Regulation Of Hematopoietic Stem Cell Trafficking By The Coagulation Pathway

Hematopoeitic stem and progenitor cells (HSPC) dynamically switch between a quiescent, non-motile mode in the bone marrow (BM), to an active state, in which they proliferate, differentiate and egress to the circulation. Injection of the coagulation protease thrombin induced rapid HSPC mobilization to the blood via activation of its major receptor, protease activated receptor 1 (PAR1) on BM hematopoietic and stromal cells. We hypothesized that coagulation factors control stem cells fate in the BM. We examined if thrombin is generated in the murine BM and found by immunohistochemistry prothrombin associated with bone lining osteoblasts in the endosteum region. These cells also highly express osteopontin which induces stem cell quiescence and retention. Cleavage of osteopontin by thrombin or by osteoclast derived cathepsin K induces stem cell mobilization. In addition, a unique structure of multinucleated CD45⁺ cell clusters in the trabecular-rich area of the murine femoral metaphysis express the cell surface receptor Tissue Factor (TF), a potent initiator of the coagulation cascade leading to thrombin generation. These clusters were found adjacent to multinucleated TRAP (tatrate resistant acid phosphate) positive active osteoclasts. In vitro, we found that immature osteoclasts expressed TF in cell fusion areas, suggesting that osteoclast maturation also activates the coagulation thrombin/PAR1 axis, thus mediating HSPC recruitment to the circulation. Supporting this notion, bleeding which prompts a hemostatic response and thrombin production, is a strong inducer of osteoclasts activation and HSPC mobilization. In addition, injection of bacterial lipopolysaccharides (LPS) is known to activate osteoclasts and induce HSPC mobilization (Kollet et al Nat Med 06). We found that LPS injection upregulated TF expression by CD45⁺ myeloid cells in the murine BM. LPS treatment provoked massive HSPC mobilization, which was attenuated by PAR1 inhibition. To further address the role of thrombin in stem cell maintenance, we targeted prothrombin in vivo by applying Antisense Oligonucleotides (ASO) knockdown technology, previously shown to induce a dose- and time-dependent up to 90% reduction of prothrombin mRNA levels in the murine liver (Monia et al Blood 2010). Prothrombin depletion altered the BM niche microenvironment by expanding the mesenchymal stem and progenitor (MSPC) population and the long-term repopulating CD34^-/ROS^low/LSK HSPC population in the BM. In untreated mice, TF was also expressed by a small MSPC population, suggesting that the bone stomal compartment may also contribute to the regulation of HSPC mobilization upon demand. To further asses the role of thrombin generation in HSPC development, we examined the involvement of the endothelial cell receptor Thrombomodulin (TM) that is pivotal for the anticoagulant pathway which mediates activation of protein C. TM protein is expressed by BM small blood vessels resembling sinusoids and by neighboring MSPC. By immunohistochemistry, we also detected activated protein C on the same blood vessels. A mouse model with a mutation in the TM gene (TM^Pro/Pro) is characterized by reduced capacity for activated protein C generation which in turn increases thrombin levels in these mice. We found increased circulating hematopoietic stem cells in TM^Pro/Pro mice, suggesting that chronically increased basal levels of thrombin generation can promote HSC egress. Conversely, short term (5 day) intermittent treatment of mice with low dose thrombin that mainly causes activated protein C formation in vivo, display higher levels of CD34^-/ROS^low/LSK and EPCR⁺LSK stem cells in the BM, indicating additional roles for the anticoagulant pathway in BM stem cell pool maintenance.

In summary, our results provide evidence that the activator of the coagulation cascade, TF, and coagulation factors Thrombin and activated protein C are present in the BM and regulate and integrate functions of hematopoietic stem and progenitor cells and BM stromal progenitor cells.