BTK and Syk inhibitors reduce oxLDL potentiation of platelet hemostatic and procoagulant activity. (A) Replicate samples (n = 4) of washed human platelets (2 × 108/mL; in modified HEPES/Tyrode buffer without addition of calcium) were treated with BTK inhibitor ibrutinib (10 μM) or Syk inhibitor R406 (10 μM) for 10 minutes and stimulated with GPVI-specific agonist CRP-XL (0.5 μg/mL) in combination with oxLDL (20 μg/mL). Platelets were monitored at 37°C under continuous stirring at 1200 rpm and the changes in light transmission were measured. Representative platelet aggregation traces of platelet samples treated with each select inhibitor are shown. (B) Platelets were stained with FITC-lactadherin to monitor for platelet surface PS exposure. Platelet samples were incubated with CaCl2 (8.3 mM) and citrated PPP (33% final). Fibrin formation was measured as a change in turbidity at an absorbance of 405 nm and the lag time (C) was quantified. (D) Platelets were monitored using the luciferase enzyme CHRONO-LUME to detect the luminescence of ATP released as a measure of platelet dense granule secretion, stained with APC-CD62P to monitor for platelet surface expression of P-selectin as a marker of α-granule secretion (E), and stained with FITC-PAC-1 to monitor for platelet surface integrin activation (F). Following the platelet aggregation assays shown in panel A, platelet supernatants were extracted and analyzed for thromboxane generation by ELISA (G). Additional platelet supernatant samples were similarly prepared from platelets treated with oxLDL, CRP-XL, and inhibitors of interest to measure levels of indicated oxylipin metabolite levels with mass spectrometry (H). Statistical analysis was performed using a one-way ANOVA test and the corresponding P values of significance are as indicated: ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. ELISA, enzyme-linked immunosorbent assay.
