Hematopoietic Size Control: A Novel Role for Factor VIII Via the Thrombin/PAR1 Axis in Regulating the Interplay Between Dynamic Bone Structure and Long Term Survival and Mobilization of Hematopoietic Stem Cells

Abstract 19

Despite great progress in embryogenesis, the underlying mechanism that controls tissues and organs size remains a mystery. We previously showed that transplanted pig embryonic tissues are markedly enlarged in Factor VIII knockout (F8KO) mice, and identified a regulatory role for thrombin. Here, we describe a novel potential pathway through which coagulation factors might regulate size control of the hematopoietic system.

The hematopoietic stem cell (HSC) pool of F8KO mice exhibited instability manifested by three parameters: 1) Reduced proportion of CD34^low cells within the Lin⁻Sca⁺Kit⁺ (LSK) progenitors. Median CD34 staining of F8KO LSK cells was 40±4% higher compared to WT controls (n=8 mice, p<0.001). 2) Reduced long term repopulating capacity measured by competitive transplantation. While 6 weeks post transplantation of F8KO or WT BM similar chimerism levels were found, markedly reduced F8KO donor chimerism levels were detected at 22 weeks (22.2±4.4% vs 38.9±9.9%; p=0.008). 3) Hyper responsiveness to G-CSF. We used G-CSF to reveal pre-existing abnormalities in the HSC pool. G-CSF treated F8KO mice exhibited significantly enlarged spleens compared to WTs (256±149mg, n=25 vs 181±61mg, n=32; p=0.01). This difference was corrected upon F8 treatment. We further used splenectomized mice to test the effect of G-CSF on BM alone. The LSK cell number in the peripheral blood (PB) of G-CSF treated splenectomized F8KO mice was 3 fold higher compared to WT splenectomized mice (380±127 cells/ml, n=3 vs 64±6 cells/ml, n=3, P=0.01).

Since factor VIII has no known physiological function other than in coagulation, it was tempting to assume that down-stream coagulation factors, in particular thrombin, are responsible for the observed distorted HSC regulation. Indeed, F8KO mice exhibit a 2 fold decrease in peak plasma thrombin concentration compared to WT mice. Furthermore, thrombin receptor 1 knockout mice (PAR1 KO), exhibited enhanced responsiveness to G-CSF, with higher PB LSK cell numbers compared to WT mice (766±180 cells/ml, n=7 vs 489±189 cells/ml, n=5; p<0.005). Likewise, G-CSF induced splenomegaly and LSK mobilization were enhanced in WT mice upon treatment with thrombin inhibitors such as Clexane and Dagibitran, or by the PAR1 antagonist, palmitoylated peptide (pal-RCLSSSAVANRS).

To investigate the thrombin site of action, a reciprocal transplantation was performed. Interestingly, chimeras generated by transplantation of WT BM into PAR1KO recipients showed enhanced responsiveness to G-CSF compared to WT recipients. Thus, chimeras lacking PAR1 on their stroma cells only, were more prone to induced splenomegaly than controls (114.4±26.4mg; n=10, vs 162.6±19.34mg; n=5; p<0.003). A less dramatic but significant enhancement was observed in WT recipients of PAR1 KO BM, lacking PAR1 on their hematopoietic cells (141.4±23.5mg, n=10, p=0.026). Our results suggest that thrombin-PAR1 signalling in BM stromal cells has a predominant role in regulating HSC maintenance.

Surprisingly, we found that coagulation factors also influence the dynamic bone structure. We evaluated the status of bone formation and remodelling in F8KO, PAR1KO and WT mice at different ages after birth. A decrease in bone density and in trabeculae number was first evident at 2 months of age and was more pronounced at 4 months in both F8KO and PAR1KO mice. In addition, micro CT analysis of distal tibia metaphyses harvested at 4 months revealed reduced bone/tissue volume ratio and trabecular number in F8KO and PAR1KO mice (Fig.1). Thus, reduced thrombin activity is associated with decreased bone mass and compromises bone architecture. This in turn can affect the long-term maintenance of repopulating HSC.

Fig. 1

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Representative micro-CT analyses of tibial metaphyses revealing reduced bone/tissue volume ratio and trabeculae numbers by 57.13±6.53%, and 73.1±4.7% in F8KO and by 44.83±15.2% and 37.11±8.07% in PAR1KO mice, respectively.

Fig. 1

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Representative micro-CT analyses of tibial metaphyses revealing reduced bone/tissue volume ratio and trabeculae numbers by 57.13±6.53%, and 73.1±4.7% in F8KO and by 44.83±15.2% and 37.11±8.07% in PAR1KO mice, respectively.

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Taken together, these results show a critical and novel role for the coagulation cascade, in part via the Thrombin/PAR1 axis, in regulating the reciprocal interplay between the dynamic bone structure and HSC maintenance and mobilization. While further studies in Hemophilia are warranted, our new insights also suggest that thrombin inhibitors such as Clexane and Dabigatran could potentially enhance HSC mobilization in G-CSF treated donors and increase the number of cells harvested.