Fig 2: Interaction of HAPPI with platelets and their preferable binding ability.(A) FACS analysis of HAPPI-mediated platelet activation as measured by coexpression of CD62P/CD41 after 10 min of incubation. Data are presented as percentage of platelets positive for both epitopes. (B) FACS analysis of attachment of AF 647–HA and HAPPI to platelets in their nonactivated and activated states [n = 3, one-way analysis of variance (ANOVA) test and Tukey’s multiple comparisons test, ****P < 0.0001]. (C) Representative dot plots for expression of AF 647–HA and HAPPI to indicate binding of platelets to HA and HAPPI following 10 min of incubation. (D) Human platelet aggregation assay of HAPPI, HA-VBP, and HA-CBP with saline as control. (E) Schematic of experimental setup for dynamic binding studies using PPFC to validate preferable binding ability of HAPPI to wound-specific proteins, collagen, and vWF, under flow. (F) Representative fluorescence images (×20 magnification) of (F1) DiOC6-stained platelets (green), (F2) AF 647–HAPPI (red), and (F3) overlay of F1 and F2 in the same field of view, demonstrating colocalization of platelets with HAPPI at the collagen + vWF surface (scale bars, 100 µm). ns, not significant; ADP, adenosine diphosphate; FACS, fluorescence-activated cell sorting.

Fig 3: Influence of CXCR4 and CXCR7 inhibition in platelet–neutrophil complex (PNC) formation and expression of PNC-related molecules in wild-type animals. (A) PNC formation and PNC-related adhesion molecules in the blood samples of wild-type mice (black circles) with and without zymosan administration (n = 8-12 mice per group). Effects of CXCR4 (AMD3100; red circles) and CXCR7 (CCX771; yellow circles) inhibition on PNC formation and on the surface expression of PNC-related selectins CD62P, CD162, and CD62L. (B) PNC sequestration into the peritoneal tissue, (C) lung, and (D) liver tissue was evaluated by immunohistochemical staining. Polymorphonuclear neutrophils (PMNs) and platelets were tackled with specific antibodies, so that PMNs appeared blue and PNCs appeared blue/black (black arrows) (images are representative slides of n = three slides from each animal; n = four mice per group; 63× magnification). (E) PNC counts were enumerated by light microscopy. PNCs from three representative high-power fields were counted from three different slides (n = four mice per group). Data are presented as mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Multiple group comparison was analyzed by one-way ANOVA and Bonferroni correction or Kruskal–Wallis test.

Fig 4: Role of CXCR4 and CXCR7 inhibition in platelet-neutrophil complex (PNC) formation during fecal-induced peritonitis and sepsis in wild-type animals. (A) Polymicrobial-induced PNC formation and expression of PNC-related molecules in the blood of wild-type mice (black circles) with and without fecal injection (n = 8 mice per group). CXCR4 (AMD3100; red circles) and CXCR7 (CCX771; yellow circles) inhibition ameliorated PNC formation and CD62P, CD162, and CD62L surface expression. (B) PNC migration into the peritoneal tissue, (C) lung, and (D) liver tissue was evaluated by immunohistochemical staining. Polymorphonuclear neutrophils (PMNs) and platelets were tackled with specific antibodies, so that PMNs appeared blue and PNCs appeared blue/black (black arrows) (images are representative slides of n = three slides from each animal; n = four mice per group; 63× magnification). (E) PNC infiltration was quantified by light microscopy. PNC formation from three representative high-power fields were counted from three different slides (n = three mice per group). Data are presented as mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Multiple group comparison was analyzed by one-way ANOVA and Bonferroni correction or Kruskal–Wallis test.

Fig 5: Platelet characteristics and protein expression in PyMT mice.(A) Platelet counts, mean platelet volume (MPV), platelet distribution width (PDW), and immature platelet fraction in 7- to 13-week-old PyMT and WT mice. n = 10 mice per condition. *P < 0.05 and **P < 0.01, as determined by one-way ANOVA. (B) Thiazole orange (TO) staining of platelets from PyMT and WT mice. n = 6 to 7 mice per condition. P values were determined by two-sample t tests. (C) Proportion of biotin+ platelets isolated from PyMT and WT mice following in vivo labeling with biotin (0 hours). Dashed line depicts basal signal from unlabeled platelets. n = 5 mice per condition. Error bars depict SEM. (D) Plasma levels of platelet factor 4 (PF4) from PyMT and WT mice. n = 9 to 11 mice per condition. P values were determined by two-sample t tests. (E) Surface expression of P-selectin (CD62p) on the surface of platelets isolated from PyMT or WT mice following thrombin stimulation. n = 4 to 5 mice per condition. Error bars depict SEM. (F) S100A8 and S100A8/A9 levels in platelets from PyMT and WT mice. n = 9 to 19 mice per condition. P values were determined by two-sample t tests. (G) S100A8/A9 from the plasma of PyMT and WT mice. n = 4 to 7 mice per condition. (H) Lcn-2 levels of platelets from 13-week-old PyMT and WT mice. n = 5 mice per condition. P values were determined by two-sample t tests. (I) Lcn-2 levels from the plasma of PyMT and WT mice. n = 3 mice per condition. P values were determined by two-sample t tests.