PF4 induces aggregation through binding to c-Mpl and activation of the JAK2-STAT3/5 pathway. (A) PF4 dose-response assessed by light transmission aggregometry. Prewarmed platelets (2 × 10⁸/mL) at 37 °C were stirred at 1200 rpm for 1 minute before addition of PF4. (Ai) Representative platelet aggregation traces. (Aii) Quantification of aggregation (area under the curve [AUC] per minute) for 30 minutes (n = 4-13). (B) Phosphorylation of STAT3, STAT5, JAK2, and c-Mpl in PF4-stimulated platelets. Washed platelets (4 × 10⁸/mL) were preincubated with eptifibatide (9 μM) for 10 minutes before PF4 addition. After 10 minutes, platelets were lysed. Protein was separated by sodium dodecyl sulfate gel electrophoresis and analyzed for panphosphotyrosine (4G10), phosphorylated STAT3 (pSTAT3; Tyr705), phosphorylated STAT5a/b (pSTAT5a/b; Tyr694/699), phosphorylated c-Mpl (pc-Mpl; Tyr626), and Syk. For JAK2, samples were immunoprecipitated (IP) before western blotting for panphosphotyrosine with 4G10 (pJAK2). (Bi) Representative western blots using monoclonal antibodies to phosphotyrosine (4G10) and to pSTAT3 and pSTAT5a/b. (Bii) Representative western blot using 4G10 after JAK2 IP. (Biii) Representative western blot using an antibody to pc-Mpl. (Biv) Quantification of pixel lane intensities for phosphorylation of STAT3 (dark blue triangles), STAT5 (red circles), c-Mpl (light blue diamonds), and JAK2 (green squares) measured as fold change relative to resting (n = 3). Values are normalized for loading controls. (C) Surface plasmon resonance (SPR) showing binding of PF4 to c-Mpl. C-Mpl was conjugated directly onto the chip and PF4 flowed over. All sensograms shown are double reference subtracted, and at least 2 replicates were injected per cycle with experimental replicates of n = 3. RU: response units. Statistical analyses by a 1-way analysis of variance; ∗P < .05, ∗∗∗P < .001.