Platelet response to agonists and spreading. (A) Platelet response to CRP, thrombin, and ADP was assessed by flow cytometry in whole blood, measuring fibrinogen binding and P-selectin expression. Although the response to CRP and thrombin was significantly increased in JAK2V617F platelets, the reverse was observed in response to ADP. For each experiment, blood from control and JAK2V617F mice was tested in duplicate at each agonist concentration. P-selectin expression and fibrinogen binding are presented as a percentage of the maximal response obtained in control blood for each agonist. Error bars represent SD. (B) Platelet stimulation in response to collagen relies on the existence of shear. This was therefore measured in classical light transmission aggregometry at 3 different concentrations of collagen in at least 3 experiments comparing a control and JAK2V617F sample. Aggregation in response to collagen was significantly increased at a medium concentration (2 μg/mL) but not at higher concentrations. Error bars represent SD. (C) Platelet spreading was assessed by adhering control and JAK2V617F platelets onto a fibrinogen-coated surface in static conditions for 45 minutes at 37°C in 5 different experiments. Pictures were taken with a DIC microscope (scale bars represent 10 μm), and spreading measured using ImageJ software, counting at least 50 platelets for each sample. JAK2V617F platelets showed significantly greater spreading, including some platelets displaying full lamellipodia formation (white arrowheads) even in the absence of exogenous agonists. Error bars represent SD. (D) Outside-in signaling downstream of integrin αIIbβ3 was studied in platelets adhered to fibrinogen (compared with unstimulated platelets plated onto BSA-coated surface). The western blots show increased phosphorylation of Plcγ2 and Syk in JAK2V617F platelets adherent to fibrinogen compared with control platelets. Densitometry analysis confirmed that the difference observed was statistically significant (P < .05).