![JAK2 and c-Mpl blockade inhibit platelet responses to PF4. (A) Inhibition of PF4-induced platelet aggregation with ruxolitinib (Rux). Prewarmed platelets (2 × 108/mL) at 37 °C were incubated with Rux (100 nM) or vehicle (dimethyl sulfoxide [DMSO] 0.01%) for 5 minutes and stirred at 1200 rpm before addition of PF4 (50 μg/mL). (Ai) Representative aggregation traces to PF4 (50 μg/mL) and Rux (100 nM). (Aii) Quantification of aggregation (area under the curve [AUC] per minute) for 30 minutes (n = 8) and analyzed by a paired t-test. (B) Enhancement of platelet aggregation to CRP by PF4 and inhibition by Rux. Prewarmed platelets were incubated with Rux (100 nM) for 5 minutes, followed by PF4 (10 μg/mL) for 5 minutes before CRP (0.03 μg/mL) addition. (Bi) Representative aggregation traces. (Bii) Quantification of aggregation (AUC per minute) for 15 minutes and analyzed by a 1-way analysis of variance (ANOVA). (C) Rux inhibits STAT3 and STAT5a/b phosphorylation induced by PF4 and TPO. Prewarmed platelets (4 × 109/mL) were preincubated with eptifibatide (9 μM) for 10 minutes, then Rux (10-100 nM) for 5 minutes before stirring for an additional minute and PF4 (50 μg/mL) or TPO (100 ng/mL) 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. (Ci) Representative western blots. (Cii-iii) Quantification of pixel lane intensities for phosphorylation of (Cii) STAT3 and (Ciii) STAT5a/b, measured as percentage change relative to resting (n = 3). Values are normalized for loading controls. (D) PF4 enhancement of platelet aggregation to IgG isolated from a patient with VITT (IgG) and inhibition by Rux. Prewarmed platelets (2 × 108/mL) at 37 °C were incubated with Rux (100 nM) or vehicle (DMSO 0.01%) for 5 minutes, PF4 (10 μg/mL) or vehicle (phosphate-buffered saline) for 5 minutes, then stirred at 1200 rpm before IgG (100 μg/mL) addition. (Di) Representative platelet aggregation traces. (Dii) Quantification of aggregation (AUC per minute) for 30 minutes (n = 7) and analyzed by a 1-way ANOVA. (E) PF4 enhancement of platelet aggregation to VITT serum (Serum) and inhibition by Rux. Platelets were prepared as for D with the addition of serum instead of IgG. (Ei) Representative platelet aggregation traces. (Eii) Quantification of aggregation (AUC per minute) for 30 minutes (n = 7) and analyzed by a 1-way ANOVA. (F) As for E but with serum from a different patient. (G) Effect of polyclonal anti–c-Mpl blocking antibody (Ab) on platelet aggregation to PF4 and PF4 + VITT IgG. (Gi-ii) Washed platelets were incubated with anti–c-Mpl antibody (10 μg/mL) for 5 minutes and stirred for an additional minute before PF4 (10 μg/mL) addition. (Gi) Representative platelet aggregation traces. (Gii) Quantification of aggregation (AUC per minute) for 30 minutes (n = 6) and analyzed by a paired t-test. (Giii-iv) As for Gi-Gii but preincubated with PF4 (10 μg/mL) for 5 minutes after incubation with c-Mpl antibody before stirring and IgG addition. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/143/1/10.1182_blood.2023020872/4/blood_bld-2023-020872-gr2.jpeg?Expires=1719324001&Signature=auu2QnXzYT1RpjqAMu46X2P21C8elZxK22yYguXrvoxvCnnXKwfgwp5yolYvJg~7ugRavixtf0lGK3tkBrSFKyqLpWWmpbU~JTZ7ATErOUIFnA6qdO9luNtk9-Cx5dkaJzhb~z4SPe6jPfAfEJBZsYp-cD2lSppnGBYzTw9m6giNBnqnf7zh4gQgKqytd3DaCUN6tVm1yzsW47mczTNXnWCmZABcQROUEp2wM4FYKlSmZ5tgpi8uy2tMJcKkCOetc1C6E~kJ~CM8bonaHDLNwheVvs75DhMSCyqNfCWW861uTwZv-4DUGmlQELKcW5FZZv2UWW5Z6XN1TpN1aFCi6A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
JAK2 and c-Mpl blockade inhibit platelet responses to PF4. (A) Inhibition of PF4-induced platelet aggregation with ruxolitinib (Rux). Prewarmed platelets (2 × 108/mL) at 37 °C were incubated with Rux (100 nM) or vehicle (dimethyl sulfoxide [DMSO] 0.01%) for 5 minutes and stirred at 1200 rpm before addition of PF4 (50 μg/mL). (Ai) Representative aggregation traces to PF4 (50 μg/mL) and Rux (100 nM). (Aii) Quantification of aggregation (area under the curve [AUC] per minute) for 30 minutes (n = 8) and analyzed by a paired t-test. (B) Enhancement of platelet aggregation to CRP by PF4 and inhibition by Rux. Prewarmed platelets were incubated with Rux (100 nM) for 5 minutes, followed by PF4 (10 μg/mL) for 5 minutes before CRP (0.03 μg/mL) addition. (Bi) Representative aggregation traces. (Bii) Quantification of aggregation (AUC per minute) for 15 minutes and analyzed by a 1-way analysis of variance (ANOVA). (C) Rux inhibits STAT3 and STAT5a/b phosphorylation induced by PF4 and TPO. Prewarmed platelets (4 × 109/mL) were preincubated with eptifibatide (9 μM) for 10 minutes, then Rux (10-100 nM) for 5 minutes before stirring for an additional minute and PF4 (50 μg/mL) or TPO (100 ng/mL) 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. (Ci) Representative western blots. (Cii-iii) Quantification of pixel lane intensities for phosphorylation of (Cii) STAT3 and (Ciii) STAT5a/b, measured as percentage change relative to resting (n = 3). Values are normalized for loading controls. (D) PF4 enhancement of platelet aggregation to IgG isolated from a patient with VITT (IgG) and inhibition by Rux. Prewarmed platelets (2 × 108/mL) at 37 °C were incubated with Rux (100 nM) or vehicle (DMSO 0.01%) for 5 minutes, PF4 (10 μg/mL) or vehicle (phosphate-buffered saline) for 5 minutes, then stirred at 1200 rpm before IgG (100 μg/mL) addition. (Di) Representative platelet aggregation traces. (Dii) Quantification of aggregation (AUC per minute) for 30 minutes (n = 7) and analyzed by a 1-way ANOVA. (E) PF4 enhancement of platelet aggregation to VITT serum (Serum) and inhibition by Rux. Platelets were prepared as for D with the addition of serum instead of IgG. (Ei) Representative platelet aggregation traces. (Eii) Quantification of aggregation (AUC per minute) for 30 minutes (n = 7) and analyzed by a 1-way ANOVA. (F) As for E but with serum from a different patient. (G) Effect of polyclonal anti–c-Mpl blocking antibody (Ab) on platelet aggregation to PF4 and PF4 + VITT IgG. (Gi-ii) Washed platelets were incubated with anti–c-Mpl antibody (10 μg/mL) for 5 minutes and stirred for an additional minute before PF4 (10 μg/mL) addition. (Gi) Representative platelet aggregation traces. (Gii) Quantification of aggregation (AUC per minute) for 30 minutes (n = 6) and analyzed by a paired t-test. (Giii-iv) As for Gi-Gii but preincubated with PF4 (10 μg/mL) for 5 minutes after incubation with c-Mpl antibody before stirring and IgG addition. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.
