The 420Q-GNE variant is associated with platelet hyposialylation and enhanced hepatic clearance. (A) Peripheral blood smear of P1. (B) Relative expression of the glycoprotein Ib-V-IX complex (GPIbα), fibrinogen receptor αIIbβ3 (αIIb), and collagen receptors glycoprotein VI (GPVI) and α2β1 (α2) in P1 (red dots) and 49 healthy controls measured with flow cytometry. Data of healthy controls (green) are expressed in box and whiskers. Average median fluorescent intensity values in controls were set at 100%. Whiskers represent 2.5th and 97.5th percentiles. (C) Platelet reactivity measured with flow cytometry in P1 (red dot) and 49 healthy controls (green box and whiskers). Platelets were stimulated with 60 μM ADP, 25 μM protease-activated receptor (PAR)-1 activating peptide (PAR1-AP) SFLLRN, 250 μM PAR4-AP AYPGKV, 1 μg/mL cross-linked collagen-related peptide (CRP-XL), or 5 μM U-46619, fixated and analyzed for expression of P-selectin as marker for granule secretion and fibrinogen binding as marker for αIIbβ3 activation. Average median fluorescent intensity values in controls were set at 100%. Whiskers represent 2.5th and 97.5th percentiles. (D) Wild-type (WT), GNE-deficient (KO), and GNE-deficient HEK293 cells overexpressing either recombinant WT-GNE, or recombinant 420Q-GNE (RQ) were incubated with fluorescein-conjugated SNA lectin for analysis of sialic acid expression, or fluorescein-conjugated RCA-1 lectin for analysis of galactose expression. Lectin binding was assessed with flow cytometry. Data were normalized on lectin binding in WT HEK293 cells (n = 3). *P < .05, error bars represent the standard deviation. (E) Platelet sialic acid exposure was measured with fluorescein-SNA lectin and galactose exposure was measured with fluorescein-RCA-1 lectin on a flow cytometer in both P1 and P2, their parents, 4 thrombocytopenic patients, and 68 healthy controls. MFI, median fluorescent intensity. (F) Autologous platelets were labeled with ¹¹¹In-tropolone and injected into P1. Platelets were collected at different time points to determine platelet half-life. The fraction ¹¹¹In-labeled platelets at 30 minutes after injection was set at 100%. Data represent the relative proportion of ¹¹¹In-labeled platelets at each time point. (G) Anterior static single-photon emission computed tomography scans of the abdomen of P1 were made with a Symbia T2 γ camera (Siemens, Erlangen, Germany) at indicated time points to quantify platelet sequestration. Radioactivity in liver and spleen regions (thick black lines) was assessed as the percentage of total radioactivity. Spleen:liver radioactivity ratio <0.8 indicates hepatic sequestration. (H) Celltracker Deep Red-labeled platelets of P1 (red) (n = 2) and healthy controls (green) were incubated with THP-1 macrophages (n = 4) and HepG2 hepatocytes (n = 3). Data are normalized on the number of platelet-binding cells with control platelets. (I) Platelets from P1 (red) (n = 3) or healthy controls (green) (n = 3) were labeled with fluorophore conjugated anti-GPIbα Fab′-fragments (6B4). Fluorophore lifetime in presence and absence of an acceptor was assessed and used to calculate fluorescence resonance energy transfer efficiency. (J) Change in platelet count in P1 and P2 after initiation of treatment with romiplostim (black arrow).