Analysis of protein Tyr phosphorylation, cytosolic [Ca²⁺] increase, and aggregation triggered by low thrombin concentration in normal and pathological platelets. (A-B) Platelets from healthy donors (lanes 1-4), and PV (lanes 5-8) or ET patients (lanes 9-12), were stimulated with either 20 mU/mL (A) or 100 mU/mL (B) of thrombin for the indicated times. Cells were then lysed and analyzed by Western blot with anti–P-Tyr and β-actin antibodies. (C) [Ca²⁺]_c increase was determined as detailed in “Determination in cytosolic [Ca²⁺] concentration” in platelets challenged with either 20 mU/mL or 100 mU/mL thrombin. The traces are representative of experiments performed with 10 normal (N), 7 PV, and 10 ET JAK2^V617F-negative or -positive samples. (D) Aggregation was determined as detailed in “Platelet aggregation” in platelets pretreated with either vehicle (V) or PP2 and then challenged with 20 mU/mL thrombin. The traces are representative of experiments performed with 4 normal (N) and 4 ET samples.