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: CLEC4F is critical to the Kupffer cell-mediated phagocytosis of desialylated platelets.A Representative confocal microscopic images of desialylated platelets (CD41, red) and Kupffer cells (F4/80, blue) in liver sections of different mouse models. B Quantification of platelets with liver Kupffer cells of different mouse models 2 h after Clostridium perfringens-derived neuraminidase injection. n = 5–10 images/20 × fields/mouse. Data represent means ± SEM from three experiments. *P < 0.05. ns, not significant.

Fig 4: Blood cell profiles in mice 12–18 months post-irradiation. (A) Lymphocytes (LYM) were enumerated using flow cytometry as CD41- and CD11b- cells in the peripheral blood leukocytes and compared between radiation-exposed (N = 36) and non-exposed (N = 11) mice. The LYM percentages were significantly different (P < 0.01) based on linear-regression analysis after adjusting for age and sex. The blue and red dots represent male and female animals, respectively. Inter-quartile ranges are shown with gray-shaded areas. (B) Myeloid cell percentages (MYE, %) in the peripheral blood leukocytes were evaluated with CD41- and CD11b+ populations, including Ly6C-low granulocytes and Ly6C-high monocytes. The same statistical analysis indicated a significant difference between radiation-exposed (N = 36) and non-exposed (N = 11) mice (P < 0.01). (C) RDW values of peripheral blood erythrocytes were measured using a hematological autoanalyzer in mice after irradiation. Three data were missing for irradiated mice because of sampling errors. The RDW values significantly differed between radiation-exposed (N = 33) and non-exposed (N = 11) mice (P < 0.01). RDW, red blood cell distribution width.

Fig 5: Kupffer cell CLEC4F is a dominant receptor in mediating the rapid clearance of desialylated platelets in the liver.A Flow cytometry analysis of galactose exposure (RCA I binding) on platelets of either WT or Clec4f−/− recipients treated with Arthrobacter ureafaciens-derived neuraminidase at different time points. MFI, Mean fluorescence intensity. B Percentage of platelets of either WT or Clec4f−/− recipients treated with Arthrobacter ureafaciens-derived neuraminidase at different time points. Platelet count was measured using flow cytometric analysis of peripheral blood based on anti-CD41. C Representative immunofluorescent images showing association of platelets (CD41, red) with Kupffer cells (F4/80, blue) in the liver sections. Endothelium, (CD31, green). D, E Quantification of association of platelets with liver Kupffer cells or Kupffer cell (KC) number at 2 h after α neuraminidase injection. n = 5–10 images/10 × fields/experiment. Data represent means ± SEM from three experiments. *P < 0.05.