Elevated platelet–leukocyte complexes are associated with, but dispensable for myocardial ischemia–reperfusion injury

Figures

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  Fig. 1. Platelet–leukocyte complexes surge in peripheral blood post-myocardial ischemia and reperfusion injury (I/R). A Representative dot plots depicting blood neutrophils, monocytes, platelet–neutrophil complexes (PNC), and platelet–monocyte complexes (PMC), respectively. B Time course for PNC (top) and PMC (bottom) fractions in blood before (t = 0) and after myocardial ischemia (35 min) with reperfusion over 72 h (black data points). *p < 0.05 denote statistically significant changes before vs after myocardial I/R (*) n = 4 per time point, Kruskal–Wallis test, Dunn’s multiple comparison, §p < 0.05 denote statistically significant changes between myocardial I/R (black) and sham (red) at 24 h of reperfusion, n = 4 per group, Mann–Whitney. C Representative ImageStreamX image PNC and PMC defining platelets as CD41 + , neutrophils as Ly6G + , monocytes as CD115 + and F-actin enrichment at the contact zone. D Correlation of plasma troponin and blood PMC or PNC levels following 35 min and 45 min of myocardial ischemia with reperfusion or permanent LAD ligation 24 h post-surgery. *p < 0.05 denote statistically significant changes, determination of correlation by Pearson’s r

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  Fig. 2. Surface expression of P-selectin (CD62P) and formation of platelet–leukocyte complexes in irradiated bone marrow chimeras, and its influence on leukocyte rolling and adhesion. Wild-type (WT) and P-selectin-deficient (KO) mice were lethally irradiated and reconstituted with either WT bone marrow in WT recipients (WTWT), KO bone marrow in WT recipients (KOWT), or WT bone marrow in KO recipients (WTKO). A Representative histograms showing selective loss of CD62P expression on platelets in KOWT chimeras and loss of CD62P expression on endothelial cells in WTKO chimeras. B Representative dot plots showing that selective loss of CD62P on platelets in KOWT chimeras reduces both platelet–neutrophil (PNC) and platelet–monocyte complexes (PMC) in blood. C Intravital microscopy of mesenteric venules in WTWT, KOWT, and WTKO chimeras 4 h post-intraperitoneal (i.p.) TNFα (200 ng) injection. Representative images of rhodamine-labeled leukocytes within the venules are shown on the left. Quantification of rolling and adherent leukocyte numbers are shown on the right. Results are presented as mean ± SEM, n = 6 for WTWT, KOWT and n = 4 for WTKO. *p < 0.05 denote statistically significant changes…

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  Fig. 3. P-selectin expression on platelets is dispensable for leukocyte recruitment to sites of sterile inflammation. A In vivo quantification of the percentage of rolling platelet–leukocyte complexes (PLC, see representative image) in mesenteric venules of WTWT and KOWT chimeras 4 h post-TNFα i.p. stimulation. Results are presented as mean ± SEM; WTWT n = 8, KOWT n = 7. B Total blood monocyte and neutrophil counts and fractions of platelet–monocyte (PMC) and platelet–neutrophil complexes (PNC) in WTWT and KOWT chimeras undergoing TNFα stimulation i.p. Results are presented as mean ± SEM, n = 6 per group. *p < 0.05 denotes statistically significant changes between groups using 2-way ANOVA, Sidak’s multiple comparison test. C Representative dot plot showing gating strategy for peritoneal neutrophils and macrophages (left), and quantification of neutrophil and macrophage cell numbers in peritoneal lavage (right). Results are presented as mean ± SEM; n = 7 per group

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  Fig. 4. PLC have no major impact on infarct size and cardiac function post-myocardial infarction. A Fractions of PMC, and PNC before and 24 h after myocardial I/R injury in WTWT and KOWT chimeras. WTWT chimeras, *p < 0.05 denote statistically significant differences between time points within the same group and §p < 0.05 between groups at the same time point. Results are presented as mean ± SEM, n = 8 per group, two-way ANOVA). B Blood counts for monocytes (Mono) and neutrophils (Neutro) 24 h after myocardial I/R injury. Results are presented as mean ± SEM, n = 8 per group. C Representative dot plots showing gating for leukocyte populations in enzymatically digested infarcted myocardium (left) and quantification of Ly6Chigh monocytes, neutrophils, PMC and PNC, and macrophages. Results are presented as mean ± SEM, *p < 0.05 denote statistically significant differences between groups, n = 9 for WTWT and n = 8 for KOWT, t test). D High-sensitive Troponin T plasma levels in WTWT and KOWT chimeras 24 h after myocardial I/R injury. Results are presented as mean ± SEM, n = 8 per group, t test. E Representative images of left ventricle (LV) sections stained with 2,3,5-triphenyltetrazolium…