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 invitro 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.

The A1 domain in Von Willebrand Factor (VWF) initiates coagulation through binding to platelet glycoprotein GPIbα receptors. Von Willebrand Disease (VWD)-Mutations in A1 that either impair (type 2M) or enhance (type 2B) platelet adhesion to VWF can locally destabilize and even misfold the domain. We leveraged misfolding in the gain-of-function type 2B VWD phenotype as a target, distinct from the normal conformation. Two nuclease-resistant 2′-fluoropyrimidine RNA aptamers were selected to discriminate normal A1 domains from a type 2B V1314D A1 variant in a glycosylated A1A2A3 tri-domain VWF-fragment. Two aptamers, W9 and V1, were isolated that selectively recognize, bind, and inhibit the A1–GPIbα interaction with WT A1A2A3 and V1314D A1A2A3, respectively. These aptamers were tested against their respective recombinant targets, plasma VWF, VWF concentrates, and patient plasma with the heterozygous type 2B VWD R1306W variant using clinical assays, surface plasmon resonance and inhibition assays of platelet adhesion to recombinant A1 and A1A2A3 domains under shear stress. The specificity of W9 and V1 aptamers confirms that pathological conformations of VWD Type 2B proteins are different from normal VWF. The availability of aptamers that distinguish normal plasma-derived VWF from VWD suggests potential applicability in clinical diagnosis of severe gain-of-function phenotypes.

Background and Aims: von Willebrand disease (VWD) is brought on by hereditary impairments in the von Willebrand factor (VWF). It has been determined that most of the molecular abnormalities are present in exon 28 of the VWF gene qualitative variations. In this regard, investigating mutations in exon 28 of the VWF coding gene can help identify the VWD type and can be used to manage patients by using appropriate strategies. Materials and Methods: 10 suspected VWD type 2B patients were investigated for the genotype of exon 28. Routine coagulation tests were performed for the patients. Molecular sequencing was also used for the evaluation of mutations in exon 28. Determining the tertiary structure of VWF can aid in understanding its functional residues and interactions. The nsSNVs’ pathogenicity was examined utilizing potent bioinformatics methods. Step-by-step testing of the high-risk mutations was done using SIFT, PolyPhen-2, I-Mutant 2.0, PHD-SNPg, and SNPs&GO. Then, the secondary structure, amino acid conservation, and feature of amino acids were investigated via Protparam, Cofactor, Interprosurf, ConSurf, NetSurfP-2.0, and HOPE. Results: N1231T, V1229G, V1316M, and P1266Q missense mutations in VWF were detected. 3D structure of VWF predicted and evaluated. Putative conserved domains were identified. P1266Q and V1316M amino acid substitutions are predicted as “Not tolerated substitution” damaging and Disease, while V1229G and N1231T amino acid substitutions are predicted as “tolerated substitution” benign and Neutral. Conclusion: V1316M and P1266Q amino acid substitutions were determined as high-risk mutations using powerful bioinformatics tools in VWD patients.

Efficacy of platelet-inspired hemostatic nanoparticles on bleeding in von Willebrand disease murine models

This report describes the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B (VWD2B) with chronic thrombocytopenia, successfully treated with nonoperative management including von Willebrand factor (VWF) replacement therapy, and platelet transfusions relayed by a thrombopoietin receptor agonist (TPO-RA, Eltrombopag). Eltrombopag was initially introduced to rescue an unusual post-platelet-transfusion reaction exacerbating the thrombocytopenia. In-depth analysis of the dramatic platelet count drop and VWF measurements timeline ruled out an allo-immune reaction and supported an alternative hypothesis of a sudden platelet clearance as a consequence of stress-induced release of abnormal VWF. One year later, a second life-threatening bleeding episode required urgent surgery successfully managed with VWF replacement therapy and platelet transfusions. Eltrombopag was further introduced in the post-surgery period to allow bleeding-free and platelet-transfusion-free successful recovery. Treatment decisions are particularly challenging in patients with VWD2B, and this case highlights how such decisions can benefit from understanding the molecular origin of platelet count fluctuations observed in these patients. Here, we successfully used a new therapeutic approach combining VWF-replacement therapy and initial platelet-transfusion relayed by TPO-RA to optimize patient management.Plain language summaryA combination of von Willebrand factor replacement and thrombopoietin receptor agonist in thrombocytopenic patients with von Willebrand disease type 2B: a new therapy approach to optimize patient management?Therapeutic management of patients with von Willebrand disease type 2B are particularly challenging in case of severe thrombocytopenia.Treatment includes von Willebrands factor replacement therapy and iterative platelet transfusions.We describe the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B successfully treated with nonoperative management including von Willebrand factor replacement therapy and platelet transfusions relayed by a thrombopoietin receptor agonist.We showed that the unusual post-platelet-transfusion reaction associated with a dramatic platelet count drop was a consequence of stress-induced release of abnormal von Willebrand factor.The combination of von Willebrand factor replacement therapy and thrombopoietin receptor agonist may offer a new therapeutic approach to optimize patient management.

患儿 女，8日龄，因“血小板减少5 d”于2020年4月收入北京儿童医院。血常规示血小板减少，外院予人免疫球蛋白治疗无效，血小板输注暂时有效，外周血及骨髓涂片可见巨大血小板，低浓度瑞斯托霉素诱导血小板聚集阳性，二代测序提示血管性血友病因子（vWF）基因c.3946G>A（p.V1316M）杂合突变，诊断为血管性血友病2B型。本例系自发性突变，我国尚无该突变位点报道。本病以预防出血为主要处理原则，患儿无活动性出血，病情平稳，未输注血制品（含成分血）。.

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both "loss as well as gain of function" mutations observed in this domain. Naturally occurring "gain of function" mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These "gain of function" mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the "gain of function" effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.

The physical spacing between the von Willebrand factor D'D3 and A1 domains regulates platelet adhesion in vitro and in vivo

Ristocetin-induced platelet aggregation (RIPA) is used as an in vitro test to determine the presence and integrity of the platelet glycoprotein (GP) Ibα-V-IX complex and von Willebrand factor (VWF) interaction and is usually performed using platelet-rich plasma (PRP). Impairment in the response of VWF/GPIbα-V-IX is measured with reference to several established concentrations of ristocetin and may indicate defects in VWF or in GPIbα-V-IX function. RIPA-based mixing studies comprise an additional approach to testing this interaction to help define whether defects identified by RIPA lie in VWF or in GPIbα-V-IX. For example, the correction of an abnormal RIPA trace after mixing PRP with normal plasma and rechallenging with ristocetin at 1.0mg/mL suggests VWF function/quantity defect. RIPA mixing studies at lower doses of ristocetin (0.5mg/mL) are recommended for discrimination of von Willebrand disease type 2B (VWD2B) from the rarer platelet-type (PT) VWD and for the phenotypic laboratory diagnosis of VWD2B. The demonstration of a plasma factor capable of inducing platelet aggregation at such low doses of ristocetin represents the hallmark for the phenotypic laboratory diagnosis of VWD2B. Moreover, since both VWD2B and PT-VWD may present with thrombocytopenia, RIPA-based mixing studies are also useful in thrombocytopenic patients in whom RIPA testing is difficult to assess.

von Willebrand disease type 2B (VWD2B) expresses gain-of-function mutations that enhance binding of an individual's von Willebrand factor (VWF) to its platelet ligand, glycoprotein Ib (GPIb), and which are usually identified by increased ristocetin-induced platelet aggregation (RIPA). We describe here the phenotypic profile of 38 genotypically selected VWD2B-affected family members (AFMs) belonging to 19 unrelated families. Major bleeding was observed in 68.4% of AFMs (previous to their diagnosis and registered by lifetime interviews), with a total of 46 episodes (1.21/patient), and was found to be highly related to the individual bleeding score and presence of thrombocytopenia, but otherwise unrelated to other laboratory parameters. Excessive muco-cutaneous bleeding symptoms were often reported, the most frequent of which comprised menorrhagia, epistaxis, easy bruising, and bleeding after teeth extraction/in oral cavity. Eight unaffected family members were also studied. The prevalence of VWD2B within families was 0.826, and the penetrance of mutations was complete, making it mandatory to study entire family sets to complete diagnostic profiles. Seven heterozygous missense mutations were found, the most common being p.V1316M. In the p.R1308C group, 75% of the AFMs showed absence of RIPA at 0.5 mg/mL, 66.6% of whom had VWF:RCo < 10 IU/dL, and 50% of whom had VWF:CB < 10 IU/dL. In the p.S1310F group, none of the AFMs had VWF:RCo/VWF:Ag < 0.6 (RCo/Ag), but 100% had VWF:CB/VWF:Ag < 0.6/(CB/Ag). Patients with p.P1266L and p.R1304V were characterized as atypical VWD2B. Two de novo mutations were found in four AFMs belonging to two families. We also describe a novel mutation: p.Y1258C. Of our patients, 70.5% had O blood group. In conclusion, a normal RCo/Ag and a negative RIPA at 0.5 mg/mL do not necessarily rule out a diagnosis of VWD2B.

We present here a 63-year old woman with a long history of immune thrombocytopenia. She was hospitalized for a traumatic intracranial hemorrhage with thrombocytopenia. Following inefficient treatment of four platelet transfusions, immunoglobulins, and corticosteroids, we initiated treatment with a thrombopoietin (TPO) receptor agonist (eltrombopag 25 mg/d) with a good efficacy. Her mother and sister also had chronic thrombocytopenia. Clinical history, hemostasis results, and gene analysis revealed von Willebrand disease (VWD) type 2B with the mutation (c.3946G>A; p.V1316M), which combines a von Willebrand factor defect with severe thrombocytopenia, as well as a thrombocytopathy. The efficacy of TPO receptor agonists appears to counterbalance, at least to some extent, the thrombocytopathy associated with this mutation. As such, the use of TPO receptor agonists could represent an alternative therapeutic approach in cases of VWD type 2B with severe thrombocytopenia.

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

Mechanisms and Therapeutic Modulation of the Bleeding Tendency in Genetically-Engineered Von Willebrand Disease Type 2B Mice

Characterisation of the p.A1461D mutation causing von Willebrand disease type 2B with severe thrombocytopenia, circulating giant platelets, and defective α-granule secretion -

Binding Of VWF To Type IV Collagen: An Additional Collagen Binding Mechanism Beyond Types I, III, and VI Collagen?

An elderly female with a positive bleeding history (personal and family) and prior but undocumented diagnosis of von Willebrand disease (vWD) presented for surgery. Testing showed mild thrombocytopenia, decreased ristocetin cofactor activity to von Willebrand factor antigen ratio, and absent high‐molecular‐weight multimers, suggesting a qualitative (type 2) subtype. Low‐dose ristocetin‐induced platelet aggregation demonstrated abnormally increased aggregation, consistent with either type 2B vWD or platelet‐type vWD (PT‐vWD). Differentiation between these subtypes guides therapy. Optimal therapy for type 2B vWD consists of vWF replacement, while platelet transfusions are used to treat PT‐vWD. Traditional options for differentiation include vWF/platelet binding assays or specialized platelet aggregation studies. However, these subspecialized tests have limited availability and sample stability. Gene sequencing performed in our laboratory allowed for detection of 1 of 4 known mutations in GP1BA, (c.763A&gt;;G, p.Met255Val, using standard nomenclature) responsible for PT‐VWD. This finding confirmed her diagnosis and appropriate therapy and facilitates diagnosis for affected family members in this disorder with an autosomal dominant inheritance pattern. Gene sequencing is a useful tool to aid in diagnosis and subtyping of type 2 vWD. kristi.smock@aruplab.com

The Co-Influence of VWD Type 2B and Type 2M Mutations and Platelet GPIbα On the Susceptibility of VWF to ADAMTS13 Under Shear Stress

Inhibition of Von Willebrand Factor (VWF) by ARC1779 Normalizes Von Willebrand Factor/FVIII Levels and Corrects Thrombocytopenia in VWD Type 2b Patients,

Abnormal VWF modifies megakaryocytopoiesis: studies of platelets and megakaryocyte cultures from patients with von Willebrand disease type 2B

Platelet morphological changes in 2 patients with von Willebrand disease type 3 caused by large homozygous deletions of the von Willebrand factor gene