TPO treatment of platelets results in tyrosine phosphorylation and activation of PI 3-kinase.

(A) Phosphorylation of p85 subunit of PI 3-kinase. Platelet samples were treated with 10 μg/mL collagen (2 minutes), 50 ng/mL TPO (3 minutes), or the combination of both agents (TPO for 3 minutes, followed by collagen for 2 minutes). The platelets were then lysed under nondenaturing conditions in 1% NP-40 and protein immunoprecipitated with p85 antiserum. The immunoprecipitates were submitted to SDS-PAGE and, after transfer, the membrane was blotted with the antiphosphotyrosine monoclonal antibody (mAb) 4G10 and exposed by ECL (upper panel). The same membrane was stripped and reprobed with anti-p85 serum (lower panel). Results are from 1 experiment representative of 5; the extent of phosphorylation varied widely between donors. (B) TPO stimulates an increase in PI 3-kinase in p85 immunoprecipitates. Activity was measured by monitoring formation of phosphatidylinositol 3-monophosphate as described in “Materials and methods.” Results are performed in duplicate and are representative of 2 experiments.