A flk-independent pathway of megakaryopoiesis is differentially responsive to stress and can produce high proportions of functionally distinct platelets Free

Platelets and megakaryocytes (Mks) are increasingly recognized as diverse and dynamic cells that serve as critical components of thrombus formation and as regulators of immune responses. Mks are found in bone marrow (BM), spleen, and lungs. While BM and spleen Mks are phenotypically similar, we and others have determined that lung Mks have low ploidy and are predominantly localized in the extra-vascular tissue space. In addition to diversity of location and function, distinct pathways of thrombopoiesis have recently been described based on lineage tracing using Flk2/Flt3-Cre. Most platelets are derived from a “canonical' pathway via Flk2 expressingmultipotential progenitors (MPP) that progressively differentiate to Mk progenitors (MkP) and platelets (Flk-dep). A second, Flk2/Flt3-independent (Flk-indep) pathway is less well elucidated and variably defined but is commonly described as a “direct” or “alternate” pathway via a subclass of HSCs to MkP, without going through a Flk2/Flt3 expressing intermediate. We recently reported that the vast majority of lung Mks in young mice arise from the Flk-indep pathway. Flk-indep platelets are generated from platelet-biased, vWF+ HSCs. Flk-indep Mks in the BM express lower levels of CD48 suggesting that there is a correlation of the Flk-indep pathway with other models of alternate thrombopoiesis.

Flk-indep platelets were identified in aged Flk/Switch mice where Flk-cre activity switches cells from tomato to GFP expressing. Utilizing this model, we report here that the Flk-indep pathway contributes a low, but significant, population of platelets in young mice. We specifically identified Flk-indep MPP2 (CD48+CD150+), MkP, and Mk in the BM of young mice. Flk-indep Mk in the BM were skewed to lower ploidy than Flk-dep MKs and expressed higher levels of the migration and adhesion proteins CCR7 and ICAM. We next investigated the responses of the Flk-dep and Flk-indep pathways to stress. Acute loss of 50% blood via phlebotomy caused an increase in proportion of circulating Flk-indep platelets, which persisted after recovery for at least 50 days. This was accompanied by increased proportion of Flk-indep MPP2, MkP, and Mk in the marrow. We also challenged mice with partial myeloablation using 150 mg/kg 5-fluorouracil (5-FU) or sublethal 4Gy total body irradiation (TBI) to determine the role of the Flk-indep pathway in rebuilding the hematopoietic system. By 2 to 3 weeks after each of these treatments, the majority of circulating platelets were provided by the Flk-indep pathway. This response rapidly rebalanced following 5-FU exposure, although slightly increased levels of Flk-indep platelets were still observed at 80 days post partial myeloablation. The contribution of Flk-indep platelets following TBI decreased more gradually and remained markedly elevated (40%) at 80 days. Interestingly, Flk-indep young red blood cells (reticulocytes), as well as granulocytes, were present in the bloodstream at 2 to 3 weeks post 5-FU and even more so following TBI. Examination of the BM revealed not only increased proportion of Flk-indep MPP2, MkP, and Mk, but also Flk-indep megakaryocyte/erythroid progenitors (MEP), erythroid progenitors (EP), and granulocyte-monocyte progenitors (GMP). Additionally, after TBI, there was an increase in Flk-indep myeloid-biased MPP3 (CD48+, CD150-), although no Flk-indep MPP4 were observed. By 80 days post 5-FU treatment, Flk-indep MPP2, MkP and Mk, remained elevated at low levels as we had observed long term after bleeding. However, TBI treated mice retained increased Flk-indep MPP2, MkP, and Mk, as well as MEP, EP, and to a lesser degree MPP3 and GMP. To determine if there are functional differences in platelets derived from the Flk-dep and Flk-indep pathways, we investigated the response of platelets to specific agonists. This analysis revealed increased sensitivity of Flk-indep platelets to stimulation by TLR7 (Loxoribine) and thromboxane/P2Y12 receptor agonists (U46619/ADP) in healthy young mice and after TBI treatment. Together, these data indicate that the Flk-independent pathway of hematopoietic differentiation is more complex than previously recognized, operational in healthy young adult mice, and rapidly upregulated by different hematopoietic stresses to modulate the proportions of circulating Flk-indep platelets, as well as the contribution to other lineages, which may have both short-term and long-term functional consequences.