Figure 2
Figure 2. Analysis of JAK2 activation state in resting and thrombin-stimulated platelets. (A) Tyrosine kinase activity of wild-type or V617F-mutated JAK2. Platelets obtained from healthy donors (lanes 1-3), PV patients homozygous for JAK2V617F (lanes 4-6), and ET patients heterozygous for JAK2V617F (lanes 7-9) were lysed in IP-buffer and immunoprecipitated with anti-JAK2 antibody. The immunocomplexes (IPs) were then analyzed for JAK2 activity tested in vitro toward the substrate polyGlu4Tyr in the presence of [γ33P]ATP and vehicle (lanes 1,4,7), or 10μM AG490 (lanes 2,5,8), or 1μM JAK2-inhibitor-I (lanes 3,6,9) as described in “In vitro tyrosine kinase assays.” Samples were subjected to SDS/PAGE and transferred to nitrocellulose sheets, which were analyzed for the radioactivity incorporated in polyGlu4Tyr by a Packard Cyclone (columns of the panel) and then immunostained with anti-JAK2 antibody (Wb of the panel). Experiments with AG490 and PP2 were performed with 8 normal, 5 JAK2V617F homozygous PV, and 7 JAK2V617F heterozygous ET samples. Experiments with AG490, JAK-inhibitor-I, and PP2 were performed with 4 normal, 3 JAK2V617F homozygous PV, and 4 JAK2V617F heterozygous ET samples. **P < .001 versus JAK2 activity tested in the absence of inhibitors. (B-C) Platelets, isolated from healthy donors, and PV or ET patients, were treated without (odd lanes) or with 100 mU/mL thrombin (even lanes) for 1 minute in the absence (lanes 1-2) or presence of 10μM AG490 (lanes 3-4), or 1μM JAK2-inhibitor-I (lanes 5-6), or 10μM PP2 (lanes 7-8). (B) Platelet lysates were analyzed by Western blot with an anti–phospho-JAK2 (P-JAK2) antibody that recognizes the activated form of the kinase. Blots were then stripped and reprobed with anti-JAK2 antibody. (C) Platelet lysates were analyzed by Western blot with anti–phospho-STAT5 (P-STAT5) antibody and reprobed with anti-STAT5 antibody. Means of densitometric values are reported above the bands relative to P-JAK2 (B) or P-STAT5 (C). Experiments in panels B and C were performed with 12 normal, 8 PV (JAK2V617F homozygous and heterozygous), and 15 ET samples. Experiments with JAK-inhibitor-I were performed with 4 normal, 3 JAK2V617F homozygous PV, and 4 JAK2V617F heterozygous ET samples. (B) **P < .001 versus P-JAK2 in the absence of JAK2 inhibitors. (C) ** P < .001 and *P < .01 versus P-STAT5 in the absence of inhibitors. #P < .001 PV and ET versus normal platelets.

Analysis of JAK2 activation state in resting and thrombin-stimulated platelets. (A) Tyrosine kinase activity of wild-type or V617F-mutated JAK2. Platelets obtained from healthy donors (lanes 1-3), PV patients homozygous for JAK2V617F (lanes 4-6), and ET patients heterozygous for JAK2V617F (lanes 7-9) were lysed in IP-buffer and immunoprecipitated with anti-JAK2 antibody. The immunocomplexes (IPs) were then analyzed for JAK2 activity tested in vitro toward the substrate polyGlu4Tyr in the presence of [γ33P]ATP and vehicle (lanes 1,4,7), or 10μM AG490 (lanes 2,5,8), or 1μM JAK2-inhibitor-I (lanes 3,6,9) as described in “In vitro tyrosine kinase assays.” Samples were subjected to SDS/PAGE and transferred to nitrocellulose sheets, which were analyzed for the radioactivity incorporated in polyGlu4Tyr by a Packard Cyclone (columns of the panel) and then immunostained with anti-JAK2 antibody (Wb of the panel). Experiments with AG490 and PP2 were performed with 8 normal, 5 JAK2V617F homozygous PV, and 7 JAK2V617F heterozygous ET samples. Experiments with AG490, JAK-inhibitor-I, and PP2 were performed with 4 normal, 3 JAK2V617F homozygous PV, and 4 JAK2V617F heterozygous ET samples. **P < .001 versus JAK2 activity tested in the absence of inhibitors. (B-C) Platelets, isolated from healthy donors, and PV or ET patients, were treated without (odd lanes) or with 100 mU/mL thrombin (even lanes) for 1 minute in the absence (lanes 1-2) or presence of 10μM AG490 (lanes 3-4), or 1μM JAK2-inhibitor-I (lanes 5-6), or 10μM PP2 (lanes 7-8). (B) Platelet lysates were analyzed by Western blot with an anti–phospho-JAK2 (P-JAK2) antibody that recognizes the activated form of the kinase. Blots were then stripped and reprobed with anti-JAK2 antibody. (C) Platelet lysates were analyzed by Western blot with anti–phospho-STAT5 (P-STAT5) antibody and reprobed with anti-STAT5 antibody. Means of densitometric values are reported above the bands relative to P-JAK2 (B) or P-STAT5 (C). Experiments in panels B and C were performed with 12 normal, 8 PV (JAK2V617F homozygous and heterozygous), and 15 ET samples. Experiments with JAK-inhibitor-I were performed with 4 normal, 3 JAK2V617F homozygous PV, and 4 JAK2V617F heterozygous ET samples. (B) **P < .001 versus P-JAK2 in the absence of JAK2 inhibitors. (C) ** P < .001 and *P < .01 versus P-STAT5 in the absence of inhibitors. #P < .001 PV and ET versus normal platelets.

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