* em P /em 0.05; ** em P /em 0.01 by unpaired em t /em -test. the platelet surface, and induces ligand-independent GPIb-IX signaling in human VX-770 (Ivacaftor) and murine platelets. These results suggest that desialylation of O-glycans of GPIb induces unfolding of the mechanosensory domain, subsequent GPIb-IX signaling including amplified desialylation of N-glycans, and eventually rapid platelet clearance. This new molecular mechanism of GPIb-facilitated clearance could potentially resolve many puzzling and seemingly contradicting observations associated with clearance of desialylated or hyposialylated platelet. Introduction More than 100 billion platelets are cleared every day from a human body through a highly efficient and tightly regulated process. Exogenous agents could commandeer the process and accelerate platelet clearance, leading to thrombocytopenia and hemorrhage. One such agent is neuraminidase, which hydrolyzes the glycosidic linkages of sialic acids to the host glycoprotein and as a consequence exposes the penultimate galactoses. Injection of neuraminidase causes thrombocytopenia in mice or rats within a few hours, followed by a gradual rise in the platelet count back to the normal level in 4-5 days due to continuous thrombopoiesis in the body.1,2 Certain bacterial infection involves the release of bacterial neuraminidase in the blood and thrombocytopenia, VX-770 (Ivacaftor) often before the onset of septic shock.3,4 Moreover, there is accumulating evidence to support the involvement of endogenous neuraminidase in platelet clearance. For example, cold storage of murine platelets induces presentation of lysosomal neuraminidases on the plasma membrane, accelerating their clearance upon infusion into a recipient mouse.5,6 Many antibodies targeting the N-terminal ligand-binding domain (LBD) of platelet glycoprotein (GP)Ib induce platelet signaling and surface presentation of lysosomal neuraminidase 1 (Neu1), leading to thrombocytopenia.7-9 Binding of plasma von Willebrand factor (VWF) to GPIb on the platelet produces similar signaling events including desialylation.10-12 Therefore, it appears that neuraminidase is critically involved in platelet clearance. It has been suggested that after platelet desialylation the exposed galactose residues on the platelet are recognized by the Ashwell-Morell VX-770 (Ivacaftor) receptor (AMR), also known as the asialoglycoprotein receptor, on the surface of liver macrophages and hepatocytes, thereby inducing internalization of the desialylated platelet by these cells and its clearance from the circulation. In support of this model, injection of neuraminidase does not reduce platelet counts in mice lacking AMR and fast clearance of desialylated platelets is significantly reduced in mice lacking AMR.3,5,13 Consistently, genetic ablation of certain sialyltransferases such as ST3Gal-IV in mice results in constitutive exposure of galactoses on the platelet, accelerated platelet clearance, and VX-770 (Ivacaftor) a significantly lower platelet count.14 Upon transfusion, St3gal4-/- or desialylated platelets are cleared at a faster rate than wild-type (WT), unless the recipient mice were pretreated with asialofetuin, a competitive inhibitor of AMR.5,15 While the involvement of the AMR in mediating clearance of desialylated platelets has been established, the underlying molecular mechanism remains controversial, as several FGF1 studies reported seemingly contradictory or confusing observations. AMR is a multi-subunit receptor complex that contains several lectin domains for binding of galactose or galactosamine.16-18 It exhibits a much higher binding affinity and ligand preference for tetra- or triantennary galactoses than di- or mono-antennary ones.17,19,20 In other words, AMR binds primarily exposed galactose residue on N-glycans instead of O-glycans because only the former supports a tetra- or tri-antennary sugar structure. It was observed that proteolytic removal of the N-terminal ligand-binding domain (LBD) of GPIb enhanced survival of transfused St3gal4-/- platelets or coldstored WT platelets.5,15 Since the LBD of human GPIb contains 2 N-glycans,21-23 desialylated N-glycans on the LBD were suggested as the ligands for AMR.5,15 However, these experiments were performed on murine platelets, yet murine GPIb does not contain any N-glycosylation sites (i.e., Asn-X-Ser/Thr) and therefore should have no Nglycans for AMR binding (was from New England Biolabs (Ipswich, MA, USA). C57BL/6J and VWF-/- mice were purchased from Jackson Laboratories (stock ns. 000664 and 003795, respectively). Transgenic hTg, GPIb-/-, and IL4R-IbTg mice have been described previously.28,29 St3gal1fl/fl mice carrying LoxP sites on exon 2 (stock n. 006897) were backcrossed in to the C57BL/6J background and paired with Pf4-Cre mice to delete St3gal1 in the megakaryocyte lineage (St3Gal1MK-/-). Similarly, Adam17fl/fl mice (stock n. 009597) were paired with Pf4-Cre mice to delete Adam17 in the megakaryocyte lineage (Adam17MK-/-). Six- to 8-week-old mice of both sexes were.