Figure 3.
Aged platelets show increased thromboinflammatory potential in vitro. (A) Bar graphs representing flow cytometric analysis of baseline surface markers of platelet age cohorts in whole blood (n = 4 per group; P = .0106, .0291, .0484, .0216, .0047, and .0216, respectively). (B) Bar graph depicting the percentage of single-labeled platelets that are PS+P-selectin–positive (P = .0023). (C) Representative micrographs and quantification of procoagulant activation of single-labeled platelets seeded on collagen I/fibrinogen matrix (n = 3 per group); graph showing percentage of single-labeled procoagulant platelets (P = .0145). (D) Bar graphs showing PLA (percentage of single-labeled platelets aggregating with leukocytes; P < .0001) and PNA (percentage of single-labeled platelets aggregating with neutrophils; P = .0318) in mouse whole blood (n = 4 per group). (E) Schematic outline, representative micrographs, and quantification of isolated platelets from pulse-labeled mice coincubated with isolated neutrophils (n = 3 per group); bar graph representing percentage of single-labeled platelets of total platelets aggregating with neutrophils (P = .0038). (F) Schematic outline of isolated platelet-rich plasma from pulse-labeled C57BL/6J mice coincubated (n = 4 per group) with isolated neutrophils (nonlabeled C57BL/6J mice, n = 2); bar graph representing the percentage of single-labeled platelets out of total platelets aggregating with neutrophils relative to their percentage in circulation (P = .0322); bar graphs depicting surface marker expressions in neutrophils post aggregation with pulse-labeled platelets: CD11b (P = .0030), CD66a (P = .0054), CD177 (P = .0143); statistical tests, unpaired t tests, 2-tailed. (G) Platelets isolated from mice pulse-labeled 108 hours before experiment treated with anti-GPIb, anti-GPIIbIIIa, anti-PSGL, and anti-CD40 Fab fragments/antibodies coincubated with isolated neutrophils; quantification of single-labeled platelets aggregating with neutrophils relative to control depicted in a bar graph (P < .0001). Statistical tests for panels A-E and 3G, ordinary 1-way ANOVA with the post hoc Dunnett multiple comparisons test. (H) Schematic outline shows pulse-labeled platelets coincubated with methicillin-susceptible S aureus prestained with SYTO 41 dye (5 μM), followed by staining with Live-or-Dye NucFix of killed bacteria; bar graphs depicting percentage of pulse-labeled platelets aggregating to methicillin-susceptible S aureus (MSSA) relative to their percentage in circulation (<0.0001) and the percentage of dead bacteria represented by the percentage of MSSA that are positive for NucFix (0.0408); statistical tests, unpaired t tests, 2-tailed. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. h, hour; ns, nonsignificant.

Aged platelets show increased thromboinflammatory potential in vitro. (A) Bar graphs representing flow cytometric analysis of baseline surface markers of platelet age cohorts in whole blood (n = 4 per group; P = .0106, .0291, .0484, .0216, .0047, and .0216, respectively). (B) Bar graph depicting the percentage of single-labeled platelets that are PS+P-selectin–positive (P = .0023). (C) Representative micrographs and quantification of procoagulant activation of single-labeled platelets seeded on collagen I/fibrinogen matrix (n = 3 per group); graph showing percentage of single-labeled procoagulant platelets (P = .0145). (D) Bar graphs showing PLA (percentage of single-labeled platelets aggregating with leukocytes; P < .0001) and PNA (percentage of single-labeled platelets aggregating with neutrophils; P = .0318) in mouse whole blood (n = 4 per group). (E) Schematic outline, representative micrographs, and quantification of isolated platelets from pulse-labeled mice coincubated with isolated neutrophils (n = 3 per group); bar graph representing percentage of single-labeled platelets of total platelets aggregating with neutrophils (P = .0038). (F) Schematic outline of isolated platelet-rich plasma from pulse-labeled C57BL/6J mice coincubated (n = 4 per group) with isolated neutrophils (nonlabeled C57BL/6J mice, n = 2); bar graph representing the percentage of single-labeled platelets out of total platelets aggregating with neutrophils relative to their percentage in circulation (P = .0322); bar graphs depicting surface marker expressions in neutrophils post aggregation with pulse-labeled platelets: CD11b (P = .0030), CD66a (P = .0054), CD177 (P = .0143); statistical tests, unpaired t tests, 2-tailed. (G) Platelets isolated from mice pulse-labeled 108 hours before experiment treated with anti-GPIb, anti-GPIIbIIIa, anti-PSGL, and anti-CD40 Fab fragments/antibodies coincubated with isolated neutrophils; quantification of single-labeled platelets aggregating with neutrophils relative to control depicted in a bar graph (P < .0001). Statistical tests for panels A-E and 3G, ordinary 1-way ANOVA with the post hoc Dunnett multiple comparisons test. (H) Schematic outline shows pulse-labeled platelets coincubated with methicillin-susceptible S aureus prestained with SYTO 41 dye (5 μM), followed by staining with Live-or-Dye NucFix of killed bacteria; bar graphs depicting percentage of pulse-labeled platelets aggregating to methicillin-susceptible S aureus (MSSA) relative to their percentage in circulation (<0.0001) and the percentage of dead bacteria represented by the percentage of MSSA that are positive for NucFix (0.0408); statistical tests, unpaired t tests, 2-tailed. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. h, hour; ns, nonsignificant.

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