Total body irradiation (TBI) is associated with dysfunction of radiosensitive organs 2–5
. To identify novel genes and pathways protecting hematopoietic stem and progenitor cells (HSPCs) against radiation injury we performed retroviral insertional mutagenesis screens with a replication deficient virus bearing a strong internal promoter expressing enhanced green fluorescent protein (EGFP) 6
(Supplementary Fig. 1a
). At week 4, 7 and 10 following BM transfer, recipients were exposed to a single dose of 3 Gy TBI, resulting in three consecutive cycles of radiation-induced contraction and subsequent re-expansion of the hematopoietic system. Viral integration sites in genomic DNA in BM cells from animals in which post-transplant TBI had resulted in a significantly augmented relative abundance of EGFP-positive cells in PB or BM were determined by ligation mediated (LM)-PCR 6
(Supplementary Fig. 1b-e
, Supplementary file 1
). Loci targeted by integration included genes known to play a role in radioprotection of either hematopoietic or neuronal cells 2,7,8
, such as PUMA
(Supplementary Fig. 2b-d
) and c-Jun
(data not shown). In animal 9 ( and Supplementary Fig. 1b
) LM-PCR revealed integration of the virus 31.6 kb upstream of the Thrombomodulin (Thbd
) gene (), which was associated with increased abundance of endogenous Thbd transcript and protein in radio-selected EGFP-positive cells, while the integration had little effect on expression of other neighboring genes like the somatostatin receptor 4 (SSTR4
) or the C-type lectin transmembrane receptor CD93
Elevated expression of Thbd selects for primitive hematopoietic cells upon irradiation in vivo
To ascertain whether augmented Thbd expression in HSPCs was sufficient for conferring a competitive selection advantage to hematopoietic cells in response to TBI, HSPCs were transduced with lentiviral Thbd-expression constructs, and Thbd over-expressing cells were subsequently transplanted into pre-conditioned C57BL/6-CD45.1 recipients ( and Supplementary Fig. 3
), followed by one 3 Gy TBI administered 4 weeks post-transplant and analysis of EGFP chimerism in PB at 3 weeks post-TBI. Cells over-expressing Thbd were 1.5-fold enriched in PB as compared to vector-only controls (), demonstrating that elevated expression of Thbd in hematopoietic cells was sufficient to confer a selective advantage after radiation injury. However, Thbd over-expressing HSPCs were not protected from the effects of ionizing radiation in vitro
, as determined by survival, apoptosis, and proliferation of progenitor cells in response to irradiation (Supplementary Fig. 4
), indicating that the beneficial effects of Thbd on HSPCs in vivo
required additional cells or molecules.
Endogenous Thbd is a multifunctional cell surface-associated receptor that regulates the activities of several physiological protease systems, including complement, fibrinolysis, and blood coagulation 9
. Biochemically, Thbd functions as a high-affinity receptor for thrombin. The Thbd/thrombin complex activates thrombin activatable fibrinolysis inhibitor (TAFI) and also converts the plasma zymogen protein C (PC) into the natural anticoagulant, activated protein C (aPC)10–12
. aPC inhibits blood coagulation via proteolysis of blood coagulation factors V and VIII, promotes indirectly the activity of the fibrinolytic system and exerts potent anti-inflammatory and cytoprotective effects on endothelial cells, neurons and various innate immune cell populations 13
that are mediated through the interaction of aPC with signaling-competent receptors, such as Par1, Par2, and Par3, integrins, and the endothelial protein C receptor (Epcr)13,14
As the beneficial effects of Thbd in vivo
could not be attributed to functions of Thbd intrinsic to HPCs, we hypothesized that extrinsically and thus systemically administered Thbd might promote systemic beneficial effects in response to radiation injury. Administration of recombinant soluble forms of THBD to baboons and humans is safe and exhibits anticoagulant and antithrombotic activities15–17
Administration of an oxidation-resistant form of soluble, recombinant human THBD (solulin, INN sothrombomodulin alpha, Supplementary Fig. 5
) up to 30 minutes post-TBI at 8.5 or 9.5 Gy resulted in significant radioprotection of wild type mice, compared to vehicle-treated controls, with a 40%-80% survival benefit (). Solulin has been shown to serve as the cofactor for conversion of the plasma zymogen protein C (PC) into the natural anticoagulant, activated protein C (aPC) 10,16,18
. To determine whether the protective effects of soluble THBD could be related to the activation of protein C, we investigated whether infusion of recombinant aPC could reproduce the radio-protective effect of soluble THBD. In independent experiments conducted in three different laboratories, administration of recombinant murine aPC to C57BL/6 mice (at 5 μg/mouse i.v., equal to 0.4 mg kg−1
) conferred a significant survival benefit compared to vehicle-treated controls (). Similar data were obtained with genetically distinct CD2F1 mice (at 0.35 mg kg−1
i.v., 30 minutes post-TBI, data not shown), indicating that the aPC effect was not dependent on genetic background. At 10 Gy, still 40% of animals survived after multiple injections of aPC (at 30 minutes, 1 hour, and 2 hours post-TBI) (). Remarkably, even when the first injection of aPC was delayed until 24 hours post-TBI, with a second injection at 48 hours post-TBI, significant radiation mitigation was observed ().
Solulin and recombinant aPC confer mitigation of radiation toxicity after TBI
Given that the radiation doses used in our experiments result in death occurring 12–20 days post radiation exposure primarily due to failure of hematopoietic cells within the bone marrow, likely candidate cellular targets of solulin as well as aPC in radiation mitigation include epithelial and/or endothelial structures of the gut or bone marrow or bone marrow hematopoietic cells. Administration of solulin or aPC to lethally irradiated mice had no detectable effect on basic blood cell parameters at day 3 or 10 after radiation exposure (Supplementary Fig. 6a
and data not shown), except for marginally elevated numbers of white blood cells in BM at day 10 in aPC treated animals (). Hematopoietic progenitor cells in BM, determined by flow cytometry (Lin−
cells, ) or functionally via CFU-C assays (), were almost undetectable at 3 days post irradiation (data not shown), but at 10 days the were significantly increased in aPC-treated mice compared to controls. The frequency of animals presenting with more than 10 CFU-C colonies per 5×104
cells in BM at day 10 post TBI was likewise significantly higher in the aPC-treated group (data not shown), and correlated with the frequencies of aPC-treated animals surviving exposure to lethal radiation doses (see ). Similar observations were made in lethally irradiated mice receiving solulin (), consistent with the notion that activation of protein C constitutes a relevant downstream effector of soluble THBD. Infusion of aPC did not alter biomarkers of radiation-induced gut injury, such as plasma citrulline levels and integrity of the gut epithelial surface (Supplementary Fig. 6c,d,e
), indicating that mitigation of radiation damage to the intestine was unlikely to significantly contribute to the efficacy of aPC.
Mechanisms of action of mitigation by soluble Thbd and aPC
To gain more insight on molecular mechanisms of aPC action, we compared distinct recombinant variants of aPC with respect to their radio-mitigating activity. The mouse 5A-aPC variant, which exhibits full Epcr- and Par1-mediated cytoprotective function, but only residual (~8%) anticoagulant activity 19,20
, did not prevent radiation-induced mortality. In contrast, infusion of E149A-aPC with augmented anticoagulant activity but with deficient signaling activities, (e.g., only ~5% of normal anti-apoptotic activity) 21
conferred a significant survival benefit that was comparable to that of wild type aPC (). The biological activity of aPC that mediates radiomitigation is thus preserved in the E149A-aPC variant, but compromised in the 5A-aPC variant.
Both Par1 and Epcr are expressed on primitive BM cells 22–25
. HSPC lacking Par1 26
or expressing greatly diminished levels of Epcr 27
were not impaired but rather slightly favored compared to wild type controls in competitive transplantation/radiation-injury experiments (Supplementary Fig. 7a,b,c
), providing additional support that the radio-mitigation activity of aPC does not involve Epcr and Par1-dependent signaling on HSPCs which is consistent with the failure of the signaling-selective 5A-aPC variant to afford radio-protection.
Functions preserved by the E149A-aPC variant, but deficient in the 5A-aPC molecule potentially include the anticoagulant effect of aPC and potentially coagulation-independent aPC effects, such as the degradation of cytotoxic histone-DNA complexes released from damaged cells 28
. However, inhibition of cytotoxic histones 3 and 4 with the function-blocking BWA3 antibody, using conditions shown previously to reduce mortality in sepsis 28,29
, did not result in radioprotection (). Similarly, inhibition of the intrinsic coagulation pathway with anti-fXI antibody 14E11 30
() or with low molecular weight heparin were both ineffective in radioprotection. Biomarkers indicative of the activation state of the blood coagulation system in PB (plasma thrombin-antithrombin complexes and fibrin D-dimer) were unaltered over a 24h time window following exposure to lethal radiation doses (Supplementary Fig. 8
), suggesting that the radioprotective effect of the E149A-aPC variant is not due to its antithrombotic actions. While it is possible that optimized dosing with antibodies blocking thrombosis or histone-induced inflammation might reveal some beneficial effect of these reagents on radiation injury, it seems unlikely that the pathological mechanism inhibited by these antibodies are the critical targets of aPC that mediate the accelerated recovery of HPC activity and survival of radiation injury. The targets of aPC mediating accelerated recovery of HPC activity and survival of radiation injury therefore remain presently unknown and likely involve novel functions associated with wild type aPC and the E149A-aPC variant 20,31
The above observations raised the question whether the endogenous Thbd-PC pathway might play a previously unrecognized role in mitigating the lethal consequences of radiation-induced bone marrow failure. In adult mice and humans, Thbd is expressed ubiquitously in endothelial cells of small blood vessels except for low levels in certain brain microvascular beds32
. Within the human hematopoietic system, THBD is expressed in a subpopulation of human dendritic cells, in monocytes, and in a small subset of neutrophils33,34
. Western Blot analysis of BM from radiation-exposed animals indicated the presence of Thbd protein in BM cells (, GFP- control) and Thbd transcript was detected in differentiated BM cells, in HPCs, in eHPCs (Lin−
cells) of BM (), in BM derived CD45−
endothelial cells and in the CD45−
stroma cell compartment in BM (). An in situ survey of β-galactoside expression in the femur of ThbdlacZ
knock-in mice indicated abundant Thbd expression within the endosteal region, as well as in femoral blood vessel endothelial cells supplying the marrow (). Flow cytometry confirmed Thbd expression in Ly-6GNEG
BM macrophages as well as in B220/CD19 positive B-cell precursors (Supplementary Fig. 9a
and data not shown). Thbd-expressing macrophages are distinct from the two previously described populations of BM resident macrophage-like cells involved in maintenance of the hematopoietic niche in BM, i.e. cells with the surface phenotype CD169POS
(Supplementary Fig. 9b
). Within the CD45NEG
endothelial population in BM, Thbd expression is detected in Sca-1 NEG
sinusoidal endothelium, but is absent from Sca-1POS
arterial endothelium (Supplementary Fig. 9c
). This combined analysis of Thbd-mRNA, Thbd-antigen, and lacZ-reporter gene expression is consistent with the presence of Thbd on hematopoietic cells and non-hematopoietic cells within the BM.
A role of endogenous Thbd in radiation protection
We next investigated whether the selective genetic disruption of protein C activation by endogenous Thbd which results in minimal residual Thbd function modified TBI survival35,36,37
. Mice (ThbdPro/LacZ
carrying only one functional Thbd-allele that encodes a Thbd variant (ThbdPro
) with severely reduced ability to activate protein C showed increased sensitivity to TBI, with the dose of radiation eliciting 50% lethality shifted from ~8.75 Gy in wild type mice to ~ 7.5 Gy in Thbd-deficient mice (), while ThbdPro/Pro
mice, which show a less severe Thbd deficiency than ThbdPro/LacZ
mice still presented with elevated sensitivity to TBI (). In contrast, aPCHI
transgenic mice with constitutively elevated plasma aPC levels due to expression of a variant human protein C that is efficiently activated by thrombin even in the absence of Thbd 38
, were protected against radiation-induced BM failure to a similar extent as wild type mice treated with recombinant aPC (). Expression of the aPCHI
transgene also rescued the increased radiation sensitivity of Thbd-deficient ThbdPro/Pro
mice (), providing direct genetic evidence that the increased radiation sensitivity of Thbd-deficient mice is caused by inadequate activation of endogenous PC.
Competitive hematopoietic reconstitution of lethally irradiated wild type recipients with BM from ThbdProPro and wild type mice, followed by exposure to 3 Gy TBI given 8 weeks after transplantation (), showed a significantly reduced recovery of ThbdProPro cells when compared to wild type cells (). The initially lower contribution of ThbdProPro BM cells to chimerism before irradiation might owe to not yet investigated additional functions of Thbd in HSC biology. Non-competitive reconstitution of irradiated ThbdProPro recipients with wild type BM (), followed by a second exposure to a LD50-dose of TBI, resulted in significantly increased 30-day mortality compared to wild type animals reconstituted with wild type BM (). Hence, endogenous Thbd expression on hematopoietic cells as well as on non-hematopoietic stroma cells affords protection against radiation mirroring the protection conferred by forced Thbd over-expression in HSPC or by therapeutic administration of soluble Thbd/aPC.
In summary, the current work identified the Thbd-protein C pathway as a physiologically relevant mechanism that accelerates HSPC recovery in response to lethal TBI to an extent that results in significant radio-mitigation. Our data are consistent with a mechanism in which endogenous Thbd expressed on stromal endothelial cells promotes protein C activation and release of aPC into the BM microenvironment, followed by aPC-stimulated recovery from radiation-induced hematopoietic suppression. Judged by the effect of Thbd-deficiency on 30-day mortality (), Thbd expression on stromal endothelial cells appears to make a more important contribution to overall survival than hematopoietically expressed Thbd. Nevertheless, Thbd expression on HSPC supports hematopoietic recovery upon TBI in an apparent cell-autonomous manner, with as yet unknown effects on whole animal survival of radiation injury. While the cellular and molecular mechanism underlying the effect of soluble THBD or aPC on the recovery of HPC activity still need to be studied in more detail, they are likely distinct from previously explored pathways, including those mediated by agonists of toll-like receptor 5 39
, inhibitors for CDK4/6, or various antioxidant compounds 40
. While the effect of endogenous aPC may largely remain confined to it’s site of formation, i.e. the local microenvironment of Thbd-expressing cells, systemic administration of soluble THBD or of aPC can reproduce and augment the radio-protective effect of the endogenous protein C pathway.
Recombinant human aPC has undergone extensive clinical testing in patients, and recombinant soluble human THBD is currently being investigated for efficacy in antithrombotic therapy in man. Our data encourage the further evaluation of these proteins for their radio-mitigating activities.. Moreover, these agents, possibly in combinations with other compounds targeting other pathways, may provide novel medical countermeasures against radiation induced pathologies that may arise in environmental or therapeutic settings that involve exposure to high levels of radiation.