Iron and Megakaryopoiesis

Inflammatory bowel diseases and several other conditions are associated with high platelet counts, platelet activation and an increased risk of thromboembolism. Megakaryopoiesis is mainly stimulated by thrombopoietin that acts synergistically with IL-3, stem cell factor, IL-11 and IL-6 and is characterized by an increase in nuclear DNA content through specific (endomitotic) cell division cycles. However, thrombocytosis also comes along with chronic iron deficiency. Iron is an essential constituent of mammalian cells. It is required for growth and development (as iron-deficient cells arrest in the G1/S transition of the cell cycle) and regulates the expression of an array of proteins (including some that control cell cycle progression). For this project we hypothesize that megakaryocytic endomitosis has evolved to maintain platelet production despite iron deficiency. Endomitosis may be an iron-preserving strategy of platelet production that would maintain or even enhance the body`s coagulation abilities in the case of blood loss and consecutive Fe deficiency. Iron deficiency may trigger endomitotic cell cycles by regulation of cell cycle protein(s). This project will establish a rat model of iron-deficient megakaryopoiesis and study endomitosis. In addition, human megakaryopoietic cells (both cell lines and primary stem cells) will be cultured under iron-deficient conditions and analyzed for the induction of polyploidy. Differential expression, immuno-fluorescence microscopy and gene expression studies are proposed to identify regulators of endomitotic cell cycles. Understanding the role of iron in the regulation of megakaryopoiesis may improve treatment of platelet disorders but also help preventing thrombocytosis or possibly thromboembolism in the setting of iron deficiency or postoperative.

Inflammatory bowel diseases (IBD) and other chronic conditions such as cancer are as-associated with high platelet counts (thrombocytosis), increased activity of platelets and an increased risk of thromboembolism which is a leading cause of premature death. It is believed that thrombocytosis comes along with iron deficiency or anemia. In fact, iron supplementation normalized platelet counts in IBD-associated thrombocytosis in a randomized controlled trial (ThromboVIT). In addition to the platelet number, iron supplementation also counteracted platelet activation. In support of clinical observations, data from this project provide a novel mechanism that iron deficiency itself can be an independent trigger for thrombocytosis. Iron an essential participant in cellular processes and thus required for cell growth and development. Proteins involved in iron uptake, storage, utilization, and export are regulated by the cellular iron content. The molecular changes that ultimately lead to thrombocytosis upon iron deficiency can occur at several stages of platelet production. Megakaryocytes (blood cells, which give rise to platelets after maturation) undergo an increase in DNA content. In fact, megakaryocytes are one of the rare cell types, in which DNA polyploidy is a physiological process. For this project we hypothesized that endomitosis (the process of increasing DNA content in megakaryocytes) contributes to maintain platelet production despite iron deficiency. We were successful to establish an animal model that displayed thrombocytosis upon iron deficiency. The platelets produced in such animals were higher in number, and displayed increased activity. Moreover, bone marrow studies revealed subtle alterations, suggestive of expansion of megakaryocyte progenitors, an increase in megakaryocyte DNA ploidy and accelerated megakaryocyte maturation. In addition we were also able to establish cell culture model of megakaryopoiesis in iron deficiency. This helped us to validate our hypothesis and examine the underlying molecular mechanisms. Overall this project was highly successful in accomplishing all the goals and laid the ground to further investigate the role of iron deficiency in the regulation of platelet production and function. Moreover, we will to extend our investigations to identify a biomarker or a signature protein expressed by platelets produced under iron-deficient megakaryopoiesis, which might aid to measure the risk for thromboembolic events arising in IBD or cancer. Final Report / Page 1 of 14