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Platelet desialylation in type 2B von Willebrand disease
meabilized platelets, and was able to translocate to the platelet membrane.9 Plasma-membrane-bound NEU1 modulates a plethora of receptors by desialylation. At the plasma membrane, NEU1 has been shown to be required for signal transduction, and recent results have provided new insights in the molecular organization of membrane-bound NEU1. Indeed, the protein has two potential transmembrane domains that may anchor NEU1 to the membrane and control its dimerization and sialidase activity.19 It has been reported that the sialidase NEU1 is involved in platelet desialylation.20 In the pres- ent study, we found a translocation of NEU1 at the platelet surface after stimulation with the p.V1316M/vWF, suggesting a reorganization of platelet membrane. However, neither platelet activation nor P- selectin exposure were found after p.V1316M/vWF treat- ment (data not shown). These results are in agreement with previous reports. Indeed, patients with type 2B vWD have a bleeding tendency that is linked to the loss of vWF multimers and platelet dysfunction.21 Platelet functions were diminished due to the inhibition of inte- grin aIIbβ3 and of the small GTPase Rap1 by vWF/p.V1316M following exposure to platelet agonists. These data indicate that the type 2B mutation p.V1316M is associated with severe thrombocytopathy, and that the addition of 2B/vWF leads to platelet inhibition rather than activation. The mode of action of NEU1 and its localization and also the potential link between NEU1 activation and platelet dysfunction require further inves- tigations.
We determined which mode of desialylation was induced by type 2B vWF. We studied the binding of MALII lectin (which reportedly binds to a-2,3-sialylation on O- glycans) in the light of a recent study showing that O-gly- can desialylation is important for platelet clearance.7 The level of MALII lectin binding did not change in the pres- ence of type 2B/vWF (relative to WT/vWF). We next determined whether the desialylation induced by the 2B/vWF occurred on N-glycans. After the desialylation induced by 2B/vWF, we removed N-glycans by incubation with PNGase F. This treatment reduced RCA and ECL binding but not MALII binding. Taken as a whole, our results demonstrate for the first time that the p.V1316M mutation of vWF specifically induces desialylation on platelet N-glycans. One important question was the iden-
tification of targets. We ruled out desialylation of GPIba and GPVI. Indeed, we found that treatment with O-sialo- glycoprotein endopeptidase (OSGE, which removes GPIba and, partially, GPVI) did not change RCA and ECL binding, suggesting that the desialylation induced by 2B/vWF did not occur on GPIba and GPVI. Interestingly, assessment of baseline desialylation of GPIba-/- platelets (using RCA) revealed an unexpected elevation in desialy- lation, relative to WT platelets.15 Furthermore, treatment of GPIba-/- platelets with neuraminidase was associated with a 10-fold relative increase in RCA binding.15 On the other hand, mouse GPIba contains the highest levels of sialic acid on O-glycans but no sialic acid was predicted on N-glycans.7 The next obvious candidates were the inte- grins aIIb and β3 carrying sialic acid both on N- and O- glycans and in mouse platelets the integrins aIIbβ3 is one of the most sialylated glycoprotein on N-glycans.7 We per- formed RCA pull-down and our results demonstrated for the first time that p.V1316M/vWF induced aIIb and β3 desialylation. No study has yet demonstrated that the integrin aIIbβ3 is a target of desialylation.
We then looked at whether a minimum level of desialy- lation was required to significantly affect platelet count. To investigate the threshold of platelelet desialylation that is linked or not to thrombocytopenia, the reference inter- val was determined in the WT population, and the RCA value corresponding to the low platelet count was calcu- lated. We found a RCA value of 6.2, suggesting that, under this threshold, the thrombocytopenia observed is likely to be independent of desialylation, while above this thresh- old, the thrombocytopenia is likely to be desialylation dependent. However, this critical platelet desialylation threshold required to induce thrombocytopenia in vivo was not achieved in either 2B patients or 2B mice with the p.V1316M mutation (giving 2.1-fold and 2-fold differ- ences, respectively) and so does not explain the low platelet count.
To go further, the observed weak correlation between RCA binding and the platelet count in patients (Figure 1A) suggests that under stressful conditions where thrombo- cytopenia may be exacerbated (e.g. pregnancy or surgery), desialylation could be associated with other mechanisms involved in thrombocytopenia, such as platelet production defects in megakaryocytes,4 and the accelerated uptake of vWF/platelet complexes in macrophages.1 However, this
ABC
Figure 5. A threshold of platelet desialylation is required to affect the platelet count. (A) Relative whole-blood platelet counts (dashed red line) and platelet RCA mean fluorescence intensity (MFI) (solid black line) in WT mice 1 hour after in vivo treatment with neuraminidase (n=3 mice for each concentration, values are quot- ed as the mean±Standard Deviation). The increase in RCA binding was calculated for each mouse as the ratio between the RCA MFI after treatment and the RCA MFI before treatment. (B) The panel focuses on the stability of the relative platelet count and the increase in RCA binding after treatment with low doses of neu- raminidase (0, 2.5 and 5 mU/g of body weight). (C) The correlation between the relative platelet count and RCA binding after neuraminidase treatment (r2=0.95 in a linear regression).
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