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Background: von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects in von Willebrand factor (VWF). The p.M771V VWF variant leads to a severe bleeding phenotype in homozygous patients. However, the exact molecular mechanism remains unclear, which prevents personalized treatment of those VWD patients., Objectives: This study aimed to characterize the underlying molecular mechanisms of the p.M771V variant in multiple representative ex vivo cell models., Methods: Endothelial colony-forming cells (ECFCs) were isolated from venous blood of VWD patients from the Willebrand in the Netherlands cohort carrying homozygous and heterozygous p.M771V VWF variants. The p.M771V variant was also introduced in cord blood-derived ECFCs (CB-ECFCs) through adenine base editing and was overexpressed in HEK293 cells. Biosynthesis, storage, and secretion of VWF was studied using biochemical methods and confocal microscopy., Results: Two unrelated homozygous p.M771V patients presented with very low VWF activity and antigen levels in plasma. Patient ECFCs showed impaired uncleaved VWF processing into mature VWF, with secreted VWF being severely reduced when compared to ECFCs of healthy donors. Multimer analysis of p.M771V ECFCs showed a deficiency of high molecular weight VWF multimers. Immunofluorescent staining revealed VWF retention in the endoplasmic reticulum; this was confirmed in various populations of base-edited CB-ECFCs harboring the p.M771V variant., Conclusion: The severe endothelial phenotype observed in patient-derived p.M771V ECFCs, HEK293 cells, and an original base-edited CB-ECFC modeling system show that endoplasmic reticulum retention of VWF and failure to undergo subsequent proteolytic processing underpins the severe bleeding phenotype of patients with homozygous variants at M771., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract: Treatment options for the bleeding disorder von Willebrand disease type 2B (VWD2B) are insufficient and fail to address the negative effects of circulating mutant von Willebrand factor (VWF). The dominant-negative nature of VWD2B makes functionally defective VWF an interesting therapeutic target. Previous in vitro studies have demonstrated the feasibility of allele-selective silencing of mutant VWF using small interfering RNAs (siRNAs) targeting common single nucleotide polymorphisms (SNPs) in the human VWF gene, an approach that can be applied irrespective of the disease-causing VWF mutation. This study aims to extend this concept to a heterozygous VWD2B mouse model (c.3946G>A; p.Val1316Met) here using mouse strain-specific genetic differences as proxy for human SNPs. Homozygous VWD2B C57BL/6J (2B-B6) mice were crossed with homozygous wild-type 129S1/SvImJ (129S) mice to create heterozygous 2B-B6.129S F1 offspring. These 2B-B6.129S mice were intravenously injected with endothelial-specific lipid nanoparticles loaded with the allele-selective siVwf.B6 or control and 96 hours later, lung Vwf messenger RNA, plasma VWF levels, and phenotypic characteristics were evaluated. Treatment with siVwf.B6 reduced total VWF levels by 50%, with an expected selective reduction in mutant VWF protein. This coincided with normalization of multimeric structure, improved VWF collagen binding/VWF antigen ratio, and normalized bleeding times in two-thirds of heterozygous 2B-B6.129S mice. Being a novel approach in the field of hemostasis, we proved, for VWD, in mice, the concept of selectively inhibiting a mutant dominant-negative allele with siRNAs targeting a single nucleotide variation rather than the disease-causing mutation. For dominant-negative VWD, this offers potential for a customized therapeutic strategy., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In Immune-mediated Thrombotic Thrombocytopenic Purpura (iTTP), patients develop antibodies against ADAMTS13. The majority of patients exhibit inhibitory anti-spacer antibodies. Non-inhibitory antibodies binding to the carboxy-terminal CUB domains have been suggested to enhance the clearance of ADAMTS13 in iTTP. Furthermore, anti-CUB antibodies induce an open conformation, which has been shown to be an important biomarker for disease severity and relapse risk. We explored whether introduction of N-glycans in the CUB domains of ADAMST13 can reduce the binding of pathogenic anti-CUB autoantibodies. The binding of a panel of iTTP patient derived anti-CUB monoclonal antibodies to newly designed N-glycan modified ADAMTS13 CUB domain variants was assessed by ELISA. Additionally, a subset of these variants was screened against plasma samples of iTTP patients which primarily contain antibodies directed towards the carboxy-terminal domains of ADAMTS13. Introduction of N-glycans at amino acid positions of 1251, 1255 and 1368 in the CUB1/2 domains of ADAMTS13 can effectively reduce binding of 6 out of 7 iTTP patient-derived anti-CUB antibodies. Reduced binding to CUB N-glycan variants was observed in 8 out of 9 patient samples. Binding was decreased from 81% to 47% for NGLY3+CUB-NGLY and 60% to 28% for 5ALA+CUB-NGLY variants. Collectively our findings show that the introduction of N-glycans within the CUB-domain of ADAMTS13 is able to prevent the binding of anti-CUB antibodies in patients with iTTP. Based on these findings, we propose that CUB-NGLY modified ADAMTS13 variants can be used for improved treatment of patients with iTTP., (Copyright © 2025 American Society of Hematology.)

Adeno-associated virus-based gene therapy for hemophilia has emerged as a revolutionary treatment option, offering potential correction of clotting factor deficiency through a single intravenous infusion of functional genes directed to hepatocytes. With 3 gene therapies recently approved, this approach shows promise in transforming the lives of individuals with hemophilia. However, the complexity of gene therapy and the lack of standardization of methods in different components of this therapy can lead to unique challenges for clinical implementation. This manuscript follows literature reviews and structured discussions by the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Working Group on Gene Therapy that identified specific areas requiring standardization of methods, including viral vector production, liver function assessment, quantification of factor (F)VIII and FIX expression levels, assessment of antiadeno-associated viral antibodies, and genomic integration detection methods. Standardization strategies aim to achieve consistent vector quality, effective patient selection, and uniform assessment methods by implementing advanced laboratory techniques and standardized protocols. Standardizing these parameters is essential for improving the understanding of short-term and long-term safety and efficacy of gene therapy in hemophilia. This effort aims to enhance the predictability of individual responses, address variability in outcomes, and ultimately provide more effective, safer, and personalized treatment options for individuals with hemophilia., (© 2024 The Author(s).)

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) poses a major challenge in hemophilia A (HA) treatment. The formation of FVIII inhibitors is a CD4+ T-cell dependent mechanism which includes antigen presenting cells (APCs), B- and T-helper lymphocytes. APCs present FVIII derived peptides on major histocompatibility complex class II (MHC-II) to CD4+ Tcells. We previously established a mass spectrometry based approach to delineate the FVIII repertoire presented on HLA-DR and HLA-DQ. In this study, specific attention was directed towards the identification of FVIII peptides presented on HLA-DP. A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocytes-derived dendritic cells (moDCs) from HLA-typed healthy donors. Using this method, we identified 176 to 1352 different HLA-DP presented peptides per donor, including 26 different FVIII derived peptides. The most frequently presented peptides derived from the A3, and C2 domains of FVIII. Comparison of the FVIII repertoire presented on HLA-DP with that presented on HLA-DR revealed considerable overlap but also suggested preferential presentation of specific peptides on either HLA-DR or HLA-DP. Fourteen FVIII peptides presented on HLA-DP were synthesized and evaluated for their binding ability to the commonly expressed HLA-DP4 molecule which is highly prevalent in the Caucasian population. Peptide binding studies showed that 7 of 14 peptides competed with a reference peptide to HLADP4. Interestingly, an A3 domain derived peptide bound with high affinity to HLA-DP4 positioning this peptide as a prime candidate for the development of novel peptide-based tolerogenic strategies for FVIII inhibitors.

Background: The main complication in hemophilia A treatment is the development of inhibitory antibodies against factor (F)VIII. Immune tolerance induction, the gold standard for eradicating anti-FVIII antibodies, is efficient in only 60% to 80% of cases. This underscores the need for more efficient induction of tolerance in patients with hemophilia A with FVIII inhibitors., Objectives: In this study, we explored whether red blood cells (RBCs) can be utilized as antigen delivery system to modulate the immune response against FVIII., Methods: Two promiscuously HLA-DR-presented peptides derived from the A2 and C1 domains of FVIII were fused to the TAT cell-penetrating peptide and incubated with RBCs., Results: Biotinylated TAT-A2 and TAT-C1 peptides were found to interact with RBCs as shown by flow cytometry and imaging flow cytometry. Moreover, macrophages efficiently phagocytosed TAT-FVIII peptide-treated RBCs. Using mass spectrometry-based immunopeptidomics we established that TAT-FVIII peptides were presented on major histocompatibility complex class II of macrophages that phagocytosed TAT peptide-pulsed RBCs. Specifically, the TAT-A2 peptide exhibited efficient processing and presentation on HLA-DR molecules. Importantly, incubation of TAT-C1 peptide-treated RBCs-loaded macrophages with a FVIII-specific T-cell hybridoma led to a significant increase in IL-2 production, suggesting functional presentation of TAT-C1-derived peptides by macrophages., Conclusion: Our findings indicate that RBCs can serve as effective vehicle for the delivery of FVIII-derived peptides to antigen-presenting cells. The successful display of T-cell epitopes on antigen-presenting cell using ex vivo-loaded RBC may be potentially utilized to modulate pathogenic immune responses such as observed in a subset of patients with hemophilia A., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Inhibitor eradication to restore factor (F)VIII efficacy is the treatment goal for persons with severe hemophilia A (HA) and inhibitors. Immune tolerance induction (ITI) is demanding and successful in about 70% of people. Until now, it has remained difficult to quantify the probability of ITI success or failure, complicating the decision to initiate or not initiate ITI. Estimating the individual chance of ITI success allows clinicians, patients, and their families to support shared decision-making., Objectives: We aimed to identify clinical predictors of ITI success and to develop a clinical prediction model to estimate the chance of successful ITI in persons with severe HA., Methods: This multicenter study included persons with severe HA who received ITI. Clinical data were collected. Successful ITI was defined by a negative inhibitor titer and an adequate response to FVIII concentrates. A multivariable logistic regression model was developed. Model performance and internal validation were performed., Results: Of 206 participants with a median age of 19.8 months (IQR, 12.1-38.8) at ITI start, 148 (71.8%) achieved ITI success. Our clinical prediction model included 4 predictors of ITI success: cumulative number of FVIII exposure days at inhibitor development, peak inhibitor titer, ethnicity, and F8 mutation type. The C statistic was 0.801 (95% CI, 0.70-0.87)., Conclusion: In our study, including 206 people with severe HA and inhibitors, we developed a clinical prediction model to estimate the chance of successful ITI. After future external validation, this clinical prediction model may be useful for informing clinicians and families., (© 2024 The Author(s).)

Independent data collection is crucial in addressing the challenges associated with gene therapy for hemophilia, which is a promising treatment option but requires careful monitoring and management of short-term and potential long-term safety concerns. The International Society on Thrombosis and Haemostasis has identified a minimum efficacy and safety data set included in the World Federation of Hemophilia Gene Therapy Registry that should be collected on a national basis at specific time points for each patient who has been treated with the gene therapy products. This Gene Therapy Minimum Data Set (GT-MDS) was developed to facilitate data collection and to ensure capturing the most relevant data and most known and unknown safety and efficacy parameters recently cited by the European Medicine Agencies. The concept of assembling a minimum data set is not about creating a new data set but rather about identifying a subset of critical and essential topics that should always be included. The GT-MDS is structured into 3 sections and comprises an abridged list of 6 topics during routine gene therapy follow-up, keeping the number of data points low but allowing for rapid and independent data evaluation. The World Federation of Hemophilia Gene Therapy Registry data set, developed by the World Federation of Hemophilia, the International Society on Thrombosis and Haemostasis, and other organizations, including industry partners in 2020, is comprehensive. The GT-MDS reports the minimum relevant information that should not be lost and is mandatory to be collected for all patients who undergo gene therapy. Therefore, the implementation of the gene therapy registry and the minimum data set empowers and enhances data collection at a global level., Competing Interests: Declaration of competing interests W.M.: Bayer, BioMarin, Biotest, CSL Behring, Chugai, Freeline, LFB, Novo Nordisk, Octapharma, Pfizer, Regeneron, Roche, Sanofi, Sigilon, Sobi, Takeda/Shire, uniQure. B.K.: Grant/research support from CSL Behring, Pfizer, Sanofi, Spark Therapeutics, Takeda, and uniQure; Consultant fees from Be Biopharma, BioMarin, Regeneron, Pfizer, Sanofi; Leadership roles: World Federation for Hemophilia, Foundation for Women and Girls with Blood Disorders. P.C.: Bayer, Boehringer Ingelheim, CSL Behring, Chugai, Freeline, NovoNordisk, Pfizer, Roche, Sanofi, Spark, Sobi, and Takeda. R.K.: Grants from Bayer; consulting fees from BioMarin, Pfizer; honoraria for lectures from Pfizer; participation on a data safety monitoring board or advisory board: BioMarin, Pfizer. F.L.: Grant/research support from CSL Behring, Takeda, uniQure, Sobi; consultant fees from uniQure, Sobi, BioMarin; Research support from CLE Behring, Takeda, Sobi, uniQure. J.M.: Grant/research support from BioMarin, Novartis, Novo Nordisk, Pfizer, F. Hoffmann-La Roche Ltd, Sanofi, Spark Therapeutics, and uniQure; consultant fees for BioMarin, Novo Nordisk, F. Hoffmann-La Roche Ltd, Sanofi, Spark Therapeutics and Takeda; Speaker bureau fees from Novo Nordisk, Pfizer, F. Hoffmann-La Roche Ltd, Sanofi, Takeda, WFH, and ISTH. M.M.: Leadership roles: EAHAD council member 2018-2022. ISTH council member 2018-2022. S.W.P.: Consulting fees from Apcintex, ASC Therapeutics, Bayer, BioMarin, CSL Behring, Equilibra Bioscience, HEMA Biologics, Freeline, LFB, Novo Nordisk, Pfizer, Regeneron/Intellia, Roche/Genentech, Sanofi, Takeda, Spark Therapeutics, and uniQure; Scientific Advisory Board member: Equilibra Bioscience, GeneVentiv. G.P.: consultant to ASC Therapeutics, BioMarin, Pfizer, Regeneron, and Spark Therapeutics and on the scientific advisory boards of Be Bio, Frontera, and Metagenomic. A.S.: speaker: Novo Nordisk, Roche, Sanofi, Takeda, Octapharma, and BioMarin; advisory board: Novo Nordisk, Roche, Sanofi, Takeda, Pfizer, BioMarin, and Spark; research grant: Roche, Novo Nordisk, Sanofi, and Pfizer. J.V.: no personal fees. F.P.: consulting fees from CSL Behring, Biomarin, Roche, Sobi, and Sanofi; payment or honoraria for lectures: Takeda and Spark., (Copyright © 2024 International Society on Thrombosis and Haemostasis. All rights reserved.)

Objectives: Anti-factor VIII (FVIII) antibodies have been reported to exhibit both neutralizing and non-neutralizing characteristics. This is the first study investigating the full spectrum of FVIII-specific antibodies, including non-neutralizing antibodies, very-low titer inhibitors, and inhibitors, in a large nationwide population of persons with hemophilia A of all severities., Methods: All persons with hemophilia A (mild (FVIII > 5-40 IU/dL)/moderate [FVIII 1-5 IU/dL)/severe (FVIII < 1 IU/dL)] with an available plasma sample who participated in the sixth Hemophilia in the Netherlands study between 2018 and 2019 were included. The presence of anti-FVIII antibodies of the immunoglobulin A, M, and G isotypes and IgG subclasses, along with antibody titer levels, were assessed using direct-binding ELISAs. FVIII specificity was assessed using a competition-based ELISA approach. The inhibitor status was determined using the Nijmegen ultra-sensitive Bethesda assay (NusBA) and the Nijmegen Bethesda assay (NBA)., Results: In total, 788 persons with hemophilia A (336 (42.6%) mild, 123 (15.6%) moderate, 329 (41.8%) severe hemophilia) were included. The median age was 45 years (IQR 24-60), and the majority (50.9%) had over 150 exposure days to FVIII concentrates. Within our population, 144 (18.3%) individuals had non-neutralizing FVIII-specific antibodies, 10 (1.3%) had very low-titer inhibitors (NusBA positive; NBA negative), and 13 (1.6%) had inhibitors (both NusBA and NBA positive). IgG1 was the most abundant FVIII-specific antibody subclass, and the highest titer levels were found for IgG4. In individuals without a reported history of inhibitor development, no clear differences were observed in antibody patterns between those who were minimally or highly exposed to FVIII concentrates. IgG4 subclass antibodies were only observed in persons with a reported history of FVIII inhibitor or in those with a currently detected (very low-titer) inhibitor., Conclusion: In this cross-sectional study, we identified non-neutralizing antibodies in a relatively large proportion of persons with hemophilia A. In contrast, in our population, consisting of persons highly exposed to FVIII concentrates, (very low-titer) inhibitors were detected only in a small proportion of persons, reflecting a well-tolerized population. Hence, our findings suggest that only a small subpopulation of non-neutralizing FVIII-specific antibodies is associated with clinically relevant inhibitors., Competing Interests: IO was supported by an unrestricted research grant from SOBI, the EAHAD research grant, the Heimburger Award, and the Martin Villar research grant. MM received funding from the European Community H2020-MSCA-ITN-2019 project 859974 EDUC8. SG received unrestricted research funding from SOBI, EAHAD, CSL Behring Heimburger Award, Grifols Martin Villar Award, and the Netherlands Organization for Scientific Research NWO. KF has received unrestricted research grants from CSL Behring, SOBI, and Novo Nordisk and consultancy fees from SOBI, Novo Nordisk, and Roche all fees to the institution. FL has received grants/research funding from CSL Behring, UniQure, SOBI, and Takeda and consultancy fees from Biomarin, CSL Behring, Takeda, and Uniqure all fees to the institution. FL also served as a DSMB member for a study sponsored by Roche. JE received research funding from CSL Behring funds to the institution. MC has received financial support for research from Anthos, Bayer, CSL Behring, Novo Nordisk, and Roche, as well as an honoraria for lecturing or consultancy from Alexion, Bayer, CSL Behring, Daiichi Sankyo, Octapharma, Pfizer, SOBI, and Viatris. Nonfinancial conflict of interest: member of the gene therapy working group of the European Association for Haemophilia and Allied Disorders EAHAD, member of the European Reference Network ERN EuroBloodNet, chair of the working group thrombosis and hemostasis of the Dutch Society of Vascular Medicine NVIVG, part of the Dutch Internist’s Society NIV. JV is listed as an inventor on a patent application on ADAMTS13 variants. SS has received a research grant from Bayer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Oomen, Verhagen, Miranda, Allacher, Beckers, Blijlevens, Bom, Coppens, Driessens, Eikenboom, Fijnvandraat, Hassan, van Heerde, Hooimeijer, Jansen, Kaijen, Leebeek, Meijer, Paul, Rijpma, Rosendaal, Smit, van Vulpen, Voorberg, Schols and Gouw.)

Neutrophil released peptidyl arginine deiminase 4 (PAD4) converts arginine residues on plasma proteins into citrulline. Here, we developed an assay to quantify citrullinated fibrinogen. We employed a biotin-conjugated phenylglyoxal (biotin-phenylglyoxal (PG)) compound that selectively labels citrulline. Patient samples were derived from a multicenter prospective cohort study that aimed to identify cancer patients at high risk for venous thromboembolism (VTE). Our data show that cancer patients have higher (median 2-fold increased) citrullinated fibrinogen levels when compared to normal human plasma and a cohort of healthy donors. Our results show that citrullination of fibrinogen is a common posttranslational modification in patients with cancer., Competing Interests: None of the authors have conflict of interest to report., (© 2023 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

Background: Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic disorder, with 1.5 to 6.0 cases per million per year. The majority of patients with TTP develop inhibitory autoantibodies that predominantly target the spacer domain of ADAMTS-13. ADAMTS-13 is responsible for cleaving von Willebrand factor (VWF) multimers, thereby regulating platelet adhesion at sites of high-vascular shear stress. Inhibition and/or clearance of ADAMTS-13 by pathogenic autoantibodies results in accumulation of VWF multimers that promotes the formation of platelet-rich microthrombi. Previously, we have shown that insertion of a single N-glycan (NGLY) in the spacer domain prevents the binding of antispacer domain antibodies., Objectives: To explore whether NGLY mediated shielding of the ADAMTS-13 spacer domain effectively prevents binding of pathogenic antispacer autoantibodies in patients with immune-mediated TTP (iTTP)., Methods: We screened 5 NGLY-ADAMTS-13 variants (NGLY3, NGLY7, NGLY8, NGLY3+7, and NGLY3+8) for binding of autoantibodies and for their activity in the presence and absence of 50 samples derived from patients with iTTP., Results: NGLY variants showed greatly reduced antibody binding, down to 27% of wild-type (wt) ADAMTS-13 binding. Moreover, NGLY variants of ADAMTS-13 remained more active in FRETS-VWF73 assay in the presence of the plasma samples from these 50 patients with acute phase iTTP when compared with wtADAMTS-13. On average, wtADAMTS-13 activity was reduced to 37% of regular levels in the presence of plasma, while NGLY3 and NGLY3+7 remained 69% and 81% active, respectively., Conclusion: These results reinforce our previous findings that NGLYs shield ADAMTS-13 from antibody binding and hence restore ADAMTS-13 activity in the presence of autoantibodies., Competing Interests: Declaration of competing interests N.A.G.G., B.E., and J.V. are listed as inventors on a patent application for NGLY modified ADAMTS-13. At the time of the study N.A.G.G., J.F.d.S., and B.M.L. were employed by Sanquinnovate, which is fully owned by Sanquin Blood Supply Foundation, a not-for-profit organisation. K.V. is a member of the advisory board of Takeda. P.C. is a member of the clinical advisory board for Alexion, Sanofi, Shire, Takeda, and Janssen. A.V. is a member of the advisory boards for Sanofi, Takeda, and LFB biomedicaments. The other authors have no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Immune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against class I human leukocyte antigens (HLA). Opsonization of donor platelets with these alloantibodies can result in rapid clearance after transfusion via multiple mechanisms, including antibody dependent cellular phagocytosis (ADCP). Interestingly, not all alloimmunized patients develop PR to unmatched platelet transfusions, suggesting variation in HLA-specific IgG responses between patients. Previously, we observed that the glycosylation profile of anti-HLA antibodies was highly variable between PR patients, especially with respect to Fc galactosylation, sialylation and fucosylation. In the current study, we investigated the effect of different Fc glycosylation patterns, with known effects on complement deposition and FcγR binding, on phagocytosis of opsonized platelets by monocyte-derived human macrophages. We found that the phagocytosis of antibody- and complement-opsonized platelets, by monocyte derived M1 macrophages, was unaffected by these qualitative IgG-glycan differences.

Background: Von Willebrand factor (VWF) and VWF propeptide (VWFpp) are stored in eccentric nanodomains within platelet alpha-granules. VWF and VWFpp can undergo differential secretion following Weibel-Palade body exocytosis in endothelial cells; however, it is unclear if the same process occurs during platelet alpha-granule exocytosis. Using a high-throughput 3-dimensional super-resolution imaging workflow for quantification of individual platelet alpha-granule cargo, we studied alpha-granule cargo release in response to different physiological stimuli., Objectives: To investigate how VWF and VWFpp are released from alpha-granules in response to physiological stimuli., Methods: Platelets were activated with protease-activated receptor 1 (PAR-1) activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, secreted protein acidic and cysteine rich (SPARC), and fibrinogen were imaged using 3-dimensional structured illumination microscopy, followed by semiautomated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content., Results: VWFpp overlapped with VWF in eccentric alpha-granule subdomains in resting platelets and showed a higher degree of overlap with VWF than SPARC or fibrinogen. Activation of PAR-1 (0.6-20 μM PAR-1 ap) or glycoprotein VI (GPVI) (0.25-1 μg/mL CRP-XL) signaling pathways caused a dose-dependent increase in alpha-granule exocytosis. More than 80% of alpha-granules remained positive for VWF, even at the highest agonist concentrations. In contrast, the residual fraction of alpha-granules containing VWFpp decreased in a dose-dependent manner to 23%, whereas SPARC and fibrinogen were detected in 60% to 70% of alpha-granules when stimulated with 20 μM PAR-1 ap. Similar results were obtained using CRP-XL. Using an extracellular anti-VWF nanobody, we identified VWF in postexocytotic alpha-granules., Conclusion: We provide evidence for differential secretion of VWF and VWFpp from individual alpha-granules., Competing Interests: Declaration of competing interests F.W.G.L. received research support from CSL Behring, Takeda, uniQure, and Sobi and is a consultant for uniQure, Biomarin, CSL Behring, and Takeda, of which the fees go to the institute. He was a DSMB member for a study sponsored by Roche. A.J.G.J. received speaker fees and travel cost payments from 3SBio, Amgen, and Novartis, is on the international advisory board at Novartis, and received research support from CSL Behring, Principia, and Argenx. None of the other authors have conflicts of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Patients with gray platelet syndrome (GPS) and Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles and store the hemostatic protein von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are lysosome-related organelles of endothelial cells and also store VWF and P-selectin. In megakaryocytes, NBEAL2 links P-selectin on AGs to the SNARE protein SEC22B on the endoplasmic reticulum, thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from the endoplasmic reticulum to Golgi and promotes the formation of elongated WPBs, but it is unclear whether this requires NBEAL2., Objectives: To investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2-deficient endothelial cells., Methods: The interaction of SEC22B with NBEAL2 in endothelial cells was investigated by interatomic mass spectrometry and pull-down analysis. Endothelial colony forming cells were isolated from healthy controls and 3 unrelated patients with GPS and mutations in NBEAL2 ., Results: We showed that SEC22B binds to NBEAL2 in ECs. Endothelial colony forming cells derived from a patient with GPS are deficient in NBEAL2 but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion is altered in the absence of NBEAL2., Conclusions: Although NBEAL2 deficiency causes the absence of AGs in patients with GPS, it does not impact WPB functionality in ECs. Our data highlight the differences in the regulatory mechanisms between these 2 hemostatic storage compartments., (© 2023 The Author(s).)

Background: Immune tolerance induction (ITI) aims to eradicate anti-factor VIII (FVIII) antibodies (inhibitors) in persons with hemophilia A. However, this burdensome treatment fails in 10% to 40%. To estimate the chance of ITI success in clinical decision making, it is important to identify the predictors of ITI success., Objectives: We performed a systematic review and meta-analysis to summarize the current evidence on determinants of ITI outcome in persons with hemophilia A., Methods: A literature search was conducted to identify randomized controlled trials, cohort, or case-control studies reporting on the predictors for ITI outcome in persons with hemophilia A. The main outcome was ITI success. Methodological quality was assessed using an adapted Joanna Briggs Institute checklist, rating as high if ≥11 of 13 criteria were met. Pooled odds ratios (ORs) for ITI success were calculated for each determinant. ITI success was defined as negative inhibitor titer (<0.6 BU/mL), FVIII recovery ≥66% of expected, and FVIII half-life ≥6 hours in 16 (59.3%) studies., Results: We included 27 studies, involving 1,734 participants. Methodological quality of 6 (22.2%) studies (418 participants) was rated as high. Twenty different determinants were assessed. Historical peak titer ≤100 BU/mL (compared with >100 BU/mL, OR, 1.7; 95% CI, 1.4-2.1), pre-ITI titer ≤10 BU/mL (compared with >10 BU/mL, OR, 1.8; 95% CI, 1.4-2.3), and peak titer during ITI ≤100 BU/mL (compared with >100 BU/mL, OR, 2.7; 95% CI, 1.9-3.8) were associated with a higher chance of ITI success., Conclusion: Our results suggest that determinants related to the inhibitor titer are associated with ITI success., (© 2022 The Author(s).)

Background: The formation of alloantibodies directed against class I human leukocyte antigens (HLA) continues to be a clinically challenging complication after platelet transfusions, which can lead to platelet refractoriness (PR) and occurs in approximately 5%-15% of patients with chronic platelet support. Interestingly, anti-HLA IgG levels in alloimmunized patients do not seem to predict PR, suggesting functional or qualitative differences among anti-HLA IgG. The binding of these alloantibodies to donor platelets can result in rapid clearance after transfusion, presumably via FcγR-mediated phagocytosis and/or complement activation, which both are affected by the IgG-Fc glycosylation., Objectives: To characterize the Fc glycosylation profile of anti-HLA class I antibodies formed after platelet transfusion and to investigate its effect on clinical outcome., Patients/methods: We screened and captured anti-HLA class I antibodies (anti-HLA A2, anti-HLA A24, and anti-HLA B7) developed after platelet transfusions in hemato-oncology patients, who were included in the PREPAReS Trial. Using liquid chromatography-mass spectrometry, we analyzed the glycosylation profiles of total and anti-HLA IgG1 developed over time. Subsequently, the glycosylation data was linked to the patients' clinical information and posttransfusion increments., Results: The glycosylation profile of anti-HLA antibodies was highly variable between patients. In general, Fc galactosylation and sialylation levels were elevated compared to total plasma IgG, which correlated negatively with the platelet count increment. Furthermore, high levels of afucosylation were observed for two patients., Conclusions: These differences in composition of anti-HLA Fc-glycosylation profiles could potentially explain the variation in clinical severity between patients., (© 2022 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Von Willebrand factor (VWF) is a glycoprotein that is secreted into the circulation and controls bleeding by promoting adhesion and aggregation of blood platelets at sites of vascular injury. Substantial inter-individual variation in VWF plasma levels exists among the healthy population. Prior to secretion, VWF polymers are assembled and condensed into helical tubules, which are packaged into Weibel-Palade bodies (WPBs), a highly specialized post-Golgi storage compartment in vascular endothelial cells. In the inherited bleeding disorder Von Willebrand disease (VWD), mutations in the VWF gene can cause qualitative or quantitative defects, limiting protein function, secretion, or plasma survival. However, pathogenic VWF mutations cannot be found in all VWD cases. Although an increasing number of genetic modifiers have been identified, even more rare genetic variants that impact VWF plasma levels likely remain to be discovered. Here, we summarize recent evidence that modulation of the early secretory pathway has great impact on the biogenesis and release of WPBs. Based on these findings, we propose that rare, as yet unidentified quantitative trait loci influencing intracellular VWF transport contribute to highly variable VWF levels in the population. These may underlie the thrombotic complications linked to high VWF levels, as well as the bleeding tendency in individuals with low VWF levels., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Approximately 20% of patients receiving multiple platelet transfusions develop platelet alloantibodies, which can be directed against human leukocyte antigens (HLA) and, to a lesser extent, against human platelet antigens (HPA). These antibodies can lead to the rapid clearance of donor platelets, presumably through IgG-Fc receptor (FcγR)-mediated phagocytosis or via complement activation, resulting in platelet refractoriness. Strikingly, not all patients with anti-HLA or -HPA antibodies develop platelet refractoriness upon unmatched platelet transfusions. Previously, we found that IgG Fc glycosylation of anti-HLA antibodies was highly variable between patients with platelet refractoriness, especially with respect to galactosylation and sialylation of the Fc-bound sugar moiety. Here, we produced recombinant glycoengineered anti-HLA and anti- HPA-1a monoclonal antibodies with varying Fc galactosylation and sialylation levels and studied their ability to activate the classical complement pathway. We observed that anti-HLA monoclonal antibodies with different specificities, binding simultaneously to the same HLA-molecules, or anti-HLA in combination with anti-HPA-1a monoclonal antibodies interacted synergistically with C1q, the first component of the classical pathway. Elevated Fc galactosylation and, to a lesser extent, sialylation significantly increased the complement-activating properties of anti-HLA and anti-HPA-1a monoclonal antibodies. We propose that both the breadth of the polyclonal immune response, with recognition of different HLA epitopes and in some cases HPA antigens, and the type of Fc glycosylation can provide an optimal stoichiometry for C1q binding and subsequent complement activation. These factors can shift the effect of a platelet alloimmune response to a clinically relevant response, leading to complement-mediated clearance of donor platelets, as observed in platelet refractoriness.

Background: Recombinant human ADAMTS13 (rADAMTS13) is a key protein in fundamental research for investigating its mode of action and the pathophysiology of thrombotic thrombocytopenic purpura (TTP). However, the expression of rADAMTS13 is quite low in mammalian cells, which makes the production of the protein time-consuming and labor-intensive., Objectives: We aimed at increasing the yield of rADAMTS13 by (1) using a more optimal signal peptide (SP) and (2) constructing an N-terminal fusion protein of ADAMTS13 with human serum albumin domain 1 (AD1-ADAMTS13)., Methods: Six SPs were investigated to select the most optimal SP. Expression plasmids containing the most optimal SP and ADAMTS13 cDNA or the fusion construct AD1-ADAMTS13 were generated and transiently transfected into CHOEBNALT85 cell-line. Expression levels of rADAMTS13 in expression medium were analyzed and compared with the expression level of rADAMTS13 with native SP (nat-SP)., Results: Expression of rADAMTS13 with coagulation factor VII (FVII) SP was 3-fold higher (16.00 μg/ml) compared with the expression with nat-SP (5.03 μg/ml). The highest yields were obtained with AD1-ADAMTS13 protein with a 15-fold higher concentration (78.22 μg/ml) compared with the expression with nat-SP. The rADAMTS13 expressed with FVII-SP retained its activity (104.0%) to cleave von Willebrand factor, whereas AD1-ADAMTS13 demonstrated even higher activity (144.3%)., Conclusion: We succeeded in generating expression vectors that yield (1) rADAMTS13 at higher levels because of more optimal FVII-SP and (2) high levels of AD1-ADAMTS13 N-terminal fusion protein. The highest expression levels were obtained with AD1-ADAMTS13 N-terminal fusion protein, which is paving the way for highly efficient protein production., (© 2022 International Society on Thrombosis and Haemostasis.)

Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells. VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPB), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde endoplasmic reticulum-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in endothelial cells using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted endothelial cells exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. In brief, we identified SNARE protein STX5 as a novel regulator of WPB biogenesis.

Background: Treatment choices for individual patients with an inborn bleeding disorder are increasingly challenging due to increasing options and rising costs for society. We have initiated an integrated interdisciplinary national research programme., Objectives: The SYMPHONY consortium strives to orchestrate personalized treatment in patients with an inborn bleeding disorder, by unravelling the mechanisms behind inter-individual variations of bleeding phenotype., Patients: The SYMPHONY consortium will investigate patients with an inborn bleeding disorder, both diagnosed and not yet diagnosed., Results: Research questions are categorized under the themes: 1) Diagnosis; 2) Treatment; and 3) Fundamental research and consist of workpackages addressing specific domains. Importantly, collaborations between patients and talented researchers from different areas of expertise promise to augment the impact of the SYMPHONY consortium, leading to unique interactions and intellectual property., Conclusions: SYMPHONY will perform research on all aspects of care, treatment individualization in patients with inborn bleeding disorders as well as diagnostic innovations and results of molecular genetics and cellular model technology with regard to the hemostatic process. We believe that these research investments will lead to health care innovations with long-term clinical and societal impact. This consortium has been made possible by a governmental, competitive grant from the Netherlands Organization for Scientific Research (NWO) within the framework of the NWA-ORC Call grant agreement NWA.1160.18.038., (This article is protected by copyright. All rights reserved.)

Von Willebrand disease (VWD) is a bleeding disorder caused by quantitative (type 1 or 3) or qualitative (type 2A/2B/2M/2N) defects of circulating von Willebrand factor (VWF). Circulating VWF levels not always fully explain bleeding phenotypes, suggesting a role for alternative factors, like platelets. Here, we investigated platelet factor 4 (PF4) in a large cohort of patients with VWD. PF4 levels were lower in type 2B and current bleeding phenotype was significantly associated with higher PF4 levels, particularly in type 1 VWD. Based on our findings we speculate that platelet degranulation and cargo release may play a role across VWD subtypes., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Rationale: von Willebrand factor (vWF) mediates platelet adhesion during thrombosis. While chronic thromboembolic pulmonary hypertension (CTEPH) is associated with increased plasma levels of vWF, the role of this protein in CTEPH has remained enigmatic. Objectives: To identify the role of vWF in CTEPH. Methods: CTEPH-specific patient plasma and pulmonary endarterectomy material from patients with CTEPH were used to study the relationship between inflammation, vWF expression, and pulmonary thrombosis. Cell culture findings were validated in human tissue, and proteomics and chromatin immunoprecipitation were used to investigate the underlying mechanism of CTEPH. Measurements and Main Results: vWF is increased in plasma and the pulmonary endothelium of CTEPH patients. In vitro , the increase in vWF gene expression and the higher release of vWF protein upon endothelial activation resulted in elevated platelet adhesion to CTEPH endothelium. Proteomic analysis revealed that nuclear factor (NF)-κB2 was significantly increased in CTEPH. We demonstrate reduced histone tri-methylation and increased histone acetylation of the vWF promoter in CTEPH endothelium, facilitating binding of NF-κB2 to the vWF promoter and driving vWF transcription. Genetic interference of NFκB2 normalized the high vWF RNA expression levels and reversed the prothrombotic phenotype observed in CTEPH-pulmonary artery endothelial cells. Conclusions: Epigenetic regulation of the vWF promoter contributes to the creation of a local environment that favors in situ thrombosis in the pulmonary arteries. It reveals a direct molecular link between inflammatory pathways and platelet adhesion in the pulmonary vascular wall, emphasizing a possible role of in situ thrombosis in the development or progression of CTEPH.

The 100th anniversary of the first description of Thrombotic Thrombocytopenic Purpura (TTP) as a disease by Dr. Eli Moschcowitz approaches. For many decades, TTP remained mostly a mysterious fatal condition, where diagnosis was often post-mortem. Initially a pentad of symptoms was identified, a pattern that later revealed to be fallible. Sporadic observations led to empiric interventions that allowed for the first impactful breakthrough in TTP treatment, almost 70 years after its first description: the introduction of plasma exchange and infusions as treatments. The main body of knowledge within the field was gathered in the latest three decades: patient registries were set and proved crucial for advancements; the general mechanisms of disease have been described; the diagnosis was refined; new treatments and biomarkers with improvements on prognosis and management were introduced. Further changes and improvements are expected in the upcoming decades. In this review, we provide a brief historic overview of TTP, as an illustrative example of the success of translational medicine enabling to rapidly shift from a management largely based on empiricism to targeted therapies and personalized medicine, for the benefit of patients. Current management options and present and future perspectives in this still evolving field are summarized., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26-34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.

von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B, and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. In this study, we identified MAP kinase-activating death domain (MADD) as the guanine nucleotide exchange factor for Rab27A and both Rab3 isoforms in primary human endothelial cells. Rab activity assays revealed a reduction in Rab27A, Rab3B, and Rab3D activation upon MADD silencing. Rab activation, but not binding, was dependent on the differentially expressed in normal and neoplastic cells (DENN) domain of MADD, indicating the potential existence of 2 Rab interaction modules. Furthermore, immunofluorescent analysis showed that Rab27A, Rab3B, and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting. Artificial mistargeting of MADD using a TOMM70 tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through previous activation of Rab27A by MADD. MADD silencing did not affect WPB morphology, but it did reduce VWF intracellular content. Furthermore, MADD-depleted cells exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. In conclusion, MADD acts as a master regulator of VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Immune thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder of which the etiology is not fully understood. Autoantibodies targeting ADAMTS13 in iTTP patients have extensively been studied, the immunological mechanisms leading to the breach of tolerance remain to be uncovered. This review addresses the current knowledge on genetic factors associated with the development of iTTP and the interplay between the patient's immune system and environmental factors in the induction of autoimmunity against ADAMTS13. HLA-DRB1*11 has been identified as a risk factor for iTTP in the Caucasian population. Interestingly, HLA-DRB1*08:03 was recently identified as a risk factor in the Japanese population. Combined in vitro and in silico MHC class II peptide presentation approaches suggest that an ADAMTS13-derived peptide may bind to both HLA-DRB1*11 and HLA-DRB1*08:03 through different anchor-residues. It is apparent that iTTP is associated with the presence of infectious microorganisms, viruses being the most widely associated with development of iTTP. Infections may potentially lead to loss of tolerance resulting in the shift from immune homeostasis to autoimmunity. In the model we propose in this review, infections disrupt the epithelial barriers in the gut or lung, promoting exposure of antigen presenting cells in the mucosa-associated lymphoid tissue to the microorganisms. This may result in breach of tolerance through the presentation of microorganism-derived peptides that are homologous to ADAMTS13 on risk alleles for iTTP., Competing Interests: NG and JV are inventors on a patent-application describing novel compounds for the treatment of immune TTP. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Laghmouchi, Graça and Voorberg.)

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) is a multidomain metalloprotease for which until now only a single substrate has been identified. ADAMTS13 cleaves the polymeric force-sensor von Willebrand factor (VWF) that unfolds under shear stress and recruits platelets to sites of vascular injury. Shear force-dependent cleavage at a single Tyr-Met peptide bond in the unfolded VWF A2 domain serves to reduce the size of VWF polymers in circulation. In patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), a rare life-threatening disease, ADAMTS13 is targeted by autoantibodies that inhibit its activity or promote its clearance. In the absence of ADAMTS13, VWF polymers are not adequately processed, resulting in spontaneous adhesion of blood platelets, which presents as severe, life-threatening microvascular thrombosis. In healthy individuals, ADAMTS13-VWF interactions are guided by controlled conversion of ADAMTS13 from a closed, inactive to an open, active conformation through a series of interdomain contacts that are now beginning to be defined. Recently, it has been shown that ADAMTS13 adopts an open conformation in the acute phase and during subclinical disease in iTTP patients, making open ADAMTS13 a novel biomarker for iTTP. In this review, we summarize our current knowledge on ADAMTS13 conformation and speculate on potential triggers inducing conformational changes of ADAMTS13 and how these relate to the pathogenesis of iTTP., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Platelets play a key role in hemostasis through plug formation and secretion of their granule contents at sites of endothelial injury. Defects in von Willebrand factor (VWF), a platelet α-granule protein, are implicated in von Willebrand disease (VWD), and may lead to defective platelet adhesion and/or aggregation. Studying VWF quantity and subcellular localization may help us better understand the pathophysiology of VWD., Objective: Quantitative analysis of the platelet α-granule compartment and VWF storage in healthy individuals and VWD patients., Patients/methods: Structured illumination microscopy (SIM) was used to study VWF content and organization in platelets of healthy individuals and patients with VWD in combination with established techniques., Results: SIM capably quantified clear morphological and granular changes in platelets stimulated with proteinase-activated receptor 1 (PAR-1) activating peptide and revealed a large intra- and interdonor variability in VWF-positive object numbers within healthy resting platelets, similar to variation in secreted protein acidic and rich in cysteine (SPARC). We subsequently characterized VWD platelets to identify changes in the α-granule compartment of patients with different VWF defects, and were able to stratify two patients with type 3 VWD rising from different pathological mechanisms. We further analyzed VWF storage in α-granules of a patient with homozygous p .C1190R using electron microscopy and found discrepant VWF levels and different degrees of multimerization in platelets of patients with heterozygous p .C1190 in comparison to VWF in plasma., Conclusions: Our findings highlight the utility of quantitative imaging approaches in assessing platelet granule content, which may help to better understand VWF storage in α-granules and to gain new insights in the etiology of VWD., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Trauma-induced organ failure is characterized by endothelial dysfunction. The aim of this study was to investigate the role of von Willebrand factor (VWF) and its cleaving enzyme, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13) in the occurrence of endothelial permeability and organ failure in trauma. In an observational study in a level-1 trauma center, 169 adult trauma patients with clinical signs of shock and/or severe injuries were included. Trauma was associated with low ADAMTS13 and high VWF antigen levels, thus generating an imbalance of ADAMTS13 to VWF. Patients who developed organ failure (23%) had greater ADAMTS13-to-VWF imbalances, persistently lower platelet counts, and elevated levels of high-molecular-weight VWF multimers compared with those without organ failure, suggesting microthrombi formation. To investigate the effect of replenishing low ADAMTS13 levels on endothelial permeability and organ failure using either recombinant human ADAMTS13 (rhADAMTS13) or plasma transfusion, a rat model of trauma-induced shock and transfusion was used. Rats in traumatic hemorrhagic shock were randomized to receive crystalloids, crystalloids supplemented with rhADAMTS13, or plasma transfusion. A 70-kDa fluorescein isothiocyanate-labeled dextran was injected to determine endothelial leakage. Additionally, organs were histologically assessed. Both plasma transfusion and rhADAMTS13 were associated with a reduction in pulmonary endothelial permeability and organ injury when compared with resuscitation with crystalloids, but only rhADAMTS13 resulted in significant improvement of a trauma-induced decline in ADAMTS13 levels. We conclude that rhADAMTS13 and plasma transfusion can reduce organ failure following trauma. These findings implicate the ADAMTS13-VWF axis in the pathogenesis of organ failure., (© 2021 by The American Society of Hematology.)

Anti-A Disintegrin and Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) autoantibodies cause a severe ADAMTS13 deficiency in immune-mediated thrombotic thrombocytopenic purpura (iTTP). ADAMTS13 consists of a metalloprotease (M), a disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). We recently developed a high-throughput epitope mapping assay based on small, nonoverlapping ADAMTS13 fragments (M, DT, CS, T2-T5, T6-T8, CUB1-2). With this assay, we performed a comprehensive epitope mapping using 131 acute-phase samples and for the first time a large group of remission samples (n = 50). Next, samples were stratified according to their immunoprofiles, a field that is largely unexplored in iTTP. Three dominant immunoprofiles were found in acute-phase samples: profile 1: only anti-CS autoantibodies (26.7%); profile 2: both anti-CS and anti-CUB1-2 autoantibodies (12.2%); and profile 3: anti-DT, anti-CS, anti-T2-T5, anti-T6-T8, and anti-CUB1-2 autoantibodies (8.4%). Interestingly, profile 1 was the only dominant immunoprofile in remission samples (52.0%). Clinical data were available for a relatively small number of patients with acute iTTP (>68), and no correlation was found between immunoprofiles and disease severity. Nevertheless, profile 1 was linked with younger and anti-T2-T5 autoantibodies with older age and the absence of anti-CUB1-2 autoantibodies with cerebral involvement. In conclusion, identifying acute phase and remission immunoprofiles in iTTP revealed that anti-CS autoantibodies seem to persist or reappear during remission providing further support for the clinical development of a targeted anti-CS autoantibody therapy. A large cohort study with acute iTTP samples will validate possible links between immunoprofiles or anti-domain autoantibodies and clinical data., (© 2021 by The American Society of Hematology.)

Background: Peptidyl arginine deiminase 4 (PAD4) is an enzyme that converts arginine into citrulline. PAD4 is expressed in neutrophils that, when activated, can drive the formation of neutrophil extracellular traps (NETs). Uncontrolled activation of PAD4 and subsequent citrullination of proteins is increasingly recognized as a driver of (auto)immune diseases. Currently, our understanding of PAD4 structure-function relationships and activity control in vivo is incomplete., Aims: To provide the current state-of-the-art on PAD4 structure-activity relationships and involvement of PAD4 in autoimmune disorders as well as in thrombo-inflammatory disease., Materials & Methods: Literature review and molecular modelling Results: In this review, we used molecular modelling to generate a three-dimensional structure of the complete PAD4 molecule. Using our model, we discuss the catalytic conversion of the arginine substrate to citrulline. Besides mechanistic insight into PAD4 function, we give an overview of biological functions of PAD4 and mechanisms that influence its activation. In addition, we discuss the crucial role of PAD4-mediated citrullination of histones during the formation of NETs. Subsequently, we focus on the role of PAD4-mediated NET formation and its role in pathogenesis of rheumatoid arthritis, sepsis and (immune-)thrombosis. Finally, we summarize current efforts to design different classes of PAD4 inhibitors that are being developed for improved treatment of autoimmune disorders as well as thrombo-inflammatory disease., Discussion: Advances in PAD4 structure-function are still necessary to gain a complete insight in mechanisms that control PAD4 activity in vivo. The involvement of PAD4 in several diseases signifies the need for a PAD4 inhibitor. Although progress has been made to produce an isotype specific and potent PAD4 inhibitor, currently no PAD4 inhibitor is ready for clinical use., Conclusion: More research into PAD4 structure and function and into the regulation of its activity is required for the development of PAD4 specific inhibitors that may prove vital to combat and prevent autoimmune disorders and (thrombo)inflammatory disease., (© 2021 School for Cardiovascular Diseases Maastricht University. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Induced pluripotent stem cells (iPSCs) were generated from blood outgrowth endothelial cells (BOECs) obtained from a healthy donor and from a patient diagnosed with Hermansky Pudlak Syndrome type 2 (HPS2), caused by compound heterozygous AP3B1 mutations (c.177delA and c.1839-1842delTAGA). BOECs were reprogrammed with a hOKSM self-silencing polycistronic lentiviral vector, where the generated iPSCs showed normal karyotype, expression of pluripotency associated markers and in vitro spontaneous differentiation towards the three germ layers. The generated iPSCs can be used to study HPS2 pathophysiology and the basic functions of AP3B1 protein in different cell types., (Copyright © 2021. Published by Elsevier B.V.)

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by the development of autoantibodies targeting different domains of ADAMTS13. Profiling studies have shown that residues R568, F592, R660, Y661, and Y665 within exosite-3 of the spacer domain provide an immunodominant region of ADAMTS13 for pathogenic autoantibodies that develop in patients with iTTP. Modification of these 5 core residues with the goal of reducing autoantibody binding revealed a significant tradeoff between autoantibody resistance and proteolytic activity. Here, we employed structural bioinformatics to identify a larger epitope landscape on the ADAMTS13 spacer domain. Models of spacer-antibody complexes predicted that residues R568, L591, F592, K608, M609, R636, L637, R639, R660, Y661, Y665, and L668 contribute to an expanded epitope within the spacer domain. Based on bioinformatics-guided predictions, we designed a panel of N-glycan insertions in this expanded epitope to reduce the binding of spacer domain autoantibodies. One N-glycan variant (NGLY3-ADAMTS13, containing a K608N substitution) showed strongly reduced reactivity with TTP patient sera (28%) as compared with WT-ADAMTS13 (100%). Insertion of an N-glycan at amino acid position 608 did not interfere with processing of von Willebrand factor, positioning the resulting NGLY3-ADAMTS13 variant as a potential novel therapeutic option for treatment of iTTP., (© 2021 by The American Society of Hematology.)

The main complication of hemophilia A treatment is the development of neutralizing antibodies (inhibitors) against factor VIII (FVIII). Immune tolerance induction (ITI) is the prescribed treatment for inhibitor eradication, although its working mechanism remains unresolved. To clarify this mechanism, we compared blood samples of hemophilia A patients with and without inhibitors for presence of immunoregulatory cells and markers, including regulatory B-cells (Bregs), regulatory T-cells (Tregs), myeloid-derived suppressor cells (MDSCs), and expression of regulatory markers on T-cells (programmed cell death protein 1 [PD1], inducable T-cell costimulator, cytotoxic T-lymphocyte-associated protein 4 [CTLA4]), by use of flow cytometry. By cross-sectional analysis inhibitor patients (N = 20) were compared with inhibitor-negative (N = 28) and ex-inhibitor (N = 17) patients. In another longitudinal study, changes in immunoregulatory parameters were evaluated during ITI (N = 12) and compared with inhibitor-negative hemophilia A patients (N = 36). The frequency of Bregs, but not of Tregs nor MDSCs, was significantly reduced in inhibitor patients (3.2%) compared with inhibitor-negative (5.9%) and ex-inhibitor patients (8.9%; P < 0.01). CTLA4 expression on T-cells was also reduced (mean fluorescence intensity 133 in inhibitor versus 537 in inhibitor-negative patients; P < 0.01). Fittingly, in patients followed during ITI, inhibitor eradication associated with increased Bregs, increased Tregs, and increased expression of CTLA4 and PD1 on CD4+ T-cells. In conclusion, inhibitor patients express significantly lower frequency of Bregs and Tregs marker expression, which are restored by successful ITI. Our findings suggest that an existing anti-FVIII immune response is associated with deficits in peripheral tolerance mechanisms and that Bregs and changes in immunoregulatory properties of CD4+ T-cells likely contribute to ITI in hemophilia A patients with inhibitors., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles, so-called Weibel-Palade bodies (WPBs). Hemostatic activity of VWF is strongly tied to WPB length, but how endothelial cells control the dimensions of their WPBs is unclear. In this study we used a targeted shRNA screen to identify the longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology along with disintegration of the Golgi and dilation of rough ER (rER) cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rER and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPBs, and thus adhesive activity of its cargo VWF, is determined by the rate of anterograde transport between ER and Golgi, which depends on Sec22b-containing SNARE complexes.

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is caused by anti-ADAMTS13 autoantibodies inducing a severe deficiency of ADAMTS13. Epitope mapping studies on samples obtained during acute iTTP episodes have shown that the iTTP immune response is polyclonal, with almost all patients having autoantibodies targeting the spacer domain of ADAMTS13., Objectives: To identify the immunogenic hotspots in the spacer domain of ADAMTS13., Patients/methods: A library of 11 full-length ADAMTS13 spacer hybrids was created in which amino acid regions of the spacer domain of ADAMTS13 were exchanged by the corresponding region of the spacer domain of ADAMTS1. Next, the full-length ADAMTS13 spacer hybrids were used in enzyme-linked immunosorbent assay to epitope map anti-spacer autoantibodies in 138 samples from acute and remission iTTP patients., Results: Sixteen different anti-spacer autoantibody profiles were identified with a similar distribution in acute and remission patients. There was no association between the anti-spacer autoantibody profiles and disease severity. Almost all iTTP samples contained anti-spacer autoantibodies against the following three regions: amino acid residues 588-592, 602-610, and 657-666 (hybrids E, G, and M). Between 31% and 57% of the samples had anti-spacer autoantibodies against amino acid regions 572-579, 629-638, 667-676 (hybrids C, J, and N). In contrast, none of the samples had anti-spacer autoantibodies against amino acid regions 556-563, 564-571, 649-656, and 677-685 (hybrids A, B, L, and O)., Conclusion: We identified three hotspot regions (amino acid regions 588-592, 602-610, and 657-666) in the spacer domain of ADAMTS13 that are targeted by anti-spacer autoantibodies found in a large cohort of iTTP patients., (© 2020 International Society on Thrombosis and Haemostasis.)

Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura (iTTP) commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced here to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported Gain-of-Function (GoF, KYKFF) and truncated 'MDTCS' variants were also included. Sera of 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues show a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aromatic residues are the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoantibodies targeting the spacer RFRYY epitope have preponderance compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest activity reductions were observed in the most autoantibody-resistant variants (15-35% residual activity in FRETS-VWF73). Among these, a triple-alanine mutant RARAA showed activity in a VWF multimer assay. This study shows that non-conservative and alanine modifications of residues within the exosite-3 spacer RFRYY epitope in full-length ADAMTS13 resist the binding of autoantibodies from iTTP patients, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.

Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14 of 18 (78%) samples, proving that anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n = 197) that also included plasma samples from iTTP patients in remission in whom ADAMTS13 activity was <50%. The open ADAMTS13 conformation was found during acute iTTP, as well as in patients in remission with ADAMTS13 activity <50% and in half of the patients with ADAMTS13 activity >50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus, open ADAMTS13 is a hallmark of acute iTTP, as well as a novel biomarker that can be used to detect subclinical iTTP in patients in remission. Finally, a long-term follow-up study in 1 iTTP patient showed that the open conformation precedes a substantial drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management., (© 2020 by The American Society of Hematology.)

Background: In immune-mediated thrombotic thrombocytopenic purpura (iTTP), patients develop an immune response against the multidomain enzyme ADAMTS13. ADAMTS13 consists of a metalloprotease (M) and disintegrin-like (D) domain, 8 thrombospondin type 1 repeats (T1-T8), a cysteine-rich (C), a spacer (S), and 2 CUB domains (CUB1-2). Previous epitope mapping studies have used relatively large overlapping ADAMTS13 fragments., Objectives: We aimed at developing small nonoverlapping ADAMTS13 fragments to fine map anti-ADAMTS13 autoantibodies in iTTP patients., Methods: A library of 16 ADAMTS13 fragments, comprising several small (M, DT, C, S, T2-T5, T6-T8, CUB1, CUB2), and some larger fragments with overlapping domains (MDT, MDTC, DTC, CS, T2-T8, CUB1-2, MDTCS, T2-C2), were generated. All fragments, and ADAMTS13, were expressed as a fusion protein with albumin domain 1, and purified. The folding of the fragments was tested using 17 anti-ADAMTS13 monoclonal antibodies with known epitopes. An epitope mapping assay using small ADAMTS13 fragments was set up, and validated by analyzing 18 iTTP patient samples., Results: Validation with the monoclonal antibodies demonstrated that single S and CUB1 were not correctly folded, and therefore CS and CUB1-2 fragments were selected instead of single C, S, CUB1, and CUB2 fragments. Epitope mapping of antibodies of patients with iTTP confirmed that 6 nonoverlapping ADAMTS13 fragments M, DT, CS, T2-T5, T6-T8, and CUB1-2 were sufficient to accurately determine the antibody-binding sites., Conclusion: We have developed a tool to profile patients with iTTP according to their anti-ADAMTS13 antibodies for a better insight in their immune response., (© 2020 Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Megakaryocyte-derived platelets and endothelial cells store their hemostatic cargo in α- and δ-granules and Weibel-Palade bodies, respectively. These storage granules belong to the lysosome-related organelles (LROs), a heterogeneous group of organelles that are rapidly released following agonist-induced triggering of intracellular signaling pathways. Following vascular injury, endothelial Weibel-Palade bodies release their content into the vascular lumen and promote the formation of long VWF (von Willebrand factor) strings that form an adhesive platform for platelets. Binding to VWF strings as well as exposed subendothelial collagen activates platelets resulting in the release of α- and δ-granules, which are crucial events in formation of a primary hemostatic plug. Biogenesis and secretion of these LROs are pivotal for the maintenance of proper hemostasis. Several bleeding disorders have been linked to abnormal generation of LROs in megakaryocytes and endothelial cells. Recent reviews have emphasized common pathways in the biogenesis and biological properties of LROs, focusing mainly on melanosomes. Despite many similarities, LROs in platelet and endothelial cells clearly possess distinct properties that allow them to provide a highly coordinated and synergistic contribution to primary hemostasis by sequentially releasing hemostatic cargo. In this brief review, we discuss in depth the known regulators of α- and δ-granules in megakaryocytes/platelets and Weibel-Palade bodies in endothelial cells, starting from transcription factors that have been associated with granule formation to protein complexes that promote granule maturation. In addition, we provide a detailed view on the interplay between platelet and endothelial LROs in controlling hemostasis as well as their dysfunction in LRO related bleeding disorders.

Development of neutralizing antibodies against biotherapeutic agents administered to prevent or treat various clinical conditions is a longstanding and growing problem faced by patients, medical providers and pharmaceutical companies. The hemophilia A community has deep experience with attempting to manage such deleterious immune responses, as the lifesaving protein drug factor VIII (FVIII) has been in use for decades. Hemophilia A is a bleeding disorder caused by genetic mutations that result in absent or dysfunctional FVIII. Prophylactic treatment consists of regular intravenous FVIII infusions. Unfortunately, 1/4 to 1/3 of patients develop neutralizing anti-FVIII antibodies, referred to clinically as "inhibitors," which result in a serious bleeding diathesis. Until recently, the only therapeutic option for these patients was "Immune Tolerance Induction," consisting of intensive FVIII administration, which is extraordinarily expensive and fails in ~30% of cases. There has been tremendous recent progress in developing novel potential clinical alternatives for the treatment of hemophilia A, ranging from encouraging results of gene therapy trials, to use of other hemostatic agents (either promoting coagulation or slowing down anti-coagulant or fibrinolytic pathways) to "bypass" the need for FVIII or supplement FVIII replacement therapy. Although these approaches are promising, there is widespread agreement that preventing or reversing inhibitors remains a high priority. Risk profiles of novel therapies are still unknown or incomplete, and FVIII will likely continue to be considered the optimal hemostatic agent to support surgery and manage trauma, or to combine with other therapies. We describe here recent exciting studies, most still pre-clinical, that address FVIII immunogenicity and suggest novel interventions to prevent or reverse inhibitor development. Studies of FVIII uptake, processing and presentation on antigen-presenting cells, epitope mapping, and the roles of complement, heme, von Willebrand factor, glycans, and the microbiome in FVIII immunogenicity are elucidating mechanisms of primary and secondary immune responses and suggesting additional novel targets. Promising tolerogenic therapies include development of FVIII-Fc fusion proteins, nanoparticle-based therapies, oral tolerance, and engineering of regulatory or cytotoxic T cells to render them FVIII-specific. Importantly, these studies are highly applicable to other scenarios where establishing immune tolerance to a defined antigen is a clinical priority., (Copyright © 2020 Lacroix-Desmazes, Voorberg, Lillicrap, Scott and Pratt.)

Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 β1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1 Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1 -/- endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2 +- mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 β1, also the μ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8 -/- endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8., (Copyright© 2019 Ferrata Storti Foundation.)

The endothelium stores the hemostatic protein Von Willebrand factor (VWF) in endothelial storage organelles called Weibel-Palade bodies (WPBs). During maturation, WPBs recruit a complex of Rab GTPases and effectors that associate with components of the SNARE machinery that control WPB exocytosis. Recent genome wide association studies have found links between genetic variations in the SNAREs syntaxin-2 (STX2) and syntaxin binding protein 5 (STXBP5) and VWF plasma levels, suggesting a role for SNARE proteins in regulating VWF release. Moreover, we have previously identified the SNARE proteins syntaxin-3 and STXBP1 as regulators of WPB release. In this study we used an unbiased iterative interactomic approach to identify new components of the WPB exocytotic machinery. An interactome screen of syntaxin-3 identifies a number of SNAREs and SNARE associated proteins (STXBP2, STXBP5, SNAP23, NAPA and NSF). We show that the VAMP-like domain (VLD) of STXBP5 is indispensable for the interaction with SNARE proteins and this capacity of the VLD could be exploited to identify an extended set of novel endothelial SNARE interactors of STXBP5. In addition, an STXBP5 variant with an N436S substitution, which is linked to lower VWF plasma levels, does not show a difference in interactome when compared with WT STXBP5. SIGNIFICANCE: The hemostatic protein Von Willebrand factor plays a pivotal role in vascular health: quantitative or qualitative deficiencies of VWF can lead to bleeding, while elevated levels of VWF are associated with increased risk of thrombosis. Tight regulation of VWF secretion from WPBs is therefore essential to maintain vascular homeostasis. We used an unbiased proteomic screen to identify new components of the regulatory machinery that controls WPB exocytosis. Our data expand the endothelial SNARE protein network and provide a set of novel candidate WPB regulators that may contribute to regulation of VWF plasma levels and vascular health., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)