MK migration and proplatelet formation. (A) Cultured MKs from control and JAK2V617F mice were isolated by BSA gradient, and migration was assessed in a fibronectin-coated Dunn chamber under an SDF1α gradient. Each track represents an individual MK movement over a period of 4 hours. (B) Analysis of the data showed that JAK2V617F MKs migrated farther over the 4-hour time period compared with control MKs (n = 7 control MKs and 9 Jak2V617F MKs from 3 different cultures; error bars represent SD). (C) Proplatelet formation was assessed by plating culture-derived MKs from JAK2V617F and control animals onto fibrinogen-coated coverslips and fixing the cells at various time points before analysis using DIC microscopy (scale bars represent 50 μm). (D) The percentage of JAK2V617F MKs forming proplatelet at different time points was significantly increased compared with control MKs. Results are shown for 5 different MK cultures, with a minimum of 100 MKs analyzed in 5 different high-power fields for each individual experiment. Error bars represent SD. (E) Outside-in signaling downstream of integrin αIIbβ3 was studied in MKs adhered to fibrinogen (compared with unstimulated MKs plated onto BSA). The western blots show increased phosphorylation of Plcγ2 and Syk in JAK2V617F MKs adherent to fibrinogen compared with control MKs.