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Therapeutic management of acquired von Willebrand syndrome (AVWS) can be challenging, particularly in cases of AVWS associated with monoclonal IgM such as Waldenström macroglobulinemia (WM) where several therapeutic options may be ineffective. Here, we describe the case of an 88-year-old patient who developed AVWS during follow-up for WM. The presence of a severe bleeding symptomatology not controlled by several therapies (plasma-derived von Willebrand factor, plasmapheresis) led us to introduce a supplementation with recombinant von Willebrand factor, vonicog α (Veyvondi, Takeda, Japan), starting at a dose of 50 IU/kg/d. This supplementation allowed clinical (no further bleeding) and biological (hemoglobin level, von Willebrand factor parameters) improvements. Because of the persistence of bleeding risk factors, the treatment was maintained at a prophylactic dose (20 UI/kg three times a week), without recurrence of bleeding events for a period of 9 months., Competing Interests: A.B. has received financial support from BMS, Takeda, Sobi, and LFB for registration fees and accommodation for scientific congress. S.C. has received financial support from CSL Behring for registration fees and accommodation for scientific congress. O.L. has received financial supports from MSD, BMS, AbbVie, and Boehringer for registration fees, education, and consultancy fees. C.L-B. has received financial supports from CSL Behring, Sobi, Novo Nordisk, Takeda, Octapharma, and LFB for registration fees and education fees. N.C. declares no conflict of interest., (Thieme. All rights reserved.)

Background: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization., Objectives: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results., Methods: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied., Results: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine., Conclusion: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents., Competing Interests: Declaration of competing interests M.L. has received speaker fees from Bayer, participated in industry-funded trials from Idorsia, served on advisory boards for Servier and JAMP/Orimed Pharma, and received in-kind support for investigator-initiated grants from Fujimori Kogyo. M.C.A. has received fees from Novo Nordisk, participated in industry-funded trials from Agrobio, participated in PIA-funded project in collaboration with Stago and Agrobio, and served on advisory boards for Novo Nordisk. U.J.S. has received research grants from Octapharma; consulting fees from Bayer, SOBI, CSL Behring, and Pfizer; and travel support from Bayer, SOBI, CSL Behring, Biotest, Takeda, and Leo Pharma. D.F. has received honorarium and travel support from Viatris. S.V. has received travel support from Stago. F.M. has received speaker fees from Fresenius, Technoclone, and Werfen. P.F. has received travel support from SOBI and Novo Nordisk. A.G. reports personal fees from Aspen, Bayer Vital, Chromatec, Instrumentation Laboratory, Portola, Sanofi-Aventis, Roche, and GTH e.V.; grants from Ergomed, Boehringer Ingelheim, Rovi, Sagent, Biokit, Fa. Blau Farmaceutics, Prosensa/Biomarin, DRK-BSD Baden-Würtemberg/Hessen, Deutsche Forschungsgemeinschaft, Robert-Koch-Institut, Dilaflor, and GIZ Else-Körner-Stiftung; grants and personal fees from Macopharma; grants and other fee from DRK-BSD NSTOB; and nonfinancial support from Veralox, Vakzine Projekt Management GmbH, AstraZeneca, and Janssen Vaccines & Prevention B.V. outside the submitted work. In addition, A.G. has a patent—screening methods for transfusion-related acute lung injury (TRALI)—with royalties paid to EP2321644, 18.05.2011. M.C. has received research grant from Agios Pharmaceuticals, Genentech Inc, and Novartis Inc; consulting fees from Novo Nordisk; and honoraria for board participation with Novo Nordisk, Takeda Inc, Genentech Inc, BPL Inc, CSL Behring Inc, and Genzyme Corp Agios Pharmaceuticals. C.P. has received research grant from Stago. I.D. has received travel support from Sobi, Takeda, and Novonordisk and speaker fees from Novonordisk. A.B. has received fees from Aguettant, Alexion, and Viatris. J.R. has received support from Instituto de Salud Carlos (PI20/00926 [ISCIII&Feder], PMP21/00052 [ISCIII&NG EU], and CB15/00055) and Sociedad Española de Trombosis y Hemostasia (Ayuda a Grupo Español de Alteraciones Plaquetarias Congénitas). P.G. has received speaker’s fees from Sanofi and Roche, and honoraria for board participation with Viatris. M.R.C., M.C., C.L.-B., M.I.K., L.G., B.H., A.S., C.C., A.N.S., M.B., M.F., K.D., W.H., M.F.H., A.N.M., J.P.F., P.S., F.P., A.C., N.B., J.R., and D.L. have no conflict of interest to declare., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Women with hereditary fibrinogen disorders (HFDs) seem to be at an increased risk of adverse obstetrical outcomes, but epidemiologic data are limited., Objectives: We aimed to determine the prevalence of pregnancy complications; the modalities and management of delivery; and the postpartum events in women with hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia., Methods: We conducted a retrospective and prospective multicentric international study., Results: A total of 425 pregnancies were investigated from 159 women (49, 95, and 15 cases of hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia, respectively). Overall, only 55 (12.9%) pregnancies resulted in an early miscarriage, 3 (0.7%) resulted in a late miscarriage, and 4 (0.9%) resulted in an intrauterine fetal death. The prevalence of live birth was similar among the types of HFDs (P = .31). Obstetrical complications were observed in 54 (17.3%) live birth pregnancies, including vaginal bleeding (14, 4.4%), retroplacental hematoma (13, 4.1%), and thrombosis (4, 1.3%). Most deliveries were spontaneous (218, 74.1%) with a vaginal noninstrumental delivery (195, 63.3%). A neuraxial anesthesia was performed in 116 (40.4%) pregnancies, whereas general or no anesthesia was performed in 71 (16.6%) and 129 (44.9%) pregnancies, respectively. A fibrinogen infusion was administered in 28 (8.9%) deliveries. Postpartum hemorrhages were observed in 62 (19.9%) pregnancies. Postpartum venous thrombotic events occurred in 5 (1.6%) pregnancies. Women with hypofibrinogenemia were at an increased risk of bleeding during the pregnancy (P = .04)., Conclusion: Compared with European epidemiologic data, we did not observe a greater frequency of miscarriage, while retroplacental hematoma, postpartum hemorrhage, and thrombosis were more frequent. Delivery was often performed without locoregional anesthesia. Our findings highlight the urgent need for guidance on the management of pregnancy in HFDs., Competing Interests: Declaration of competing interests A.C. reports grants and fees paid to his institution from CSL Behring, Octapharma, Sobi, LFB, Takeda, and Novo Nordisk. A.L. reports grants and fees from Octapharma and LFB. C.L.B reports receiving grants and fees from LFB, Octapharma, CSL Behring, and Novo Nordisk. M.F. reports grants and fees from LFB. All other authors have no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Glanzmann thrombasthenia (GT) is a genetic bleeding disorder characterised by severely reduced/absent platelet aggregation in response to multiple physiological agonists. The severity of bleeding in GT varies markedly, as does the emergency situations and complications encountered in patients. A number of emergency situations may occur in the context of GT, including spontaneous or provoked bleeding, such as surgery or childbirth. While general management principles apply in each of these settings, specific considerations are essential for the management of GT to avoid escalating minor bleeding events. These recommendations have been developed from a literature review and consensus from experts of the French Network for Inherited Platelet Disorders, the French Society of Emergency Medicine, representatives of patients' associations, and Orphanet to aid decision making and optimise clinical care by non-GT expert health professionals who encounter emergency situations in patients with GT., (© 2023. The Author(s).)

Background: Neurointerventionists lack guidelines for the use of antithrombotic therapies in their clinical practice; consequently, there is likely to be significant heterogeneity in antithrombotic use between centers. Through a nationwide survey, we aimed to obtain an exhaustive cross-sectional overview of antithrombotic use in neurointerventional procedures in France., Methods: In April 2021, French neurointerventional surgery centers were invited to participate in a nationwide 51-question survey disseminated through an active trainee-led research collaborative network (the JENI-RC)., Results: All 40 centers answered the survey. Fifty-one percent of centers reported using ticagrelor and 43% used clopidogrel as premedication before intracranial stenting. For flow diversion treatment, dual antiplatelet therapy was maintained for 3 or 6 months in 39% and 53% of centers, respectively, and aspirin was prescribed for 12 months or more than 12 months in 63% and 26% of centers, respectively. For unruptured aneurysms, the most common heparin bolus dose was 50 IU/kg (59%), and only 35% of centers monitored heparin activity for dose adjustment. Tirofiban was used in 64% of centers to treat thromboembolic complications. Fifteen percent of these comprehensive stroke centers reported using tenecteplase to treat acute ischemic strokes. Cangrelor appeared as an emergent drug in specific indications., Conclusion: This nationwide survey highlights the important heterogeneity in clinical practices across centers. There is a pressing need for trials and guidelines to further evaluate and harmonize antithrombotic regimens in the neurointerventional field., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

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

Background:  Heparin-induced thrombocytopenia (HIT) is a rare complication of heparin treatments, and only a few large patient cohorts have been reported. In this study, biological and clinical data from 144 French patients with HIT were analyzed in comparison with the literature., Methods:  The diagnosis of HIT was confirmed in all patients by an immunoassay combined with serotonin release assay. In the literature, only cohorts of at least 20 HIT patients published from 1992 were selected for a comparative analysis., Results:  Two-thirds of patients were hospitalized in surgery and most were treated with unfractionated heparin (83.2% vs. 16.8% with low molecular weight heparin only). Thrombotic events in 54 patients (39.7%) were mainly venous (41/54). However, arterial thrombosis was more frequent after cardiac surgery (13.2% vs. 2.4% in other surgeries, p  = 0.042) with a shorter recovery time (median = 3 vs. 5 days, p  < 0.001). The mortality rate was lower in our series than in the 22 selected published studies (median = 6.3% vs. 15.9%). Three genetic polymorphisms were also studied and homozygous subjects FcγRIIA RR were more frequent in patients with thrombosis (37.8 vs. 18.2% in those without thrombosis, p  = 0.03)., Conclusion:  This study shows that the mortality rate due to HIT has recently decreased in France, possibly due to earlier diagnosis and improved medical care. It also confirms the strong association between polymorphism FcγRIIA H131R and thrombosis in HIT., Competing Interests: Y.G.: Consulting and/or travel fees from Octapharma, Shire, CSL Behring, Novo Nordisk, Bayer, LFB, and Léo Pharma. J.R.: Travel fees from Octapharma, Shire, and CSL Behring. C.V.: Travel fees from Sobi, Roche, Shire, and CSL Behring. D.F.: Consulting and/or travel fees from Aspen, Boehringer, Werfen, Stago, and Léo Pharma. A.B.: Travel fees from Aspen, Boehringer, Werfen, Pfizer, and LFB. A.L.: Consulting and/or travel fees from Bayer, LFB, Pfizer, Sobi, and Octapharma. E.D.M.: Travel fees from Sobi, Bayer, Novo Nordisk, and Pfizer. B.T.P.: Consulting and/or travel fees from Sobi, Bayer, Shire, CSL Behring, and Pfizer. I.E.: Consulting and/or travel fees from BMS, Aspen, Léo Pharma, Daiichi Sankyo, Pfizer, Sanofi-Aventis, and Shire. C.L.B.: Travel fees from Sobi, Octapharma, CSL Berhing, and Novo Nordisk. C.M.: Consulting and/or travel fees from BMS, Aspen, Pfizer, and Bayer. C.B.: Consulting and/or travel fees from CSL, Sobi, Bayer, and Novo Nordisk. C.T.: Consulting and travel fees from Sobi, Roche, Octapharma. F.N.G.: Travel fees from Sobi, Bayer, BMS, and Boehringer. J.D.: Travel fees from CSL and Novo Nordisk. T.B.A.: Travel fees from Servier and MSD. E.D.R.: Consulting and/or travel fees from Shire, Alexion, Sobi, Bayer, and Novo Nordisk. I.G.T.: Consulting and/or travel fees from Bayer, MSD, Pfizer, Sanofi-Aventis, and BMS. L.R.: Consulting and/or travel fees from CSL, LFB, Novo Nordisk, Sobi, and Bayer. B.T.: Consulting and/or travel fees from Sobi, Bayer, Novo Nordisk Aspen, CSL Behring, and Pfizer. C.P.: Consulting and/or travel fees from Sobi, Roche, Novo Nordisk, and CSL Behring. P.W., M.A.G., S.V., V.G.G., J.P., and B.G. have no conflict to disclose. All the authors did not receive any personal honorarium or funds related to the study reported in this manuscript. Y.G. reports grants from Ministère de la Santé (Government), during the conduct of the study; personal fees and nonfinancial support from CSL Behring, personal fees and nonfinancial support from Octapharma, nonfinancial support from Shire, personal fees from Léo Pharma, personal fees and nonfinancial support from LFB, nonfinancial support from Bayer, nonfinancial support from Novo Nordisk, outside the submitted work., (Georg Thieme Verlag KG Stuttgart · New York.)

von Willebrand disease (VWD) type 2B is characterized by gain-of-function mutations in von Willebrand factor (VWF), enhancing its binding affinity for the platelet receptor glycoprotein (GP)Ibα. VWD type 2B patients display a bleeding tendency associated with loss of high-molecular-weight VWF multimers and variable thrombocytopenia. We recently demonstrated that a marked defect in agonist-induced activation of the small GTPase, Rap1, and integrin αIIbβ3 in VWD (p.V1316M) type 2B platelets also contributes to the bleeding tendency. Here, we investigated the molecular mechanisms underlying impaired platelet Rap1 signaling in this disease. Two distinct pathways contribute to Rap1 activation in platelets: rapid activation mediated by the calcium-sensing guanine nucleotide exchange factor CalDAG-GEF-I (CDGI) and sustained activation that is dependent on signaling by protein kinase C (PKC) and the adenosine 5'-diphosphate receptor P2Y12. To investigate which Rap1 signaling pathway is affected, we expressed VWF/p.V1316M by hydrodynamic gene transfer in wild-type and Caldaggef1 -/- mice. Using αIIbβ3 integrin activation as a read-out, we demonstrate that platelet dysfunction in VWD (p.V1316M) type 2B affects PKC-mediated, but not CDGI-mediated, activation of Rap1. Consistently, we observed decreased PKC substrate phosphorylation and impaired granule release in stimulated VWD type 2B platelets. Interestingly, the defect in PKC signaling was caused by a significant increase in baseline PKC substrate phosphorylation in circulating VWD (p.V1316M) type 2B platelets, suggesting that the VWF-GPIbα interaction leads to preactivation and exhaustion of the PKC pathway. Consistent with PKC preactivation, VWD (p.V1316M) type 2B mice also exhibited marked shedding of platelet GPIbα. In summary, our studies identify altered PKC signaling as the underlying cause of platelet hypofunction in p.V1316M-associated VWD type 2B., (© 2018 by The American Society of Hematology.)

Interaction between von Willebrand factor (VWF) and platelet GPIbα is required for primary haemostasis. Lack or loss-of-function in the ligand-receptor pair results in bleeding complications. Paradoxically, gain-of-function mutations in VWF or GPIbα also result in bleeding complications as observed in type 2B von Willebrand disease (VWD) and platelet-type- (PT-) VWD, respectively. A similar phenotype is observed with increased ristocetin-induced platelet agglutination and disappearance of the highest molecular weight multimers of VWF. We evaluated a patient with a bleeding disorder and a biological presentation compatible with type 2B VWD. VWF and platelet functional assays, sequencing of the VWF and GP1BA genes, and expression studies in HEK cells were performed. Sequencing of the VWF gene in the propositus revealed a heterozygous p.Pro1266Leu mutation previously found in type 2B VWD Malmö/New York. These variants are characterised by a mild phenotype and a normal VWF multimer composition suggesting the presence of a second mutation in our propositus. Sequencing of the GP1BA gene revealed a heterozygous c.765G>A substitution changing Met at position 255 of GPIbα to Ile. This new mutation is located in the β-switch domain where five other gain-of-function mutations have been reported in PT-VWD. Expression of GPIbα Ile255 in HEK GPIb-IX cells resulted in enhanced VWF binding compared to wild-type, similar to known PT-VWD mutations (p.Val249, p.Ser249 and p.Val255) indicating that it contributes to the propositus defects. This first report associating PT- with type 2B VWD illustrates the importance of combining biological assays with genetic testing to better understand the clinical phenotype.

Von Willebrand disease-type 2A (VWD-2A) and acquired von Willebrand syndrome (AVWS) due to aortic stenosis (AS) or left ventricular assist device (LVAD) are associated with an increased proteolysis of von Willebrand factor (VWF). Analysis of VWF multimeric profile is the most sensitive way to assess such increased VWF-proteolysis. However, several technical aspects hamper a large diffusion among routine diagnosis laboratories. This makes early diagnosis and early appropriate care of increased proteolysis challenging. In this context of unmet medical need, we developed a new ELISA aiming a quick, easy and reliable assessment of VWF-proteolysis. This ELISA was assessed successively in a LVAD-model, healthy subjects (n=39), acquired TTP-patients (n=4), VWD-patients (including VWD-2A(IIA), n=22; VWD-2B, n=26; VWD-2A(IIE), n=21; and VWD-1C, n=8) and in AVWS-patients (AS, n=9; LVAD, n=9; and MGUS, n=8). A standard of VWF-proteolysis was specifically developed. Extent of VWF-proteolysis was expressed as relative percentage and as VWF proteolysis/VWF:Ag ratio. A speed-dependent increase in VWF-proteolysis was assessed in the LVAD model whereas no proteolysis was observed in TTP-patients. In VWD-patients, VWF-proteolysis was significantly increased in VWD-2A(IIA) and VWD-2B and significantly decreased in VWD-2A(IIE) versus controls (p< 0.0001). In AVWS-patients, VWF-proteolysis was significantly increased in AS- and LVAD-patients compared to controls (p< 0.0001) and not detectable in MGUS-patients. A significant increase in VWF-proteolysis was detected as soon as three hours after LVAD implantation (p< 0.01). In conclusion, we describe a new ELISA allowing a rapid and accurate diagnosis of VWF-proteolysis validated in three different clinical situations. This assay represents a helpful alternative to electrophoresis-based assay in the diagnosis and management of AVWS with increased VWF-proteolysis.

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.

Advantages of dabigatran, a thrombin inhibitor, for stroke prevention in patients with atrial fibrillation are numerous. Elderly patients with impaired renal function are at high risk of bleeding. Recommendations about the renal monitoring in elderly patients are not precise enough. The hemoclot direct thrombin inhibitor (HTI) assay measures accurately the dabigatran activity. Both could help managing serious bleeding events in selected populations. Four elderly patients recently treated with appropriate doses of dabigatran were hospitalized for major bleeding. Three patients were very elderly (> 80 years) and three had impaired renal function (clearance < 50 ml/min) before treatment initiation. Serious bleeding events occurred shortly after dabigatran initiation (< 2 months). In all cases, there was a documented dabigatran plasma overdose associated with a renal function impairment concomitant with the bleeding. Why should physicians be aware of this finding?: A close follow-up of the renal function in clinically fragile elderly patient, before and during the weeks following dabigatran initiation, could help to detect the risk of major bleeding event. The HTI dosage could help managing the treatment in case of severe bleeding event.

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., (© 2014 WILEY PERIODICALS, INC.)

Rationale: The pathophysiology of acute chest syndrome (ACS) in patients with sickle cell disease is complex, and pulmonary artery thrombosis (PT) may contribute to this complication., Objectives: To evaluate the prevalence of PT during ACS using multidetector computed tomography (MDCT)., Methods: We screened 125 consecutive patients during 144 ACS episodes. One hundred twenty-one MDCTs (in 103 consecutive patients) were included in the study., Measurements and Main Results: Twenty MDCTs were positive for PT, determining a prevalence of 17% (95% confidence interval, 10-23%). Revised Geneva clinical probability score was similar between patients with PT and those without. D-dimer testing was very often positive (95%) during ACS. A precipitating factor for ACS was less frequently found in patients with PT as compared with those without. Patients with PT exhibited significantly higher platelet counts (517 [273-729] vs. 307 [228-412] 10(9)/L, P < 0.01) and lower bilirubin (28 [19-43] vs. 44 [31-71] μmol/L, P < 0.01) levels at the onset of ACS as compared with others. In addition, patients with PT had a higher platelet count peak (537 [345-785] vs. 417 [330-555] 10(9)/L, P = 0.048) and smaller bilirubin peak (36 [18-51] vs. 46 [32-83] μmol/L, P = 0.048)and lactate dehydrogenase peak (357 [320-704] vs. 604 [442-788] IU/L, P = 0.01) during hospital stay as compared with others., Conclusions: PT is not a rare event in the context of ACS and seems more likely in patients with higher platelet counts and lower hemolytic rate during ACS. Patients with sickle cell disease presenting with respiratory symptoms suggestive of ACS may benefit from evaluation for PT.

Unlabelled: We undertook a survey of French university hospital hematological laboratories to ascertain the clinical characteristics of patients with suspected HIT, the laboratory tests performed, and the therapeutic strategy adopted in current practice., Methods: A standardized medical records database for patients with suspected HIT was sent to 19 laboratories. During two months, all consecutive patients for whom a biological test was performed were included., Results: 169 patients were included, 27 (16%) patients having a final diagnosis of HIT. At the time HIT was suspected, the heparin duration and the level of thrombocytopenia were similar in HIT- positive and HIT-negative groups. The use of unfractionated heparin, a therapeutic heparin dose regimen and the presence of thrombotic complications were significantly more frequent in HIT-positive patients. When the heparin dose regimen was taken into account, only thrombotic complications under a therapeutic dose regimen were significantly increased in HIT-positive patients. Eighty-six percent of patients presented at least one alternative diagnosis of thrombocytopenia without significant difference between the two groups. Laboratory tests were performed after a mean of 0.3days and mainly consisted of antigen assays. At the time HIT was suspected, heparin was stopped in 56 (33%) patients, being replaced mainly by danaparoid. Only three laboratories declared they usually received all the necessary clinical information to establish the likelihood of HIT., Conclusion: In current practice in France, the clinical probability of HIT is rarely established, leading to systematic requests for laboratory HIT tests., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Methods routinely used for investigating the molecular basis of antithrombin (AT) deficiency do not detect large SERPINC1 rearrangements. Between 2000 and 2008, 86 probands suspected of having AT-inherited type I deficiency were screened for SERPINC1 mutations in our laboratory. Mutations causally linked to the deficiency were identified by sequencing analysis in 63 probands. We present here results of multiplex ligation-dependent probe amplification (MLPA) analysis performed in 22 of the 23 remaining probands, in whom sequencing had revealed no mutation. Large deletions, present at the heterozygous state, were detected in 10 patients: whole gene deletions in 5 and partial deletions removing either exon 6 (n = 2), exons 1-2 (n = 1) or exons 5-7 (n = 2) in 5 others. Exon 6 partial deletions are a 2,769-bp deletion and a 1,892-bp deletion associated with a 10-bp insertion, both having 5' and/or 3' breakpoints located within Alu repeat elements. In addition, we identified the 5' breakpoint of a previously reported deletion of exons 1-2 within an extragenic Alu repeat. Distinct mutational mechanisms explaining these Alu sequence-related deletions are proposed. Overall, in this series, large deletions detected by MLPA explain almost half of otherwise unexplained type I AT-inherited deficiency cases.

Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short-lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral-blood mobilized CD34(+) cells were transduced with a human immunodeficiency virus-derived self-inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIbalpha, GpIIb, or EF1alpha gene regulatory sequences. Both GpIbalpha and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIbalpha promoter was strictly MK-specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328-base-pair fragment of the human GpIbalpha gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells. Disclosure of potential conflicts of interest is found at the end of this article.

Myelofibrosis is characterized by excessive deposits of extracellular matrix proteins, which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably developing myelofibrosis has allowed demonstration of the crucial role of transforming growth factor (TGF)-beta1 released by hematopoietic cells in the onset of myelofibrosis. The purpose of this study was to investigate whether TGF-beta1 inhibition could directly inhibit fibrosis development in a curative approach of this mice model. An adenovirus encoding for TGF-beta1 soluble receptor (TGF-beta-RII-Fc) was injected either shortly after transplantation (preventive) or 30 days post-transplantation (curative). Mice were transplanted with syngenic bone marrow cells transduced with a retrovirus encoding for murine TPO. All mice developed a myeloproliferative syndrome. TGF-beta-RII-Fc was detected in the blood of all treated mice, leading to a dramatic decrease in TGF-beta1 level. Histological analysis show that the two approaches (curative or preventive) were successful enough to inhibit bone marrow and spleen fibrosis development in this model. However, lethality of TPO overexpression was not decreased after treatment, indicating that in this mice model, myeloproliferation rather than fibrosis was probably responsible for the lethality induced by the disorder.

Megakaryocytic alpha-granules contain secretory proteins relevant to megakaryocyte and platelet functions. Understanding alpha-granule biogenesis is hampered because human primary megakaryocytes are difficult to manipulate. Existing promegakaryocytic cell lines do not spontaneously exhibit mature alpha-granules. Dami cells, transfected with the megakaryocytic platelet factor 4, fused to GFP (PF4-GFP), were induced in the presence of thrombopoietin (TPO), a megakaryocyte cytokine and PMA. Using confocal microscopy, PF4-GFP colocalized with von Willebrand Factor (vWF) in newly formed storage granules. Immunoelectron microscopy demonstrated alpha-granule-like features, including a dense core or parallel tubules and colocalization of PF4-GFP and vWF. Hence, TPO-treated Dami cells are a suitable model to study alpha-granules and their biogenesis.

Using a transgenic approach, we studied the role of GATA-3 in T cells. As previously shown, enforced GATA-3 expression in transgenic mice inhibits Th1 differentiation of CD4 T cells, but unexpectedly, both type 1 (interferon gamma) and type 2 (interleukin (IL)-4 and IL-13) cytokine genes were activated in the transgenic CD8 T cells. Because IL-13 gene expression was highly enhanced in vivo by GATA-3 expression, we studied the human and the mouse IL-13 gene promoters and found an evolutionary-conserved association of a consensus GATA binding site and two GATG motifs. We showed that efficient GATA-3 binding to this regulatory sequence required these three motifs and that the affinity of the GATA zinc fingers for this association was five times higher than for the consensus GATA binding site alone. Transfections in a T cell line or transactivation by GATA-3 showed that the combination of the three sites was required for full transcriptional activity of the IL-13 gene promoter. Finally we showed that this association of binding sites causes a very high sensitivity of the IL-13 gene promoter to small variations in the level of GATA-3 protein. Altogether, these results indicate an important role of GATA-3 in CD8 cytokine gene expression and demonstrate that a critical network of GATA binding sites highly modulates GATA-3 activity.