Search

BackgroundNeurointerventionists 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.MethodsIn 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).ResultsAll 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.ConclusionThis 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.

BACKGROUND Women with hereditary fibrinogen disorders (HFDs) seem to be at increased risk of adverse obstetrical outcomes, but epidemiologic data are limited Patients/methods: We conducted a retrospective and prospective international study to determine the prevalence of pregnancy complications, the modalities and management of delivery, and the postpartum events. RESULTS A total of 425 pregnancies were investigated from 159 women (49 hypofibrinogenemia, 95 dysfibrinogenemia, 15 hypodysfibrinogenemia). Overall, only 55 (12.9%) pregnancies resulted in an early miscarriage, 3 (0.7%) in a late miscarriage and 4 (0.9%) in an intrauterine fetal death. Prevalence of live birth was similar among the types of HFD (p=0.31). Obstetrical complications were observed in 54 (17.3%) of live birth pregnancies, including vaginal bleeding (14, 4.4%), retroplacental hematoma (13, 4.1%), and thrombosis (4, 1.3%). Most 56deliveries were spontaneous (218, 74.1%) with a vaginal non-instrumental delivery (195, 63.3%). A neuraxial anesthesia was performed in 116 (40.4%) pregnancies, while 71 (16.6%) and 129 (44.9%) were under general or no anesthesia, respectively. A fibrinogen infusion was administered in 28 (8.9%) deliveries. Postpartum hemorrhages were observed in 62 (19.9%) of pregnancies. Postpartum venous thrombotic events occurred in 5 (1.6%) pregnancies. Women with hypofibrinogenemia were more at risk of bleeding during the pregnancy (p=0.04). CONCLUSIONS Compared to 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 management of pregnancy in HFDs.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet ɑ-granules, splenomegaly and bone marrow (BM) fibrosis. Due to its rarity, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathological features, we performed a detailed clinical genotypic and phenotypic study of 47 GPS patients. We identified 33 new causal variants in NBEAL2. Our GPS patient cohort exhibited known phenotypes, including macro-thrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. We also observed novel clinical phenotypes; these include 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 demonstrate that, in addition to the well-described platelet defects in GPS, there are also immune defects. The abnormal immune cells may be the drivers of systemic abnormalities, such as autoimmune disease.

Les granules denses plaquettaires sont des organites contenant des molecules non peptidiques : calcium, serotonine, nucleotides et phosphates inorganiques. Lors de l’activation plaquettaire, ils fusionnent avec la membrane plasmique afin de secreter leur contenu dans le milieu exterieur. La maladie du pool vide δ-plaquettaire correspond a une anomalie du nombre et/ou du contenu en granules denses. Les etiologies de cette maladie sont diverses et variees. On distingue (1) les formes constitutionnelles, qui peuvent etre isolees ou s’inscrire dans le cadre de syndromes plus complexes (syndromes d’Hermansky–Pudlak, Chediak–Higashi, ou Griscelli) ; et (2) les formes acquises, qui sont le plus souvent associees a des hemopathies malignes (syndrome myelodysplasique, leucemie aigue, syndrome myeloproliferatif, ou autres). La prevalence de la maladie du pool vide δ-plaquettaire dans sa forme isolee est probablement sous-estimee, principalement parce que cette pathologie est peu connue et que son diagnostic biologique est relativement complexe. Les patients souffrant de deficit en granules denses ont generalement un phenotype hemorragique modere. Neanmoins, certains d’entre eux peuvent avoir des manifestations hemorragiques severes notamment en situation chirurgicale ou traumatique. C’est donc une thrombopathie qui doit etre diagnostiquee et prise en compte, en particulier lorsqu’il est necessaire de realiser des gestes a risque hemorragique. In vitro, les deficits en granules denses sont associes a une reduction de l’agregation dans les conditions ou celle-ci est particulierement sensible, c’est-a-dire aux faibles concentrations d’agonistes. Cependant, le profil d’agregation peut ne pas etre altere ou donner des resultats atypiques et dissocies. C’est pourquoi, la confirmation diagnostique necessite la realisation d’examens tres specialises afin de demontrer la diminution du nombre ou du contenu en granules denses. En l’etat actuel des connaissances, il est recommande, dans la mesure du possible, de realiser des dosages de serotonine, une quantification des nucleotides, ainsi qu’une etude des granules denses par microscopie electronique (whole mount). La maladie du pool vide δ-plaquettaire est donc une pathologie complexe presentant des aspects cliniques et biologiques tres divers, requerant une grande expertise afin de ne pas ignorer son diagnostic.

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 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.

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. ispartof: BLOOD vol:136 issue:17 pages:1956-1967 ispartof: location:United States status: published

SummaryIntroduction Factor XIII (FXIII) deficiency is a rare hemorrhagic disorder whose early diagnosis is crucial for appropriate treatment and prophylactic supplementation in cases of severe deficiency. International guidelines recommend a quantitative FXIII activity assay as first-line screening test. FXIII antigen measurement may be performed to establish the subtype of FXIII deficiency (FXIIID) when activity is decreased. Methods The aim of this multicenter study was to evaluate the analytical and diagnostic levels of performance of a new latex immunoassay, K-Assay® FXIII reagent from Stago, for first-line measurement of FXIII antigen. Results were compared to those obtained with the Berichrom® FXIII chromogenic assay for measurement of FXIII activity. Of the 147 patient plasma samples, 138 were selected for analysis. Results The accuracy was very good, with intercenter reproducibility close to 7%. Five groups were defined on FXIII activity level ( 0.05). Correlation of the K-Assay® with the Berichrom® FXIII activity results was excellent (r = 0.919). Good agreement was established by the Bland and Altman method, with a bias of +9.4% on all samples, and of −1.4% for FXIII levels lower than 30%. One patient with afibrinogenemia showed low levels of Berichrom® FXIII activity but normal antigen level and clot solubility as expected. Conclusions The measurement of FXIII antigen using the K-Assay® is a reliable first-line tool for detection of FXIII deficiency when an activity assay is not available.

SummaryInteraction 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.

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.

SummaryVon 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 speeddependent increase in VWF-proteolysis was assessed in the LVAD model whereas no proteolysis was observed in TTP-patients. In VWDpatients, 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 VWFproteolysis 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.Supplementary Material to this article is available online at www.thrombosis-online.com.

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.

Summary 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 0.3 days 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.

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 GpIbα, GpIIb, or EF1α gene regulatory sequences. Both GpIbα 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 GpIbα 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 GpIbα 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.

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.

Introduction Anti-thrombin antibodies are a rare cause of acquired bleeding disorder. We report a case of a patient with a monoclonal gammapathy of unknown significance (MGUS) referred to us for an acquired bleeding tendency. We found that he had an isolated anti-thrombin antibody and showed that this antibody inhibit platelet accumulation and fibrin generation in vivo in a mouse model of arterial thrombus formation. Patient A 40-year-old man with no personal or familial history of bleeding was referred to us for multiple bleeding episodes (rectal bleeding, hematoma) for 18 years. He was diagnosed 12 years previously with an IgG Kappa MGUS which was stable. Complete blood counts were normal. Activated partial thromboplastin time (APTT) and thrombin time (TT) were prolonged at 76 seconds (control 35) and > 120 seconds (control 18) respectively. The prolonged TT was observed using human and bovine thrombin. A TT mixing study was performed by measuring the TT of serial dilutions of patient’s plasma in normal pooled plasma. This study confirmed an acquired anti-thrombin activity of patient’s plasma, as TT of the mix was prolonged from dilutions 1:1 to 1:16 (>120 to 25 seconds). Other coagulation factors were normal. No other auto-antibodies were detected except an isolated lupus anticoagulant. Thrombin generation in patient’s platelet rich plasma measured using the Calibrated Automated Thrombogram (CAT) showed a prolonged lag time (15.5 versus 4.7 minutes) associated with a decreased endogenous thrombin potential (ETP, 834 versus 1730 nmol/L min). The diagnosis of an acquired anti-thrombin antibody associated with a MGUS was proposed. The patient received multiple treatments, including immunosuppressive agents, anti-CD20 monoclonal antibody therapy and bortezomib, in order to eradicate the antibody with no success. Methods Anti-thrombin IgG antibodies were affinity purified from the patient’s plasma using activated agarose beads covalently coupled to human alpha thrombin (Aminolink, ThermoScientific). A mouse model of thrombosis was used to test the effect of the infusion of the purified anti-thrombin IgG on thrombus formation in vivo. Platelet accumulation and fibrin generation at the site of injury was imaged after laser-induced injury of cremaster arterioles using intravital microscopy in wild type mice. Platelet and fibrin labeling were performed using anti-CD42 antibody conjugated to DyLight 488 (Emfret) and an anti-fibrin antibody conjugated with AlexaFluor 647 (Sekisui). Results Immunoblotting using human and bovine alpha-thrombin thrombin confirmed the purified IgG from the patient’s plasma (yield 86 µg/ml) to be anti-thrombin antibodies. At a concentration of 2.45 mg/mL, these anti-thrombin IgG were able to prolong APTT and TT (59 versus 33 seconds and >120 versus 19 seconds, respectively). Control IgG (2.5 mg/mL) purified from healthy individuals had no effect on APTT and TT. Using the mouse model of laser-induced arteriolar injury, we found that patient-derived anti-thrombin IgG (690 microg) caused a significant inhibition of platelet accumulation and fibrin generation of more than 70% at the site of vessel wall injury. Conclusion We report a case of severe acquired bleeding tendency secondary to an anti-thrombin antibody in a patient with an IgG Kappa MGUS. Anti-thrombin IgG antibodies purified from the patient’s plasma retain the anti-thrombin activity in vitro. Additionally, infusion of purified anti-thrombin IgG from the patient induced a dramatic decrease in platelet thrombus accumulation and fibrin generation at the site of injury in a mouse model of arterial injury, proving the etiology of patient’s bleeding tendency. This is the first description of the anti-thrombotic effect of anti-thrombin antibodies directly in vivo. Disclosures: Turhan: BMS, Novartis: Honoraria, Research Funding.