CHAPTER 1 Basic sc*ience of ITP Editor: John W. Semple 1.1 Megakaryocyte differentiation and platelet produc*tion A.S. Weyrich 1.2 Autoimmune mechanisms and T regula*tory cell disturbances in ITP K. Yazdanbakhsh 1.3 Mouse models o*f ITP J.W. Semple 1.4 Peptide therapy for patients with* ITP S.J. Urbaniak * Basic science of ITP 1.1 Megakaryocyte differen*tiation and platelet production Andrew S. Weyrich CHAPTER 1.1 • Megakaryocyte differentiation and platelet production 1. Introduction Platelets are anucleate cells that circulate in the bloodstream for approximately 10 days. The average adult must produce roughly 1 x 10 11 platelets per day to maintain normal platelet counts, a level of production that increases dramatically in a variety of clinical scenarios (1). In 1906, Wright provided the first evidence that megakaryocytes give rise to blood platelets. Since then, our understanding of the molecular basis of thrombopoiesis has progressed substantially and is arguably in a logarithmic growth phase. This chapter will review our current understanding of thrombopoiesis and highlight how the field is evolving. The history of megakaryocytes and platelets is fairly young. In 1841, Addison first described platelets and in 1882, Bizzozero named and identified platelets in the circulation and determined that they could induce clotting. In 1890, Howell named megakaryocytes, and 16 years later, Wright discovered that these were actually the precursors of platelets. Thus, the late 1880’s and early 1900’s were a period of prolific activity in the elucidation of megakaryocytes. Several discoveries were made about platelets and also about erythropoietin, which implied that a humoral substance also regulated platelet production though its exact nature was not yet known. In 1958, the term “thrombopoietin” (TPO) was coined, which means stimulator of “thrombopoiesis” or “platelet production”. In the early 1990’s, this substance was identified, and in 1994, several articles published in Science and Nature demonstrated that TPO can, in fact, increase platelet counts. Since that time, recombinant TPO and, more recently, TPO mimetics have been used clinically, as discussed in later chapters. 2. Megakaryocytes and thrombopoietin Platelets sprout from the cytoplasm of megakaryocytes (2, 3). Megakaryocytes are rare cells in the bone marrow; there is also literature to suggest that megakaryocytes can be found in peripheral blood and in the lungs. In vivo , it has been shown that bone marrow derived megakaryocytes produce platelets. Megakaryocytes originate from a stem cell progenitor in the bone marrow ( Figure 1 ) (4). In the presence of cytokines that include TPO, interleukins such as IL-3 and IL-6, and SDF (stromal-cell derived factor) the pluripotent haematopoietic stem cell (HPC) goes through endomitosis and DNA replication to produce a mature megakaryocyte. From the cytoplasm of the mature megakaryocyte sprouts numerous platelets. Though all above-stated cytokines are important, TPO has been found to be critical in this process (1). TPO signals via c-Mpl, which is expressed by megakaryocyte progenitors, megakaryocytes and their progeny. TPO is an acidic glycoprotein produced mainly 13 IMMUNE THROMBOCYTOPENIA Figure 1: Regulation of megakaryopoiesis Megakaryopoiesis Cytokines and chemokines F ) 4 S T HSC R C E C - l X M a C i / l G i 1 , - m CFU-GEMM 2 F a 1 f D - S , , T 1 M 1 A CFU-EM - C , ( 6 l - p , 3 M BFU-EM - / L O I P 4 T F CFU-Meg P Mature megakaryocyte Platelets Modified from (4) by the liver and the kidneys, and also by some stromal cells of the bone marrow. It stimulates thrombopoiesis by both enhancing proliferation of megakaryocyte progenitors and supporting differentiation of these progenitors into platelet producing cells. The discovery of TPO has led to the development of new drugs to treat thrombocytopenia (5) and the evolvement of megakaryocyte cultures that reconstitute platelet formation in vitro (2, 6). In conditions of thrombocytosis or thrombocytopenia, TPO levels are altered appropriately ( Figure 2 ) (1). In fact, platelets in the periphery help to adjust TPO levels. In this regard, when TPO levels are too high, the platelets take up TPO, which then leads to a reduction in megakaryocytes in the bone marrow and reduced platelet production. In conditions of thrombocytopenia, the bone marrow senses increased levels of TPO, as the platelets are not present in high enough concentrations to “quench” it. This leads to increased numbers of bone marrow megakaryocytes, raising the platelet count. Thus, this delicate balance is controlled both in the periphery and in the bone marrow, with TPO serving as a sensor. 3. Proplatelet formation The proplatelet theory, put forth by Joseph Italiano and his group (2, 7, 8), is 14 THE HANDBOOK FIRST EDITION CHAPTER 1.1 • Megakaryocyte differentiation and platelet production Figure 2: Thrombopoietin regulates platelet production Liver Kidney TPO Thrombocytosis Thrombocytopenia Bone marrow Megakaryocyte Reduced Enhanced thrombopoiesis thrombopoiesis Modified from (1) currently well accepted. According to this theory, proplatelet formation is a prerequisite step used by megakaryocytes to package granules and organelles into platelets. Haematopoietic progenitors mature and eventually reach a stage where they begin to take on platelet and megakaryocyte phenotypic markers and have granules in their cytoplasm. In the final stages of maturation, they undergo endomitosis, where the DNA replicates but the cell does not divide. There is maturation of the cytoplasm and then microtubules start moving towards the periphery. In the final stages of this process, cytoplasmic extensions are generated into which organelles and granules are targeted into bulbs that become individual platelets. The extensions eventually reach into venous sinusoids and are released into the circulation and are clipped off, a process that releases individual platelets into the circulation. Proplatelets are characteristically rich in tubulin and have the appearance of beads linked by thin cytoplasmic bridges. Microtubules propel proplatelet formation and granules ride microtubular-rich shafts before entering nascent platelet buds. Microtubules also facilitate platelet release (3, 8). Our group has looked at this process by studying platelet formation in megakaryocytes generated from pluripotent HPC isolated from human cord blood (9). Human progenitors have a very small amount of cytoplasm, but are rich in ribosomes and translational machinery so they can support cell division and proliferation. When TPO is added to these cultured HPC, together with other cytokines, such as IL-3, 15 IMMUNE THROMBOCYTOPENIA the haematopoietic progenitor proliferates and begin to differentiate into a megakaryocytes that express platelet markers such as integrin IIb (Figure 3 ). Thus, one of the key things that TPO does is to increase the number of megakaryocyte progenitor cells present in the bone marrow. In human in vitro culture systems, progenitor cells differentiate into megakaryocytes over a period of twelve to thirteen days. During the final stages of maturation, the differentiated megakaryocytes have distinct organelles, granules and long proplatelet extensions sprout from their cytoplasm ( Figure 4 ) (6, 10). Thus, the process in humans is very similar to those originally described in murine model systems. Figure 3: Haematopoietic CD34 + cells differentiate into megakaryocytes that a express integrin IIb CD34 + cells Megakaryocyte Megakaryocytes precursors This research was originally published in Blood. Foulks et al. PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids. Blood 2009;113:6699-6706. © the American Society of Hematology (9). It shows freshly isolated CD34 + cells (culture day 0), megakaryocyte precursors (culture day 7), or differentiated megakaryocytes (culture day 13) that are adherent to immobilised fibrinogen for 1 hour. The cells are stained for sialic acids (wheat germ agglutinin [WGA], green) and integrin aIIb (red). Scale bar = 50 mm. 4. RNAs and translational machinery in platelets Recent evidence shows that platelets are not as simple as has been thought. Like all other cells they have ongoing protein synthesis and protein turnover to allow them to maintain equilibrium and homeostasis under normal conditions. Our group and others have shown, that during proplatelet production, in addition to the platelets receiving granules, cytoskeletal and tubular elements, messenger RNAs (mRNA), microRNAs, and translational machinery are also delivered to the platelets (Figure 4) (6, 10, 11). Our hypothesis has been that megakaryocytes transfer mRNA to anuclear platelets. In the case of megakaryocytes, it was thought for a long time that the mRNA got 16 THE HANDBOOK FIRST EDITION CHAPTER 1.1 • Megakaryocyte differentiation and platelet production Figure 4: Haematopoietic CD34 + cells that differentiate into megakaryocytes develop proplatelet extensions Day 8 Day 14 Transition phase Proplatelets Adherent 1 hr 6 S / A G W This research was originally published in Blood. Weyrich et al. mTOR-dependent synthesis of Bcl-3 controls the retraction of fibrin clots by activated human platelets. Blood 2007;109:1975-1983. © the American Society of Hematology (6). It shows that megakaryocytes transfer granules to proplatelets during thrombopoiesis. The megakaryocytes were cultured from CD34 + haematopoietic stem cells and stained with WGA, which localises to sialic acid-rich granules. The panel shows