertheless, some atypical cases should evoke the hypothesis of genetic thrombocytopenia. Indeed, in the past years, 30 new genes had been described in the field of inherited thrombocytopenia. We report a series of 11 cases of a newly diagnosed entity: ACTN1-related macrothrombocytopenia. Mutations in the gene ACTN1 cause mild macrothrombocytopenia characterized by elevated mean platelet volume and elevated immature platelet fraction, and low bleeding tendency. Its transmission is autosomal dominant. Molecular diagnosis is made by sequencing the ACTN1 gene. Its potential role in hematological malignancy predisposition remains unclear and should be clarified.We identified 11 patients with ACTN1-related macrothrombocytopenia diagnosed through pediatric probands. The aim was to underline the specificities of this entity, especially in children, and bring it to the knowledge of pediatricians.

Excessive bleeding at surgery is a feared complication in patients with inherited platelet disorders. However, very few studies have evaluated the frequency of surgical bleeding in these hemorrhagic disorders. We performed a worldwide, multicentric, retrospective study to assess the bleeding complications of surgery, the preventive and therapeutic approaches adopted, and their efficacy in patients with inherited platelet disorders: the Surgery in Platelet disorders And Therapeutic Approach (SPATA) study. We rated the outcome of 829 surgical procedures carried out in 423 patients with well-defined forms of inherited platelet disorders: 238 inherited platelet function disorders and 185 inherited platelet number disorders. Frequency of surgical bleeding was high in patients with inherited platelet disorders (19.7%), with a significantly higher bleeding incidence in inherited platelet function disorders (24.8%) than in inherited platelet number disorders (13.4%). The frequency of bleeding varied according to the type of inherited platelet disorder, with biallelic Bernard Soulier syndrome having the highest occurrence (44.4%). Frequency of bleeding was predicted by a pre-operative World Health Organization bleeding score of 2 or higher. Some types of surgery were associated with a higher bleeding incidence, like cardiovascular and urological surgery. The use of pre-operative pro-hemostatic treatments was associated with a lower bleeding frequency in patients with inherited platelet function disorders but not in inherited platelet number disorders. Desmopressin, alone or with antifibrinolytic agents, was the preventive treatment associated with the lowest bleedings. Platelet transfusions were used more frequently in patients at higher bleeding risk. Surgical bleeding risk in inherited platelet disorders is substantial, especially in inherited platelet function disorders, and bleeding history, type of disorder, type of surgery and female sex are associated with higher bleeding frequency. Prophylactic pre-operative pro-hemostatic treatments appear to be required and are associated with a lower bleeding incidence.

Less than 50 patients with FPD/AML (OMIM 601309) have been reported as of today and there may an underestimation. The purpose of this study was to describe the natural history, the haematological features and the genotype-phenotype correlations of this entity in order to, first, screen it better and earlier, before leukaemia occurrence and secondly to optimize appropriate monitoring and treatment, in particular when familial stem cell transplantation is considered., We have investigated 41 carriers of RUNX1 alteration belonging to nine unrelated French families with FPD/AML and two syndromic patients, registered in the French network on rare platelet disorders from 2005 to 2015., Five missense, one non-sense, three frameshift mutations and two large deletions involving several genes including RUNX1 were evidenced. The history of familial leukaemia was suggestive of FPD/AML in seven pedigrees, whereas an autosomal dominant pattern of lifelong thrombocytopenia was the clinical presentation of two. Additional syndromic features characterized two large sporadic deletions. Bleeding tendency was mild and thrombocytopenia moderate (>50 x10(9)/L), with normal platelet volume. A functional platelet defect consistent with a δ-granule release defect was found in ten patients regardless of the type of RUNX1 alteration. The incidence of haematological malignancies was higher when the mutated RUNX1 allele was likely to cause a dominant negative effect (19/34) in comparison with loss of function alleles (3/9). A normal platelet count does not rule out the diagnosis of FPD/AML, since the platelet count was found normal for three mutated subjects, a feature that has a direct impact in the search for a related donor in case of allogeneic haematopoietic stem cell transplantation., Platelet dysfunction suggestive of defective δ-granule release could be of values for the diagnosis of FPD/AML particularly when the clinical presentation is an autosomal dominant thrombocytopenia with normal platelet size in the absence of familial malignancies. The genotype-phenotype correlations might be helpful in genetic counselling and appropriate optimal therapeutic management.

The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr(419) phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC-positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors. ispartof: Science Translational Medicine vol:8 issue:328 pages:328- ispartof: location:United States status: published

Inherited bleeding, thrombotic and platelet disorders (BPDs) are diseases affecting approximately 300 individuals per million births. With the exception of haemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialised tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing (HTS) platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants (SNVs), short insertions/deletions (indels) and large copy number variants (CNVs), though not inversions, which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples respectively from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology while the remainder had ana priorihighly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only eight of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

The mechanisms behind the hereditary thrombocytosis induced by the thrombopoietin (THPO) receptor MPL P106L mutant remain unknown. A complete trafficking defect to the cell surface has been reported, suggesting either weak constitutive activity or nonconventional THPO-dependent mechanisms. Here, we report that the thrombocytosis phenotype induced by MPL P106L belongs to the paradoxical group, where low MPL levels on platelets and mature megakaryocytes (MKs) lead to high serum THPO levels, whereas weak but not absent MPL cell-surface localization in earlier MK progenitors allows response to THPO by signaling and amplification of the platelet lineage. MK progenitors from patients showed no spontaneous growth and responded to THPO, and MKs expressed MPL on their cell surface at low levels, whereas their platelets did not respond to THPO. Transduction of MPL P106L in CD34(+) cells showed that this receptor was more efficiently localized at the cell surface on immature than on mature MKs, explaining a proliferative response to THPO of immature cells and a defect in THPO clearance in mature cells. In a retroviral mouse model performed in Mpl(-/-) mice, MPL P106L could induce a thrombocytosis phenotype with high circulating THPO levels. Furthermore, we could select THPO-dependent cell lines with more cell-surface MPL P106L localization that was detected by flow cytometry and [(125)I]-THPO binding. Altogether, these results demonstrate that MPL P106L is a receptor with an incomplete defect in trafficking, which induces a low but not absent localization of the receptor on cell surface and a response to THPO in immature MK cells. Stefan N. Constantinescu and William Vainchenker are co-last Author

FPD/AML is a familial platelet disorder characterized by platelet defects, predisposition to acute myelogenous leukemia (AML) and germ-line heterozygous RUNX1 alterations. Here we studied the in vitro megakaryopoiesis of 3 FPD/AML pedigrees. A 60% to 80% decrease in the output of megakaryocytes (MKs) from CD34+ was observed. MK ploidy level was low and mature MKs displayed a major defect in proplatelet formation. To explain these defects, we focused on myosin II expression as RUNX1 has been shown to regulate MYL9 and MYH10 in an inverse way. In FPD/AML MKs, expression of MYL9 and MYH9 was decreased, whereas MYH10 expression was increased and the MYH10 protein was still present in the cytoplasm of mature MKs. Myosin II activity inhibition by blebbistatin rescued the ploidy defect of FPD/AML MKs. Finally, we demonstrate that MYH9 is a direct target of RUNX1 by chromatin immunoprecipitation and luciferase assays and we identified new RUNX1 binding sites in the MYL9 promoter region. Together, these results demonstrate that the defects in megakaryopoiesis observed in FPD/AML are, in part, related to a deregulation of myosin IIA and IIB expression leading to both a defect in ploidization and proplatelet formation.

RUNX1 gene alterations are associated with acquired and inherited hematologic malignancies that include familial platelet disorder/acute myeloid leukemia, primary or secondary acute myeloid leukemia, and chronic myelomonocytic leukemia. Recently, we reported that RUNX1-mediated silencing of nonmuscle myosin heavy chain IIB (MYH10) was required for megakaryocyte ploidization and maturation. Here we demonstrate that runx1 deletion in mice induces the persistence of MYH10 in platelets, and a similar persistence was observed in platelets of patients with constitutional (familial platelet disorder/acute myeloid leukemia) or acquired (chronic myelomonocytic leukemia) RUNX1 mutations. MYH10 was also detected in platelets of patients with the Paris-Trousseau syndrome, a thrombocytopenia related to the deletion of the transcription factor FLI1 that forms a complex with RUNX1 to regulate megakaryopoiesis, whereas MYH10 persistence was not observed in other inherited forms of thrombocytopenia. We propose MYH10 detection as a new and simple tool to identify inherited platelet disorders and myeloid neoplasms with abnormalities in RUNX1 and its associated proteins.

Nous rapportons un nouveau cas de syndrome lupus anticoagulant-hypoprothrombinemie (LAHPS) detecte lors de l’exploration biologique d’une enfant de 11 ans admise aux urgences pour des petechies, une hemorragie de la cavite buccale persistante apres une extraction dentaire realisee deux jours auparavant et une hematurie macroscopique. Outre ce syndrome, le bilan biologique revele une anemie severe peu regenerative, une thrombopenie, une lymphopenie, une insuffisance renale avec hypoalbuminemie. Ces anomalies font evoquer un LAHPS secondaire a un lupus severe. Un traitement comprenant culots globulaires, plasma frais congele (PFC), immunoglobulines IgIV (400 mg/kg/pendant 5 j) associe a des bolus de corticoides (500 mg/m 2) puis cyclophosphamide (800 mg/m 2) est debute en urgence. Une retrocession du syndrome est obtenue en 13 jours. Le diagnostic de lupus est confirme par la presence d’anticorps anti-ADN et la biopsie renale. L’evolution sera marquee par deux rechutes precoces mais apres un suivi de 2 ans, la maladie est maitrisee sous mycophenolate mofetil seul (1 200 mg/m 2/j) et le LAPHS n’est pas reapparu.

Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MK). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping and similarity regression. We describe two unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 p.R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was associated with reduced proplatelet formation from cultured MKs, cell clustering and abnormal cortical filamentous actin. Similarly, in platelets there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Over-expression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insights into the autoregulation of DIAPH1 activity.

This study evaluated 65 pregnancies in 34 women with five different inherited platelet function disorders. Gestation was similar to that of the general population. Severe bleeds requiring blood transfusions were observed in 50% of deliveries in Glanzmann thrombasthenia (GT), but not in the patients with delta storage pool disease, Hermansky-Pudlak syndrome, P2Y12 defect or defect of thromboxane A2 receptor. Of note, severe haemorrhage also occurred in women with GT who had received prophylactic platelet transfusions, suggesting that better preventive treatments are required. Diagnosis and degree of spontaneous bleeding tendency before pregnancy were reliable parameters to predict the delivery-related bleeding risk.

Secondary to the detection of a chronic anemia with a slightly increased Hb F level in a 7-year-old boy carrying a hemoglobin (Hb) variant, we investigated the members of his family and found that they were related to the original case of Hb Calais. In the present study, we report the clinical and biological impacts of this Hb variant in various members of three generations of this family.

We performed a prospective study of 110 patients (75 not previously published) with the 11q terminal deletion disorder (previously called Jacobsen syndrome), diagnosed by karyotype. All the patients have multiple dysmorphic features. Nearly all the patients (94%) have Paris-Trousseau syndrome characterized by thrombocytopenia and platelet dysfunction. In total, 56% of the patients have serious congenital heart defects. Cognitive function ranged from normal intelligence to moderate mental retardation. Nearly half of the patients have mild mental retardation with a characteristic neuropsychiatric profile demonstrating near normal receptive language ability, but mild to moderate impairment in expressive language. Ophthalmologic, gastrointestinal, and genitourinary problems were common, as were gross and fine motor delays. Infections of the upper respiratory system were common, but no life-threatening infections were reported. We include a molecular analysis of the deletion breakpoints in 65 patients, from which genetic "critical regions" for 14 clinical phenotypes are defined, as well as for the neuropsychiatric profiles. Based on these findings, we provide a comprehensive set of recommendations for the clinical management of patients with the 11q terminal deletion disorder.

International audience; Point mutations in the 5′ UTR of ankyrin repeat domain 26 (ANKRD26) are associated with familial throm-bocytopenia 2 (THC2) and a predisposition to leukemia. Here, we identified underlying mechanisms of ANKRD26-associated thrombocytopenia. Using megakaryocytes (MK) isolated from THC2 patients and healthy subjects, we demonstrated that THC2-associated mutations in the 5′ UTR of ANKRD26 resulted in loss of runt-related transcription factor 1 (RUNX1) and friend leukemia integration 1 transcription factor (FLI1) binding. RUNX1 and FLI1 binding at the 5′ UTR from healthy subjects led to ANKRD26 silencing during the late stages of megakaryopoiesis and blood platelet development. We showed that persistent ANKRD26 expression in isolated MKs increased signaling via the thrombopoietin/myeloproliferative leukemia virus oncogene (MPL) pathway and impaired proplatelet formation by MKs. Importantly, we demonstrated that ERK inhibition completely rescued the in vitro proplatelet formation defect. Our data identify a mechanism for development of the familial thrombocytopenia THC2 that is related to abnormal MAPK signaling.

The main molecular basis of essential thrombocythemia and hereditary thrombocytosis is acquired, and germ-line-activating mutations affect the thrombopoietin signaling axis. We have identified 2 families with hereditary thrombocytosis presenting novel heterozygous germ-line mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, whereas the other presents 2 JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of Janus kinase 2 (JAK2) R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-thrombopoietin receptor (MPL) but not of Ba/F3-human receptor of erythropoietin cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed thrombopoietin-independent phosphorylation of signal transducer and activator of transcription 1. The JAK2 R867Q and S755R/R938Q proteins had significantly longer half-lives compared with JAK2 V617F. The longer half-lives correlated with increased binding to the heat shock protein 90 (HSP90) chaperone and with higher MPL cell-surface expression. Moreover, these mutants were less sensitive to JAK2 and HSP90 inhibitors than JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL while inducing a decreased sensitivity to clinically available JAK2 inhibitors.

Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy and the course of pregnancy did not differ from that of healthy subjects in terms of miscarriages, fetal bleeding and pre-term births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but 2 of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8% to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of abnormal blood loss was increased with respect to the general population. However, no mother died or had to undergo hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery below 50 × 10(9)/L. Fil: Noris, Patrizia. Istituti di Ricovero e Cura a Carattere Scientifico. Policlinico San Matteo di Pavia; Italia. Università degli Studi di Pavia; Italia Fil: Schlegel, Nicole. Université Paris Diderot - Paris 7; Francia Fil: Klersy, Catherine. Istituti di Ricovero e Cura a Carattere Scientifico. Policlinico San Matteo di Pavia; Italia Fil: Heller, Paula Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Civaschi, Elisa. Università degli Studi di Pavia; Italia Fil: Pujol Moix, Nuria. Universitat Autònoma de Barcelona; España Fil: Fabris, Fabrizio. Università di Padova; Italia Fil: Favier, Remi. Inserm; Francia. Armand Trousseau Children’s Hospital; Francia. French Reference Center for Inherited Platelet disorders; Francia Fil: Gresele, Paolo. Università di Perugia; Italia Fil: Latger Cannard, Véronique. Centre Hospitalo-Universitaire. Service d’Hématologie Biologique; Francia. Reference French Centre. Centre de Compétence Nord-Est des Pathologies Plaquettaires; Francia Fil: Cuker, Adam. University of Pennsylvania; Estados Unidos Fil: Nurden, Paquita. Hôpital Xavier Arnozan; Francia Fil: Greinacher, Andreas. Institut für Immunologie und Transfusionsmedizin; Alemania Fil: Cattaneo, Marco. Università degli Studi di Milano; Italia Fil: De Candia, Erica. Università Cattolica del Sacro Cuore; Italia Fil: Pecci, Alessandro. Università degli Studi di Pavia; Italia Fil: Hurtaud Roux, Marie Françoise. Université Paris Diderot - Paris 7; Francia Fil: Glembotsky, Ana Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Muñiz Diaz, Eduardo. Banc de Sang i Teixits de Catalunya. Immunohematology Department; España Fil: Randi, Maria Luigia. Università di Padova; Italia Fil: Trillot, Nathalie. Centre Hospitalier Régional Universitaire de Lille. Pôle Biologie Pathologie Génétique. Institut d’Hématologie-Transfusion; Francia Fil: Bury, Loredana. Università di Perugia; Italia Fil: Lecompte, Thomas. Hôpitaux Universitaires de Genève; Suiza. Université de Genève. Faculté de Médecine; Suiza Fil: Marconi, Caterina. Università di Bologna; Italia Fil: Savoia, Anna. Università degli Studi di Trieste; Italia Fil: Balduini, Carlo L.. Istituti di Ricovero e Cura a Carattere Scientifico Burlo Garofolo. Institute for Maternal and Child Health; Italia. Università degli Studi di Pavia; Italia Fil: European Hematology Association Scientific Working Group on Thrombocytopenias and Platelet Function Disorders. No especifica

MYH9-related disease (MYH9-RD) is one of the most frequent autosomal-dominant forms of inherited macrothrombocytopenias and is caused by mutations in MYH9 (nonmuscle myosin IIA), the gene coding for the heavy chain of the nonmuscle myosin IIA. Affected individuals can present with isolated thrombocytopenia, and whereas only some will have bleeding events requiring intervention, nearly all will require the use of prophylactic platelet transfusions before surgery. Here we report the first prophylactic use of eltrombopag before surgery in a child with MYH9-RD. Our patient was a 13-year-old girl with an MYH9 S96L missense mutation who required a tympanoplasty due to chronic otitis media. Pretreatment microscopic platelet count was 10 × 109/L. The child was treated with eltrombopag starting 4 weeks before her planned surgery. On the day of surgery her platelet count was 70 × 109/L. She required no platelet transfusions and no abnormal bleeding was reported either during surgery or postoperatively. Given these results, the first reported in a child, we suggest that the use of this thrombopoietic agent should be further evaluated as a useful presurgical prophylactic option in this hereditary thrombocytopenia, thus avoiding the use of platelet transfusions and their associated risks, which include alloimmunization and the transmission of infectious agents.

Objective— We examined platelet functions in 4 unrelated patients with filaminopathy A caused by dominant mutations of the X-linked filamin A ( FLNA ) gene. Methods and Results— Patients P1, P2, and P4 exhibited periventricular nodular heterotopia, heterozygozity for truncating FLNA mutations, and thrombocytopenia (except P2). P3 exhibited isolated thrombocytopenia and heterozygozity for a p.Glu1803Lys FLNA mutation. Truncated FLNa was undetectable by Western blotting of P1, P2, and P4 platelets, but full-length FLNa was detected at 37%, 82%, and 57% of control, respectively. P3 FLNa (p.Glu1803Lys and full-length) was assessed at 79%. All patients exhibited a platelet subpopulation negative for FLNa. Platelet aggregation, secretion, glycoprotein VI signaling, and thrombus growth on collagen were decreased for P1, P3, and P4, but normal for P2. For the 2 patients analyzed (P1 and P4), spreading was enhanced and, more markedly, in FLNa-negative platelets, suggesting that FLNa negatively regulates cytoskeleton reorganization. Platelet adhesion to von Willebrand factor under flow correlated with platelet full-length FLNa content: markedly reduced for P1 and P4 and unchanged for P2. Interestingly, von Willebrand factor flow adhesion was increased for P3, consistent with a gain-of-function effect enhancing glycoprotein Ib-IX-V/von Willebrand factor interaction. These results are consistent with a positive role for FLNa in platelet adhesion under high shear. Conclusion— FLNA mutation heterogeneity correlates with different platelet functional impacts and points to opposite regulatory roles of FLNa in spreading and flow adhesion under shear.

Item does not contain fulltext The exon-junction complex (EJC) performs essential RNA processing tasks. Here, we describe the first human disorder, thrombocytopenia with absent radii (TAR), caused by deficiency in one of the four EJC subunits. Compound inheritance of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this inheritance mechanism explained 53 of 55 cases (P < 5 x 10(-228)) of the rare congenital malformation syndrome. Of the 53 cases with this inheritance pattern, 51 carried a submicroscopic deletion of 1q21.1 that has previously been associated with TAR, and two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in diminished RBM8A transcription in vitro and that Y14 expression is reduced in platelets from individuals with TAR. Our data implicate Y14 insufficiency and, presumably, an EJC defect as the cause of TAR syndrome.

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.

Various components of the coagulation and fibrinolytic pathways are involved in normal embryonic implantation, trophoblast invasion, placentation, and recurrent miscarriages are characterized by defective placentation and microthrombi in the placental vasculature. Although recurrent miscarriage is a heterogeneous condition the relationship between abnormalities in the haemostatic pathways and pregnancy outcome is increasingly recognized. The challenge we face is how to discriminate between women who are destined to miscarry from those whose pregnancy will be successful. Considering the crucial role of thrombomodulin and tissue factor in coagulation and in embryonic development, we have performed a study using specific assays for thrombomodulin, tissue factor activity and procoagulant phospholipids in association with other parameters in 30 early (under 12weeks) and 32 late (over 22weeks) pregnancy loss women and compared them with 62 normal pregnancy women and 35 non-pregnant women. Plasma levels of tissue factor activity, thrombomodulin activity, and procoagulant phospholipids were significantly higher in patients than in control subjects. In addition the tissue factor activity/free tissue factor pathway inhibitor ratio was higher in patients than in controls. Interestingly, patients with late pregnancy loss had higher tissue factor activity/free tissue factor pathway inhibitor ratios than patients with early pregnancy loss. The combinations of these different parameters reveal an increase in procoagulant activity which could be secondary to endothelial damage or coagulation activation and then are involved in the pathogenesis of pregnancy loss. Their simultaneous measurement of these activities might provide a new tool to assess the prognosis of pregnancy loss.

This report is of a 14-month-old girl affected with severe hemophilia A. Both her parents had normal values for factor VIII activity, and von Willebrand disease type 2N was excluded. Karyotype analysis demonstrated no obvious alteration, and BclI Southern blot did not reveal F8 gene inversions. Direct sequencing of F8 gene exons revealed a frameshift-stop mutation (Q565delC/ter566) in the heterozygous state in the proposita only. F8 gene polymorphism analysis indicated that the mutation must have occurred de novo in the paternal germline. Furthermore, analysis of the pattern of X chromosome methylation at the human androgen receptor gene locus demonstrated a skewed inactivation of the derived maternal X chromosome from the lymphocytes of the proband's DNA. Thus, the severe hemophilia A in the proposita results from a de novo F8 gene frameshift-stop mutation on the paternally derived X chromosome, associated with a nonrandom pattern of inactivation of the maternally derived X chromosome.

Background: cblC disease is a cause of hemolytic uremic syndrome (HUS), which has been primarily described in neonates and infants with severe renal and neurological lesions. Patients: Two sisters aged 6 and 8.5 years presented with a latent hemolytic process characterized by undetectable or low plasma haptoglobin, respectively, associated with renal failure and gross proteinuria. Renal biopsies performed in both patients found typical findings of thrombotic microangiopathy suggesting the diagnosis of HUS. Both patients were free of neurologic signs. Results: Biochemical investigations found a cobalamin processing deficiency of the cblC type. Search for additional factors susceptible to worsen endothelial damage revealed homozygosity 677C→ T mutation in the methylenetetrahydrofolate reductase gene as well as heterozygosity for a 3254T→ C mutation in factor H in the patient with the most severe clinical presentation. Long-term subcutaneous administration of hydroxocobalamin in combination with oral betaine and folic acid resulted in clinical and biological improvement in both patients. Conclusion: cblC disease may be a cause of chronic HUS with delayed onset in childhood. Superimposed mutation of factor H gene might influence clinical severity.

SummaryParis-Trousseau syndrome (PTS) is an inherited disorder char-acterized by mild hemorragic tendency associated with 11q chromosome deletion. Here we report ten new patients (5 boys,5 girls) with complete clinical history, biological data, ultra-structural and molecular investigations. Thrombocytopenia is chronic in all the patients except two boys in whom it disappeared during the two first years of life. On Romanovsky stained peripheral blood smears, abnormal platelets with giant granules were detected in all the children and confirmed by electron microscopy (EM). On bone marrow smears, dysmega-karyopoiesis with many micromegakaryocytes was constantly observed. Abnormal alpha-granules were virtually absent from bone marrow and cultured megakaryocytes, while EM detected numerous images of granule fusion within blood platelets. Molecular analyses evidenced that the fli-1 gene is deleted in all the patients except one confirming the crucial role of the transcription factor FLI-1 in megakaryopoiesis. In summary, this study documents ten new cases of PTS with characteristic alpha-granule abnormalities, and shows the putative pathogenic role of fli-1 gene in the pathophysiology of this syndrome.

In type 2N von Willebrand disease (VWD), von Willebrand factor (VWF) is characterized by a markedly decreased affinity for Factor VIII (FVIII), and the mutations responsible are essentially located in the D' domain of VWF. We report the identification, in seven unrelated French families, of two novel type 2N VWD mutations, Q1053H and C1060R (Gln290His and Cys297Arg in mature VWF sequence), in exon 24 of the VWF gene. These missense mutations have been identified in the heterozygous, homozygous or hemizygous states. Using site-directed mutagenesis and transient expression in COS-7 cells, we showed that both mutations, although located in the D3 domain of VWF, outside the tryptic fragment containing the FVIII domain, dramatically decrease the binding of VWF to FVIII. In contrast, the R924Q substitution, which was identified in a patient who was heterozygous for C1060R, was shown to be a polymorphism.

8 pages., This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (≤ 10% of control) platelet content of GPIIb–IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3'-UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb-mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb-mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady-state level of GPIIb-mRNA. The reduced availability of platelet GPIIb-mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in-frame STOP codon with the result of activation of nonsense-mediated mRNA decay, or both., This work was supported in part by grants from the Dirección General de Investigación (PM99-0095, PB97-1240 and SAF 2000-0127), Fondo de Investigaciones Sanitarias (96/2014), and Comunidad Autónoma de Madrid (08.4/0031/1998). R.F. was supported in part by the French Society of Pediatrics Pathology.

The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which thrombocytopenia is associated with increased platelet size and decreased α-granule content. This report describes 3 new pediatric cases presenting with the classical platelet abnormalities of GPS within one family with normal parents. Examination of blood smears of the 3 patients demonstrated not only gray platelets, but also gray polymorphonuclear neutrophils (PMNs) with decreased or abnormally distributed components of secretory compartments (alkaline phosphatase, CD35, CD11b/CD18). Secondary granules were also decreased in number as assayed by immunoelectron microscopy. These data confirm that the secretory compartments in neutrophils were also deficient in this family. Megakaryocytes (MKs) were cultured from the peripheral blood CD34+ cells of the 3 patients for 14 days, in the presence of thrombopoietin and processed for immunoelectron microscopy. Although von Willebrand factor (vWF) was virtually undetectable in platelets, vWF immunolabeling was conspicuous in cultured maturing MKs, particularly within Golgi saccules, but instead of being packaged in α-granules, it was released into the demarcation membrane system. In contrast, P-selectin followed a more classical pathway. Double-labeling experiments confirmed that vWF was following an intracellular pathway distinct from the one of P-selectin. In these 3 new cases of GPS, the MKs appeared to abnormally process vWF, with secretion into the extracellular space instead of normal α-granule packaging. Furthermore, the secretory compartment of another blood cell line, the neutrophil, was also affected in this family of GPS.

SummaryPlasmin inhibitor (PI) is a major physiological inhibitor of plasminmediated fibrinolysis; hence, its deficiency results in a severe haemorrhagic diathesis. We analyzed the PI gene of a French boy apparently homozygous for PI deficiency and his heterozygous parents. Both alleles of the homozygous patient had a novel G to A transition at the consensus splicing donor site in the intron 2 of the PI gene. In an expression assay using the heterologous cells transfected with the mutant PI expression vector, 3 types of aberrant transcripts using a cryptic splicing donor site within the intron 2 were detected. All of these mRNAs had a stop codon upstream of the cryptic splicing site and encode only 25 amino acids, comprising the first 21 amino acids of the signal peptide (27 amino acids) plus 4 new amino acids. This mutant was designated as PI-Paris-Trousseau.

Idiopathic thrombocytopenic purpura (ITP) is characterized by the presence of autoantibodies reacting with platelet-specific antigens resulting in thrombocytopenia. In the antiphospholipid antibody syndrome (APS), clinical manifestations are associated with the presence of antiphospholipid