Integrin activation, P-selectin exposure, and RAP1 activation of RASGRP2 KO MKs in response to short treatment with platelet agonists. (A-B) Day 13 RASGRP2 CRISPR KO and control MKs were treated for 2 minutes with thrombin (THR, 1 U/mL) or 3 minutes with convulxin (CVX, 2 μg/mL) or CRP (2 μg/mL), stained for activated αIIb/β3 (PAC-1) and P-selectin, and analyzed by flow cytometry according to the gating strategy described in the legend for Figure 2B. Normalized mean ± SEM summaries of % PAC-1–positive MKs (A) or % P-selectin–positive MKs (B) (3 independent cords per group). (C-D) Western blot and densitometry analysis of total RAP1B protein in control and GP6 or RASGRP2 KO MKs (3 independent cords per group). One-way analysis of variance (ANOVA) with Dunnett’s adjustment for multiple comparisons. (E) RAP1-GTP was precipitated from equal numbers of negative control, GP6, or RASGRP2 KO MKs activated with convulxin, lysed at the time points indicated, and analyzed by western blot. Shown is a representative of 2 independent experiments from 2 different cords, which are summarized by the densitometry analysis in panel F. Paired Student t tests: *P < .05; **P < .01.