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Platelet desialylation in type 2B von Willebrand disease
Thrombocytopenia in type 2B mice is independent of platelet desialylation
Since desialylation is mediated by sialidases, we next treated 2B and WT mice with two sialidase inhibitors (DANA or oseltamivir phosphate) or Hank's balanced salt solution as a control. Treatment with each inhibitor was associated with significantly lower platelet desialylation in vivo (as measured by RCA binding) in 2B mice but not in WT mice (Figure 4A and B). After 6 h of treatment, the level of RCA binding was much the same as in the WT mice (Figure 4A and B). Surprisingly, the platelet counts remained low and unchanged in 2B mice for up to seven days after the infusion (168 h) (Figure 4A). Our observa- tion was especially surprising because both sialidase inhibitors have been reported to correct platelet counts in immune thrombocytopenia, with a desialylation profile after a single administration of a lower dose than that used in our experiments.9,16 Our findings indicate for the first time that the level of desialylation observed in type 2B vWD mice has only a minor role in platelet clearance or is not sufficient to induce thrombocytopenia. This lack of effect might be attributable to the type of desialylation. Although elevated platelet clearance has been reported in mice lacking N-glycan sialylation,17 a recent study provid- ed insights into the essential role of O-glycan sialylation in
A
platelet clearance.7 Furthermore, it has been reported that both mouse and human platelets contain high levels of O- glycans, with more sialic acids on the latter than on N-gly- cans.7,18
Threshold of platelet desialylation and thrombocytopenia
We then looked at whether the sialidase inhibitors’ apparent lack of effect in 2B mice was due to a threshold effect, i.e. whether a minimum level of desialylation was required to significantly affect platelet count. Hence, WT mice were treated with various concentrations of neu- raminidase, and the desialylation efficiency was moni- tored by RCA binding (Figure 5A). We observed a clear relationship between desialylation and the fall in the platelet count (Figure 5A). Importantly, the platelet count was not affected when the RCA ratio was around 2 or less (Figure 5B). We observed a strong and inverse correlation between the platelet count and RCA ratio (r2=0.95) and the circulating platelet count was about half the control value, corresponding to a RCA ratio of 10 (Figure 5C). This strongly indicates that the RCA ratio observed in patients and mice with the severe p.V1316M mutation (giving 2.1- fold and 2-fold differences, respectively) does not explain the low platelet count.
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C
Figure 3. Type 2B von Willebrand disease (vWF) induces desialylation of the integrin aIIbβ3. (A) Flow-cytometric analy- sis of wild-type (WT) platelets after being stained for GPIba, GPVI, aIIbβ3, RCA and ECL with or without (w/o) OSGE treat- ment. Graphs are representative of three independent exper- iments. (B) RCA pull-down and western blot. Washed WT mouse platelets were treated with WT or 2B mouse vWF dur- ing 1 hour and then lyzed and prepared to RCA pull-down. Samples were subjected to western blot to analyze: (B) GPIba and GPVI, (C) aIIb and β3. A histogram of aIIb and β3 in the
pull-down expressed in
pull-down)/total form (from lysate). The mean±Standard Deviation values (n=3 experiments) were compared using a one-way ANOVA and Dunnett’s post-test: *P<0.05; ***P<0.001.
desialylated form
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