Platelets Protect Cardiomyocytes from Ischemic Damage

Fig. 1. Factors released from platelet secretory vesicles protect cardiomyocytes from ischemic injury. ( A and D ) Cardiomyocytes from wild-type (WT) hearts preconditioned with released factors from vehicle-treated (control) and collagen-related peptide (CRP)-treated (stimulated) platelets from WT or Unc13d Jinx mice (as indicated) were subjected to ischemic injury using a cell pelleting method. Samples from the pellet were analyzed for cell viability every 30 minutes, for up to 240 minutes, with nonischemic (oxygenated control) samples monitored at 0- and 240-minute time points. Data are mean ± SD ( n = 7 for ( A ), n = 5 for ( D )), ( A ) * p < 0.05, *** p < 0.001 versus WT-controlled releasate and ( D ) ΦΦ p < 0.01, ΦΦΦ p < 0.001 versus Unc13d Jinx stimulated releasate. ( B ) Representative images of trypan blue stained and nonstained cardiomyocytes at 60-minute intervals throughout ischemia assay (0–240 minutes). ( C ) Washed platelets (2 × 10 8 /mL) from WT and Unc13d Jinx mice were treated with vehicle control (Tyrode's) or 10 µg/mL CRP and monitored for α-granule secretion by CD62P surface expression. Data are mean ± SD; n = 3, ** p < 0.01. Two-way ANOVA with Bonferroni post hoc…

Fig. 2. Role for platelet α-granule proteins, SDF-1α and TGF-β1, in mediating the protective effects of platelet secreted factors. ( A–F ) Cardiomyocytes were preconditioned with releasates from vehicle-treated (control) WT platelets: ( A ) filtered, <3 kDa and retained, >3 kDa releasates from CRP-treated (stimulated) WT platelets; ( B ) recombinant SDF-1α (100 ng/mL); ( C ) recombinant TGF-β1 (100 ng/mL) and ( D–F ) unfiltered releasate from CRP-treated (stimulated) WT platelets, then monitored for cardiomyocyte viability for up to 240 minutes of ischemic injury, with nonischemic (oxygenated control) samples monitored at 0- and 240-minute time points. ( D–F ) Cardiomyocytes were pretreated with vehicle control (0.1% DMSO) or inhibitors: ( D ) 0.5 µM AMD3100 (CXCR4 antagonist); ( E ) 1 µM SB431542 (TGFBR1 antagonist), and ( F ) 0.5 µM AMD3100 and 1 µM SB431542 for 30 minutes prior to releasate preconditioning. Data are mean ± SD ( n = 4 for ( A ) and ( F ), n = 3 for B–E ); * p < 0.05, ** p < 0.01, *** p < 0.001 versus control releasate ( A, D–F )/vehicle ( B,C ) and Φ p < 0.05, ΦΦ p < 0.01, ΦΦΦ p < 0.001 versus stimulated releasate (<3 kDa ( A )/ + AMD3100 ( D )/ + SB431542 ( E…

Fig. 3. Protein kinase C (PKC) activity is essential for platelet releasate-mediated cardioprotection. ( A ) Cardiomyocytes preconditioned with releasates from vehicle- (control) and CRP- (stimulated) treated WT platelets were subjected to ischemic injury, with samples removed from the pellet and lysed at indicated ischemic time points. Samples were monitored for changes in PKC activity (Phospho serine PKC substrate), phospho Akt (Ser473), and Erk 1/2 (Thr202/Tyr204) by immunoblotting with specific antibodies followed by appropriate loading controls. Blots shown are representative of three independent experiments. ( B ) Cardiomyocytes were pretreated in the absence (+ DMSO vehicle) or presence of 10 µM BIM I for 30 minutes prior to preconditioning with releasates from vehicle- (control) and CRP-treated (stimulated) WT platelets, then monitored for cardiomyocyte viability for up to 240 minutes of ischemic injury, with nonischemic (oxygenated control) samples monitored at 0- and 240-minute time points. Data are mean ± SD; n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001 versus control releasate, ΦΦ p < 0.01, ΦΦΦ p < 0.001 versus stimulated releasate (+ BIM I). Two-way ANOVA with Bonferroni…

Fig. 4. Targeting platelet P2Y 12 , but not cyclooxygenase, reduces platelet secretion and cardioprotection. ( A ) Washed platelets (2 × 10 8 /mL) from WT mice were pretreated with vehicle (Tyrode's) or inhibitors; 30 µM aspirin (cyclooxygenase) and 1 µM AR-C 66096 (P2Y 12 antagonist) for 10 minutes, then stimulated with 10 µg/mL CRP and analyzed for α-granule secretion by surface CD62P exposure. Data are mean ± SD; n = 4, *** p < 0.001, one-way ANOVA with Bonferroni post hoc test. ( B–D ) Cardiomyocytes preconditioned with releasates from vehicle-treated (control) or CRP-treated (stimulated) WT platelets in the absence (+ Tyrode's vehicle) or presence of ( B ) 30 µM aspirin, ( C ) 1 µM AR-C 66096, and ( D ) 30 µM aspirin + 1 µM AR-C 66096 were monitored for changes in viability during ischemic injury, with nonischemic (oxygenated control) samples monitored at 0 and 240 minutes time points. Data are mean ± SD; n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001 versus WT-controlled releasate (+ vehicle ( B–D )) and Φ p < 0.05, ΦΦ p < 0.01, ΦΦΦ p < 0.001 versus stimulated releasate (+Aspirin ( B )/ + ARC66096 ( C )/ + Aspirin + AR-C66096 ( D )), two-way ANOVA with Bonferroni post hoc test.