The mutation N528S in the von Willebrand factor (VWF) propeptide causes defective multimerization and storage of VWF

Abstract

We characterized a consanguineous Turkish family suffering from von Willebrand disease (VWD) with significant mucocutaneous and joint bleeding. The relative reduction of large plasma von Willebrand factor (VWF) multimers and the absent VWF triplet structure was consistent with type 2A (phenotype IIC) VWD. Surprisingly, platelet VWF was completely deficient of multimers beyond the VWF protomer, suggesting defective alpha-granular storage of larger multimers. Patients were nearly unresponsive to desmopressin acetate, consistent with a lack of regulated VWF release from endothelial cell Weibel-Palade bodies, suggesting defective storage also in endothelial cells. We identified an N528S homozygous mutation in the VWF propeptide D2 domain, predicting the introduction of an additional N-glycosylation site at amino acid 526 in close vicinity to a "CGLC" disulphide isomerase consensus sequence. Expression studies in mammalian cells demonstrated that N528S-VWF was neither normally multimerized nor trafficked to storage granules. However, propeptide containing the N528S mutation trafficked normally to storage granules. Our data indicate that the patients' phenotype is the result of defective multimerization, storage, and secretion. In addition, we have identified a potentially novel pathogenic mechanism of VWD, namely a transportation and storage defect of mature VWF due to defective interaction with its transporter, the mutant propeptide.

Figure 1

Pedigree and laboratory data of a family diagnosed with von Willebrand disease. Affected patients are represented by black symbols; unaffected persons, by half-filled symbols; squares, males; and circles, females. The laboratory test results are listed as follows: VWF:Ag (U/dL), VWF:CB (U/dL), platelet (plt) VWF:Ag (U/10¹¹ plts), and FVIII:C (U/dL); nd indicates not determined. Affected patients (black symbols) were found to have a homozygous N528S VWF mutation, whereas unaffected (half-filled symbols) were heterozygous for the mutation. Affected, N528S VWF homozygous patients had low VWF:Ag with substantially reduced VWF:CB and platelet VWF:Ag.

Figure 2

Desmopressin acetate test of patient II-3. VWF laboratory parameters were determined before (t = 0) and after administration of desmopressin acetate. Almost no response of VWF parameters was observed, except for FVIII:C.

Figure 3

Plasma and platelet VWF multimers in unaffected and affected family members. Plasma and platelet VWF multimers were assessed by SDS-agarose gel electrophoresis and Western blotting. Vertical line(s) have been inserted to indicate a repositioned gel lane. Lane 1 in both frames: platelet and plasma VWF of unaffected heterozygous family member, II-1. Lanes 2-4: affected family members II-2, II-3, and II-4 with a homozygous N528S VWF mutation. Affected family members have an abnormal plasma multimer distribution with a relative decrease of large multimers and pronounced oligomers and either a complete loss (II-2, II-3) or a severely decreased concentration (II-4) of large platelet VWF multimers.

Figure 4

Multimer distribution and intracellular localization of recombinant N528S-VWF. (A) Mutant N528S-VWF (lane 1) alone and coexpressed with wt VWF (lane 2) in 293 EBNA cells in the conditioned medium was assessed by SDS-agarose electrophoresis and Western blotting. Vertical line(s) have been inserted to indicate a repositioned gel lane. Recombinant wt VWF multimers are shown for comparison (lane 3). N528S-VWF multimers lack HMWM completely, whereas N528S coexpressed with wt VWF could not be distinguished from wt VWF alone. (B) AtT-20 cells were transiently transfected with (top panel) wild-type VWF or (bottom panel) N528S-VWF. Cells were immunostained for VWF and examined by confocal microscopy. N528S-VWF was not trafficked to storage granules.

Figure 5

Trafficking of N528S-VWFpp and coexpressed mature VWF in AtT-20 cells. (A) AtT-20 cells were transiently transfected with wild-type VWFpp or N528S-VWFpp. Transfected cells were fixed and dual-stained for VWFpp (i, iv) and ACTH (ii, v) and examined by confocal microscopy. The merged image of the 2 stains is shown in the last column (iii, vi) with colocalization shown in yellow. Both wild-type VWFpp and N528S-VWFpp appear to traffic to endogenous ACTH-containing storage granules. (B) Wild-type VWFpp and N528S-VWFpp were coexpressed with propeptide-deleted VWF (Δpro) in AtT-20 cells. Transfected cells were dual-stained for VWFpp (vii, x) and VWF (viii, xi). The merged image shown in the last column (ix, xii) with colocalization shown in yellow. For wild-type VWFpp coexpressed with Δpro, both VWFpp and VWF were colocalized in granules, whereas for N528S-VWFpp with Δpro, only VWFpp was localized in granules, and VWF demonstrated diffuse staining. (C) Wild-type and N528S-VWF were expressed and cells were dual-stained for VWFpp (xiii, xvi, xix) and VWF (xiv, xvii, xx). The merged image shown in the last column (xv, xviii, xxi) with colocalization shown in yellow. For wild-type VWF, both VWFpp and VWF were colocalized in granules. Cells expressing N528S-VWF showed either granular VWFpp staining with diffuse VWF staining or diffuse staining for both proteins.

Figure 6

VWF granule biogenesis in HEK293 cells transfected with mutant and wild-type VWF. (A) K9-VWD-AECs were transiently transfected with N528S-VWF, wild-type VWF, or pCIneo (Mock). Cells were immunostained for VWF (green; i, iv, vii) and P-selectin/CD62P (red; ii, v, viii) and intracellular localization examined by confocal microscopy. The merged image is shown in the last column with colocalization shown in yellow (iii, vi, ix). Wild-type VWF formed granules and recruited CD62P, whereas N528S-VWF failed to form granules. (B) HEK293 cells were transiently transfected with N528S-VWF, wild-type VWF-myc-his, or heterozygous N528S-VWF/wild-type VWF-myc-his. Cells were immunostained using monoclonal AVW-5 that does not recognize wild-type VWF-myc-his (green; x, xiii, xvi, xix) and rabbit antimyc that does not recognize the N528S-VWF lacking a myc-his tag (red; xi, xiv, xvii, xx). Intracellular localization was determined by confocal microscopy and the merged image shown in panels xii, xv, xviii, and xxi. When expressed alone, wild-type VWF formed granules, whereas N528S-VWF did not. When coexpressed, both N528S-VWF and wild-type VWF are localized in granules.