Ligand binding and spreading of CRISPR KO MKs in response to platelet agonists. Day 13 CRISPR KO and control MKs were treated for 10 minutes with convulxin (CVX, 2 μg/mL) in the presence of Alexa-Fluor 647–labeled fibrinogen and analyzed by flow cytometry. (A) Representative flow cytometry plots of unstimulated vs agonist-stimulated negative control or CRISPR KO MKs as indicated. Viable MKs were gated as described in the legend for Figure 2B and on MK marker CD42a-phycoerythrin (y-axis). The positive gate for labeled fibrinogen (x-axis) bound MKs was arbitrarily set with reference to unstimulated MKs. (B-C) Normalized mean ± SEM summaries of PAC-1–positive (B) or P-selectin–positive (C) cells (3 independent cords per group). Mixed effects analysis with Dunnett’s adjustment for multiple comparisons. (D) Representative image of spread and unspread control and GP6 KO MKs plated on collagen for 1 hour. (E-F) Mean ± SEM of the percentage of negative control or CRISPR KO MKs spread on collagen (E) or fibrinogen (F) for 1 hour. Shown are data from 3 cords with each sample plated in duplicate wells; >20 fields and >200 MKs were scored (blinded to treatment) per sample. Repeated measures ANOVA with Dunnett’s adjustment for multiple comparisons: *P < .05; **P < .01; ***P < .001; ****P < .0001.