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A. Dupont et al.
To investigate the threshold of platelelet desialylation linked or not to thrombocytopenia, reference interval has to be determined in the WT population. With the para- metric approach, the central 95% boundaries are specified by the mean±2SD, if the data follow a Gaussian (normal) distribution. After having confirmed if our values come from a Gaussian distribution by using a D’Agostino- Pearson omnibus normality, we calculated, in WT mice population, a mean platelet count of 803x109/L with SD=159 (n=129) and mean-2SD of 485x109/L. Under this value of platelet count, mice could be considered as thrombocytopenic. Based on the graph in Figure 5C, a minimal platelet count of 485x109/L corresponds to a RCA ratio of 6.2. Under this threshold, the thrombocytopenia observed is likely to be independent of desialylation, and above this threshold, the thrombocytopenia is likely to be desialylation dependent.
Discussion
In the present study, we tested the hypothesis whereby accelerated platelet clearance (and thus thrombocytope- nia) in type 2B vWD is caused by desialylation. Indeed, Deng et al. recently proposed a novel original mechanism
of platelet clearance mediated by active vWF.10 More specifically, authors demonstrated that type 2B vWF led to platelet desialylation, and predicted that this process might be responsible (at least in part) for the thrombocy- topenia observed in type 2B vWD. This hypothesis has yet to be tested in vivo.
We had access to blood samples from a cohort of 36 patients with three different mutations (p.R1341Q, p.R1306Q, and the severe p.V1316M) and from 35 healthy controls. We also studied our novel mouse model bearing a point mutation (p.V1316M) in the endogenous Vwf gene; we recently validated this mouse as a relevant model of type 2B vWD.4,11
Our present results indicated that even though the level of desialylation was abnormally high in both human and murine models, it was not sufficient to mediate thrombo- cytopenia. Indeed, treatments of 2B mice with the siali- dase inhibitors oseltamivir (Tamiflu®) and DANA did not correct the thrombocytopenia.
How does gain-of-function vWF mediate platelet desialylation? In platelets, the sialidase activity is due to the activity of neuraminidases. Neuraminidase 1 (NEU1) is a lysosomal sialidase catalysing the removal of termi- nal sialic acids from sialyloconjugates. Furthermore, this NEU1 was found to be abundant in the granules of per-
AB
Figure 4. Effect of platelet desialylation on the platelet count. (A) Platelet RCA mean fluorescence intensity (MFI) (left) and whole-blood platelet counts (right) in 2B (red line) and wild-type (WT) (black line) mice were measured at the indicated time points before and after treatment with a sialidase inhibitor (DANA or oseltamivir phosphate) or HBSS as a control (2B: n=4 mice for the control, n=11 mice for DANA, n=6 mice for oseltamivir phosphate, WT: n=4 mice for the control, n=3 mice for DANA, n=3 mice for oseltamivir phosphate). The mean±Standard Deviation values were compared using a one-way ANOVA and Dunnett’s post-testin a pre-/post- treatment comparison: *P<0.05; **P<0.01. (B) A histogram of RCA lectin binding (top) and the platelet count (bottom) before and six hours (h) after the injection of the drugs into WT and 2B mice. **P<0.01.
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haematologica | 2019; 104(12)