Effect of antiplatelet agents and tyrosine kinase inhibitors on oxLDL-mediated procoagulant platelet activity

Figures

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  Figure 1.. Elevated oxLDLand enhancedGPVI-mediated procoagulant platelet generation in obese nonhuman primates. Blood was collected from a cohort of lean (n = 6) and diet-induced obese (n = 6) rhesus macaques to prepare serum for analysis of triglycerides (A), total cholesterol (B), high-density lipoprotein cholesterol (C), LDL cholesterol (D), and oxLDL immunoreactivity (E). Citrate-anticoagulated whole blood from lean and obese animals was stimulated with GPVI agonist (0.1 μg/mL CRP-XL) or a combination of GPVI and PAR1 agonist (1 μg/mL CRP-XL, 20 μg/mL TRAP-6) before flow cytometry analysis of platelet α-granule secretion (F) and platelet PS exposure (G). Statistical analysis was performed using a one-way analysis of variance (ANOVA) test and the corresponding P values of significance are as indicated: ∗P < .01, ∗∗P < .001, and ∗∗∗P < .0001. CRP-XL, crosslinked collagen-related peptide.

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  Figure 2.. oxLDL potentiates GPVI-mediated platelet aggregation, platelet PS exposure, and platelet-driven fibrin formation. Replicate samples of washed human platelets (2 × 108/mL, in modified HEPES/Tyrode buffer without additional calcium) were incubated with the GPVI-specific agonist CRP-XL) alone (0.1, 0.5, 1 μg/mL), oxLDL alone (20 μg/mL), and oxLDL in combination with CRP-XL. Platelet samples were monitored at 37°C under continuous stirring at 1200 rpm and changes in light transmission were measured (A-D). (E) Washed platelets (2 × 108/mL) were incubated with oxLDL (0, 20 μg/mL) in combination with CRP-XL (0, 0.1, 0.5, 1 μg/mL) and monitored using luciferase enzyme activity (CHRONO-LUME) to detect ATP released as a measure of platelet dense granule secretion. (F) Platelets were stained with APC-CD62P to monitor for platelet surface expression of P-selectin as a marker of α-granule secretion and (G) stained with FITC-PAC-1 to monitor for platelet surface integrin activation. (H) Platelets were incubated with oxLDL (0, 20 μg/mL) in combination with GPVI-specific agonist CRP-XL (0, 0.1, 0.5, 1 μg/mL) before fixation and staining with FITC-lactadherin to monitor for platelet surface PS…

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  Figure 3.. P2YR antagonists but not COX inhibitors reduce oxLDL potentiation of platelet GPVI responses. (A) Replicate samples (n ≥ 3) of washed human platelets (2 × 108/mL; in modified HEPES/Tyrode buffer without addition of calcium) were treated with P2YR inhibitors ticagrelor (750 nM) and ARC/MRS (10 μM), and COX inhibitors aspirin (1 mM) and indomethacin (10 μM) for 10 minutes and stimulated with oxLDL (20 μg/mL) in combination with GPVI-specific agonist CRP-XL (0.5 μg/mL). Platelet samples were monitored using the luciferase enzyme CHRONO-LUME to detect the luminescence of ATP released as a measure of platelet dense granule secretion. (B) Platelets were stained with APC-CD62P to monitor for platelet surface expression of P-selectin and (C) stained with FITC-PAC-1 to monitor for platelet surface integrin activation. Statistical analysis was performed using a one-way ANOVA test and the corresponding P values of significance are as indicated: ∗∗∗P < .001. (D) Replicate samples (n = 4) of washed human platelets (2 × 108/mL) were treated with P2YR inhibitors ticagrelor (750 nM) and ARC/MRS (10 μM), and COX inhibitors aspirin (1 mM) and indomethacin (10 μM) for 10 minutes and stimulated with…

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  Figure 4.. Antiplatelet agents and TKIs differentially reduce oxLDL potentiation of platelet GPVI/ITAM signaling. Replicate samples (n = 3) of washed human platelets (1 × 109/mL; in modified HEPES/Tyrode buffer without addition of calcium) were pretreated with P2YR inhibitor ticagrelor, BTK inhibitor ibrutinib (10 μM), or Syk inhibitor R406 (10 μM) for 10 minutes and then stimulated with oxLDL (20 μg/mL) in combination with GPVI-specific agonist CRP-XL (0.5 μg/mL) for 5 minutes at 37°C. After collection into sample buffer, platelet lysates were separated by capillary electrophoresis and stained with indicated antisera for quantitative immunoblot analysis on a ProteinSimple Jess western blotting system. (A) Representative immunoblot analysis and (B) quantitation of signal intensities with indicated antisera; α-tubulin serves as a loading control for total protein levels. Positions of molecular weight (kD) markers are indicated.

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  Figure 5.. 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…

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  Figure 6.. oxLDL enhances platelet procoagulant activity in vivo. (A) Wild-type C57BL/6J mice (median age, 6 weeks) were intravenously injected with vehicle (phosphate-buffered saline) alone or oxLDL 10 minutes before blood collection into citrate (n = 5 per condition). Whole blood samples were analyzed for platelet-monocyte aggregate formation as previously described.35,49 (B) Whole blood samples from vehicle- and oxLDL-injected animals were subsequently stimulated with GPVI agonist (0.1 μg/mL CRP-XL) before flow cytometry analysis of CD62P as a marker of platelet α-granule secretion. (C) Sets of mice (n = 5 each) were administered by vehicle (dimethyl sulfoxide), ticagrelor (100 mg/kg), aspirin (30 mg/kg), or ibrutinib (10 mg/kg) by intraperitoneal injection before oxLDL injection, blood collection, and flow cytometry analysis of FITC-lactadherin to follow platelet PS exposure. (D) Aliquots of citrate-anticoagulated whole blood from obese rhesus macaques (detailed in Figure 1, in previous section) were pretreated with vehicle (dimethyl sulfoxide), ticagrelor, aspirin, or ibrutinib before stimulation with GPVI agonist (0.1 μg/mL CRP-XL), lactadherin-FITC staining, and flow cytometry…