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Background: Rare coagulation factor deficiencies and disorders of fibrinolysis (defined as rare bleeding disorders [RBDs]) present with a heterogeneous bleeding phenotype, and bleeding severity is difficult to predict., Objectives: Describe underlying rare genetic variants in the Dutch RBD population and investigate the relationship between genotype, laboratory phenotype, and clinical phenotype., Methods: The Rare Bleeding Disorders in the Netherlands is a cross-sectional, nationwide study conducted between October 1, 2017, and November 30, 2019. Bleeding scores and blood samples were collected during a single study visit. Coagulation factor levels were measured centrally, and targeted exome analysis was performed on 156 genes involved in thrombosis and hemostasis. Pathogenicity was assigned according to the Association for Clinical Genetic Science guidelines., Results: Rare genetic variants specific to the diagnosed RBD were found in 132 of 156 patients (85%). Of the 214 rare genetic variants identified, 57% ( n  = 123) were clearly pathogenic, 19% ( n  = 40) were likely pathogenic, and 24% ( n  = 51) were variants of unknown significance. No explanatory genetic variants were found in patients with plasminogen activator inhibitor type 1 deficiency or hyperfibrinolysis. A correlation existed between factor activity levels and the presence of a genetic variant in the corresponding gene in patients with rare coagulation factor deficiencies and alpha-2-antiplasmin deficiency. Co-occurrence of multiple genetic variants was present in a quarter of patients, but effect on phenotype remains unclear., Conclusion: Targeted exome analysis may offer advantages over single-gene analysis, emphasized by a number of combined deficiencies in this study. Further studies are required to determine the role of co-occurring hemostasis gene variants on the bleeding phenotype in RBDs., (© 2024 The Author(s).)

Background: Heavy menstrual bleeding (HMB) is associated with a reduced quality of life and limitations in social and physical functioning. Data on HMB in women with rare bleeding disorders (RBDs), including coagulation factor deficiencies and fibrinolytic disorders, are scarce., Objectives: To analyze the prevalence, severity, and treatment of HMB in Dutch women with an RBD., Methods: The Rare Bleeding Disorders in the Netherlands (RBiN) study included 263 patients with an RBD from all 6 hemophilia treatment centers (October 2017-November 2019). In this analysis, data of 111 women aged ≥16 years were studied. According to the International Society on Thrombosis and Haemostasis bleeding assessment tool, HMB symptoms were scored from 0 (no/trivial) to 4 (severe symptoms requiring medical intervention). HMB was defined as a score ≥1. Age at RBD diagnosis was extracted from patient files., Results: HMB was reported by 80% of women (89/111) and was more prevalent in women with a fibrinolytic disorder (33/35; 94%) than in women with a coagulation factor deficiency (56/76; 74%) (P = .011). Of the 89 women with HMB, 82% (n = 73) ever required treatment. Multiple treatment modalities were frequently used, both in severe and mild deficiencies. Hormonal treatment was mostly used (n = 64; 88%), while antifibrinolytics were prescribed less frequently (n = 18; 25%). In women with HMB since menarche (n = 61; 69%), median age at RBD diagnosis was 28 years (IQR, 14-41)., Conclusion: HMB is common in women with RBDs. Women with mild deficiencies also frequently reported HMB. Only a minority of women were treated with hemostatic agents. A significant diagnostic delay was observed after the onset of HMB symptoms., Competing Interests: Declaration of competing interests K. Meijer reports speaker fees from Bayer and Alexion, participation in trial steering committee for Bayer, consulting fees from Uniqure, participation in data monitoring and endpoint adjudication committee for Octapharma. M.H. Cnossen's institution has received investigator-initiated research and travel grants as well as speaker fees over the years from the Netherlands Organisation for Scientific Research (NWO) and Netherlands National research Agenda (NWA), the Netherlands Organization for Health Research and Development (ZonMw), the Dutch Innovatiefonds Zorgverzekeraars, Stichting Haemophilia, Baxter/Baxalta/Shire/ Takeda, Pfizer, Bayer Schering Pharma, CSL Behring, Sobi Biogen, Novo Nordisk, Novartis, Roche, and Nordic Pharma, and for serving as a steering board member for Roche, Bayer and Novartis. All grants and fees go to the Erasmus MC as an institution. She is coordinator of Erasmus MC as a Health Care Provider within the European Reference Network (ERN) for rare hematological diseases EuroBloodNet and (co)leader of the local Erasmus MC Expert Centers for Rare Bleeding Disorders and Sickle Cell and Thalassemia Comprehensive Care Center. R.E.G. Schutgens reports grants from Bayer, Baxalta, Pfizer, and Novo Nordisk outside the submitted work. M. Peters reports a grant from Pfizer outside the submitted work. W.L. van Heerde reports financial support from Takeda, Bayer, Sobi and CSL Behring, and funding from Takeda and Bayer for Enzyre. The remaining authors declare no competing financial interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Women with rare bleeding disorders (RBDs), including coagulation factor deficiencies and fibrinolytic disorders, may have a higher risk of postpartum hemorrhage (PPH). Information on this patient category is lacking in the existing PPH guidelines because data on PPH in patients with RBDs are scarce., Objective: To describe the prevalence of PPH in women with an RBD and evaluate the use of peripartum hemostatic prophylaxis., Methods: In the Rare Bleeding Disorders in the Netherlands (RBiN) study, patients with RBDs (n = 263) were included from all 6 Dutch hemophilia treatment centers. Patient-reported information on delivery, peripartum hemostatic prophylaxis, and occurrence of PPH was collected retrospectively. If available, information about the precise volume of postpartum blood loss was extracted from electronic patient files. PPH was defined as blood loss ≥500 mL (World Health Organization guideline)., Results: A total of 244 pregnancies, including 193 livebirths, were reported by 85 women. A considerable proportion of these women experienced PPH, ranging from 30% in factor V deficiency to 100% in hyperfibrinolysis. Overall, PPH was reported in 44% of deliveries performed with and 53% of deliveries performed without administration of peripartum hemostatic prophylaxis. Blood loss was significantly higher in deliveries without administration of hemostatic prophylaxis (median 1000 mL) compared to deliveries with administration of prophylaxis (median 400 mL) (p = 0.011). Patients with relatively mild deficiencies also frequently experienced PPH when peripartum hemostatic prophylaxis was omitted., Conclusion: PPH is common in rare coagulation factor deficiencies, both severe and mild, and fibrinolytic disorders, especially when peripartum prophylactic hemostatic treatment was not administered. The use of prophylactic hemostatic treatment was associated with less postpartum blood loss., Competing Interests: Declaration of competing interests K.M. reports speaker fees from Bayer and Alexion, participation in trial steering committee for Bayer, consulting fees from Uniqure, and participation in data monitoring and endpoint adjudication committee for Octapharma. M.H.C. received investigator–initiated research and travel grants, as well as speaker fees, over the years from the Netherlands Organisation for Scientific Research (NWO), the Netherlands Organization for Health Research and Development (ZonMw), the Dutch “Innovatiefonds Zorgverzekeraars,” Baxter, Baxalta, Shire, Takeda, Pfizer, Bayer Schering Pharma, CSL Behring, Sobi Biogen, Novo Nordisk, Novartis, and Nordic Pharma and has served as a steering board member for Roche, Bayer, and Novartis. All grants, awards, and fees go to the Erasmus MC as institution. R.E.G.S. reports grants from Bayer, Baxalta, Pfizer, and Novo Nordisk outside the submitted work. M.P. reports a grant from Pfizer outside the submitted work. W.L.v.H. reports financial support from Takeda, Bayer, Sobi, and CSL Behring and funding from Takeda and Bayer for Enzyre. The remaining authors declare no competing financial interests., (Copyright © 2022 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Background: Patients with rare inherited bleeding disorders (RBDs) exhibit hemorrhagic symptoms, varying in type and severity, often requiring only on-demand treatment. Prolonged bleeding after invasive procedures is common. Adequate peri-procedural therapy may reduce this bleeding risk., Objective: To describe general treatment plans of RBD patients and evaluate the use of peri-procedural hemostatic therapy., Methods: In the Rare Bleeding Disorders in the Netherlands (RBiN) study, RBD patients from all six Dutch Hemophilia Treatment Centers were included. General treatment plans were extracted from patient files. Patients with a dental or surgical procedure in their history were interviewed about use of peri-procedural treatment and bleeding complications., Results: Two-hundred sixty-three patients with a rare coagulation factor deficiency or fibrinolytic disorder were included. Eighty-four percent had a documented general treatment plan. General treatment plans of patients with the same RBD were heterogeneous, particularly in factor XI deficiency. Overall, 308 dental and 408 surgical procedures were reported. Bleeding occurred in 50% of dental and 53% of surgical procedures performed without hemostatic treatment and in 28% of dental and 19% of surgical procedures performed with hemostatic treatment. Not only patients with severe RBDs, but also patients with mild deficiencies, experienced increased bleeding without proper hemostatic treatment., Conclusion: Large heterogeneity in general treatment plans of RBD patients was found. Bleeding after invasive procedures was reported frequently, both before and after RBD diagnosis, irrespective of factor activity levels and particularly when peri-procedural treatment was omitted. Improved guidelines should include uniform recommendations for most appropriate hemostatic products per RBD and emphasize the relevance of individual bleeding history., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Background: In recent years, more awareness is raised about sex-specific dilemmas in inherited bleeding disorders. However, no large studies have been performed to assess differences in diagnosis, bleeding phenotype and management of men and women with bleeding disorders. Therefore, we investigated sex differences in a large cohort of well-defined patients with autosomal inherited bleeding disorders (von Willebrand disease (VWD), rare bleeding disorders (RBDs) and congenital platelet defects (CPDs))., Methods: We included patients from three nationwide cross-sectional studies on VWD, RBDs and CPDs in the Netherlands, respectively the WiN, RBiN and TiN study. In all studies a bleeding score (BS) was obtained, and patients filled in an extensive questionnaire on the management and burden of their disorder., Findings: We included 1092 patients (834 VWD; 196 RBD; 62 CPD), of whom 665 (60.9%) were women. Women were more often referred because of a bleeding diathesis than men (47.9% vs 36.6%, p  = 0.002). Age of first bleeding was similar between men and women, respectively 8.9 ± 13.6 (mean ±sd) years and 10.6 ± 11.3 years ( p  = 0.075). However, the diagnostic delay, which was defined as time from first bleeding to diagnosis, was longer in women (11.6 ± 16.4 years) than men (7.7 ± 16.6 years, p  = 0.002). Similar results were found when patients referred for bleeding were analyzed separately. Of women aging 12 years or older, 469 (77.1%) had received treatment because of sex-specific bleeding., Interpretation: Women with autosomal inherited bleeding disorders are more often referred for bleeding, have a longer diagnostic delay, and often require treatment because of sex-specific bleeding., Funding: The WiN study was supported (in part) by research funding from the Dutch Hemophilia Foundation (Stichting Haemophilia), Shire (Takeda), and CSL Behring (unrestricted grant)., Competing Interests: F. Atiq received the CSL Behring-professor Heimburger Award 2018 and a travel grant from Sobi. J.L. Saes and S.E.M. Schols received travel support from Bayer, Takeda and Sobi. F.W.G. Leebeek received research support from CSL Behring and Shire/Takeda for performing the Willebrand in the Netherlands (WiN) study, and from UniQure and Sobi for other studies. He is consultant for UniQure, Novo Nordisk, BioMarin and Shire/Takeda, of which the fees go to the institution, and has received a travel grant from Sobi. He is also a DSMB member for a study by Roche. H.C.J. Eikenboom received research support from CSL Behring and he has been a teacher on educational activities of Roche. K.P.M. van Galen received unrestricted research support from CSL Behring and Bayer and speakers fee from Takeda, CSL Behring and Bayer. J.G. van der Bom has been a teacher on educational activities of Bayer. M.H. Cnossen has received grants from governmental research institutes, such as the Dutch Research institute (NOW), ZonMW, Innovation fund, NWO-NWA and unrestricted investigator initiated research grants as well as educational and travel funding from various companies over the years (Pfizer, Baxter/Baxalta/Shire, Bayer Schering Pharma, CSL Behring, Sobi Biogen, novo Nordisk, Novartis and Nordic Pharma), and has served as a member on steering boards of Roche and Bayer. All grants, awards and fees go to the Erasmus MC as an institution. The institution of K. Fijnvandraat has received unrestricted research grants from CSL Behring, Sobi and NovoNordisk and her institution received consultancy fees from Grifols, Takeda, Novo Nordisk and Roche. K. Meijer received research support from Bayer, Sanquin and Pfizer; speaker fees from Bayer, Sanquin, Boehringer Ingelheim, BMS and Aspen; consulting fees from Uniqure, of which all fees go to the institution. B.A.P. Laros‐van Gorkom has received unrestricted educational grants from Baxter and CSL Behring. M.J.H.A. Kruip received grants from governmental research institutes, such as the Dutch Research institute (ZonMW/NWO), Dutch Thrombosis Foundation, Innovation fund, unrestricted grants from Bayer, Pfizer, Daiichi Sankyo, Sobi and Boehringer Ingelheim and speakers fee from Bayer. R.E.G. Schutgens received grants from Bayer, grants from Baxalta, Pfizer, NovoNordisk. The institution of M. Peters received an unrestricted research grant from Pfizer. F.J.M. van der Meer received grants from CSL Behring, Pfizer, Bayer, Novo Nordisk, Sobi, Roche, and OctaPharma. W.L. van Heerde reports speaker and consultant and travel fees from Takeda, Bayer, CSL Behring, and Sobi. He is also founder and co-owner of Enzyre. None of the other authors has a conflict of interest to declare., (© 2021 The Authors.)

Patients with hereditary rare bleeding disorders (RBDs) present with diverse hemorrhagic symptoms. Correlation between factor activity levels and clinical bleeding severity is poor for most RBDs. Threshold factor activity levels have been previously described in relation to bleeding severity but have not yet been validated. The Rare Bleeding Disorders in the Netherlands (RBiN) study is a nationwide cross-sectional study of patients registered in all 6 Dutch Haemophilia Treatment Centers with a known RBD and who are age 1 to 99 years. Bleeding scores were determined, and laboratory and clinical data were extracted from patient files. In all, 263 patients were included, of whom 202 (77%) attended the scheduled study visit. The median International Society of Thrombosis and Haemostasis (ISTH) bleeding assessment tool (BAT) score was 9. Correlations between baseline factor activity levels and ISTH BAT scores were strong for deficiencies in factor II (FII) (r = -0.792) and FX (r = -0.838) and were moderate for deficiencies of fibrinogen (r = -0.683), FV (r = -0.623), FVII (r = -0.516), FXIII (r = -0.516), and α2-antiplasmin (r = -0.594). There was no correlation for FXI deficiency (r = -0.218). The RBD BAT identified more women (94% vs 83%) and children (100% vs 71%) with an RBD than the ISTH BAT did. Importantly, 48% of patients had more severe bleeding than predicted for their baseline factor activity level. In addition, 34% of patients were predicted to be asymptomatic, but they actually had grade 2 (31%) or 3 (3%) bleeding. Bleeding severity in patients with RBDs is more pronounced than previously anticipated. The previously determined threshold factor activity levels to ensure no (spontaneous) bleeding in patients with an RBD are inaccurate. This trial was registered at www.clinicaltrials.gov as #NCT03347591., (© 2020 by The American Society of Hematology.)

A supramolecular approach was undertaken to create functionally activatable cell-penetrating peptides. Two tetra-arginines were assembled into an active cell-penetrating peptide by heterodimerizing leucine zippers. Three different leucine-zipper pairs were evaluated: activation was found to depend on the association constant of the coiled-coil peptides. The weaker-binding peptides required an additional disulfide linkage to induce cell-penetrating capability, whereas for the most-stable coiled-coil no additional stabilization was needed. The latter zipper pair was used to show that the induced formation of the coiled coils allows control over the uptake of an oligoarginine CPP-conjugated cargo protein., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have developed an integrated solution for the site-specific immobilization of proteins on a biosensor surface, which may be widely applicable for high throughput analytical purposes. The gold surface of a biosensor was coated with an anti-fouling layer of zwitterionic peptide molecules from which leucine zipper peptides protrude. Proteins of interest, the autoantigenic proteins La and U1A, were immobilized via a simple incubation procedure by using the complementary leucine zipper sequence as a genetically fused binding tag. This tag forms a strong coiled-coil interaction that is stable during multiple consecutive measurements and under common regeneration conditions. Visualization of the immobilized proteins of interest via antibody binding with multiplex surface plasmon resonance imaging demonstrated 2.5 times higher binding responses than when these proteins were randomly attached to the surface via the commonly applied activated ester-mediated coupling. The proteins could also be immobilized in a leucine zipper-dependent manner directly from complex mixtures like bacterial lysates, eliminating the need for laborious purification steps. This method allows the production of uniform functional protein arrays by control over immobilized protein orientation and geometry and is compatible with high-throughput procedures.