Figure 6. aPC-EPCR signaling limits NO production and promotes LT-HSC retention.

(a) NO (green) levels in BM LSK EPCR⁺ (purple) and EPCR⁻ lineage negative Sca-1⁺ (yellow) c-Kit⁺ (red) cells. Scale bar, 7 µm. (b) Percentage of NO^low SK SLAM cells following in vitro treatment of bone marrow cells with EPCR neutralizing or non-neutralizing antibody; n = 4. (c) Percentage of NO^low cells and NO levels in wild-type or Procr^low bone marrow LSK SLAM cells, with or without in vitro aPC treatment for 1 hour; n = 3, P values, two-way ANOVA with Bonferroni post-test. (d) Kinetics of eNOS phosphorylation on Ser1177 or Thr495 in bone marrow SK SLAM cells following in vitro aPC stimulation; n = 4. (e) eNOS phosphorylation on Thr495 in bone marrow LSK SLAM cells following in vitro stimulation with aPC for 15 minutes, active-site blocked aPC, FVIIa, or active-site blocked FVIIa; n = 4, P values, one-way ANOVA with Tukey’s post-test. (f) Circulating LSK cells in wild-type or Procr^low chimeric mice following prolonged l-NAME treatment for 5 days; n = 5, P values, two-way ANOVA with Bonferroni post-test. (g) Adhesion and migration of Procr^low bone marrow LSK cells following in vitro l-NAME or PBS treatment for 2 hours; n = 6. (h) Circulating LSK (n = 8), bone marrow EPCR⁺ SK SLAM cells (n = 4) and representative FACS plot for EPCR expression following prolonged l-NAME or PBS treatment for 5 days. (i) Competitive repopulation of bone marrow cells obtained from donors treated with l-NAME or PBS, assessed 16 weeks following transplantation; each dot represents an individual recipient mouse; P values, one-way ANOVA with Tukey’s post-test. (j) Survival after 5-FU treatment, with or without SNAP treatment (n = 9, *** P value = 0.0003), in wild-type vs F2r−/− mice (n = 5, ** P value= 0.0010) or co-treated with EPCR neutralizing or non-inhibitory antibody (n = 9, *** P value < 0.0001).