Thrombopoietin enhances SFLLRN-stimulated aggregation, fibrinogen binding, intracellular signaling, and Rap1 activation in a JAK2/PI3 kinase-dependent manner. Platelets were incubated for 10 minutes with 10μM JAK2i IV (A,C,H) or 100nM wortmannin (D,E,H), followed by 5 minutes incubation with 100 ng/mL TPO (A,C,H) or 100nM IGF-1 (B). Platelets were subsequently stimulated with 0.5μM SFLLRN (A-B) or the indicated concentration of SFLLRN (C-H). Aggregation (A-B) was recorded for 5 minutes and FITC-fibrinogen binding (C-D) and P-selectin expression (E) was measured after 15 minutes stimulation as described in Figure 1A and B. Phosphorylated proteins (F-G) were analyzed by immunoblotting of platelet lysates obtained 5 minutes after stimulation and Rap1 activation (H) by GST-RalGDS pull-down was analyzed from extracts obtained 5 minutes after stimulation. (A,B,F,G,H) Representative results for at least 3 independent experiments. Alternatively, results are expressed as average ± SEM of percentage maximal fibrinogen binding of vehicle treated platelets (C-D, n = 3) or percentage of maximum P-selectin surface expression of vehicle treated platelets (E, n = 3).