Assessment of myeloid progenitor amplification and signaling in platelets from JAK2-mutated patients from families 1 and 2. Mononuclear cells from R867Q (n = 2) patients, S755R/R938Q (n = 4) patients, or control donors (n = 5) were purified and plated (A-B) in methylcellulose with SCF/IL-3/EPO to study BFU-E progenitors or (C-D) in serum-free fibrin clot with SCF and increasing doses of TPO, (E) with 10 ng/mL TPO to study CFU-MK progenitors. Data represent means ± standard deviation for each patient plated in triplicate. (F-G) Histograms represent the means ± SEM of CFU-MK progenitors in the presence of SCF and TPO (10 ng/mL) for patients (n = 5 for S755R/938Q, n = 3 for R867Q) and controls (n = 4). Two-tailed Student t test: **P < .01. (H-I) The numbers of MKs per cluster were counted. (J-K) Platelets were isolated from R867Q (n = 2) patients, S755R/R938Q (n = 4) patients, or control donors (n = 3); washed in phosphate-buffered saline; and stimulated or not with TPO (5 ng/mL). Platelets were lysed, and the phosphorylation status of JAK2, STAT1, STAT3, STAT5, AKT, and ERK1/2 was examined by western blotting with the respective anti-phospho specific antibodies, as indicated. Expressions of HSC70 and of total STAT proteins, AKT, and ERK in the samples were used as loading controls. Blots show representative results in 2 S755R/R938Q patients (P1, P3) (J) and 2 R867Q patients (P′2, P′3) (K).