Thrombocytopenia complicates many diseases and can be a life-threatening consequence of genotoxic treatments including chemotherapy and radiation therapy. It is well established that thrombopoiesis occurs in the bone marrow where mature megakaryocyte (MK) precursor cells associate with sinusoidal endothelial cells and extrude pro-platelets into the vasculature. There has been much interest in elucidating mechanisms that control megakaryopoiesis and in utilizing these pathways to increase platelet output. The leading paradigm of megakaryopoiesis centers on the ability of cytokines, chiefly thrombopoietin (TPO), to promote MK progenitor proliferation and MK precursor maturation. More recently, attention has been focused on the ability of the bone marrow microenvironment to promote MK maturation and platelet formation. The chemokine stromal-derived factor-1 (SDF-1, also known as CXCL12), signaling through its receptor CXCR4, is implicated in the chemotaxis of MKs toward sinusoidal vessels, and in vivo evidence demonstrates that sustained plasma elevation of SDF-1 can increase platelet counts (Avecilla et al. Nature Medicine, 2004). To more specifically determine the short-term effects of SDF-1, we injected mice with a single 400ng intravenous dose of SDF-1 and enumerated the progenitor, precursor, and platelet compartments of the MK lineage. At 24 hours, SDF-1 induced a 30% increase in platelets compared to vehicle control (p<0.05). However, MK progenitors, defined functionally by the formation of acetylcholinesterase-positive colonies in vitro, and MK precursors, enumerated by imaging flow cytometry, were both unchanged (p>0.7 and p>0.5). To quantitatively determine if SDF-1 regulates the physical interactions of MK precursors with sinusoidal endothelium, we developed a double immunohistochemistry assay using Gp1Bβ to distinguish MK precursors and MECA32 to identify vascular endothelial cells. In vehicle-treated mice, 39% of MKs in the marrow localized to the sinusoidal endothelium, and this increased to 53% 24 hours following SDF-1 treatment (p<0.01). Thus, a single dose of SDF-1 acutely increases the number of MKs in the vascular niche as well as peripheral platelet counts. Given these results, we tested whether a single dose of SDF-1 could improve thrombocytopenia in the setting of radiation-induced marrow injury. Mice were treated with SDF-1 4 days after sublethal 4Gy total body irradiation (TBI), when radiosensitive MK progenitors are drastically reduced and radioresistant MK precursors and platelets are just beginning to decline. At 5 days post-TBI (24 hours post-SDF-1), SDF-1 treatment increased the number of circulating platelets by 15% (p<0.01) as well as the percentage of MK precursors in the vascular niche by over 15% (p<0.02) without changing the total number of MK progenitors or precursors in the marrow compared to irradiated vehicle controls (p>0.4 and p>0.7). As the platelet response following SDF-1 was less robust in the setting of TBI injury, we hypothesized that SDF-1-induced thrombopoiesis may improve if the number of MK precursors available to move to the vascular niche is increased. To test this, we administered TPO and SDF-1 at 2 hours and at 4 days, respectively, following TBI. TPO treatment alone resulted in 15% more MK precursors than irradiated vehicle controls at 5 days post-TBI (p<0.02), but did not significantly change the platelet count (p>0.2). In contrast, mice receiving both TPO and SDF-1 had over 20% more platelets than irradiated vehicle controls (p<0.01) and over 12% more platelets than mice receiving TPO alone (p<0.05) with the same increase in MK precursors (p>0.9). Correcting for differences in MK precursor numbers, mice receiving TPO and SDF-1 post-TBI had 1.8-fold more MKs in the vascular niche than irradiated vehicle controls (p<0.03) and 1.4-fold more than mice treated with TPO only (p<0.02). Taken together, we provide quantitative data in support of the concept that SDF-1 acutely promotes increases in the number of MK precursors in the vascular niche as well as peripheral platelet counts, and this effect correlates with the number of MK precursors in the marrow. Therapeutic approaches combining agents that first increase MK number and secondly increase MKs poised for thrombopoiesis by localization within the vascular niche may be a novel strategy to maximally increase peripheral platelet counts.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.