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A. Dupont et al.
effective enzymatic method for removing N-linked oligosaccharides from glycoproteins. We first character- ized the effect of PNGase F treatment on RCA, ECL and MALII binding on platelets. PNGase F treatment reduced the binding of RCA (to 40±4% of the control value) (Figure 2F) and ECL (to 50±10%) (data not shown) to WT platelets but not that of MALII. Given that RCA and ECL were sensitive to PNGase F, the platelets were then incu- bated with WT or p.V1316M/vWF to induce desialylation and were then treated (or not) with PNGase F during 18 h. Platelet desialylation was still observed after 18 h of incu- bation in the absence of PNGase F (RCA ratio: 0.97±0.04 for WT, and 1.49±0.13 for 2B; P<0.01) (Figure 2G). Strikingly, removal of N-glycans after p.V1316M/vWF treatment was associated with much lower RCA binding (Figure 2G, P<0.001). Indeed, RCA binding in the presence of WT or 2B vWF was similar after PNGase F treatment (RCA ratio: 0.62±0.07 for WT, and 0.72±0.06 for 2B). Taken as a whole, our results demonstrated for the first time that the p.V1316M mutation in vWF induces N-gly- can-specific platelet desialylation.
2B von Wilebrand factor induces aIIb and β3 desialylation
Many platelet glycoprotein and surface receptors contain sialic acid. GPIba contains the highest levels, followed by
integrin β3, integrin aIIb, GPV and GPVI/GPIbβ/GPIX, for the main receptors.7 We first investigated whether GPIba and GPVI were desialylated-targets by the p.V1316M/vWF. We found that treatment with OSGE, which removes GPIba and, to some extent, GPVI but not aIIbβ3 did not change RCA and ECL binding (Figure 3A), suggesting that the desialylation induced by p.V1316M/vWF did not take place on GPIba and GPVI. We then confirmed that p.V1316M/vWF did not induce GPIba and GPVI desialyla- tion by performing RCA pull-down experiments followed by western blotting (Figure 3B). These data confirm our results and rule out mouse GPIba and GPVI as a specific platelet desialylation targeted by p.V1316M/vWF. Taken as a whole our present results reveal the existence of desialy- lation of platelet proteins other than GPIba and GPVI.7,15 The next candidates were the integrins aIIb and β3 carry- ing sialic acid both on N- and O-glycans.7,15 We performed RCA pull-down experiments by incubating normal washed platelets with recombinant WT/vWF or p.V1316M/vWF. Our results demonstrated that p.V1316M/vWF induced aIIb and β3 desialylation. Indeed, we observed a 1.90±0.17-fold relative increase for aIIb and a 2.02±0.36-fold relative increase for β3 compared to WT/vWF (Figure 3C). Taken as a whole, our results demonstrated for the first time that the p.V1316M muta- tion in vWF induces aIIb and β3 desialylation.
ABCD
EFG
Figure 2. Platelet desialylation by 2B von Willebrand disease (vWF) in vitro occurs on N-glycans. (A) A histogram of RCA lectin binding on wild-type (WT) mouse platelets in PRP treated with vWF-deficient plasma (vWF-dp), WT plasma or 2B plasma. The fold change in each experiment was calculated relative to the binding obtained with vWF-deficient plasma, set to 1. The mean±Standard Deviation (SD) values (n=3 experiments) were compared using a one-way ANOVA and Dunnett’s post-test; **P<0.01. (B) A histogram of RCA lectin binding to washed WT mouse platelets treated with WT or 2B mouse vWF (p.V1316M). (C) Histograms of RCA and ECL binding (for β-galactose exposure) on washed WT mouse platelets treated with 0.2 μg/mL WT or 2B mouse vWF. The fold change in each experiment was cal- culated relative to the baseline value (in the absence of vWF), set to 1. The mean±SD values (n=4 experiments) were compared using a one-way ANOVA and Dunnett’s post-test; **P<0.01). (D) Flow cytometric analysis of NEU1 expression on washed WT mouse platelets treated with 0.2 μg/mL WT or 2B mouse vWF; *P<0.05. (E) Histograms of MALII lectin binding (for α-2,3-linked sialic acids on O-glycans) on washed WT mouse platelets treated with 0.2 μg/mL WT or 2B mouse vWF. (F) Flow cytometric analysis of WT platelets after treatment (or not) with PNGase F and staining with RCA and MALII lectin. The data are representative of three independent experiments. (G) A histogram of RCA binding on washed WT mouse platelets treated with 0.2 μg/mL WT or 2B vWF. Platelets were then treated (or not) with PNGase F. The mean±SD values (n=3 experiments) were compared using a one-way ANOVA and Dunnett’s post-test. ***P<0.001.
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haematologica | 2019; 104(12)