Regulation of Hematopoiesis By the Coagulation Receptor Thrombomodulin

BACKGROUND: Pharmacologic supplementation of protein C pathway function by infusion of recombinant Thbd or activated protein C supports recovery of hematopoietic function from lethal radiation injury in mice [Geiger et al., Nature Medicine, 2012]. Partial Thbd deficiency in hematopoietic stem and progenitor cells (HSPC) or bone marrow endothelium results in augmented sensitivity towards radiation injury [Geiger et al., Nature Medicine, 2012]. The underlying cellular and molecular mechanisms of Thbd function in hematopoiesis are not yet characterized. The objective of the current study was to determine the expression pattern and functional role of Thbd in HSPC.

RESULTS: Flow cytometric analysis was employed to detect Thbd expression in defined subsets of murine HSPC. Thbd was co-expressed with the endothelial protein C receptor (Procr/EPCR) in the majority of bona fide stem cells with long-term-repopulating capacity (LT-HSC), and was also expressed on EPCR-negative stem cells with short-term repopulating capacity (ST-HSC), multipotent progenitors (MPP), common lymphoid progenitors (CLP), common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP). In contrast, only a subset of megakaryocyte-erythrocyte progenitors (MEP) expressed low levels of Thbd. In the bone marrow, Thbd was also expressed by B cells in early stages of maturation (from progenitor B cell stage till immature stage). In this lineage, the fraction of Thbd-positive cells was inversely correlated with the stage of B cell maturation. In addition, Thbd was detected in three distinct subsets of bone marrow-resident myeloid cells (CD11b+CD115+, CD11b+CD11c+ and CD11b-CD115+). Thbd expression outside the bone marrow was limited to a small fraction of hematopoietic cells (2-5% in the peripheral blood and spleen). These cells included myeloid cells (macrophage/monocyte and dendritic cells). Approximately 1-2% of all B cells in the peripheral blood and the spleen expressed Thbd, possibly reflecting recent bone marrow emigrants. Thbd expression was largely absent from splenic follicular and marginal zone B cells.

Adult mice with complete, ubiquitous ablation of Thbd gene function (Meox2Cre-ThbdloxP -mice; "Thbd-null") were generated to analyze the functional role of Thbd in hematopoiesis. Thbd-null mice exhibited low birth weight, but only a mild prothrombotic diathesis, reflected in occasional peripheral vascular occlusion limited to the tail vein. Flow cytometric analyses revealed increased frequency of LT- and ST-HSC, a trend towards reduced CLP frequency, but normal relative abundance of MPP, CMP, GMP, and MEP. Thbd deficiency was also associated with a significant increase in the absolute number of LT-HSC and a reduction in the absolute number of CLP in the bone marrow. No such derangements were observed in mice lacking EPCR. In functional assays, bone marrow from wildtype and mutant mice yielded comparable numbers of CFU-GM. In contrast, the number of CFU-GM was increased in the spleen and peripheral blood of Thbd-null animals. The absolute number and frequency of all B cell precursors, as well as mature B cells in the bone marrow was reduced by ~50%. In the spleen, the absolute number of B cells was increased, whereas other hematopoietic populations in peripheral organs of Thbd-null mice were identical to that of wildtype controls.

CONCLUSION: Thbd is expressed in the majority of hematopoietic progenitor cells in the bone marrow, including LT- and ST-HSC, and B cell precursors. Despite abundant Thbd expression in bone marrow resident cells and a modest prothrombotic phenotype, complete Thbd deficiency had only mild effects on steady-state hematopoiesis. Hematopoietic derangements were limited to the B cell compartment, and an Increased presence of CFU-GM in the spleen and peripheral blood of Thbd-null mice, possibly reflecting stimaulation of extramedullary hematopoiesis and/or altered bone marrow retention of precursors.