Alessandra, Casonato

Daniela, Regazzo

Eva, Galletta

Lisa, Gianesello

Viviana, Daidone

English

Archivio istituzionale della ricerca - Università di Padova

Received: 28 June 2023 - Revised: 7 August 2023 - Accepted: 24 August 2023https://doi.org/10.1016/j.rpth.2023.102200R E S EA R CH L E T T E RNo von Willebrand factor domains other than A1 are involvedin type 2B von Willebrand disease: what the p.R924Q andp.A2178S variants teach us1 | INTRODUCTIONvon Willebrand factor (VWF) is a high-molecular weight multimericglycoprotein with a role in primary hemostasis. It mediates plateletadhesion at the site of vascular injury [1] and serves as a carrier ofcoagulation factor VIII (FVIII). The molecule has a multimeric struc-ture, with oligomers that range from 500,000 to 20 million Dalton, andlarge VWF multimers characterized by a high-hemostatic capacity.Each VWF subunit comprises of 4 repetitive domains named from A toD, with different structural and functional roles. The A1 domain isinvolved in the interaction with platelet glycoprotein Ib (GPIb) [2].von Willebrand disease (VWD)—the most common of all theinherited bleeding disorders [3]—is associated with VWF deficiencies orabnormalities classified as type 1, 2, or 3, according to the type of VWFdefect involved. In type 2B VWD, there is a greater affinity betweenVWF and platelet GPIb [4] associated with gain-of-function mutationson exon 28 of the VWF gene [5] that encodes for the A1 domain ofVWF. In vivo, this defect prompts type 2B VWF to bind spontaneouslyto platelet; this causes a loss of large VWF multimers, and leads tomoderate or severe, persistent or transient thrombocytopenia [4]. Type2B is revealed, in vitro, by a more marked ristocetin-induced plateletaggregation, which is an indication of how well a patient’s platelet-richplasma can aggregate at very low-ristocetin concentrations [6]. TheVWF of patients with type 2B VWD may have a greater affinity forplatelets even if their multimer pattern is normal and they have mu-tations in the A1 domain, but never thrombocytopenia [7]. It wasrecently posited that domains other than A1, and especially the D’D3and D4 domains, may be involved in VWF–platelet interaction in in-dividuals carrying both the p.R924Q and the p.A2178S variants [8].Here, we describe our patients with VWD carrying the combinedp.R924Q and p.A2178S variants that present as a typical type 1 VWDand not a type 2B phenotype.2 | RESULTSFive subjects from 3 unrelated families having life-long mildbleeding symptoms were studied. The patients showed a normal© 2023 The Author(s). Published by Elsevier Inc. on behalf of International Society onNC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Res Pract Thromb Haemost. 2023;7:e102200https://doi.org/10.1016/j.rpth.2023.102200ristocetin-induced platelet aggregation at 1.2 mg/mL, and thelowest dose eliciting a platelet response to ristocetin was 1.0 mg/mL, so not dissimilar to what happens in normal subjects (Table).The patients had slightly reduced plasma VWF:antigen (VWF:Ag),VWF:collagen binding, and VWF:ristocetin cofactor levels, withnormal VWF:collagen binding to VWF:Ag ratio, and VWF: ristocetincofactor to VWF:Ag ratio (Table). These findings would suggest anormal VWF multimeric structure. Analyses of the circulating VWFmultimers in patients demonstrated a homogeneous, mild reduction ofall oligomers, with a preserved representation of high-molecularweight multimers, and no accumulation of lower molecular weightVWF forms (Figure). The patients’ platelet counts were normal,ranging from 228 × 103μL to 275 × 103/μL (Table). Their platelet VWFcontent was within normal range, suggestive of a normal VWF syn-thesis. VWF propeptide (VWFpp), measured to explore VWF half-lifeunder steady-state conditions, was found slightly shorter than normal,but the VWFpp to VWF:Ag ratio appeared to be normal in all 5 pa-tients, consistent with a normal VWF survival. The patients studiedwere thus classified as a mild form of type 1 VWD.Sanger sequencing of the whole VWF gene revealed the presenceof 2 variants: the c.2771G>A, p.R924Q, in exon 21; and thec.6532G>T, p.A2178S in exon 37, at heterozygous level. No othervariants came to light anywhere in the VWF gene. The c.2771G>Avariant induces the substitution p.R924Q in the D’D3 domain. Thec.6532G>T prompts the substitution p.A2178S in the D4 domain.These substitutions were transmitted as a dominant defect, since 3members of the same family from 2 generations were found to carryboth variants, and to express a mild VWF defect.3 | DISCUSSIONThe A1 domain plays a key part in binding VWF to platelet GPIb,promoting the adhesion and aggregation of platelets on an injuredvessel wall. However, it was recently posited that domains other thanA1, and especially the D’D3 and D4 domains, may be involved inVWF–platelet interaction, in patients with type 2B VWD carrying thep.R924Q and the p.A2178S variants [8]. This observation seemed toThrombosis and Haemostasis. This is an open access article under the CC BY-www.rpthjournal.org - 1 of 3T AB L E Main hemostatic findings in the patients with VWD carrying the p.R924Q and the p.A2178S mutations combinedFamily/subjectGender/ABO/agePlateletcount£103/uLRIPA1.2mg/mL%MADRmg/mLFVIIIU/dLVWF:AgU/dLVWF:RCoU/dLVWF:CBU/dLVWF:FVIIIBU/dLPlateletVWF:AgVWFpropeptideU/dL1/1 M/A/38 210 81.5 1.0 40.7 43.2 63.5 (1.47) 45.3 (1.05) 52 (1.20) 78.2 47.6 (1.10)1/2 F/A/10 265 NP NP 40.7 42.6 69.7 (1.64) 43.5 (1.02) 42.3 (0.99) 73.5 50.2 (1.18)1/3 F/O/3 228 79.8 1.0 42.9 30.3 25.2 (0.83) 31 (1.02) 25.5 (0.84) NP NP2/1 F/O/32 NP 93.7 1.0 40.1 46.4 43.2 (0.93) 53 (1.14) 46 (0.99) 93.1 NP3/1 M/NP/5 275 NP NP 53.5 47.3 NP 46.5 46.1 (0.97) 85 45.9 (0.96)Normalrange150-350 60-84 1.0 60-160 60-160 60-130 (>0.70) 65-150(>0.70)60-160 (≥0.74) 70-140 60-160 (0.8-1.3)The values inside the brackets represent the corresponding ratios.CB, collagen binding; MADR, minimal aggregating dose of ristocetin; NP, not performed; RCo, ristocetin cofactor; RIPA, ristocetin-induced plateletaggregation; VWF, von Willebrand factor.2 of 3 - RESEARCH LETTERcast doubts on the dogma that gain-of-function type 2B variants arealways and only located in the A1 domain of VWF.The p.R924Q and the p.A2178S variants reportedly induce a VWFpolarity change, promoting the transition toward the open conforma-tional state needed for VWF to interact with platelets, even in theabsence of shear forces [8]. Such a suggestion was strongly supported bythe in vitro functional behavior of recombinant mutated VWF, and by thedemonstrationof a partially stretched andopenconformationofmutatedVWF on molecular modeling, transmission electron microscopy, andatomic forcemicroscopy. These in vitrofindingswere fully consistentwiththe patient’ phenotype, involving hyper-responsiveness to ristocetin, theabsence of large VWF multimers [4,7], thrombocytopenia and increasedF I GUR E Plasma VWF multimeric pattern obtained on 1.8%Sodium Dodecyl Sulfate (SDS) agarose gel electrophoresis undernonreducing conditions. Patients 1, 2, and3 fromfamily 1 are identifiedby the numbers entered in the Table, as comparedwith normal plasma(NP). The multimers were detected using 125I-conjugated anti-VWFantibody, followed by autoradiography. High-molecular weightmultimers are at the top, low-molecular weight VWF forms at thebottom. VWF, von Willebrand factor.VWFpp ratio [9]—the typical features of type 2B VWD. This observationcould have opened up new scenarios for treating patients with VWD bytaking action through novel VWF domains.Unfortunately, we cannot corroborate such findings of a nonca-nonical type 2B VWD because our 5 patients (from 3 unrelated fam-ilies) carrying both the variants p.R924Q and the p.A2178S, and noothers anywhere in the VWF gene, showed a clear type 1 VWDphenotype. For a start, they did not have a low-platelet count or lack oflarge VWF multimers; they showed a homogeneous reduction in thequantity of VWF multimers and no accumulation of low-molecularweight forms. That said, it is well known that some type 2B variantsin the A1 domain do not induce the consumption of large VWF multi-mers, despite an increased affinity of VWF for platelets [7], so the pa-tient carrying the combined p.R924Q and p.A2178S variants describedby Sacco et al. [8] may belong to this group. However, our patientsshowed no platelet hyper-responsiveness to ristocetin, the phenotypichallmark of type 2B variants in patients with a normal or abnormalVWF multimer pattern [7], excluding the possibility of them being casesof type 2B VWD with a normal multimer pattern. Our patients also hadnormal platelet VWF levels, suggesting a normal VWF synthesis, andthereby ruling out any likelihood of an altered splicing site in exon 28,reportedly associated with the p.R924Q variant [10], explaining thereduction in circulating VWF levels. Finally, VWF survival seems to benormal in our patients, judging from their normal VWFpp ratio values,again in contrast with the patient findings of the study by Sacco et al.[8], and in patients with type 2B [9]. We have no explanation for thediscrepancy between the phenotype seen in our patients and the onedescribed by Sacco et al. [8]. It would be tempting to assume that thepatient described by Sacco et al. [8] had some other, unidentified mu-tation(s) that would explain this picture, but that cannot be the casebecause the authors found the recombinant VWF carrying the com-bined p.R924Q and p.A2178S variants hyper-responsive to ristocetin,especially when expressed in the homozygous state.In conclusion, our findings mean that p.R924Q and p.A2178S inthe VWF D’D3 and D4 cannot be responsible for type 2B VWD, noteven in a noncanonical form, so for now, no known variants away fromRESEARCH LETTER - 3 of 3the A1 domain can have gain-of-function effects on VWF–plateletinteraction. This highlights how complex and heterogeneous VWDcan be, and how difficult it can be to characterize these patients.FUNDINGThis work was supported by a grant from the MURST (ex 60%, 2019).AUTHOR CONTRIBUTIONSC.A. designed the study, discussed the results and wrote the paper; G.L.performed analysis and interpretation of the data; R.D. performed geneticanalysis;G.E. andD.V. performedacquisitionof thedataand interpretationand revision of the results. All the authors have reviewed the finalmanuscript, agree on its contents, and approve its submission to Researchand Practice in Thrombosis and Haemostasis for possible publication.RELATIONSHIP DISCLOSUREThere are no competing interests to disclose.Casonato AlessandraGianesello LisaRegazzo DanielaGalletta EvaDaidone VivianaDepartment of Medicine, First chair of Internal Medicine,University of Padua Medical School,Padua, ItalyHandling Editor: Dr Johnny Mahlangu.CorrespondenceAlessandra Casonato, Department of Medicine, First chair of InternalMedicine, University of Padua Medical School, via Ospedale Civile105, Padova, Italy.Email: sandra.casonato@unipd.itTWITTERRegazzo Daniela @DanielaRegazzoREFERENCES[1] Sadler JE. Biochemistry and genetics of von Willebrand factor. AnnuRev Biochem. 1998;67:395–424.[2] Fujimura Y, Titani K, Holland LZ, Russell SR, Roberts JR, Elder JH,et al. von Willebrand factor. A reduced and alkylated 52/48-kDafragment beginning at amino acid residue 449 contains the domaininteracting with platelet glycoprotein Ib. J Biol Chem. 1986;261:381–5.[3] Leebeek FWG, Eikenboom JCJ. Von Willebrand’s disease. N Engl JMed. 2016;375:2067–80.[4] Ruggeri ZM, Pareti FI, Mannucci PM, Ciavarella N, Zimmerman TS.Heightened interaction between platelets and factor VIII/von Wil-lebrand factor in a new subtype of von Willebrand’s disease. N Engl JMed. 1980;302:1047–51.[5] Randi AM, Rabinowitz I, Mancuso DJ, Mannucci PM, Sadler JE.Molecular basis of von Willebrand disease type IIB. Candidatemutations cluster in one disulfide loop between proposedplatelet glycoprotein Ib binding sequences. J Clin Invest. 1991;87:1220–6.[6] De Marco L, Mazzuccato M, Grazia Del Ben M, Budde U,Federici AB, Girolami A, et al. Type IIB von Willebrand factor withnormal sialic acid content induces platelet aggregation in theabsence of ristocetin. Role of platelet activation, fibrinogen, and twodistinct membrane receptors. J Clin Invest. 1987;80:475–82.[7] Casonato A, Daidone V, Galletta E, Bertomoro A. Type 2B vonWillebrand disease with or without large multimers: a distinction ofthe two sides of the disorder is long overdue. PLoS One. 2017;12:e0179566.[8] Sacco M, Lancellotti S, Ferrarese M, Bernardi F, Pinotti M,Tardugno M, et al. Noncanonical type 2B von Willebrand diseaseassociated with mutations in the VWF D’D3 and D4 domains. BloodAdv. 2020;4:3405–15.[9] Casonato A, Gallinaro L, Cattini MG, Pontara E, Padrini R,Bertomoro A, et al. Reduced survival of type 2B von Willebrandfactor, irrespective of large multimer representation or thrombocy-topenia. Haematologica. 2010;95:1366–72.[10] Berber E, James PD, Hough C, Lillicrap D. An assessment of thepathogenic significance of the R924Q von Willebrand factor sub-stitution. J Thromb Haemost. 2009;7:1672–9.

No von Willebrand factor domains other than A1 are involved in type 2B von Willebrand disease: what the p.R924Q and p.A2178S variants teach us

Casonato Alessandra

Gianesello Lisa

Regazzo Daniela

Galletta Eva

Daidone Viviana

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https://www.research.unipd.it/bitstream/11577/3496628/1/Gianesello%20-%20RPTH%202023.pdf

No von Willebrand factor domains other than A1 are involved in type 2B von Willebrand disease: what the p.R924Q and p.A2178S variants teach us

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