Figure 6
Dasatinib impairs MK migration in response to a SDF1α gradient, spreading, proplatelet formation, and integrin-induced tyrosine phosphorylation. (A) Purified BM-derived mature MKs adherent on a fibronectin (20 μg/mL)–coated coverslip were allowed to migrate toward a SDF1α gradient over 3 hours within the Dunn chamber in which the outer well contained SDF1α (300 ng/mL) and dasatinib (10μM), as described in “Methods.” The migration paths over 3 hours were traced. The intersection of the x and y axes was taken to be the starting point of each cell path, whereas the source of SDF1α was at the top. The net translocation distance (displacement from the start to the end point) of each cell in the absence or presence of dasatinib is represented; ***P < .005. (B) Purified BM-derived mature MKs were incubated in the presence or absence of 10μM dasatinib for 15 minutes at 37°C. MKs were plated on a fibronectin-coated surface for 3 hours at 37°C. Adherent MKs were fixed and permeabilized and actin fibers were stained with rhodamine-phalloidin. Representative images and surface area quantification from 4 independent experiments are shown (scale bar = 20 μm); **P < .01. (C) Purified BM-derived mature MKs were incubated in the presence or absence of 10μM dasatinib for 15 minutes at 37°C. MKs were plated on a fibrinogen-coated surface for 5 hours at 37°C, fixed, and labeled with an anti–mouse GPIIb-FITC antibody. Representative images, percentage of MKs forming proplatelets, and proplatelet mean area were quantified and data are presented as means ± SEM of 4 independent experiments; **P < .01. (D) Purified BM-derived mature MKs were preincubated for 15 minutes with 10μM dasatinib and plated on a fibronectin (FN)- or BSA-coated dish for 3 hours. (i) MKs were lysed and whole-cell lysates (WCL) were western blotted with SFK activation loop p-Tyr-418, Src inhibitory site p-Tyr-529, and pan Src antibodies. (ii) Syk and PLCγ2 were immunoprecipitated from equal amounts of whole-cell lysates and blotted with an anti-phosphotyrosine antibody. Membranes were subsequently stripped and reblotted with anti-Syk and anti-PLCγ2 antibodies. (iii) MKs were lysed and whole cell lysates were western blotted with MLC-P and MLC antibodies. Western blots are representative of 3 independent experiments.

Dasatinib impairs MK migration in response to a SDF1α gradient, spreading, proplatelet formation, and integrin-induced tyrosine phosphorylation. (A) Purified BM-derived mature MKs adherent on a fibronectin (20 μg/mL)–coated coverslip were allowed to migrate toward a SDF1α gradient over 3 hours within the Dunn chamber in which the outer well contained SDF1α (300 ng/mL) and dasatinib (10μM), as described in “Methods.” The migration paths over 3 hours were traced. The intersection of the x and y axes was taken to be the starting point of each cell path, whereas the source of SDF1α was at the top. The net translocation distance (displacement from the start to the end point) of each cell in the absence or presence of dasatinib is represented; ***P < .005. (B) Purified BM-derived mature MKs were incubated in the presence or absence of 10μM dasatinib for 15 minutes at 37°C. MKs were plated on a fibronectin-coated surface for 3 hours at 37°C. Adherent MKs were fixed and permeabilized and actin fibers were stained with rhodamine-phalloidin. Representative images and surface area quantification from 4 independent experiments are shown (scale bar = 20 μm); **P < .01. (C) Purified BM-derived mature MKs were incubated in the presence or absence of 10μM dasatinib for 15 minutes at 37°C. MKs were plated on a fibrinogen-coated surface for 5 hours at 37°C, fixed, and labeled with an anti–mouse GPIIb-FITC antibody. Representative images, percentage of MKs forming proplatelets, and proplatelet mean area were quantified and data are presented as means ± SEM of 4 independent experiments; **P < .01. (D) Purified BM-derived mature MKs were preincubated for 15 minutes with 10μM dasatinib and plated on a fibronectin (FN)- or BSA-coated dish for 3 hours. (i) MKs were lysed and whole-cell lysates (WCL) were western blotted with SFK activation loop p-Tyr-418, Src inhibitory site p-Tyr-529, and pan Src antibodies. (ii) Syk and PLCγ2 were immunoprecipitated from equal amounts of whole-cell lysates and blotted with an anti-phosphotyrosine antibody. Membranes were subsequently stripped and reblotted with anti-Syk and anti-PLCγ2 antibodies. (iii) MKs were lysed and whole cell lysates were western blotted with MLC-P and MLC antibodies. Western blots are representative of 3 independent experiments.

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