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Platelet desialylation in type 2B von Willebrand disease
ated increased β-galactose exposure in our study (n=2 patients) and also in vitro,10 we studied the possible role of desialylation in our recently engineered knock-in murine model of severe type 2B vWD (p.V1316M mutation) (Figure 1F). The murine disease mimics the human dis- ease, and 2B mice display thrombocytopenia4,11 (mean±SD platelet count: 355±104x109/L), relative to WT mice (803±159x109/L). Platelets from 2B mice had a significant- ly greater amount of exposed β-galactose (MFI for RCA: 7093±3156, n=67) than WT mice did (3154±1098, n=61; P<0.001) (Figure 1F). After taking platelet size into account by measuring the ratio between the MFI for RCA to the MFI for CD41 (aIIb integrin), a 2-fold elevation was still observed (RCA/CD41: 0.358±0.181 for 2B mice, n=67; 0.179±0.600 for WT mice, n=61; P<0.001) (Figure 1G). Thus, our results evidenced a link between the platelet count and platelet desialylation in type 2B vWD.
2B von Willebrand factor induces N-glycan-specific platelet desialylation
With a view to highlighting a direct link between the p.V1316M mutation in vWF and desialylation, we incu- bated plasma from 2B mice (p.V1316M) with WT platelets. In contrast to vWF-deficient or WT plasma, 2B plasma induced β-galactose exposure (with a 1.48±0.12- fold increase) (Figure 2A) to the same extent as botrocetin treatment of WT plasma (Online Supplementary Figure S1), as previously described.10 We confirmed that p.V1316M/vWF (0.2 μg/mL) induced desialylation (rela-
tive to WT/vWF) by incubating normal washed platelets with recombinant p.V1316M/vWF; we observed a 1.85±0.15-fold relative increase (Figure 2B). We confirmed this result with another lectin (ECL) that is specific for β-galactose (Figure 2C). We then investigated how gain- of-function vWF mediates platelet desialylation. Desialylation is due to the activity of neuraminidases NEU1, a sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. Relative to WT/vWF, p.V1316M/vWF treatment was able to induce the translo- cation of NEU1 to the platelet surface (Figure 2D).
Platelet glycoproteins are commonly modified by com- plex carbohydrates including N-linked glycans (N-glycans) and mucin-type O-linked glycans (O-glycans).7,14 Both N- and O-glycans are commonly 'capped' by sialic acids. We next looked at whether the desialylation induced by the p.V1316M/vWF occurred on O-glycans and/or N-gly- cans. To this end, we used MALII lectin which recognizes a-2,3-sialylation on O-glycans,7,13 and a recent study found that the absence of O-glycans (in C1galt1-/- mice) reduced MALII binding but did not change RCA binding.7 We first confirmed the specificity of MALII by using desialylated platelets showing a decrease of the MALII binding, as expected (Online Supplementary Figure S2). We found that p.V1316M/vWF did not change MALII binding to platelets, relative to WT/vWF (Figure 2E), suggesting that desialylation was not likely to be on O-glycans. We next looked at whether the desialylation induced by the 2B vWF occurred on N-glycans by using PNGase F, the most
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Figure 1. Platelet desialylation in human and murine type 2B von Willebrand disease (vWF) in vivo. (A-B) Analysis of the correlation between RCA binding and the platelet count in patients with type 2B vWD (n=36) (A) (left panel: r2=0.113 (P=0.048) and healthy controls (n=35) (B) r2=0.095 (P=0.092). Distribution of platelet counts (C) and RCA mean fluorescence intensity (MFI) (D) or RCA/MPV (E) values in healthy controls and in patients with type 2B vWD, as a function of the mutation in the vWF A1 domain (p.R1341Q, n=30, p.R1306Q, n=4, p.V1316M, n=2). A one-way ANOVA was followed by Dunnett’s test; *P<0.05. (F-G) Distribution of RCA MFI (F) or RCA/CD41 (G) values in WT (n=61) and 2B mice (n=67) (unpaired Student’s t-test; ***P<0.001).
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