Accelerated platelet clearance accounts for macrothrombocytopenia in Twf2a^−/− mice. (A-B) Platelet count (A) and size (B) were determined with an automated cell analyzer (Sysmex). (C) Platelet lifespan was assessed by flow cytometric measurement of the fluorescence-positive platelet population at the indicated time points after injection of a fluorophore-conjugated anti-GPIX antibody derivative. (D-E) Platelet counts were monitored over time after clodronate-encapsulated liposome-mediated macrophage depletion (D) and splenectomy (E). (F-G) The relative platelet content in control and Twf2a^−/− spleens was determined by immunostaining (platelet GPIb, cyan; endothelial CD105, magenta) and confocal microscopy of cryosections. Scale bars, 250 μm. Images were acquired with a TCS SP8 confocal microscope (25×/0.95 FLUOTAR VISIR water objective, Leica Microsystems) and are representative of at least 4 individuals. (H-I) The fraction of aged platelets was assessed by platelet lectin-binding (Erythrina crista-galli lectin [ECL] and Ricinus communis agglutinin [RCA]) and the fraction of young (RNA-rich) platelets by thiazole orange (TO) staining and flow cytometry. The overall gated platelet population (based on forward sideward characteristics and GPIX labeling) was set as 100%. Neuraminidase-treated platelets were used as a positive control (H) and RNase-treated platelets as a negative control (I). Values represent mean ± SD (n = 6). Each symbol represents 1 individual. Horizontal lines represent mean (I). ***P < .001, **P < .01, and *P < .05, unpaired Student t test. NS, not significant; WT, wild-type.