Analysis of PKA activity in patient platelets and megakaryocytes. (A-B) Western blot analysis and quantification of GPIbβ phosphorylated at Ser¹⁶⁶ in MKs derived in vitro from (A) blood CD34⁺ progenitors and (B) in platelets of patients homozygous (II-1 and II-2) for the PRKACG p.74I>M mutation. Two external controls (C1 and C2) were analyzed. Total GPIbβ was used as a control of protein loading. MKs were investigated at (A) day 12 of culture. (C-D) Western blot analysis and quantification of filamin A in (C) MKs and (D) platelets of patients carrying the homozygous (II-1 and II-2) mutation. Two external controls (C1 and C2) were used. Actin or HSC70 was used as a control of protein loading. (E) Analysis of cAMP level in platelets isolated from one external (C1) and one internal control (II-3) and from 2 patients homozygous (II-1 and II-2) and 1 heterozygous for the PRKACG p.74I>M mutation (III-1). Error bars represent mean ± SD of triplicate. Experiments were performed 2 times with similar results. *P < .05, 2-tailed Mann-Whitney test.