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Figure 1

From: Mild hyperlipidemia in mice aggravates platelet responsiveness in thrombus formation and exploration of platelet proteome and lipidome

Figure 1The alternative text for this image may have been generated using AI.

Multiparameter assessment of platelet activation and aggregation under arterial flow of Apoe−/− and Ldlr−/− mouse blood in the absence of coagulation. Blood was used from young (9–12 weeks) and aged (37–42 weeks) Apoe−/−, Ldlr−/− and corresponding wild-type mice, held on normal chow diet. PPACK-anticoagulated blood was perfused over collagen type I at wall shear rate of 1000 s−1. Brightfield images were captured, after which the deposited platelets were stained for integrin αIIbβ3 activation, P-selectin expression and PS exposure in different colors (see “Methods”). (A) Representative images from young mice are shown for each color per genotype. Bars indicate 20 μm. (B) Quantification (% SAC) of brightfield images of platelet deposition and fluorescence images of platelet activation parameters: phosphatidylserine (PS) exposure, P-selectin expression and integrin αIIbβ3 activation of (i) young mice and (ii) aged mice. Mean ± SEM (n = 6–15 animals/group). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. wild-type (Mann–Whitney U test). (C) Platelet activation parameters were obtained from brightfield and fluorescence images after 3.5 min: P1, morphological score; P2, platelet surface area coverage (% SAC); P3, aggregate contraction score; P4, aggregate multilayer score; P5, aggregate multilayer coverage (% SAC); P6, PS exposure (% SAC); P7, P-selectin expression (% SAC); P8, integrin αIIbβ3 activation (% SAC). Values per parameter were linearly scaled to 0–10 and a scaled heatmap with integration of age groups was generated. (D) Scaled heatmap of effect sizes per genotype. Effect size per parameter was calculated from the pooled standard deviation, Cohen’s d and regression coefficient r. Analysis based on relevant changes restricted to differences with P < 0.05 (t test, 2-sided, equal variance). Means (n = 15–20 animals/group). (E) Isolated platelets were activated by thrombin (0.5–4 nM) or cross-linked collagen-related peptide (CRP-XL, 0.5–5 μg/ml); exposure of phosphatidylserine (PS), P-selectin and activation of integrin αIIbβ3 were determined by flow cytometry using AF647-labeled annexin A5, FITC-labeled anti-CD62P mAb, and PE-labeled JON/A mAb, respectively. Means ± SEM (n = 8–12), *P < 0.05, #P < 0.01, $P < 0.001 compared to WT (Kruskall–Wallis test).

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