Fig 1: Plasma markers of immunothrombosis are increased in ischemic stroke patients.Plasma samples were obtained within 48 hours of hospital admission from stroke patients or age- and sex-matched healthy donors (HD). D-dimer (A), PF4 (B), neutrophil calprotectin (C), H3cit (D), MPO-DNA complexes (E), and DNase activity (F) were measured by ELISA. n = 27 per group. Groups were compared by Mann-Whitney test. **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 2: ELISA quantified mediators of angiogenesis in (A) purified platelets and (B) media after activation of 108 platelet/mL with collagen or thrombin compared to the resting state. Platelets and media from different HD stage patients were compared with pre-HD and hCTRL (stage 1, n=5; stage 2, n=5; stage 3, n=5; stage 4, n=4; pre-HD, n=5; hCTRL, n=6). The release of both (C) serotonin and (D) PF4 was quantified by ELISA in the activated-platelet media from different HD stage patients, pre-HD and hCTRL (stage 2, n=4; stage 4, n=4; pre-HD, n=8; hCTRL, n=9). Statistical analyses: Kruskal-Wallis with Dunn’s multiple comparison test (*p<0.05, **p<0.01) and Wilcoxon signed rank test with theoretical median set at 1 (#p<0.05). ANG-2, angiopoietin-2; FGF basic, basic fibroblast growth factor; hCTRL, healthy control group; HD, Huntington’s disease; HGF, hepatocyte growth factor; IL-8, interleukin 8; PDGFR, platelet-derived growth factor; TIMP-1, metalloproteinase inhibitor 1; TIMP-2, metalloproteinase inhibitor 2; TNFa, tumour necrosis factor; pre-HD, pre-manifest.
Fig 3: Platelet-specific HMGB1 knockout blocks platelet-induced NET formation and improves stroke outcomes.(A and B) Platelets and neutrophils were isolated from 4 healthy donors. Platelets were activated for 15 minutes with convulxin and then incubated for 2.5 hours with neutrophils in the presence of BoxA or vehicle, after which NETs were quantified using a MPO-DNA ELISA. n = 4 per group. (C and D) Platelets were isolated from 3 HMGB1fl/fl (WT) or HMGB1fl/fl PF4-cre (KO) mice, activated with convulxin, and incubated for 2.5 hours with WT neutrophils, after which NETs were quantified using an MPO-DNA ELISA. n = 3 per group. (E–J) HMGB1fl/fl (WT; n = 9) or HMGB1fl/fl PF4-cre (KO; n = 11) mice were subjected to 1 hour of tMCAO followed by 23 hours of reperfusion. Plasma was isolated and brains were analyzed for ischemic stroke brain damage by TTC staining 24 hours after stroke onset. Upon TTC staining, live brain tissue will stain red, while dead brain tissue will remain white (outlined with black dotted line). (E) Plasma HMGB1 levels were measured by ELISA. (F) Plasma NET levels were measured by MPO-DNA complex ELISA. (G and H) Infarct size was determined by TTC staining and planimetric analysis. (I) Neurological score was measured 24 hours after stroke using Bederson’s test. (J) Motor function was assessed 24 hours after stroke using the grip test. Groups were compared by unpaired t test (B, D–F, and H) or Mann-Whitney test (I and J). *P < 0.05, **P < 0.01, ***P < 0.001.
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