EPCR/PAR1 Signaling Navigates Long-Term Repopulating Hematopoietic Stem Cell Bone Marrow Homing to Thrombomodulin-Enriched Blood Vessels
Authors: Gur Cohen Shiri , Itkin Tomer , Chakrabarty Sagarika , Graf Claudine , Kollet Orit , Golan Karin , Kalinkovich Alexander , Esmon Charles T. , Ruf Wolfram , Sagi Irit , Erez Ayelet , Lapidot Tsvee 
Published: October 2019

Abstract
Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) is an active and essential first step in clinical stem cell transplantation. EPCR is expressed by murine BM LT-HSCs endowed with the highest repopulation potential and its ligand, activated protein C (aPC), has anticoagulant and anti-sepsis effects in EPCR+/PAR1+ endothelial cells. We recently found that signaling cascades, traditionally viewed as coagulation and inflammation related, also independently control EPCR+ LT-HSC BM retention and recruitment to the blood via distinct PAR1 mediated pathways. EPCR/PAR1 signaling retains LT-HSCs in the BM by restricting nitric oxide (NO) production and Cdc42 activity, promoting VLA4 affinity and adhesion. Conversely, thrombin/PAR1 signaling overcome EPCR+ LT-HSC BM retention by initiating NO production, leading to TACE-‎mediated EPCR shedding, CXCR4 and PAR1 upregulation and parallel CXCL12 secretion by PAR1+ BM stromal cells, enhancing stem cell migration and mobilization.
Since EPCR shedding is essential for BM LT-HSC recruitment, we tested EPCR role in LT-HSC BM homing. EPCR+ LT-HSC exhibited reduced in vitro migration towards CXCL12 and enhanced CXCL12-dependent adhesion to fibronectin. Unexpectedly, transplanted EPCR+ LT-HSCs preferentially homed ‎to the host BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing antibody treatment, which blocks binding to aPC, leading to attenuated EPCR+ LT-HSC homing to the BM but not to the spleen. Importantly, short term aPC pretreatment inhibited NO production and dramatically increased EPCR+ LT-HSC BM homing. Since EPCR navigates LT-HSC to the BM, we studied the role of EPCR signaling in LT-HSC BM repopulation. Mimicking EPCR signaling by in vivo NO inhibition induced preferential expansion of blood and bone-forming stem cells and gave rise to higher donor type EPCR+ LT-HSCs in competitive repopulation assays. Similarly, repeated treatment with aPC expanded BM EPCR+ stem cells and increased competitive LT-repopulation. Importantly, loss of EPCR function reduced HSC long-term repopulation ability while maintaining their short-term repopulation activity. BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with normal stem cells in competitive long-term repopulation assays. Consistent with inferior HSC BM repopulation, Procrlow mice exhibited reduced numbers of BM LT-HSC with reduced adhesion capacity. Additionally, these mice displayed increased HSC frequencies in the blood circulation and the spleen, which were pharmacologically corrected by inhibiting NO generation with L-NAME treatment. BM retention is essential for quiescent HSC protection from chemotherapy. Mice treated with NO donor SNAP, or with blocking EPCR antibody as well as Fr2-/-mice lacking PAR1 expression, were more susceptible to hematological failure and mortality induced by 5-FU treatment compared to control mice. Together, these results indicate a functional aPC/EPCR/PAR1 signaling pathway, regulating EPCR+ LT-HSC BM homing, adhesion and long-term repopulation potential. The thrombin-thrombomodulin (TM) complex converts protein C to its activated form aPC, facilitating high affinity binding to its receptor EPCR. To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by a unique BM endothelial cell (BMEC) subpopulation, but not in the spleen. Moreover, EPCR+ LT-HSCs were found adjacent to TM+/aPC+ BMECs, imposing their adhesion and retention. Interestingly, similar to BMECs, BM EPCR+ LT-HSC also express surface TM, implying the possibility of autocrine aPC generation.
Herein we define EPCR as a guidance molecule, navigating slow migrating LT-HSC in the blood flow specifically to TM+ BMEC supporting niches, maintaining NOlow stem cell retention, long-term blood production and protection from myelotoxic insult. Conversely, thrombin/PAR1 signaling oppositely increase NO generation and EPCR shedding allowing increased CXCR4-dependent LT-HSC migration and mobilization. Harnessing EPCR signaling may improve clinical stem cell transplantation, increasing LT-HSC specific BM homing and repopulation by aPC pretreatment, as well as potentially to overcome malignant stem cell chemotherapy resistance.

Disclosures
No relevant conflicts of interest to declare.