Disrupting GP1bα-flnA interaction results in aberrant megakaryocyte budding in bone marrow. Bone marrows isolated from hGPIbα^WT and hGPIbα^FW mice were immunostained with antibodies against CD41 (green, for platelets and megakaryocytes), laminin (red, for vessels), P-selectin, or VWF (magenta), in addition to a nuclear stain (Hoechst 33342 [blue]), as indicated. (A) Representative confocal images showing intravascular and interstitial platelets/buds (arrowheads) in hGPIbα^WT and hGPIbα^FW bone marrows, at low and high magnifications (63×; scale bar, 50 μm; inset scale bar, 20 μm). (B-C) Quantification of the total number of CD41⁺ buds (interstitial and intravascular) (B) and interstitial buds (C) per 370 μm² field, generated from tile scans in hGPIbα^WT and hGPIbα^FW mice. Graphs represent the mean ± SEM of 3 mice; ∗∗P < .01. (D) Representative STED image showing a granular VWF-containing interstitial bud in hGPIbα^FW bone marrow interstitium (93×; scale bar, 10 μm; inset scale bar, 3 μm). (E) Representative confocal and Airyscan (inset) image showing a granular P-selective containing interstitial bud in hGPIbα^FW bone marrow (63×; scale bar, 30 μm; inset scale bar, 5 μm). (F-G) Bone marrow MK sinusoidal localization was analyzed in hGPIbα^WT and hGPIbα^FW mice. (F) Tiled confocal images showing MK sinusoidal and interstitial localization in bone marrows of hGPIbα^WT and hGPIbα^FW mice; scale bars, 50 μm; inset scale bar, 20 μm. (G) Quantification of the percentage (%) megakaryocytes with sinusoidal contacts (SC) or without contact (BMHC) or within sinusoids (intrasinusoidal). Results depict the mean ± SEM; n = 3 mice, with at least 50 megakaryocytes analyzed per mouse; ∗P < .05, ∗∗P < .01, 2-way ANOVA with Bonferroni correction. ANOVA, analysis of variance; SEM, standard error of the mean.