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Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Hematopoietic Stem Cells IX

Regulation of long-term repopulating hematopoieticstem cells by EPCR/PAR1 signaling

Shiri Gur-Cohen,1 Orit Kollet,1 Claudine Graf,2,3 Charles T. Esmon,4 Wolfram Ruf,2,5

and Tsvee Lapidot11Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. 2Center for Thrombosis and Hemostasis andJohannes Gutenberg University Medical Center, Mainz, Germany. 3Third Medical Department, Johannes GutenbergUniversity Medical Center, Mainz, Germany. 4Coagulation Biology Laboratory, Oklahoma Medical Research Foundation andDepartments of Pathology and Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, OklahomaCity, Oklahoma. 5Department of Immunology and Microbial Science, the Scripps Research Institute, La Jolla, California

Address for correspondence: Dr. Shiri Gur-Cohen, Weizmann Institute of Science, Department of Immunology, Herzel St. 234,76100 Rehovot, Israel. [email protected] or Prof. Tsvee Lapidot, Weizmann Institute of Science, Department ofImmunology, Herzel St. 234, 76100 Rehovot, Israel. [email protected]

The common developmental origin of endothelial and hematopoietic cells is manifested by coexpression of severalcell surface receptors. Adult murine bone marrow (BM) long-term repopulating hematopoietic stem cells (LT-HSCs),endowed with the highest repopulation and self-renewal potential, express endothelial protein C receptor (EPCR),which is used as a marker to isolate them. EPCR/protease-activated receptor-1 (PAR1) signaling in endothelial cellshas anticoagulant and anti-inflammatory roles, while thrombin/PAR1 signaling induces coagulation and inflamma-tion. Recent studies define two new PAR1-mediated signaling cascades that regulate EPCR+ LT-HSC BM retentionand egress. EPCR/PAR1 signaling facilitates LT-HSC BM repopulation, retention, survival, and chemotherapy resis-tance by restricting nitric oxide (NO) production, maintaining NOlow LT-HSC BM retention with increased VLA4expression, affinity, and adhesion. Conversely, acute stress and clinical mobilization upregulate thrombin generationand activate different PAR1 signaling that overcomes BM EPCR+ LT-HSC retention, inducing their recruitment to thebloodstream. Thrombin/PAR1 signaling induces NO generation, TACE-mediated EPCR shedding, and upregulationof CXCR4 and PAR1, leading to CXCL12-mediated stem and progenitor cell mobilization. This review discusses newroles for factors traditionally viewed as coagulation related, which independently act in the BM to regulate PAR1signaling in bone- and blood-forming progenitor cells, navigating their fate by controlling NO production.

Keywords: hematopoietic stem cells; HSC mobilization; bone marrow retention; aPC/EPCR/PAR1 signaling; throm-

bomodulin; CXCL12/CXCR4; nitric oxide

Introduction

Long-term repopulating hematopoietic stem cells(LT-HSCs) maintain continued blood cell pro-duction and host immunity throughout life andrespond to demands due to inflammation, injury,and blood loss. The bone marrow (BM) is themain site of adult hematopoiesis, and the majorityof LT-HSCs remain confined to the BM microen-vironment in a quiescent nonmotile mode viaadhesive interactions.1–3 The hallmark of LT-HSCsis their durable BM repopulation potential in func-tional transplantation assays, which requires high

self-renewal ability, homing, and developmentalpotential. Previous studies revealed that a very smallnumber of hematopoietic stem and progenitor cells(HSPCs) circulate in the peripheral blood as partof homeostasis and can reconstitute hematopoiesisin lethally irradiated transplanted recipients.4 Thephysiologic process of HSPC egress from the BM tothe circulation is accelerated by mobilizing agents,which mimic inflammatory and alarm situations,as HSPCs are mostly harvested from the peripheralblood rather than the BM for clinical stem celltransplantation.5

doi: 10.1111/nyas.13013

65Ann. N.Y. Acad. Sci. 1370 (2016) 65–81 C© 2016 New York Academy of Sciences.

PAR1 signaling dictates HSC retention or migration Gur-Cohen et al.

Emerging evidence shows that hemostatic factors,traditionally viewed as coagulation and inflamma-tion related, also independently control hematopoi-etic stem cells in the murine BM.6–8 The hemostaticsystem was originally discovered as a response toacute and transient tissue injury, balancing betweenprocoagulant and anticoagulant forces to preventblood loss and to control intravascular thrombosis.9

The procoagulant cascades include platelet and fib-rin clot formation, while the anticoagulant activitiesinclude inhibitors of coagulation and the fibrinolyticsystem.

Thrombin is among the most potent proco-agulant factors acting through proteolytic cleav-age of cell surface protease-activated receptor-1(PAR1), and its activity induces proinflammatoryand proapoptotic responses. PAR1, which belongsto a family of G protein–coupled cell surface recep-tors, is expressed by many cell types, includingBM endothelial and stromal cells,10 leukocytes,11

and blood-12 and bone-forming progenitors.13

Conversely, anticoagulant and anti-inflammatoryresponses are induced by binding of protease–activated protein C (aPC) to the endothelial proteinC receptor (EPCR), enabling endothelial cell PAR1signaling that is anti-inflammatory and cytoprotec-tive, due to cleavage of PAR1 at a site different fromthrombin.14–16

EPCR was originally identified and cloned asan endothelial-specific receptor, together with itsmajor ligand aPC.17 EPCR plays a critical rolein supporting aPC-mediated anticoagulant andcytoprotective signaling. Binding of aPC to EPCRfacilitates PAR1 cleavage and signaling, initiatinganti-inflammatory and antiapoptotic responsesand protection of endothelial barrier integrity.18

Recently, it was shown that EPCR is also expressedby some BM HSCs, suggesting additional new rolesfor this receptor. It has been demonstrated thata minority of HSCs in the murine fetal liver andadult BM, endowed with the highest BM long-termrepopulation potential, also express EPCR, whichwas used as a marker to isolate them.19–21 EPCR+

LT-HSCs functionally exhibit durable self-renewalpotential at the single transplanted stem cell level.22

This review outlines the complex and multiplayerregulation of hematopoietic stem cell maintenancein the BM microenvironment. We will discussthe bifunctional roles of PAR1 signaling in thecontext of hematopoietic stem cell function (i.e.,

BM repopulation and retention versus recruitmentto the blood circulation and development). We willhighlight the importance of dynamic PAR1 signal-ing, both for hematopoietic and for bone-formingstem cell migration and development, as well asfor protection from myelotoxic insult via control ofnitric oxide (NO) generation. Cooperation betweenosteoclast-/osteoblast-mediated bone turnover andaPC/EPCR/PAR1 versus thrombin/PAR1 signalingin regulating balanced stem cell function under bothnormal and acute stress conditions will be discussed.

LT-HSCs and preservation of endothelialelements

Before circulation is established during embryonicdevelopment, primitive blood-forming stem cellsreside within the yolk sac and the aorta–gonad–mesonephros (AGM) region. Once the vascularsystem develops, the lack of sufficient stromalsupport facilitates initial hematopoietic seedingof the fetal liver, where HSCs undergo extensiveproliferation and differentiation to all fetal bloodcell lineages.23–25 In the final stages of embryonicdevelopment, HSCs migrate from the fetal liver viathe blood circulation, across the physical blood–BMendothelial cell barrier, and home to their stromalniches, dependent on the chemokine receptorCXCR4 and its ligand CXCL12.26 Close physicalassociation between the developing primary bloodcell and endothelial lineages in the yolk sac initiallysuggested the “hemangioblast” concept, in whichthe hematopoietic cells and endothelial cells sharea common precursor during development.27,28

Indeed, hematopoietic and endothelial markershave extensive overlapping protein expressionpattern in the AGM, including VE-cadherin,28

Runx1,29 eNOS,30 CD31,29 CD34, and CD45.31

New cell-tracing techniques challenged the ancestorhemangioblast concept and suggested earlier seg-regation of hematopoietic stem cells from specificblood vessel endothelial cells.32 While the hierarchyof transcription factors that guide hematopoieticdevelopment is incompletely defined, lineagetracing may provide new avenues for studying HSCdevelopmental hierarchy and offer new perspectivesinto the origins of HSCs.

Recent reports reveal that the close associationbetween the hematopoietic and vascular systemsalso continues into postnatal life. These studieshave shown that endothelial cells, including

66 Ann. N.Y. Acad. Sci. 1370 (2016) 65–81 C© 2016 New York Academy of Sciences.

Gur-Cohen et al. PAR1 signaling dictates HSC retention or migration

those isolated from adult BM, are capable ofsupporting long-term hematopoiesis in vitro.33

Importantly, in vivo studies have demonstrated thatBM endothelial cells are essential for hematopoieticrecovery from lethal total-body irradiation andfor transplanted stem cell self-renewal and BMrepopulation.34,35 Recent advances in imaging tech-nologies have greatly advanced our understandingof the association between vasculature organizationand HSC localization in the murine BM. Themarrow microenvironment is highly vascularized,containing large blood vessels and sinusoids.Interestingly, some adult BM LT-HSCs were locatedin perivascular niches, adjacent to endothelial cells,in postneonatal life.36,37 Nonetheless, these nichesare not fully characterized and could also depend oncritical contributions from nonvascular cells, suchas �SMA+ macrophages,38 stromal precursors,39

and CXCL12-expressing reticular cells.40,41

While the ultimate consequence of the endothe-lial-to-hematopoietic transition during ontogenyis downregulation of the endothelial program inblood-forming stem cells and their progeny,42 BM-retained adult LT-HSCs also preserve and expresssome endothelial markers. Vascular cell adhesionmolecule-1 (VCAM1) and endothelial cell–selectiveadhesion molecule-1 are related adhesion moleculesfirst described and identified in endothelial cells,but are also upregulated in LT-HSCs, both at thetranscript and protein levels.43 VCAM1 interactionswith the integrin �4�1 (also termed as VLA4) medi-ate cell–cell interactions in multiple cell types, bothVCAM1 and integrin �4�1 inhibition have beenimplicated in LT-HSC mobilization,44 and theiractivity is essential for their homing to the BM.45,46

Single-cell analysis showed that a minority ofphenotypically defined BM LT-HSCs also expressvon Willebrand factor (vWF), previously thoughtto be exclusively expressed by megakaryocytes,platelets, and the endothelium.47 vWF+ HSCs iden-tify a primitive BM HSC population capable of sta-ble long-term myeloid- and megakaryocyte-biasedreconstitution supporting platelet production.47

vWF is central for platelet aggregation, hemostasis,and thrombus formation. Recently, it became evi-dent that vWF plays multiple roles in vascular biol-ogy, controlling smooth muscle cell proliferation,vascular inflammation, and angiogenesis.48 Whilethe ultimate role of vWF in LT-HSCs has yet to bedetermined, it is conceivable that vWF might be

secreted by HSCs themselves to contribute to theirregulation by ITGA2B-dependent adhesion49 in aself-primed, specific niche. Providing unique adhe-sion ligands might also pave the way for LT-HSCexpansion and skewing toward injury-responsivedifferentiation with megakaryocyte- and platelet-biased progenitor expansion.

Gene array studies have revealed that the anti-coagulant and anti-inflammatory EPCR is highlyexpressed predominantly in purified LT-HSCsobtained from murine fetal liver and adult BM butnot in common lymphoid or myeloid progenitorcells.50,51 Furthermore, isolation of primitive fetalliver and adult BM LT-HSCs on the basis of surfaceEPCR expression followed by transplantationassays revealed that EPCR+ LT-HSCs have thehighest hematopoietic reconstitution activity.19–21

Single-cell transplantations of EPCR+Sca-1high/CD150+CD48– (SLAM (signaling lymphocyte acti-vation molecule family of cell surface receptors))cells isolated from adult murine BM defined ahighly purified population of LT-HSCs exhibitingdurable self-renewal potential.22 Interestingly,while EPCR expression is a clear endothelialcharacteristic,52,53 it has also been identified asa stem cell marker in other tissues,12 includingmammary stem cells,54 and its function is crucialfor regulating integrin �4�1 in breast cancer stemcells and for tumor progression.55 Of note, atypicalEPCR expression by BM stem and progenitorcells was observed in the S129 (129S1/SvlmJ)mouse strain (preliminary results, data not shown),indicating that different mouse strains might havedifferent EPCR expression and function.

Although the association of the zymogen PC withEPCR greatly enhances activation to the serine pro-tease aPC with crucial intravascular anticoagulantfunctions, ligation of EPCR by aPC is key to alter-ing signaling pathways resulting in stabilization andprotection of the endothelial blood barrier.56 Thezymogen PC is synthesized predominantly in theliver and proteolytically activated by the thrombin–thrombomodulin (TM) complex.57 Preservation ofEPCR expression among different stem cell lineagessuggests that EPCR may provide stem cell signalsthat maintain the undifferentiated stemness pheno-type. Current evidence indicates that the aPC/EPCRpathway plays an important role in both fetalstem cell survival21 and adult BM LT-HSC reten-tion and protection from myelotoxicity,6 ensuring



preservation of the BM stem cell pool throughoutadult life.

BM niches are major sites for stemcell maintenance

LT-HSCs reside in specialized niches in the BM,where stromal and hematopoietic cells provide sup-port for adhesion of the stem cell pool, maintainingtheir undifferentiated primitive state.1,38,40 The BMmicroenvironment is considered to be a negativeregulator of stem cell migration,58 proliferation,and differentiation, as part of the mechanisms topreserve the dormant BM HSC pool while main-taining their developmental potential.59 Signalsproduced as a consequence of the dynamic osteo-clast/osteoblast bone turnover by BM-derived cells,such as osteoblasts,60 mesenchymal progenitor cells,endothelial cells, and myeloid cells, coordinatelyregenerate the BM, making it the preferred site foradult LT-HSCs.1,61–63 Despite technology break-throughs in high-resolution in vivo imaging tech-niques, the exact location of BM LT-HSCs and thedynamic nature, cellular composition, and structureof their niches remain controversial topics.1

Another fundamental open question that remainsis whether different niches exist for quiescent andfor cycling LT-HSCs in the BM. Current under-standing suggests that both endosteal (provided bybone-lining cells) and perivascular niches coexistin the BM, comprising stromal and hematopoieticcells that provide membrane-bound and secretedfactors to promote LT-HSC retention, maintenance,and development.1 Considering the complexity ofthe BM architecture, it might be possible that thesetwo proposed distinct niches might be, in fact,closely associated and might cooperate to providemutual signals to promote BM LT-HSC protec-tion and regulation. Adding layers of complexity,accumulating evidence implies that several typesof endothelial cells and blood vessels make up thestem cell vascular niche.2 While the vascular sinu-soidal niche has been implicated in HSC mainte-nance and regeneration,36,64 recent studies suggestthat the HSC vascular niche may not be limitedto sinusoids, since phenotypically defined LT-HSCsare also found adjacent to nonsinusoidal vascularstructures, including arteries and arterioles adjacentor in close proximity to bone.56 While considerableoverlap exists between HSC vascular microenviron-ments, an interesting question is whether all sinu-

soids or arterioles can functionally participate inpromoting LT-HSC maintenance and protection inthe BM, or if only specialized domains within thesevascular structures can do so.

Recent observations suggest that fetal and adultBM EPCR+ LT-HSCs are located in close proximityto BM endothelial subpopulations that highlyexpress TM.6,7,21 Generated thrombin typicallyforms a tight complex with TM that converts PCto its activated form, aPC.57 Importantly, TM+

endothelial cells in the adult murine BM alsostained for aPC and EPCR+ LT-HSCs were foundto be located adjacent to aPC-enriched endothelialareas6 (Fig. 1). Interestingly, similarly to endothelialcells,14,15 binding of the anticoagulant proteaseaPC to EPCR, which is functional on the LT-HSCsurface, induced PAR1 signaling,6 most likely bycleaving a site different from the canonical throm-bin cleavage site, as has been demonstrated forendothelial cells. Signals coming from aPC throughthe EPCR/PAR1 axis promote LT-HSC retentionin the BM, thereby providing protection fromDNA-damaging agents and preservation of theHSC pool size in the murine adult BM.6 It appearsthat, within the BM endothelial microenvironment,only a specialized type of endothelial cell, definedas TM-6,7 and aPC-expressing6 cells, can efficientlyand specifically maintain EPCR+ LT-HSCs in theBM. The interplay of TM and aPC in specialized BMmicroenvironments was found to be physiologicallyrelevant to allow accelerated stem cell recovery fromradiation-induced hematopoietic suppression anddeath.7 While the relevant targets of aPC allowingradioprotective activity remain unknown,7 EPCRsignaling was found to be essential for stem cell pro-tection from chemotherapy-induced hematologyfailure and death.6

The TM/aPC pathway may also be linked to BMmegakaryocytes that have been shown to negativelyregulate BM HSC proliferation via the secretion ofplatelet factor 4 (PF4).65,66 PF4 (also termed CXCL4)stabilizes TM and promotes aPC generation,67 sug-gesting that megakaryocytes may participate in theestablishment of the stem cell niches for BM EPCR+

cells, potentially enhancing aPC generation andthereby ensuring EPCR+ LT-HSC retention andprotection.6 While the role of aPC in this pro-cess remains to be formally demonstrated, sup-portive evidence shows that phenotypically definedLin– SLAM cells are frequently located adjacent to



Figure 1. PAR1 signaling balances EPCR+ long-term repopulating stem cell BM retention and trafficking. (1) TM+/aPC+endothelial cells form an anticoagulant niche for EPCR+ LT-HSCs in the BM. Binding of the anticoagulant protease aPC to EPCRon the LT-HSC surface induces PAR1 signaling, leading to the restriction of nitric oxide (NO) production and thereby initiatingCdc42–GDP polarity, downregulation of its GTP activity, and VLA4-dependent adhesion. The TM/aPC pathway may be linked toBM megakaryocytes, which secrete PF4, stabilizing the thrombin–TM complex, thereby promoting aPC generation and inductionof EPCR+ LT-HSC retention. (2) Physiologic stress, inflammation, injury, and cytokines transiently increase thrombin generation,which, in turn, activates extrinsic metalloproteases, such as C5a and MMPs, promoting HSC detachment from the BM. (3) Thethrombin–PAR1 axis initiates NO generation, causing LT-HSCs to become NOhigh, leading to enhanced Cdc42–GTP activity andreduced VLA4 affinity and promoting TACE-mediated EPCR shedding. In parallel, thrombin also induces PAR1-dependent CXCL12secretion from BM stromal cells into the circulation, followed by PAR1 and CXCR4 upregulation on the surface of BM HSPCs,leading to increased CXCL12-induced chemoattraction of responsive HSPCs to the blood. (4) It remains to be determined whichsignals initiate aPC generation in the BM and how EPCR– stem cells home back to the BM. How do circulating and mobilized HSCs,which lack surface EPCR expression (but may express intracellular EPCR), reexpress EPCR upon their BM homing, lodgment, andrepopulation?

marrow megakaryocytes,66 and, under chemother-apeutic stress conditions, PF4 secretion by BMmegakaryocytes increased HSC levels65 (Fig. 1). Thecoexpression of TM on BM EPCR+ LT-HSCs, butnot on stem cells in the bloodstream, suggests thataPC generation might not only take place in the sur-rounding BM endothelial microenvironment, butalso by the EPCR+ LT-HSCs themselves.6,7

aPC/EPCR/PAR1 signaling regulatesLT-HSC retention in the BM

The adult bone cavity is organized in a highly com-plex architecture of cellular components, includingmature and immature leukocytes, surrounded by

mesenchymal cell lineages. The current paradigmsuggests that only a single stem cell occupies a nichein the BM microenvironment, rather than stem cellsbeing clustered together. This clonal paradigm sug-gests that a single stem cell exploits the marrowmicroenvironmental resources; therefore, stem cellexpansion is functionally and mechanically limited,maintaining their slow cycling status. Such restrictedcycling, in turn, provides protection from myelotox-icity and exhaustion due to extensive inflammatoryconditions and during age-dependent functionaldecline.

A key additional concept is that mutual interac-tions between HSCs and their stromal supporting



cells exert inhibitory feedback on their proliferationand differentiation, keeping HSCs dormant in anonmotile mode.59,68 The chemokine receptorCXCR4 is essential for quiescence of LT-HSCs inthe murine BM, suggesting that membrane-boundCXCL12 expressed by BM stromal cells inducesadhesion and retention of CXCR4+ LT-HSCs.40

Conditional deletion of CXCL1269 or CXCR440,70

leads to increased cycling and exhaustion of thestem cell pool, as well as loss of BM retention andprotection from DNA-damaging agents. WhileCXCR4 is essential for LT-HSC quiescence,40 BMEPCR+ LT-HSCs express only low levels of surfaceCXCR4,6 suggesting that low CXCR4 receptorsignaling requires additional synergistic signalingcascades to maintain EPCR+ LT-HSC BM reten-tion. EPCR-expressing LT-HSCs exhibit increasedin vitro adhesion to fibronectin (FN)-boundCXCL12 and reduced migration ability to solubleCXCL12, in correlation with their BM retention.Short in vitro aPC prestimulation further increasedtheir CXCL12-induced adhesion, demonstratingcooperation between EPCR and CXCR4 in LT-HSC adhesion and suggesting a synergistic rolefor these receptors in LT-HSC BM retention.6

Interestingly, inhibitory effects of aPC/EPCR onleukocyte migration have also been documented inhuman activated neutrophils expressing EPCR inneutrophil chemotaxis triggered by interleukin-8.71

In BM LT-HSCs, the aPC/EPCR pathway inducesa PAR1 signaling cascade leading to restriction ofNO production, thereby initiating Cdc42 down-regulation and increased VLA4-dependent adhe-sion. HSCs expressing low levels of EPCR or HSCspretreated with EPCR-neutralizing antibody failto compete with normal untreated EPCR+ LT-HSCs in competitive BM transplantation into irra-diated hosts.6 In support of the crucial role ofligand binding to EPCR in maintaining functionalLT-HSCs in the BM, mice harboring a disabling sin-gle mutation in the aPC-binding domain of EPCRdevelop splenomegaly as a result of hematologicalBM failure.72

The cell adhesion receptor integrin VLA4binds FN and VCAM-1 and is crucial forCXCL12/CXCR4-mediated BM HSC retention.44,73

VLA4 is expressed by most leukocytes, as well assome nonhematopoietic cells,74 while its expressionis higher on EPCR+ LT-HSCs as compared to EPCR–

progenitor cells. VLA4 affinity was enhanced as a

consequence of aPC/EPCR signaling in LT-HSCs,providing a clear mechanism for their preferen-tial location within the adherent BM environment6

(Fig. 1). It has long been proposed that VLA4expression by circulating and BM-retained LT-HSCsmight be important for binding and detachmentof stem cells within the human BM microenviron-ment. Strong expression of VLA4 was mainly foundon human BM HSPCs, while circulating cells follow-ing granulocyte-colony stimulating factor (G-CSF)–induced clinical stem cell mobilization express lowlevels of VLA4. Surprisingly, EPCR expression dur-ing steady state was restricted to the murine BMand also absent from circulating HSPCs. Moreover,genetically modified mice strains with only low lev-els of EPCR (EPCRlow)6 or deficient in VLA4 havehigher numbers of circulating HSPCs.6,75,76

While it has yet to be determined whether EPCRsignaling also regulates VLA4 transcription andsurface expression, these observations indicatethat, apart from regulating the VLA4 affinity state,EPCR and VLA4 might have a tightly regulatedcross talk to enable LT-HSC retention in the BM.Since adhesive interactions are crucial for retentionof HSPCs in their BM niches, targeting thesemolecules is expected to induce mobilization.Indeed, administration of neutralizing antibodiesto the integrin VLA4 induced LT-repopulating HSCmobilization.77 Similarly, blocking EPCR signalingby neutralizing EPCR antibody treatment inducedCXCR4 upregulation and reduced VLA4 affinity,inducing mobilization of HSCs that expressed highlevels of EPCR in the circulation.6 The surprisinglyoverlapping roles of EPCR and VLA4 in enhancingLT-HSC adhesion and BM retention are furthersupported by the preliminary observation demon-strating that EPCR supports LT-HSCs homingspecifically to the BM.6 Upon injection, EPCR-expressing LT-HSCs rapidly appear in the BM inan EPCR-dependent manner, while EPCR blockagehad no effect on HSC homing to other hematopoi-etic organs, such as the spleen. This unique homingpattern of LT-HSCs to the BM was previouslyreported in studies demonstrating that cells lackingthe integrin �4 exhibited impaired homing to theBM, but not to the spleen.75 In addition to thehoming defect, an impaired expansion and BMrepopulation ability were even more pronounced intransplanted �4 integrin–deficient cells, which wererestricted to the BM compartment and not to the



spleen.75 The idea that the BM and the spleen mightprovide different signals for HSC maintenanceand development requires additional mechanisticinvestigation. However, preliminary results revealthat the murine spleen lacks TM+ endothelialcells, which are needed for aPC generation.78 Thisdifference may explain the unique BM periarterialmicroenvironments that are needed to attract andto retain EPCR+ LT-HSCs with high VLA4 affinityand adhesions.6

Adhesion and retention of LT-HSCs not onlyrequire the activity of adhesion molecules but alsorequire significant changes in regulators of theactin cytoskeleton. The small GTPases Rac1, Rac2,Cdc42, and RhoA are involved in adhesion signal-ing of hematopoietic cells.79,80 Cdc42 is a memberof the Rho GTPase family that cycles between theGTP-bound active and the GDP-bound inactivestates and thus acts as a binary molecular switchto activate responses for a variety of extracellu-lar stimuli, which can be diffusible factors, sig-nals on neighboring cells, and/or signals from theextracellular matrix.81 Cdc42 activation levels influ-ence cell adhesion, migration, division, and polarityestablishment.82

Regulation of Cdc42 activity and its polardistribution in HSPCs is crucial for stem celladhesion and BM repopulation potential.83,84

Deficiency in Cdc42 results in increased HSCcycling, impaired homing, and retention, leading tomassive stem cell mobilization.85 Conversely, miceexpressing elevated levels of active Cdc42–GTPalso exhibit reduced adhesion, impaired directionalmigration, and defective short-term and long-termengraftment.83 Taking these results together, itappears that either very high or very low levels ofCdc42 correlate with impaired HSPC retention inthe BM and that balanced Cdc42 activity ultimatelydetermines HSPC retention. Low Cdc42 activitywas found to be essential to allow EPCR+ LT-HSCretention in the BM, and aPC/EPCR signalingreduced Cdc42-GTP levels (Fig. 1).6

Moreover, as Cdc42 activity was found to becorrelated with its cellular distribution,86 EPCR-expressing LT-HSCs demonstrate polar Cdc42 dis-tribution, while EPCR– progenitor cells exhibit arandom distribution of Cdc42 within the cell.6 Cel-lular polarity has been tightly linked to proper cellfunction in general and particularly to stem cellfunction.87 A study reported asymmetric segrega-

tion of Numb, which inhibits Notch signaling inmurine HSCs, suggesting that Numb polarity isimportant for HSC self-renewal.88 Moreover, thevast majority of immature human CD34+ cellsacquire a polarized cell shape when cultured in thepresence of stem cell–supporting cytokines, suchas stem cell factor and thrombopoietin.89 Further-more, this polarization pattern is accompanied by aredistribution and polarization of several lipid raftsand adhesion molecules, such as CD133 and inter-cellular adhesion molecule 1 (ICAM1).90 Polariz-able myosin IIB contributes to human CD34+ stemcell asymmetric division, thereby facilitating stemcell self-renewal without expansion. As a conse-quence, inhibiting myosin II expands LT-HSCs.91 Itis therefore conceivable that EPCR, through its abil-ity to establish Cdc42 polarity and restrict Cdc42activity, regulates not only LT-HSC retention butalso HSC self-renewal potential.

Thrombin/PAR1 signaling regulatesHSC mobilization

As part of the host defense, the BM reservoirof immature and maturing leukocytes effectivelyadjusts to address alarm situations induced byinjury, bleeding, inflammation, and DNA damage.These processes are tightly regulated to avoid over-production of leukocytes and exhaustion of theBM stem cell pool, yet to allow rapid and effi-cient generation of blood and immune cells ondemand. Bidirectional trafficking between the BMand the periphery, referred to as homing, steady-state egress, stress-induced recruitment, and clin-ical mobilization, is a hallmark of HSPC physiol-ogy. Although the vast majority of HSPCs residein the BM, a small amount of HSPCs continuouslyegress from the BM to the peripheral blood and aredramatically increased during stress conditions.92

Such systemic and stress-induced changes includethe detachment of primitive stem cells and imma-ture progenitors from their anchored BM niches,followed by increased motility and massive recruit-ment of HSPCs and maturing leukocytes to thecirculation.93 Several clinical protocols mimickingphysiological stress conditions were developed toenable the use of mobilized HSPCs in order to recon-stitute hematopoiesis in ablated patients. Clinicalmobilizing agents that induce BM HSPC migra-tion include chemotherapy drugs such as cyclophos-phamide or repeated injections of the myeloid



cytokine G-CSF, mimicking stress-induced HSPCrecruitment through proinflammatory signals.94

An expanding line of evidence suggests thatPAR1 signaling critically regulates BM HSPC traf-ficking. Physiologic stress, inflammation, injury,and cytokines transiently increase thrombin gener-ation as part of the inflammatory process.95,96 Earlyobservations indicated that the powerful mobiliz-ing cytokine G-CSF might, in fact, induce a hyper-coagulant state in healthy donors, as evidenced byincreased thrombin generation, and consequentlycarry a risk of thrombosis.97,98 The expression of themajor thrombin receptor PAR1 assessed by microar-rays was found to be 3.3-fold higher in G-CSF–mobilized human CD34+ stem and progenitor cellscompared with steady-state BM CD34+ HSPCs.99

These observations paved the way for the hypothe-sis that stress-induced, accelerated thrombin gener-ation may promote PAR1+ HSPC exit from the BM,and that the higher PAR1 surface expression by themobilized human CD34+ stem and progenitor cellshas motility-supporting functions. In the murinehematopoietic system, mimicking acute stress byinjecting high levels of thrombin into mice showsthat active thrombin enters the BM very rapidly,activating PAR1 signal transduction pathways in BMHSPCs and stromal cells in parallel, inducing PAR1-dependent HSPC mobilization and recruitment ofLT-HSCs into the bloodstream.6

The chemokine CXCL12 is a powerful chemo-tactic factor for HSPCs, which express the majorCXCL12 receptor CXCR4.100 CXCL12/CXCR4interaction is an essential signal pathway in thetightly regulated process of LT-HSC traffickingand allows quiescent BM retention and protectionfrom chemotherapy-induced cell death.40 Modulat-ing the balanced CXCL12/CXCR4 interaction by thethrombin/PAR1 pathway in vivo was found to pro-vide the essential signal to enhance HSPC recruit-ment from the BM. CXCL12 is produced by manystromal BM resident cell types,39,40,100–102 includ-ing stem cell niche–supporting mesenchymal stemcells (MSCs) and endothelial cells.103 Basal cell sur-face expression of CXCL12 by BM stromal cells isrequired for HSPC homeostatic adhesion and main-tenance, acting via CXCR4 to ensure LT-HSC qui-escence and BM retention in a nonmotile mode.40

Unlike membrane-bound stromal and endothe-lial CXCL12, which induces LT-HSC adhe-sion, retention, and quiescence, secreted soluble

CXCL12 is a potent chemotactic factor leadingto HSPCs egress and mobilization to the bloodcirculation.100,104 Perturbed CXCL12/CXCR4 sig-naling in the BM leads to mobilization, which isassociated with CXCL12 secretion by marrow stro-mal cells and its release into the circulation. In paral-lel, HSPCs in the BM gain increased surface CXCR4expression and motility, followed by their enhancedmigration to the periphery.100,104,105 Rapid HSPCmobilization is a fast process that does not involvestem cell expansion and proliferation. One agentto rapidly mobilize HSCs is AMD3100, which wascharacterized as a CXCR4 antagonist according toits ability to inhibit migration of enriched BMmononuclear cells toward a gradient of CXCL12 invitro.106,107 Consistent with the notion that detach-ment of stem cells from their nursing microenviron-ment and recruitment to the circulation is an activeprocess, AMD3100 also has a dominant agonisticeffect by mediating rapid CXCL12 secretion frommurine BM CXCR4+ stromal and endothelial cells,followed by rapid release of CXCL12 into the circu-lation and induction of CXCR4-dependent HSPCmobilization.100

Thrombin proteolytic activity in vivo inducesrapid PAR1-dependent CXCL12 secretion fromBM stromal cells into the circulation, followed byPAR1 and CXCR4 upregulation on the surface ofBM HSPCs, leading to increased CXCL12-inducedchemoattraction of responsive HSPCs to the blood6

(Fig. 1). Evidence for thrombin in the blood plasmaduring G-CSF and AMD3100 mobilization fur-ther demonstrates the complexity of HSPC motilityregulatory mechanisms.108 It is therefore conceiv-able that increased thrombin levels are a commonmechanism to switch the BM EPCR- and CXCR4-mediated retention to rapid and prolonged PAR1-mediated CXCL12 secretion and CXCR4-inducedHSPC mobilization. Clinically, the timing of stemcell harvest with respect to thrombin peak levelsin the plasma may yield higher levels of mobilizedHSPCs, leading to better therapeutic outcomes.

Current data strongly implicate metallopro-teinases and other proteolytic enzymes as part ofthe stem cell migration and mobilization machin-ery by inactivating BM-derived growth factors,chemokines, and extracellular matrix responsiblefor hematopoietic stem cell adhesion and reten-tion. Proteolytic degradation, in turn, facilitatesenhanced movement across the physical barrier of



the BM extracellular matrix.109–111 It has been sug-gested that high levels of thrombin can prime themigration of HSPCs toward a gradient of CXCL12,enabled by upregulation of proteolytic enzymes.112

Thrombin induced MMP-9 secretion and upreg-ulation of MT1-MMP expression in human cordblood CD34+ cells, leading to enhanced chemoin-vasion of HSPCs toward a low CXCL12 gradient.112

G-CSF–mobilized human CD34+ HSPCs also haveincreased surface MT1-MMP expression and func-tion and reduced levels of its inhibitor RECK, pos-sibly induced by thrombin.113 Thrombin was alsofound to be a potent inducer of MMP-9 secre-tion from human monocytes,114 facilitating recruit-ment of leukocytes to inflammatory sites,115 furtherdemonstrating the potential role of thrombin/PAR1signaling in mediating enhanced HSPC motility byregulating proteolytic enzyme activation.

Increased thrombin levels in the blood plasmacan also facilitate the activation of the complementcascade, leading to enhanced stem cell recruitmentfrom the BM.116 Interestingly, direct inhibitionof thrombin with the drug refludan significantlyattenuated HSPC mobilization after G-CSF andAMD3100 administration. Reduced AMD3100-induced HSPC mobilization following thrombininhibition may imply that thrombin/PAR1 signalingcontributes to mobilization through CXCR4.108

Notably, in another study, G-CSF–induced pro-genitor cell mobilization in PAR1-deficient F2r–/–

mice was found to be higher than in wild-typemice, which was attributed to defective bonestructure and stromal signaling.48 One shouldconsider that the altered BM microenvironmentmay induce alternative mobilization pathwaysthat can substitute for the loss of PAR1 signaling.However, since these mice have higher basal levelsof circulating HSPCs,6 the overall G-CSF–inducedmobilization index is reduced. The altered HSPCBM retention is most probably due to the defectiveBM microenvironment,8 since PAR1 signaling isalso required for EPCR+ LT-HSC BM retention aswell as for BM stromal cells.

Apart from initiating microenvironmentalchanges, such as CXCL12 secretion and complementactivation to enhance HSPC mobilization, throm-bin, through the PAR1 axis, has a major role indirecting the exit of HSCs with the highest long-term repopulation potential. Thrombin–PAR1 sig-naling rapidly induces shedding of EPCR from the

surface of LT-HSCs, dependent on the activationof the metallopeptidase TNF-� converting enzyme(TACE/ADAM17)6 (Figs. 1 and 2). Interestingly, BMEPCR+ LT-HSCs highly express TACE/ADAM17and, in particular, its nonactivated precursor con-taining the inhibitory prodomain motif. Throm-bin activation of PAR1 induces rapid activationof TACE/ADAM17, as demonstrated by reducedexpression of the inhibitory prodomain, leadingto EPCR shedding from the surface of BM LT-HSCs. This, in turn, inactivates the EPCR-mediatedretention of LT-HSCs, directing their migration tothe blood circulation.6 Of interest, mice lackingTACE/ADAM17 exhibited hypercellularity in theBM and extramedullary hematopoiesis in the spleenand liver.117 This BM-failure phenotype may suggestthat EPCR shedding and recycling on the surface ofLT-HSCs is an essential process to allow normalhematopoiesis.117 Additional work will be requiredto determine whether TACE/ADAM17 activationis also required to allow clinical G-CSF– andAMD3100-induced HSC mobilization and whetherdirect activation of TACE might be an efficientstrategy to induce specific mobilization of EPCR-expressing LT-HSCs.

NO levels balance LT-HSC retention andtrafficking

PAR1 acts as a rheostat that, when activated bythrombin or aPC, induces distinct cascades ofevents, essentially balancing HSC BM localiza-tion versus trafficking to the blood circulation. Byswitching the intracellular NO generation machin-ery to either “on” or “off,” PAR1 signaling dis-tinctly regulates LT-HSC migratory fate6 (Fig. 2).NO is a small diffusible metabolite that serves asa cellular messenger in numerous biological sys-tems. NO can either act within the cell in whichit is produced or, by short-range release, affectadjacent cells. Evidently, the aPC–EPCR–PAR1 axisrestricts NO generation, preserving the stem cellsin an NOlow state that leads to reduced surfaceCXCR4 expression, reduced Cdc42 activity, andincreased VLA4-mediated adhesion. In contrast,the thrombin–PAR1 axis initiates NO generation,leading to increased CXCR4 expression, enhancedCdc42–GTP loading, and TACE/ADAM17 activa-tion. These events cumulate in EPCR sheddingand reduced BM adhesion and retention (Fig. 2).Balancing the NO levels in stem cells through



eNOS

Ser1177

Thr495

Cdc42-GTP

NOlow

NOhigh

Thrombin/PAR1

Low VLA4 affinity

High VLA4 affinity

aPC/EPCR/PAR1

High CXCR4 expression and function

Low CXCR4

Inactive TACE/ADAM17

Activated TACE/ADAM17

High PAR1 expression and function

Low PAR1 expression

TACE prodomain inhibitory motif

Soluble EPCR

Polar & lowCdc42-GTP

Figure 2. The two faces of PAR1 signaling pathways: the nitric oxide (NO) switch in BM EPCR+ long-term repopulating stem cells.PAR1 acts as rheostat that, when activated by thrombin or aPC, induces two different signaling cascades, which balance LT-HSC BMretention versus trafficking to the blood circulation by switching the intracellular NO generation machinery. The aPC–EPCR–PAR1axis (bottom) restricts NO generation, preserving the stem cells in an NOlow state that leads to reduced surface CXCR4 expression,polar Cdc42 with reduced GTP activity, and increased VLA4-mediated adhesion, thus promoting LT-HSC retention in the BM.The thrombin–PAR1 axis (top) initiates NO generation, leading to increased PAR1 and CXCR4 expression and function, enhancedCdc42-GTP activity loading, and TACE/ADAM17 activation and EPCR shedding, thus promoting HSC trafficking.

differential PAR1 signaling is orchestrated byendothelial NO synthase (eNOS) activity.6 NO sig-naling is essential for the initiation of hematopoiesisduring development.30,118 eNOS (Nos3) is expressedby HSCs in the murine AGM30 and in adult BM,6

further linking the hematopoietic and endothelialdevelopmental programs.

Direct treatment with NO donors, such asS-nitroso-N-acetyl-dl-penicillamine (SNAP), canbypass thrombin/PAR1 signaling, leading toCXCL12 secretion, PAR1 and CXCR4 upregulation,and rapid HSPC recruitment to the bloodstream.6

In support of these results in the mouse, humancord blood CD34+ progenitor cells pretreated withNO donors also exhibited increased surface CXCR4

expression and function.119 Many effects of NO aremediated through its canonical receptor, the solu-ble guanylyl cyclase generating cGMP. Classically,elevated NO content activates the cGMP signalingpathway, rapidly downmodulating the affinity stateof VLA4 and integrin-dependent cell adhesion.120

NO plays an expanding functional role in pro-tein S-nitrosylation, a reversible posttranslationalmodification of protein cysteines. Reduced VLA4affinity may involve S-nitrosylation of cytoskeletalproteins and integrins, leading to integrin inactiva-tion and cell detachment.120 Similarly, NO has beenimplicated in the activation of TACE/ADAM17 byinducing S-nitrosylation of the inhibitory motifof the TACE prodomain.121 Thus, NO production



induced by the thrombin–PAR1 axis might activateboth canonical cGMP signaling and noncanoni-cal protein modification, allowing TACE-mediatedEPCR shedding and reduced VLA4 affinity, therebyfacilitating HSC recruitment. The stromal contri-bution of NO generation is further demonstratedin the context of endothelial progenitor cell mobi-lization, which is dependent on BM stromal eNOSactivity involving MMP-9 proteolytic activity.122

NO production by the adult bone stroma isthought to have a positive and supportive effecton hematopoiesis;123 however, a collective set ofevidence indicates that NO production is detri-mental to hematopoietic recovery and survival afterradiation124 and chemotherapy,6 causing death. Byswitching the eNOS phosphorylation status, aPC–EPCR–PAR1 signaling induces eNOS phosphoryla-tion at its negative regulatory site, which restrictsNO generation, and promotes eNOS dephospho-rylation in its positive regulatory site, thereby pre-serving the EPCR-expressing LT-HSCs in a low-NOstate. Low-NO conditions are essential to maintainthe stem cells retained in the marrow cavity and toprotect them from chemotherapy-induced death.Pharmacological inhibition of NO generation bythe eNOS inhibitor N-nitro-l-arginine methyl ester(l-NAME) expands the LT-HSC pool in the BM.6

Furthermore, exposure of mice to NOS inhibitors,either directly or after irradiation and BM trans-plantation, increases the number of stem cells in theBM.124 Adding another layer of complexity, restric-tion of NO generation was reported to block differ-entiation of human CD34+ HSCs while maintainingtheir proliferation potential.125 Future studies willbe required to determine the series of events lead-ing to LT-HSC expansion following inhibition ofNO generation and whether the increased stem cellpool size is a cell-autonomous effect or supported bythe surrounding increase in available stromal nichecells. These observations may imply that NO can bea possible target for improved stem cell expansionand BM transplantation protocols.

Bone remodeling and hemostasis crosstalk modulates HSC maintenance

Residing in bone trabecular regions with highremodeling rates, HSCs are durably exposed to sig-nals of bone formation and resorption. During fetaldevelopment, HSC lodgment to the BM is synchro-nized with BM vascularization; however, in Osx–/–

mice lacking osteolineage (bone) cells, HSCs fail todurably repopulate the marrow of ablated recipi-ents, demonstrating the need for osteolineage cellsin HSC function.126 Consistent with this concept,ectopic generation of HSC niches was successfulonly in the presence of endochondral ossification, aprocess leading to bone formation.127 Many factorsexpressed by MSCs and their maturing osteolineagecell progeny are endowed with HSC-preservationpotential.128 Thus, bone formation appears tobe associated with HSC preservation and nicheassembly. Osteoblast and osteoclast activities arecoupled to maintain net bone remodeling through-out life. Bone formation is defective in newbornoc/oc mice due to the a lack of functional bone-resorbing osteoclasts.60 These mice have reducedBM HSC pools, impaired osteoblast differentiation,and defective BM HSC niches. Consequently, hom-ing of normal HSCs to the BM of oc/oc mice isreduced. Restoring BM niches and HSC functionby rescue of osteoclast activity demonstrates thatosteoclasts are required for normal formation ofHSC niches in newborn mice.60,129

Bone resorption is accompanied by extensiveactivity of osteoclast-secreted degrading enzymes,among which cathepsin K is essential. We have pre-viously shown that cathepsin K cleaves endostealmembrane–bound stem cell factor, CXCL12, andosteopontin, all essential components of BM HSCniches with major stem cell–regulation activities.101

Destruction of these retention-mediating moleculesdeactivates the adhesion machinery and releasesHSCs from the BM. Hence, HSC mobilization (e.g.,by G-CSF, bleeding, and bacterial LPS) is associatedwith increased osteoclast maturation and activityin wild-type mice, whereas reduced mobilizationcapacity was obtained in mice exhibiting defec-tive osteoclast adhesion and activity.101 Interest-ingly, young op/op mice have normal HSCs butlack the cytokine M-CSF, osteoclast activity, andbone cavities.130 These mice had enhanced G-CSF–induced HSPC mobilization and a lack of stemcell protection and death induced by 5-fluorouracilchemotherapy, most probably due to a lack of BMstem cell niches.130

Are hemostatic factors, which are active in bone-remodeling cells, involved in regulation of HSCsin their niches? As discussed earlier in this review,aPC/EPCR signaling in HSCs facilitates their reten-tion in their established BM niches. Interestingly,



EPCR expression in the BM is not limited toLT-HSCs, as preosteoblasts and osteoblasts alsofunctionally express EPCR.131,132 EPCR expres-sion by osteoblasts might also participate in boneturnover, as aPC stimulates osteoblast prolifer-ation via EPCR132 and administration of aPCtogether with bone morphogenetic protein 2 in miceinduced ectopic bone formation with higher rates ofosteoclast differentiation and vascularization, allimplicated in bone-remodeling processes.131 Fur-ther evidence linking hemostatic regulation withbone physiology demonstrated that long-term oralanticoagulant therapy in children is associated withosteopenia and a risk of future development ofosteoporosis,133 possibly indicating the need for bal-anced production of anticoagulant factors and nor-mal bone turnover.

As for procoagulant factors, as far back in 1916,the BM was shown to be a source of the throm-bin precursor prothrombin (PT);134 however, theproducing cell types were not well characterized.Recent data show that PT is mainly present in thenewly formed bone matrix of the metaphyseal tra-becular bone (a region rich in HSCs), in closeassociation with osteoclasts. MMP-9+ osteoclastscontain PT, and PT expression is increased duringosteoclast differentiation.135 Thus, PT is availablein the plasma, in osteoclasts, and is also incorpo-rated into the newly formed bone, which makesit available as a source for thrombin generationduring bone remodeling. Of interest, BM stromalprogenitor cells, as well as bone-lining cells, alsoexpress PT (unpublished data), demonstrating anadditional source for thrombin availability in theBM. The effects of PT conversion to thrombin inthe marrow cavity on LT-HSC physiology remain tobe determined.

Tissue factor (TF) is a major initiator of throm-bin generation in vivo. TF is expressed in variousmyeloid cells, monocytes, and macrophages. Underhomeostatic conditions, TF is expressed at low lev-els in monocytes. In the context of innate immuneresponses, TF is upregulated by inflammatory medi-ators, such as LPS.136,137 In vitro, receptor activatorof nuclear factor �B ligand (RANKL)-differentiatedosteoclasts also express TF and coagulation factorXa, which convert PT to thrombin.138 TF also playsa nonhemostatic role and regulates innate immunefunction through PAR2 signaling, also implicatedin inflammation.139 Enhanced HSC mobilization

and osteoclast activation during inflammation pre-sumably involves TF activity in osteoclasts to locallyproduce thrombin, which releases HSCs from theirniches. On the other hand, thrombin inhibitsosteoclast differentiation through a nonproteolyticmechanism,140 which may be a regulatory mecha-nism to shut down the inflammatory response.

PAR1, the major thrombin receptor, also playsa role in bone repair. Reduced new bone forma-tion and increased osteoclasts invading the damagedbone were observed in PAR1-deficent mice. Throm-bin treatment dose dependently induced prolifera-tion of BM stromal cells from wild-type mice, butnot from PAR1-deficent mice, demonstrating therequirement for PAR1 for bone healing.141 SincePAR1 is activated by plasmin and the fibrinolyticsystem is necessary for bone repair,56 PAR1 signalinginvolving proteases other than thrombin may con-tribute to homeostatic maintenance of the HSCsduring bone remodeling. Indeed, Par1 knockoutmice suffer from an abnormal bone phenotype.8,142

Taken together, factors of the coagulation systemcooperate within the bones in nonhemostatic path-ways to orchestrate bone-remodeling processes andstem cell niche regulation during steady-state andinflammatory conditions, along with pro- and anti-coagulant signaling. These mediators act togetherto harness HSCs to the BM or navigate them outfrom this organ as part of host immunity andhematopoiesis.

Concluding remarks and perspectives

“Well, you can go on looking forward . . . Theremay be many unexpected feasts ahead of you.”

– J.R.R. Tolkien.

Since the first cloning and identification of EPCRas the physiological receptor for the power-ful anticoagulant aPC143 and the discovery ofEPCR/aPC/PAR1 anticoagulant signaling,14 addi-tional evidence emerged for a close interactionbetween the coagulation systems and inflamma-tion, cellular metabolism, angiogenesis, and innateimmunity.144–146 Blood coagulation and primaryhemostasis evolved as important defense mecha-nisms to prevent bleeding,147 and a primary func-tion of the vascular TM/aPC/EPCR pathway is tocounterbalance intravascular thrombosis and main-tain endothelial quiescence. With new functionsemerging in our understanding of EPCR in BM



LT-HSCs and the challenge of deciphering themechanisms of the tight regulation by distinct PAR1signaling,6 new questions arise on the relevance ofthe hemostatic system for human stem cell physiol-ogy, and future studies will enhance our understand-ing of the role of PAR1 and NO signaling in clinicalG-CSF– and AMD3100-induced HSPC mobiliza-tion. As was previously suggested, PAR1 expressionmight be a good predictor of the efficacy of stem cellmobilization,99 and further strategies to manipulatePAR1 signaling and its downstream effectors mightbe beneficial in individuals who do not respondefficiently to current mobilization protocols and aretherefore termed poor mobilizers. Another funda-mental question is whether imbalanced coagulantactivity, often observed in elderly patients, might bea relevant target to improve HSPC mobilization andBM transplantation.

Stem cells in the BM were characterized asNOlow (Ref. 6) and reactive oxygen species (ROS)low

cells.148 While it has yet to be determined whetherthere is an overlap between the two cell populations,it was previously suggested that high levels of ROSare reversible, and reduction of ROS levels in thestem cell would induce reduced cycling and restorelong-term repopulation potential.148 Since circulat-ing cells express higher levels of NO,6 one can spec-ulate that restoration of low NO levels might bebeneficial to improve stem cell retention potentialupon BM transplantation.

The leukemic stem cell paradigm refers to theability of a subpopulation of cancer cells to initiatetumorigenesis by undergoing self-renewal anddifferentiation. It becomes clear that the highfrequency of relapse after conventional cytotoxicchemotherapies predicts that leukemic stem cellsare resistant to standard therapy, suggesting thattargeting the stem cell population will likely leadto improved patient outcomes. It is also becomingevident that the BM microenvironment is linkedwith primary leukemic stem cell resistance totherapy. The potential for unraveling how EPCRsignaling and the anticoagulant microenvironmentparticipate in malignant hematological disordersmight provide new approaches for improved cancertherapy.

Acknowledgments

The studies discussed in this review were par-tially supported by the Israel Science Foundation

(851/13), the Ernest and Bonnie Beutler ResearchProgram of Excellence in Genomic Medicineand FP7-HEALTH-2010 (CELL-PID 261387), theWeizmann–University of Michigan–Technion Col-laboration Program, NIH Grant HL-60742, and theHumboldt Foundation.

Conflicts of interest

The authors declare no conflicts of interest.

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