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. 2017 Aug;15(8):1559-1566.
doi: 10.1111/jth.13742. Epub 2017 Jun 23.

Clinical and laboratory phenotype variability in type 2M von Willebrand disease

A L Doruelo  1   2 , S L Haberichter  1   2   3 , P A Christopherson  3 , L N Boggio  4 , S Gupta  5 , S R Lentz  6 , A D Shapiro  5 , R R Montgomery  1   2   3 , V H Flood  1   2   3
Affiliations

Clinical and laboratory phenotype variability in type 2M von Willebrand disease

A L Doruelo et al. J Thromb Haemost. 2017 Aug.

Abstract

Essentials The pathophysiology of type 2M von Willebrand disease (VWD) is poorly understood. Sequence variations in type 2M VWD subjects were characterized. A high degree of clinical and laboratory variability exists within type 2M VWD variants. Some type 2M variants may share features of type 2A VWD.

Summary: Background von Willebrand factor (VWF) is a multimeric coagulation factor that tethers platelets to injured subendothelium. Type 2M von Willebrand disease (VWD) is characterized by a qualitative defect in VWF with preserved multimer distribution. Objectives Through the Zimmerman Program for the Molecular and Clinical Biology for VWD, five VWF sequence variations were studied in subjects diagnosed with type 2M VWD. Methods Bleeding phenotype was assessed using the ISTH bleeding assessment tool. Full-length VWF gene sequencing was performed for each subject. Each variant was placed into a recombinant VWF vector using site-directed mutagenesis and expressed in HEK293T cells as homozygous or heterozygous VWF. Variant expression, collagen binding and platelet GPIbα binding were studied through ELISA assays. Multimer analysis was performed by gel electrophoresis. Results Bleeding scores were elevated for all subjects except for the p.P1162L and p.R1374C variants. Although all had reduced VWF ristocetin cofactor activity/VWF antigen ratios on plasma testing, recombinant VWF did not show a classic type 2M phenotype for any of the five variants. Homozygous expression of variants p.D1283Y, p.R1349C, p.R1374C and p.I1453N was consistent with type 2A VWD, although all had normal expression as heterozygous recombinant VWF. Variant p.P1162L had normal VWF expression and function, consistent with the lack of bleeding symptoms. Conclusions Although originally classified as type 2M VWD, these homozygous recombinant VWF variants do not fulfill complete 2M VWD diagnostic criteria. A better classification schema and improved testing for putative type 2M variants is needed in order to effectively diagnose and treat affected patients.

Keywords: clinical laboratory techniques; hemorrhage; platelets; von Willebrand disease; von Willebrand factor.

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Conflict of interest statement

Disclosure of Conflicts of Interests

L. N. Boggio has served as a consultant for Alnylam, Bayer, Biogen, CSL Behring, Grifols, Novo Nordisk, Octapharma, OPKO, Pfizer, and Shire. S. R. Lentz. has received grants from NIH. V. H. Flood has received grants from NIH and has served as a consultant for CSL Behring and Shire.

Figures

Figure 1
Figure 1. Type 2M variants cluster in the VWF A1 domain
Spheres in the VWF A1 domain crystal structure [30] indicate variant locations. Note that variant P1162L is located in the D3 domain and is thus not depicted in the crystal structure.
Figure 2
Figure 2. Variable in vitro secretion, collagen binding, and platelet GPIαb binding of type 2M variants
Data shown represents averages of 3–4 different assays on 3–4 separate transfections. Error bars represent 1 standard deviation. Panel A: Expression. Variants 1283Y, 1374C and 1453N show a marked decrease in expression relative to wild-type. Variants 1162L and 1349C have normal expression. Panel B: Collagen binding. Binding to collagen 3 is shown in light gray and binding to collagen 4 is shown in dark gray. Variants 1283Y, 1349C, and 1374C have decreased binding to collagen 3 and 4. 1453N has absent binding to both collagen 3 and 4. 1162L has normal binding to collagen 3 and 4. Panel C: Platelet GPIαb binding. Variants 1283Y, 1349C, 1374C, and I1453N have absent binding to platelet GPIb. 1162L has normal binding. Data represents the average of three different VWF:GPIbM ELISA assays performed on three separate transfections.
Figure 2
Figure 2. Variable in vitro secretion, collagen binding, and platelet GPIαb binding of type 2M variants
Data shown represents averages of 3–4 different assays on 3–4 separate transfections. Error bars represent 1 standard deviation. Panel A: Expression. Variants 1283Y, 1374C and 1453N show a marked decrease in expression relative to wild-type. Variants 1162L and 1349C have normal expression. Panel B: Collagen binding. Binding to collagen 3 is shown in light gray and binding to collagen 4 is shown in dark gray. Variants 1283Y, 1349C, and 1374C have decreased binding to collagen 3 and 4. 1453N has absent binding to both collagen 3 and 4. 1162L has normal binding to collagen 3 and 4. Panel C: Platelet GPIαb binding. Variants 1283Y, 1349C, 1374C, and I1453N have absent binding to platelet GPIb. 1162L has normal binding. Data represents the average of three different VWF:GPIbM ELISA assays performed on three separate transfections.
Figure 2
Figure 2. Variable in vitro secretion, collagen binding, and platelet GPIαb binding of type 2M variants
Data shown represents averages of 3–4 different assays on 3–4 separate transfections. Error bars represent 1 standard deviation. Panel A: Expression. Variants 1283Y, 1374C and 1453N show a marked decrease in expression relative to wild-type. Variants 1162L and 1349C have normal expression. Panel B: Collagen binding. Binding to collagen 3 is shown in light gray and binding to collagen 4 is shown in dark gray. Variants 1283Y, 1349C, and 1374C have decreased binding to collagen 3 and 4. 1453N has absent binding to both collagen 3 and 4. 1162L has normal binding to collagen 3 and 4. Panel C: Platelet GPIαb binding. Variants 1283Y, 1349C, 1374C, and I1453N have absent binding to platelet GPIb. 1162L has normal binding. Data represents the average of three different VWF:GPIbM ELISA assays performed on three separate transfections.
Figure 3
Figure 3. Some homozygous variants have defective multimer structure that is improved when expressed in heterozygous form
Variants 1283Y, 1349C, 1374C, and 1453N have loss of HMWM when expressed as homozygotes. Variants 1283Y, 1349C, 1374C, and 1453N have improved multimer distribution when expressed as heterozygous variant/wild-type. The multimer distribution for homozygous 1162L is similar to wild-type VWF.
Figure 4
Figure 4. Intersecting phenotypes for types 2A and 2M VWD
This diagram shows the overlap in phenotype for the variants described here, combining attributes of type 2A and type 2M VWD.
See this image and copyright information in PMC

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