EPCR Limits Nitric Oxide Levels, Mediating Human and Murine Stem Cell Adhesion and Retention In The Bone Marrow, By Conjugating PAR1 and CXCR4 Signaling

Long term repopulating hematopoietic stem cells (LTR-HSC) in the murine bone marrow (BM) highly express endothelial protein C receptor (EPCR), yet the function of EPCR in HSC is incompletely defined. EPCR is expressed primarily on endothelial cells and has anti coagulation and anti inflammatory roles. While physiological stress due to injury or bleeding is a strong inducer of HSC mobilization and leukocyte production, a role for the coagulation protease thrombin, and its major receptor PAR1 in regulation of HSC has not yet been identified. We hypothesized that thrombin plays a role in HSC mobilization in the context of injury and that, conversely, signaling involving EPCR and its ligand activated protein C (aPC) play a regulatory role in HSC maintenance. Herein, we report that murine BM EPCR^high stem cells display enhanced CXCL12 mediated adhesion and reduced migration capacitie, while motile circulating HSC in the murine blood and spleen lack high EPCR expression. Mechanistically, we found that EPCR is a negative regulator of nitric oxide (NO) levels. EPCR^high stem cells display low intracellular NO levels, low motility, and increased adhesion to BM stroma. Furthermore, EPCR^low transgenic mouse cells displayed reduced stem cell adhesion to BM stroma and increased motility, manifested by reduced EPCR^low HSC in the BM and their corresponding increased levels in the blood. In vitro stimulation with the EPCR ligand, aPC, which we found to be physiologically expressed adjacent to small murine BM blood vessels, augmented EPCR^high HSC adhesion and further limited their intracellular NO content by increasing eNOS phosphorylation at Thr495 in BM HSC, causing reduced production of NO. Conversely, administration of the pro-coagulant protease thrombin to mice induced PAR1 mediated EPCR shedding from BM HSC, followed by CXCR4 upregulation on HSC, and PAR1-mediated CXCL12 secretion by BM stromal cells. Together, these events lead to loss of retention and rapid stem cell mobilization to the blood. Interestingly, shedding of EPCR was found to be mediated by elevation of intracellular NO content, leading to EPCR co-localization with Caveolin. Correspondingly, thrombin failed to induce EPCR shedding and mobilization in eNOS and PAR1 deficient mice. Additionally, we found that BM LTR-HSC functionally express the metalloproteinase TACE (ADAM17) on the cell membrane, and that in- vitro inhibition of TACE activity by a newly developed selective inhibitor, reduces thrombin- mediated EPCR shedding, suggesting the involvement of TACE in EPCR shedding and HSC mobilization. Moreover, EPCR shedding was also CXCR4 dependent, revealing a crosstalk between EPCR, PAR1 and CXCR4. HSPC mobilized by thrombin possessed increased long-term repopulation capability following transplantation into lethally irradiated recipient mice and re-synthesis of EPCR by donor HSC in the engrafted host BM. In addition, EPCR expression was re-induced on circulating stem cells following in vitro treatment with eNOS inhibitor. Interestingly, bypassing eNOS by directly injecting NO donor, induced EPCR shedding, CXCR4 upregulation and rapid HSPC mobilization in both wild type and eNOS KO mice. Importantly, we found that similar to mice, EPCR was selectively and highly expressed by primitive human BM CD34⁺CD38^- HSC, but not in the blood circulation of clinical G-CSF mobilized stem cells or in motile cord blood stem cells. Human BM CD34⁺/CD38^- HSC are functionally EPCR^high cells, maintaining low levels of intracellular NO which mediates their increased adhesion, while EPCR shedding was important for their migration and mobilization. In the functional pre-clinical NOD/SCID mouse model, G-CSF mobilization induced EPCR shedding, up-regulation of PAR1 and CXCR4 on human stem and progenitor cells, while NO signaling inhibition blocked G-CSF induced mobilization and increased both murine and human EPCR^high stem cell accumulation in the murine BM.

Our results define functional roles for EPCR, on both human and murine HSC, and suggest that regulation of EPCR expression is linked to NO, PAR1 and CXCR4 signaling as a pivotal mechanism determining HSC localization and function. Our study reveals that activation of coagulation in the context of cell injury controls stem cells retention and motility, and suggests that targeting this system may be useful in improving clinical stem cell mobilization and transplantation protocols.