Search

Background: Valoctocogene roxaparvovec transfers a human factor (F)VIII coding sequence into hepatocytes of people with severe hemophilia A to provide bleeding protection., Objectives: To present 3-year efficacy and safety in the multicenter, open-label, single-arm, phase 3 GENEr8-1 trial., Methods: GENEr8-1 enrolled 134 adult males with severe hemophilia A who were receiving FVIII prophylaxis. Efficacy endpoints included annualized bleeding rate, annualized FVIII utilization, FVIII activity (chromogenic substrate assay; imputed as 1 IU/dL at baseline and 0 IU/dL after discontinuation), and the Haemophilia-Specific Quality of Life Questionnaire for Adults. Safety was assessed by adverse events (AEs)., Results: At week 156, 131 of 134 participants remained in the study; overall, 17 of 134 resumed prophylaxis. Mean annualized bleeding rate for treated bleeds decreased from 4.8 (SD, 6.5) bleeds/y at baseline to 0.8 (SD, 2.3; P < .0001) bleeds/y after prophylaxis (prophylaxis cessation to last follow-up) and 0.97 (SD, 3.48) bleeds/y during year 3. Annualized FVIII utilization decreased 96.8% from baseline after prophylaxis and 94.2% during year 3. At week 156, mean and median FVIII activity were 18.4 (SD, 30.8) and 8.3 IU/dL, respectively. FVIII activity decrease was lower between years 2 and 3 than between years 1 and 2. At the end of year 3, clinically meaningful improvements in the Haemophilia-Specific Quality of Life Questionnaire for Adults Total Score were observed (mean change from baseline, 6.6; 95% CI, 4.24-8.87; P < .0001). Mild alanine aminotransferase elevations remained the most common AE during year 3 (23.7% of participants). A serious AE of B-cell acute lymphoblastic leukemia was considered unrelated to treatment., Conclusion: Hemostatic efficacy was maintained, and safety remained unchanged from previous years., Competing Interests: Declaration of competing interests M.C.O. has participated in advisory boards for Bayer, BioMarin Pharmaceutical Inc, Pfizer, Sanofi, and Takeda and received honoraria from BioMarin Pharmaceutical Inc, Biotest, Novo Nordisk, Pfizer, Roche, Takeda, and Sanofi. P.R. has received grant/travel support from CSL Behring, Sobi, and Takeda and advisory honoraria from Idogen, Pfizer, Sigilon, and Sobi. E.S. has received travel grants from CSL Behring and Novo Nordisk. D.V.Q. has served on advisory boards and speakers bureaus or as a consultant for Bayer, BioMarin Pharmaceutical Inc, Genentech, Novo Nordisk, Octapharma, Sanofi, Takeda, and uniQure. A.D.L. has served as an investigator for BioMarin Pharmaceutical Inc and Pfizer. S.W.P. has served as a consultant for ApcinteX, Bayer, BioMarin Pharmaceutical Inc, CSL Behring, Equilibra Bioscience, GeneVentiv, HEMA Biologics, LFB, Novo Nordisk, Pfizer, Regeneron, Roche, Sanofi, Siemens, Spark, Takeda, and uniQure. G.L. has received honoraria for participating in educational events from Alexion, LEO Pharma, Novartis, Novo Nordisk, Sanofi, Sobi, and Takeda and consulting fees from UCB. G.K. has received grant funding from Genentech and Pfizer and served on advisory boards or as a consultant for Bayer, BioMarin Pharmaceutical Inc, Genentech, Novo Nordisk, Sanofi, Sobi, Spark, and Takeda. M.T.R. has served as an investigator for Bayer and BioMarin Pharmaceutical Inc and served on advisory boards or speakers bureaus for Bayer, CSL Behring, Genentech, HEMA Biologics, Novo Nordisk, Sanofi, and Takeda. M.W. has served as a consultant for Bayer, BioMarin Pharmaceutical Inc, Bioverativ, Catalyst, CSL Behring, Genentech, HEMA Biologics, and Novo Nordisk. A.v.D. has received research funding from Pfizer and Sanofi, is a cofounder and board member of Hematherix Inc, and served as a consultant for ASC Therapeutics, BioMarin Pharmaceutical Inc, CSL Behring, Genentech, Regeneron, Sanofi, Takeda, and uniQure. R.K. has received grants from Bayer, CSL Behring, and LEO Pharma; consulting fees from Bayer, BioMarin Pharmaceutical Inc, CSL Behring, Novo Nordisk, Octapharma, Pfizer, Roche/Chugai, Sanofi, Sobi, and Takeda; and payment of honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Bayer, BioMarin Pharmaceutical Inc, Biotest, CSL Behring, Daiichi Sankyo, Grifols, LEO Pharma, Novo Nordisk, Octapharma, Pfizer, Roche/Chugai, Sanofi, Sobi, Shire/Takeda, and uniQure. S.S. has received speaking, educational, or travel honoraria from CSL Behring, Pfizer, Roche, Sobi, and Takeda. H.C. has served as a consultant for BioMarin Pharmaceutical Inc, Novo Nordisk, Roche, and Sobi. A.L.D has received research funding from BioMarin Pharmaceutical Inc, Novo Nordisk, Sanofi, and Takeda; served as a consultant for CSL Behring, Roche, and uniQure; and is a board member of the National Hemophilia Foundation and World Federation of Hemophilia USA. J.O. has received research funding from Bayer, Biotest, CSL Behring, Octapharma, Pfizer, Swedish Orphan Biovitrum, and Takeda and consultancy, speakers bureau, honoraria, scientific advisory board, and travel expenses from Bayer, Biogen Idec, BioMarin Pharmaceutical Inc, Biotest, Chugai, CSL Behring, Freeline, Grifols, LFB, Novo Nordisk, Octapharma, Pfizer, Roche, Sanofi, Spark Therapeutics, Swedish Orphan Biovitrum, and Takeda. F.P. has served as a consultant for BioMarin Pharmaceutical Inc, Grifols, Roche, Sanofi, Sobi, and Takeda. C.M.M. has received research funding from CSL Behring, Grifols, and Takeda and has served as a speaker or consultant for CSL Behring, LFB Pharma, Novo Nordisk, Octapharma, Takeda, and Sobi. D.O., H.Y., E.D.-A., and T.M.R. are employees and shareholders of BioMarin Pharmaceutical Inc. J.Mahlangu has received research funding from BioMarin Pharmaceutical Inc, Catalyst, Roche, Novo Nordisk, Pfizer, Sandoz, Sanofi, and Spark Therapeutics. B.M., J. Mason, and S.-C.C. have no conflicts to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Von Willebrand disease (VWD) is an inherited bleeding disorder caused by quantitative or qualitative deficiencies in von Willebrand factor (VWF). People with VWD may experience excessive, recurrent or prolonged bleeding, particularly during menstruation, childbirth, surgery or following trauma. However, many VWD patients are undiagnosed, and therefore inadequately treated. Reasons for the underdiagnosis of VWD include its relatively mild symptoms, complex diagnosis, lack of awareness among non-specialist healthcare providers and the general population, and a lack of prioritisation of disorders disproportionately affecting females. The vwdtest.com platform was launched as part of a global initiative to raise awareness and improve diagnosis of VWD. Besides providing VWD-specific educational resources, the website includes an online bleeding self-assessment tool and offers diagnostic support for individuals, and their providers, who have a score suggestive of a bleeding disorder. vwdtest.com helps to address these unmet needs, especially in regions with limited access to educational and diagnostic resources., Competing Interests: Declaration of Competing Interest FFCM has received grant/research support from Bayer and Octapharma, and honoraria or consultation fees from Bayer, Octapharma and Sanofi. ABF has received research support from Baxalta/Shire/Takeda, CSL-Behring, and honoraria or consultation fees from Baxalta/Takeda, CSL Behring, Grifols, Kedrion, LFB and Octapharma. AS has no competing interests to declare. CMM has received research support from Baxter/Takeda, CSL Behring and Grifols and honoraria or consultation fees from CSL Behring, LFB, Octapharma and Takeda. She has participated in advisory boards for Takeda and CSL Behring. JCR has disclosed receiving research support from Takeda and Genentech. He has participated in advisory boards for Sanofi Genzyme, Takeda, Octapharma, Novo Nordisk, Pfizer, and CSL Behring. He is on speaker bureaus for Sanofi Genzyme, Novo Nordisk, Octapharma, and Takeda. AD has no competing interests to declare. JJ received an unrestricted educational grant from Octapharma. EB has received grant/research support from Bayer, Baxalta/Takeda, CSL Behring and Sobi, and honoraria or consultation fees from Bayer, Baxalta/Takeda, CSL Behring and Octapharma., (Copyright © 2022. Published by Elsevier Ltd.)

Factor X deficiency is a rare coagulation disorder that can be hereditary or acquired. The typology and severity of the associated bleeding symptoms are highly heterogeneous, adding to the difficulties of diagnosis and management. Evidence-based guidelines and reviews on factor X deficiency are generally limited to publications covering a range of rare bleeding disorders. Here we provide a comprehensive review of the literature on factor X deficiency, focusing on the hereditary form, and discuss the evolution in disease management and the evidence associated with available treatment options. Current recommendations advise clinicians to use single-factor replacement therapy for hereditary disease rather than multifactor therapies such as fresh frozen plasma, cryoprecipitate, and prothrombin complex concentrates. Consensus in treatment guidelines is still urgently needed to ensure optimal management of patients with factor X deficiency across the spectrum of disease severity., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: CMM has received research support from 10.13039/100004702Baxter, 10.13039/100008322CSL Behring and Grifols; speaker's fees from 10.13039/100008322CSL Behring, LFB, Octapharma and 10.13039/100008373Takeda; consultancy fees from 10.13039/100008322CSL Behring, LFB, 10.13039/501100004191Novo Nordisk and 10.13039/100008373Takeda; and educational support from 10.13039/501100004191Novo Nordisk, Octapharma and 10.13039/501100012112SOBI. RA and JC declare no conflict of interest.

The heterogeneous manifestations of MYH9-related disorder (MYH9-RD), characterized by macrothrombocytopenia, Döhle-like inclusion bodies in leukocytes, bleeding of variable severity with, in some cases, ear, eye, kidney, and liver involvement, make the diagnosis for these patients still challenging in clinical practice. We collected phenotypic data and analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder. Patients were enrolled in the BRIDGE-BPD and ThromboGenomics Projects and their samples processed by high throughput sequencing (HTS). We identified 50 patients with a rare variant in MYH9. All patients had macrothrombocytes and all except two had thrombocytopenia. Some degree of bleeding diathesis was reported in 41 of the 50 patients. Eleven patients presented hearing impairment, three renal failure and two elevated liver enzymes. Among the 28 rare variants identified in MYH9, 12 were novel. HTS was instrumental in diagnosing 23 patients (46%). Our results confirm the clinical heterogeneity of MYH9-RD and show that, in the presence of an unclassified platelet disorder with macrothrombocytes, MYH9-RD should always be considered. A HTS-based strategy is a reliable method to reach a conclusive diagnosis of MYH9-RD in clinical practice., (© 2019 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

Background: Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular dysplasia resulting in visceral arteriovenous malformations and smaller mucocutaneous telangiectasia. Most patients experience recurrent nosebleeds and become anemic without iron supplementation. However, thousands may require anticoagulation for conditions such as venous thromboembolism and/or atrial fibrillation. Over decades, tolerance data has been published for almost 200 HHT-affected users of warfarin and heparins, but there are no published data for the newer direct oral anticoagulants (DOACs) in HHT., Methods: To provide such data, a retrospective audit was conducted across the eight HHT centres of the European Reference Network for Rare Multisystemic Vascular Diseases (VASCERN), in Denmark, France, Germany, Italy, the Netherlands and the UK., Results: Although HHT Centres had not specifically recommended the use of DOACs, 32 treatment episodes had been initiated by other clinicians in 28 patients reviewed at the Centres, at median age 65 years (range 30-84). Indications were for atrial fibrillation (16 treatment episodes) and venous thromboembolism (16 episodes). The 32 treatment episodes used Apixaban (n = 15), Rivaroxaban (n = 14), and Dabigatran (n = 3). HHT nosebleeds increased in severity in 24/32 treatment episodes (75%), leading to treatment discontinuation in 11 (34.4%). Treatment discontinuation was required for 4/15 (26.7%) Apixaban episodes and 7/14 (50%) Rivaroxaban episodes. By a 4 point scale of increasing severity, there was a trend for Rivaroxaban to be associated with a greater bleeding risk both including and excluding patients who had used more than one agent (age-adjusted coefficients 0.61 (95% confidence intervals 0.11, 1.20) and 0.74 (95% confidence intervals 0.12, 1.36) respectively. Associations were maintained after adjustment for gender and treatment indication. Extreme hemorrhagic responses, worse than anything experienced previously, with individual nosebleeds lasting hours requiring hospital admissions, blood transfusions and in all cases treatment discontinuation, occurred in 5/14 (35.7%) Rivaroxaban episodes compared to 3/15 (20%) Apixaban episodes and published rates of ~ 5% for warfarin and heparin., Conclusions: Currently, conventional heparin and warfarin remain first choice anticoagulants in HHT. If newer anticoagulants are considered, although study numbers are small, at this stage Apixaban appears to be associated with lesser bleeding risk than Rivaroxaban.

Rheological forces in the blood trigger the unfolding of von Willebrand factor (VWF) and its A2 domain, exposing the scissile bond for proteolysis by ADAMTS13. Under quiescent conditions, the scissile bond is hidden by the folded structure due to the stabilisation provided by the structural specialisations of the VWF A2 domain, a vicinal disulphide bond, a calcium binding site and a N1574-glycan.The reduced circulating high MW multimers of VWF in patients with type 2A von Willebrand disease (VWD) may be associated with mutations within the VWF A2 domain and this is attributed to enhanced ADAMTS13 proteolysis. We investigated 11 VWF A2 domain variants identified in patients with type 2A VWD. In recombinant full-length VWF, enhanced ADAMTS13 proteolysis was detected for all of the expressed variants in the presence of urea-induced denaturation. A subset of the FLVWF variants displayed enhanced proteolysis in the absence of urea. The mechanism of enhancement was investigated using a novel VWF A2 domain FRET construct. In the absence of induced unfolding, 7/8 of the expressed mutants exhibited a disrupted domain fold, causing spatial separation of the N- and C- termini. Three of the type 2A mutants were not secreted when studied within the VWF A2 domain FRET construct. Urea denaturation revealed for all 8 secreted mutants reduced unfolding cooperativity and stability of the VWF A2 domain. As folding stability was progressively disrupted, proteolysis by ADAMTS13 increased. Due to the range of folding stabilities and wide distribution of VWF A2 domain mutations studied, we conclude that these mutations disrupt regulated folding of the VWF A2 domain. They enhance unfolding by inducing separation of N- and C-termini, thereby promoting a more open conformation that reveals its binding sites for ADAMTS13 and the scissile bond.

Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterized. Pathogenic variants in RASGRP2 , which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in 3 pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5422 controls. Eleven cases harbored 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included 5 high-impact variants predicted to prevent CalDAG-GEFI expression and 6 missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbβ3 signaling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical, and dental bleeding from childhood, requiring ≥1 blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to adenosine 5'-diphosphate and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a nonsyndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation., (© 2017 by The American Society of Hematology.)

Four non-vitamin K oral anticoagulants (NOACs) are now licensed and available in the UK, offering unprecedented choices in anticoagulant therapy for clinicians and patients. NOACs have many clear benefits over warfarin, the most striking being the reduction in intracranial haemorrhage. However, a number of uncertainties remain: their efficacy in certain situations, utility of drug assays, significance of drug interactions and management of bleeding. In the absence of any direct comparative trials, it is not clear that any of the NOACs is significantly better than the others in any of the licensed indications. The differential activities, pharmacokinetics, metabolism, excretion and side effects of the agents should be considered when selecting the most appropriate anticoagulant. In this article, we discuss how, with careful selection for the relevant indication, NOACs can simplify therapy while improving outcomes. We aim to provide clinicians with the information needed to select the most suitable anticoagulant drug for an individual patient in a given situation., (© Royal College of Physicians 2017. All rights reserved.)

Inhibitor formation in non-severe haemophilia A is a life-long risk and associated with morbidity and mortality. There is a paucity of data to understand real-world inhibitor screening practice. We evaluated the treatment burden, haemostatic strategies, F8 genotyping and inhibitor screening practices in non-severe haemophilia A in seven London haemophilia centres. In the 2-year study period, 44% (377/853) patients received at least one haemostatic treatment. Seventy-nine percent of those treated (296/377) received factor VIII (FVIII) concentrate. F8 genotype was known in 88% (331/377) of individuals. Eighteen per cent (58/331) had 'high-risk' F8 genotypes. In patients with 'standard-risk' F8 genotypes treated on-demand with FVIII concentrate, 51·3% episodes (243/474) were screened within 1 year. However, poor screening compliance was observed after 'high-risk' treatment episodes. In patients with 'standard-risk' F8 genotypes, 12·3% (28/227) of treatment episodes were screened in the subsequent 6 weeks after surgery or a bleed requiring ≥5 exposure days. Similarly, in the context of 'high-risk' F8 genotypes after any FVIII exposure, only 13·6% (12/88) of episodes were screened within 6 weeks. Further study is required to assess optimal practice of inhibitor screening in non-severe haemophilia A to inform subsequent clinical decisions and provide more robust prevalence data to further understand the underlying immunological mechanism., (© 2017 John Wiley & Sons Ltd.)

Introduction: Maintaining haemostasis in surgery is challenging for hereditary rare bleeding disorders in which multi-coagulation-factor concentrates are the only therapeutic option. Hereditary factor X (FX) deficiency affects 1:500 000 to 1:1 000 000 individuals, and no specific replacement FX concentrate has been available. A high-purity, plasma-derived FX concentrate (pdFX) has been developed for patients with hereditary FX deficiency., Aim: Our objective was to assess the safety and efficacy of pdFX in subjects with FX deficiency undergoing surgery., Methods: Subjects with hereditary mild-to-severe FX deficiency (basal plasma FX activity [FX:C] <20 IU dL(-1) ) undergoing surgery received pdFX preoperatively to raise FX:C to 70-90 IU dL(-1) and postoperatively to maintain levels >50 IU dL(-1) until the subject was no longer at risk of bleeding due to surgery. Efficacy of pdFX was assessed by blood loss during surgery, requirement for blood transfusion, postoperative bleeding from the surgical or other sites, and changes in haemoglobin levels. Safety was assessed by adverse events (AEs), development of inhibitors, and clinically significant changes in laboratory parameters., Results: Five subjects (aged 14-59 years) underwent seven surgical procedures (four major and three minor). Treatment duration was 1-15 days. For each procedure, pdFX treatment was assessed as "excellent" in preventing bleeding and achieving haemostasis. No blood transfusions were required, no AEs related to pdFX were observed, and no clinically significant trends were found in any laboratory parameters., Conclusion: These data demonstrate that pdFX is safe and effective as replacement therapy in five subjects with mild-to-severe FX deficiency undergoing surgery on seven occasions., (© 2016 John Wiley & Sons Ltd.)

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 (MKs). 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 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to 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. Overexpression 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 insight into the autoregulation of DIAPH1 activity., (© 2016 by The American Society of Hematology.)

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized 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 platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (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, whereas the remainder had an a priori highly 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 8 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., (© 2016 by The American Society of Hematology.)

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., (Copyright © 2016, American Association for the Advancement of Science.)

Background: Heritable bleeding and platelet disorders (BPD) are heterogeneous and frequently have an unknown genetic basis. The BRIDGE-BPD study aims to discover new causal genes for BPD by high throughput sequencing using cluster analyses based on improved and standardised deep, multi-system phenotyping of cases., Methods: We report a new approach in which the clinical and laboratory characteristics of BPD cases are annotated with adapted Human Phenotype Ontology (HPO) terms. Cluster analyses are then used to characterise groups of cases with similar HPO terms and variants in the same genes., Results: We show that 60% of index cases with heritable BPD enrolled at 10 European or US centres were annotated with HPO terms indicating abnormalities in organ systems other than blood or blood-forming tissues, particularly the nervous system. Cases within pedigrees clustered closely together on the bases of their HPO-coded phenotypes, as did cases sharing several clinically suspected syndromic disorders. Cases subsequently found to harbour variants in ACTN1 also clustered closely, even though diagnosis of this recently described disorder was not possible using only the clinical and laboratory data available to the enrolling clinician., Conclusions: These findings validate our novel HPO-based phenotype clustering methodology for known BPD, thus providing a new discovery tool for BPD of unknown genetic basis. This approach will also be relevant for other rare diseases with significant genetic heterogeneity.

Background: Hemophilia B, resulting from a deficiency of coagulation factor IX, is treated effectively with either recombinant FIX (r-FIX) or plasma-derived FIX (pd-FIX) concentrates, although differences in pharmacokinetics are observed. FIX is activated in vivo by both activated FXI (FXIa) and tissue factor (TF)-activated FVII (FVIIa); however, conventional activated partial thromboplastin time (APTT)-based assays assess only activation by FXIa., Objectives: To examine the differences between pd-FIX and r-FIX concentrates with respect to their thrombogenicity and activation., Methods and Results: FIX ELISA was used to quantify antigenic FIX. Calibrated automated thrombography was performed to evaluate the effect of FIX on thrombin generation. FIXa was quantified by the cleavage of FIXa-specific chromogenic substrate. FIX activation was studied in a purified system., Results: We found that r-FIX had ~ 1.6-fold greater specific activity than pd-FIX. r-FIX generated a markedly higher thrombin peak than pd-FIX at an equivalent antigen level when coagulation was initiated by TF, but this was not seen in contact activation-triggered thrombin generation (TG). Interestingly, the amount of FIXa in r-FIX concentrate was 10 times higher than that in pd-FIX concentrate. In a purified system, the amount of r-FIXa generated by FXIa in the first 10 min of activation was 1.37-fold that of pd-FIXa, whereas no difference between the concentrates was observed when triggered by TF-FVIIa., Conclusions: Clear differences were observed between pd-FIX and r-FIX concentrates, including the proportion of FIXa and the activation by FXIa. These may explain some of the discrepancies observed clinically, and suggest that the APTT may not reflect their resultant in vivo properties., (© 2013 International Society on Thrombosis and Haemostasis.)

In this issue of Blood, Schiele et al report the development of a monoclonal antibody that reverses the anticoagulant effect of the direct thrombin inhibitor dabigatran.

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

The identification of variant Creutzfeldt-Jakob disease (vCJD) in the UK in 1996 led to significant concerns about the possibility of secondary transmission, however the prevalence of subclinical vCJD and risks of vCJD transmission by plasma are not known. In the UK, public health precautions have been implemented in all recipients of coagulation factor concentrates manufactured from UK plasma pools between 1980 and 2001. The recent demonstration of abnormal prion protein in a spleen sample at autopsy of a UK haemophilic patient who received coagulation factor concentrates to which a donor incubating vCJD had contributed most likely represents the first case of vCJD transmission by coagulation factor concentrates. We review the uncertainties that surround risk of vCJD transmission by coagulation factor concentrates, the challenges in dealing with undefined risks, the rationale behind current policies and the implementation of vCJD surveillance and risk management measures in bleeding disorder patients in the UK., (© 2012 Blackwell Publishing Ltd.)

Background: Type 2M von Willebrand disease (VWD) results from mutations in the A1 domain of von Willebrand factor (VWF) that reduce its platelet-binding function. However, currently employed VWF functional static assays may not distinguish between clinical phenotype., Methods: Fifteen individuals from five kindreds with VWF-A1 domain mutations I1416T or I1416N, correlated with mild and moderate clinical phenotypes, respectively, were investigated. The mutations were reproduced by site-directed mutagenesis and expressed in HEK293T cells; functional studies of the recombinant mutants, including GPIbα binding using a flow-based assay, were performed., Results: Plasma from all individuals demonstrated discordant reductions in VWF antigen and platelet-binding function in the presence of high-molecular-weight VWF multimers consistent with VWD type 2M. There was lowered expression and secretion of both mutants compared with wild type (WT) recombinant (r)VWF as well as a significant reduction in GPIbα binding. Binding to collagen was normal and electrophoretic analysis demonstrated a similar multimer distribution between the mutant proteins and wt-rVWF. GPIbα binding under flow was also significantly reduced for I1416N and I1416T rVWF. Impairment of GPIbα binding was more marked for I1416N rVWF than I1416T under both static and flow conditions: this was in spite of similar VWF:Ristocetin cofactor (RCo) activities in patient plasma and is consistent with a respective clinical phenotype., Conclusions: Our findings have established for the first time that I1416N and I1416T are responsible for a type 2M VWD phenotype and demonstrate that quantification of VWF function under shear stress may provide a more accurate measure of clinical severity than the static functional measurements in current diagnostic use., (© 2012 International Society on Thrombosis and Haemostasis.)

Type 2B von Willebrand disease (VWD) is a rare, inherited bleeding disorder resulting from a qualitative defect in von Willebrand factor (VWF). There is very little published information on how to quantify bleeding risk and manage haemostasis in type 2B VWD patients during pregnancy. This article presents the changes in VWF parameters and details of patient management and delivery outcomes for four pregnancies in three women with two different mutations causing type 2B VWD. We report an unexpected rise in the VWF:Ag at 37 weeks gestation in two sisters with R1306W associated with significant thrombocytopenia. These patients were supported with platelet transfusions as well as intermediate purity VWF-FVIII plasma concentrates during the peri- and postpartum periods. No thrombocytopenia was observed in our third case with a mutation encoding an R1308C substitution; haemostatic support was with intermediate purity VWF-FVIII plasma concentrates alone. No adverse bleeding events occurred and in all cases a live healthy infant was delivered. One patient was readmitted post partum with bleeding symptoms due to retained placenta; no further haemostatic support was given at this time. This case series is the first to detail the progression of laboratory parameters, management and outcomes of pregnancy in patients with type 2B VWD. The cases illustrate some of the challenges posed by the increased production of a VWF variant with a gain-of-function effect. The rapid coagulation changes observed in this series illustrate the need for continual monitoring of VWF parameters and platelet count throughout pregnancy in women with type 2B VWD., (© 2011 Blackwell Publishing Ltd.)

The risk of variant Creutzfeldt-Jakob disease (vCJD) from potentially infected plasma products remains unquantified. This risk has been assessed for 787 UK patients with an inherited bleeding disorder prospectively followed-up for 10-20 years through the UK Haemophilia Centre Doctors' Organisation (UKHCDO) Surveillance Study. These patients had been treated with any of 25 'implicated' clotting factor batches from 1987 to 1999, which included in their manufacture, plasma from eight donors who subsequently developed clinical vCJD. Variant CJD infectivity of these batches was estimated using plasma fraction infectivity estimates and batch-manufacturing data. Total potential vCJD infectivity received by each patient has been estimated by cumulating estimated infectivity from all doses received during their lifetime. Of 787 patients, 604 (77%) were followed-up for over 13 years following exposure to an implicated batch. For these 604 patients, the estimated vCJD risk is ≥ 1% for 595, ≥ 50% for 164 and 100% for 51. This is additional to background UK population risk due to dietary exposure. Of 604 patients, 94 (16%) received implicated batches linked to donors who developed clinical vCJD within 6 months of their donations. One hundred and fifty-one (25%) had received their first dose when under 10 years of age. By 1st January 2009, none of these patients had developed clinical vCJD. The absence of clinical vCJD cases in this cohort to date suggests that either plasma fraction infectivity estimates are overly precautionary, or the incubation period is longer for this cohort than for implicated cellular blood product recipients. Further follow-up of this cohort is needed., (© 2011 Blackwell Publishing Ltd.)

Summary: All UK patients with bleeding disorders treated with any UK-sourced pooled factor concentrates between 1980 and 2001 have been informed that they may be at an increased risk of infection with variant Creutzfeldt-Jakob disease (vCJD). We describe a study to detect disease-associated, protease-resistant prion protein (PrP(res)) in 17 neurologically aymptomatic patients with haemophilia considered to be at increased risk of vCJD. Materials from 11 autopsy and seven biopsy cases were analysed for PrP(res). The tissues available from each case were variable, ranging from a single biopsy sample to a wide range of autopsy tissues. A single specimen from the spleen of one autopsy case gave a strong positive result on repeated testing for PrP(res) by Western blot analysis. This tissue came from a 73-year-old male patient with no history of neurological disease, who was heterozygous (methionine/valine) at codon 129 in the prion protein gene. He had received over 9000 units of factor VIII concentrate prepared from plasma pools known to include donations from a vCJD-infected donor, and some 400,000 units not known to include donations from vCJD-infected donors. He had also received 14 units of red blood cells and had undergone several surgical and invasive endoscopic procedures. Estimates of the relative risks of exposure through diet, surgery, endoscopy, blood transfusion and receipt of UK-sourced plasma products suggest that by far the most likely route of infection in this patient was receipt of UK plasma products.

Summary: The appearance and rapid evolution of BSE in UK cattle in the mid 1980s, with compelling data supporting variant Creutzfeldt-Jakob disease (vCJD) as its human manifestation, pose a potentially severe threat to public health. Three clinical cases and one asymptomatic case of vCJD infection have been reported in UK recipients of non-leucodepleted red cell transfusions from donors subsequently diagnosed with vCJD. Plasma from both these and other donors who later developed vCJD has contributed towards plasma pools used to manufacture clotting factor concentrate. The United Kingdom Haemophilia Centre Doctors' Organisation (UKHCDO) Surveillance Study has detected asymptomatic vCJD postmortem in a haemophilic patient treated with UK plasma products including two batches of clotting factor linked to a donor who subsequently developed vCJD. Over 4000 bleeding disorder patients treated with UK plasma products are recorded on the UKHCDO National Haemophilia Database. The risk of vCJD transmission by plasma products is not known. However, public health precautions have been implemented since 2004 in all UK inherited bleeding disorder patients who received UK-sourced plasma products between 1980 and 2001 to minimize the possible risk of onward vCJD transmission. We evaluate vCJD surveillance and risk management measures taken for UK inherited bleeding disorder patients, report current data and discuss resultant challenges and future directions.

Investigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wild-type recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecular-weight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that isolated collagen-binding defects are classified as a distinct subtype of von Willebrand disease.

Reduced plasma survival of von Willebrand factor (VWF) may contribute towards the pathogenesis of type 1 von Willebrand disease (VWD). However, little is known about mechanism(s) of VWF clearance and factors that may affect it. The half-life of VWF-related parameters following the administration of DDAVP was measured in 26 patients with type 1 VWD and 10 haemophilia A controls. Binding of lectins Ricinus communis (RCA-I) and Erythina crystagalli (ECA) agglutinins to VWF and VWF susceptibility to ADAMTS-13-mediated proteolysis were investigated. Sequence analysis of targeted regions of the VWF gene was performed to inspect for mutations that have been associated with increased clearance. Post-DDAVP clearance of VWF was increased approximately three-fold in the type 1 VWD cohort overall. However this was not shown to consistently associate with steady-state VWF antigen (VWF:Ag) levels. Furthermore, increased VWF clearance was not consistently associated with increased ratios of VWF propeptide (VWFpp) to VWF:Ag indicating that a normal ratio does not necessarily reflect normal post-DDAVP survival in type 1 VWD patients. RCA-I and ECA binding to VWF were increased in type 1 VWD patients and, although inversely correlated with VWF levels, this was independent of VWF clearance. There was no association between VWF clearance and ADAMTS-13-mediated proteolysis. Three novel candidate mutations with an increased clearance phenotype were identified. The data are consistent with heterogeneity in pathogenic mechanisms in type 1 VWD and are consistent with type 1 VWD representing a complex genetic trait.

Oligosaccharides make up approximately 20% of the mass of VWF and although their structures are well established, their functional role remains unclear. Modification of the VWF oligosaccharide structures has been shown to result in increased plasma clearance of the protein. A mutation which alters cell type-specific expression of the Galgt2 glycosyltransferase gene in the RIIIS/J mouse results in an autosomal dominant partial quantitative deficiency of VWF. Increased plasma clearance of VWF has been demonstrated in some individuals with a partial quantitative deficiency of the protein and it is possible that variation in VWF glycosylation may contribute towards this. ABH antigens occur within the oligosaccharide component of VWF and may account for the variation in plasma VWF:Ag levels observed between individuals of different ABO blood groups. The structures and functional roles of the oligosaccharide side chains of VWF and possible pathogenetic mechanisms by which they may contribute towards VWD are reviewed in this article.