Macrophage receptor SR-AI is crucial to maintain normal plasma levels of coagulation factor X by Vincent Muczynski, Amine Bazaa, Cécile Loubière, Amélie Harel, Ghislaine Cherel, Cécile V. Denis, Peter J. Lenting, and Olivier D. Christophe Blood Volume 127(6):778-786 February 11, 2016 ©2016 by American Society of Hematology

FX binding to human monocytes and macrophages. FX binding to human monocytes and macrophages. (A-B) Undifferentiated THP1 (A) or THP1-derived macrophages (B) were incubated with Alexa 488–labeled FX or Alexa 488–labeled Fab′2 as a control (10 µg/mL, 1 hour at 37°C) and subsequently analyzed by flow cytometry for FX binding. Black curves represent Fab′2-incubated cells (control) whereas red curves represent Alexa 488-FX–incubated cells. Representative plots of 3 different experiments are shown. (C) The mean FX fluorescence was quantified and is expressed in arbitrary units. Each dot represents 1 experiment (N = 5-6 in total) and bars represent the mean ± SD. ***P < .001 in a 2-way ANOVA followed by the Sidak posttest for multiple comparison. (D-E) Widefield microscopy images of immunofluorescent staining for FX (green) in undifferentiated THP1 (D) or THP1-derived macrophages (E) incubated with 10 µg/mL FX (1 hour at 37°C). Nuclei and polymerized actin were counterstained using DAPI (blue) and Alexa 647–labeled phalloidin (magenta), respectively. Arrows indicate spots of FX staining. Bars represent 10 µm; objective, ×63. DAPI, 4,6 diamidino-2-phenylindole; N.S., not significant. Vincent Muczynski et al. Blood 2016;127:778-786 ©2016 by American Society of Hematology

Colocalization of FX in human macrophages. Colocalization of FX in human macrophages. (A-E) Widefield microscopy images of Duolink-PLA assay between FX and MMR (A), DC-SIGN (B), CLEC10A (C), or SR-AI (D) in THP1-derived macrophages, or between FX and SR-AI in CD14+-derived macrophages (E) incubated with either PBS (top panels) or FX (bottom panels). Nuclei were counterstained using DAPI. Bars represent 10 µm; objective, ×40. Red spots indicate a distance <40 nm between 2 antigens. Images are representative of 3 different experiments. (F-I) Confocal analysis of immunofluorescent staining for FX (red) and MMR (F) or SR-AI (G) in a THP1-derived macrophage incubated with FX. Cell stack was reconstituted in orthoview to visualize colocalization of the 2 signals (right panels). Arrows indicate areas of colocalization. Z depth is 0.5 µm; bars represent 10 µm; objective, ×63. The mean FX fluorescence of the cells was quantified using Fiji software (H). tMC represents a statistical parameter verifying whether fluorescent signals truly overlap and was calculated using JACoP plugin in Fiji for THP1-derived macrophages incubated with FX and immunostained for FX and MMR (negative control) or SR-AI (I). ***P < .001, respectively, in the Mann-Whitney nonparametric unpaired statistical test. Dots represent each individual cell value and bars represent the mean ± SD of 7 (FX/MMR) to 12 (FX/SR-AI) cells from 3 independent experiments. PBS, phosphate-buffered saline. Vincent Muczynski et al. Blood 2016;127:778-786 ©2016 by American Society of Hematology

Differential binding of FX and Ac-LDL to SR-AI. Differential binding of FX and Ac-LDL to SR-AI. (A-B) Increasing concentration of FX (0-40 µg/mL) (A) or increasing concentration of SR-AI inhibitor (Poly[I], polyclonal anti–SR-AI antibody, or Ac-LDL; 1-50 µg/mL) along with 1 µg/mL FX (B) were incubated in microtiter wells coated with hSR-AI (0.5 μg per well). Bound FX was probed using a peroxidase-labeled polyclonal anti-FX antibody and revealed by chromogenic conversion of tetramethylbenzidine. For the negative control, hSR-AI was omitted during the coating (○ in panel A). Data represent the mean ± SD (n = 3-7). (C-H) Confocal analysis of THP1-derived macrophages incubated (1 hour at 37°C) with 150 nM Alexa 488–labeled Ac-LDL (C-D), 10 μg/mL Alexa 488–labeled FX (E-F), or preincubated with FX prior to the addition of Alexa 488–labeled Ac-LDL (G-H). Polymerized actin was counterstained using Alexa 647–labeled phalloidin (C,E,G) or cells were immunostained for EEA-1 (D,F,H). Dotted lines define cell boundaries based on phalloidin staining. Arrows indicate area of colocalization. Z depth is 0.5 µm; bars represent 10 µm; objective, ×63. OD, optical density; PoAb, polyclonal antibody; Poly[I], polyinosinic acid. Vincent Muczynski et al. Blood 2016;127:778-786 ©2016 by American Society of Hematology

Binding of FX to cellular SR-AI. Binding of FX to cellular SR-AI. (A-D) THP1-derived macrophages were preincubated with PBS (A), nonspecific IgG (B), or a polyclonal anti–SR-AI antibody (C) and further incubated with 10 µg/mL Alexa 488–labeled FX (1 hour at 37°C). Images were acquired in widefield microscopy and quantified for FX fluorescence (D). (E-I) Immunofluorescent staining of SR-AI (red) and FX (green) was performed in nontransfected HEK-293 cells (E and G, respectively) or HEK-293 cells transfected with pcDNA6/hSR-AI (F and H, respectively) incubated with 10 µg/mL FX (1 hour at 37°C). Images were acquired in widefield microscopy and subsequently quantified for FX fluorescence (I). Data are presented in mean pixel intensity per cell (D,I). Boxes represent the median and 25th to 75th percentile, and bars represent the 10th to 90th percentile (at least 5 different fields per experiment in 3 different experiments). (J) Representative images of double immunostaining for FX and SR-AI in HEK-293 pcDNA6/hSR-AI analyzed using confocal microscopy. Nuclei and polymerized actin were counterstained using DAPI (blue) and Alexa 647–labeled phalloidin (magenta), respectively. Objective, ×63; bars represent 10 µm; Z depth is 0.4 µm (J) and arrows indicate area of colocalization. ***P < .001, respectively, in the Mann-Whitney nonparametric unpaired statistical test. Vincent Muczynski et al. Blood 2016;127:778-786 ©2016 by American Society of Hematology

FX binding to murine SR-AI–deficient macrophages. FX binding to murine SR-AI–deficient macrophages. (A-C) CD115+-derived macrophages from wt (A) or SR-AI–deficient (B) C57BL/6 mice incubated with human FX (10 µg/mL, 1 hour at 37°C) were double immunostained for murine SR-AI (red) and human FX (green). Images were acquired in widefield microscopy and subsequently quantified for FX fluorescence (C). Data are presented in mean pixel intensity per cell. Boxes represent the median and 25th to 75th percentile, and bars represent the 10th to 90th percentile (at least 5 different fields per experiment in 3 different experiments). Nuclei and polymerized actin were counterstained using DAPI (blue) and Alexa 647–labeled phalloidin (magenta), respectively (A and B). Objective, ×63; bars represent 10 µm; ***P < .001, respectively, in the Mann-Whitney nonparametric unpaired statistical test. Vincent Muczynski et al. Blood 2016;127:778-786 ©2016 by American Society of Hematology

SR-AI and FX levels in vivo. SR-AI and FX levels in vivo. (A) Citrated plasma was collected from wt or SR-AI–deficient C57BL/6 mice prior or 24 hours after GdCl3 injection (50 mg/kg). FIX and FX activity were measured (A) and results are expressed in percentage of the normalized mean of wt values. (B-C) C57Bl/6 wt mice were injected in the caudal vein with FX (10 μg per mouse) in the presence of control IgG or polyclonal anti–mSR-AI antibodies (50 μg per mouse; 20 mouse per group). Plasma was collected at various time points (5 minutes, 1 hour, 3 hours, 6 hours, and 24 hours; 4-5 mice per group; 2-3 collections per mouse) and residual FX antigen was measured. Data were fitted to a biexponential decay equation to calculate pharmacokinetic parameters. Shown are the recovery at 5 minutes (B) and MRT (C) for each group. (A-B) Data represent the mean ± SD. (C) Boxes represent mean ± range of the calculated MRT, with the range being obtained from curves plotted with the minimum and maximum SD of residual FX levels. ***P < .001, respectively, in the Mann-Whitney nonparametric unpaired statistical test. Vincent Muczynski et al. Blood 2016;127:778-786 ©2016 by American Society of Hematology