Megakaryopoiesis and Thrombopoiesis

Megakaryopoiesis

1. Megakaryopoiesis and Thrombopoiesis Williams Hematology: Chapter 113 Ranjita Pallavi Fellow 1 Department of Hematology and Oncology

2. Kinetics of Thrombopoiesis The circulatory life span of a platelet is approximately 10 days in humans with normal platelet counts, but somewhat shorter in patients with moderate (7 days) to severe (5 days) thrombocytopenia, as a higher proportion of the total-body platelet mass is consumed in the day-to-day function of maintaining vascular integrity . Based on a “normal” level of 200,000 platelets/ μL , a blood volume of 5 L, and a half-life of 10 days, 1 × 10 11  platelets per day are produced. If 1 megakaryocyte produces approximately 1000 platelets, approximately 1 × 10 8  megakaryocytes are generated in the marrow each day.

3. Kinetics of Thrombopoiesis The transit time from megakaryocyte progenitor cell to release of platelets into the circulation ranges from 4 to 7 days. For example, following platelet apheresis, the platelet count falls, recovers substantially by day 4, and completely recovers by day 7. In most physiologic and pathologic states, the platelet count is inversely related to plasma thrombopoietin levels.

4. Kinetics of Thrombopoiesis Liver failure is associated with moderate thrombocytopenia as a result of splenomegaly and thrombopoietin deficiency. Within the first week following orthotopic liver transplantation, the platelet count rises substantially, with kinetics matching those of thrombopoietin infusion.

5. Cellular Physiology of Thrombopoiesis Platelets form by fragmentation of megakaryocyte membrane extensions termed proplatelets , in a process that consumes nearly the entire cytoplasmic complement of membranes, organelles, granules, and soluble macromolecules. Each megakaryocyte is estimated to give rise to 1000 to 3000 platelets  before the residual nuclear material is engulfed and eliminated by marrow macrophages. The continuum of megakaryocyte development is arbitrarily divided into four stages . The major criteria differentiating these stages are the quality and quantity of the cytoplasm and the size, lobulation , and chromatin pattern of the nucleus.

6. Cellular Physiology of Thrombopoiesis

7. Megakaryoblast Stage I megakaryocytes, also termed  megakaryoblasts , account for approximately 20 percent of all cells destined to form platelets. These cells in human marrow are 8 to 24 μm in spherical diameter, contain a relatively large, minimally indented nucleus with loosely organized chromatin and multiple nucleoli, and scant basophilic cytoplasm containing a small Golgi complex, a few mitochondria and α granules, and abundant free ribosomes.

8. Date of download: 12/21/2014 Copyright © 2014 McGraw-Hill Education. All rights reserved. Electron micrograph of a normal human megakaryoblast stained for platelet peroxidase. The small cell (<9 μm) exhibits dense platelet peroxidase in the perinuclear space and endoplasmic reticulum (arrows) (magnification ×12,150). (Inset) Enlargement of the Golgi zone. The Golgi saccules and vesicles are devoid of platelet peroxidase (open arrows), whereas the endoplasmic reticulum contains platelet peroxidase activity (closed arrow) (magnification ×25,000).
(Courtesy of Dr. J. Breton-Gorius.) Legend : From: Chapter 113. Megakaryopoiesis and Thrombopoiesis Williams Hematology, 8e, 2010

9. Megakaryoblast : Surface Adhesion Molecule Expression Integrin α IIb β 3  is an integral transmembrane protein of two subunits, but only the α subunit is megakaryocyte-lineage specific. Absence of integrin α IIb β 3  leads to Glanzmann thrombasthenia resulting from failure of the defective platelets to engage fibrinogen and other adhesive ligands during hemostasis. Megakaryocytes and platelets contain in their cytoplasmic membranes about twice the amount of integrin α IIb β 3  as is present on the cell surface.

10. Megakaryoblast : Surface Adhesion Molecule Expression The granule compartment serves as a mobilizable pool that is exteriorized upon platelet activation. During the early and mid-stages of megakaryocyte development, the granule content of integrin rises. Moreover, as developing megakaryocytes do not synthesize but contain fibrinogen in their α-granules and cells from patients with Glanzmann thrombasthenia do not, integrin α IIb β 3  clearly begins to function, at least at the level of fibrinogen binding and uptake, long before platelet formation.

11. Megakaryoblast : Surface Adhesion Molecule Expression The glycoprotein (GP) Ib -IX complex is expressed only slightly after the appearance of integrin α IIb β 3 . Although endothelial cells reportedly express GPIb , its levels are very low; otherwise, GPIb is a second megakaryocyte-specific protein. Glycoprotein V also is expressed in complex with GPIb and GPIX, in a ratio of 1:2:2.

12. Megakaryoblast : Surface Adhesion Molecule Expression However, the genetic elimination of GPV has little effect on platelet adhesion, and unlike GPIb and GPIX, no mutations of GPV are associated with Bernard- Soulier disease.  Therefore, GPV does not appear to be required for the GPIb -V-IX complex to function as a von Willebrand factor receptor. Rather, GPV is a target of thrombin, potentially playing a role in platelet activation.

13. Megakaryoblast : Demarcation Membranes Another feature of the megakaryoblast is the initial development of demarcation membranes, which begin as invaginations of the plasma membrane and ultimately develop into a highly branched interconnected system of channels that course through the cytoplasm. The demarcation membrane system is in open communication with the extracellular space, based on studies using electron dense tracers. Biochemical analysis indicates the composition of these membranes is very similar to the plasma membrane at each stage of megakaryocyte development.

14. Megakaryoblast : Demarcation Membranes Over the 72 hours required for stage III/IV cells to develop from megakaryoblasts , the demarcation membrane system grows substantially. The purpose of the demarcation membrane system has been disputed for several decades. As the term implies, many believed the demarcation membrane system acts to compartmentalize the megakaryocyte cytoplasm into “platelet territories,” which ultimately fragment into mature platelets along the cleavage planes so formed. In contrast, the current belief is that these membranes provide the material necessary for development of proplatelet processes, structures that form in stage IV megakaryocytes and give rise upon fragmentation to mature platelets.

15. Megakaryoblast : Endomitosis One of the most characteristic features of megakaryocyte development is endomitosis , a unique form of mitosis in which the DNA is repeatedly replicated in the absence of nuclear or cytoplasmic division. The resultant cells are highly polyploid . Endomitosis begins in megakaryoblasts following the many standard cell divisions required to expand the number of megakaryocytic precursor cells and is completed by the end of stage II megakaryocyte development.  During the endomitotic phase, each cycle of DNA synthesis produces an exact doubling of all the chromosomes, resulting in cells containing DNA content from 8 to 128 times the normal chromosomal complement in a single, highly lobated nucleus.

16. Megakaryoblast : Endomitosis Endomitosis is not simply the absence of mitosis but rather consists of recurrent cycles of aborted mitoses. Cell-cycle kinetics in endomitotic cells also are unusual, characterized by a short G 1  phase, a relatively normal DNA synthesis phase, a short G 2  phase, and a very short endomitosis phase. During the endomitosis phase, megakaryocytic chromosomes condense, the nuclear membrane breaks down, and multiple (at advanced stages) mitotic spindles form upon which the replicated chromosomes assemble. However, following initial chromosomal separation, individual chromosomes fail to complete their normal migration to opposite poles of the cell, the spindle dissociates, the nuclear membrane reforms around the entire chromosomal complement, and the cell again enters G 1  phase.

17. Date of download: 12/21/2014 Copyright © 2014 McGraw-Hill Education. All rights reserved. Origin and development of megakaryocytes. The pluripotential stem cell produces a progenitor committed to megakaryocyte differentiation (colony-forming unit–megakaryocyte [CFU-MK]), which can undergo mitosis. Eventually the CFU-MK stops mitosis and enters endomitosis. During endomitosis, neither cytoplasm nor nucleus divides, but DNA replication proceeds and gives rise to immature polyploid progenitors, which then enlarge and mature into morphologically identifiable, mature megakaryocytes that shed platelets. This figure does not necessarily imply that endomitosis and platelet formation are sequential but they can occur simultaneously. Meg-CFC, megakaryocyte colony-forming cells. Legend : From: Chapter 113. Megakaryopoiesis and Thrombopoiesis Williams Hematology, 8e, 2010

18. Megakaryoblast : Regulation of Gene Expression The promoters for integrin α IIb , GPIb , GPVI, GPIX, and platelet factor-4 are active at the megakaryoblast stage of development. Consensus sequences for both GATA-1 and members of the Ets family of transcription factors (e.g., Fli-1) are present in the 5′ flanking regions of these genes, deletion of which reduces or eliminates reporter gene expression, at least in mature hematopoietic cells. MafB also enhances GATA-1 and Ets activity during megakaryoblast differentiation,  induced by activation of ERK1/2, one of the primary downstream events of thrombopoietin stimulation.

19. Megakaryoblast : Cytokine Dependency The cytokines, hormones, and chemokines that affect the survival and proliferation of megakaryoblasts include thrombopoietin , interleukin (IL)-3, stem cell factor (also termed mast cell growth factor, steel factor, and  c-kit  ligand), and stromal cell-derived factor (SDF)-1. Thrombopoietin is the most critical, as genetic elimination of the  TPO  gene in mice leads to circulating platelet levels approximately 10 percent of normal. Homozygous or complex heterozygous mutation of the gene encoding the thrombopoietin receptor cMpl leads to congenital amegakaryocytic thrombocytopenia , in which platelet levels are approximately 10 percent of normal because of a near absence of megakaryocytic progenitors and megakaryoblasts .

20. Megakaryoblast : Cytokine Dependency Genetic reduction in expression of stem cell factor or its receptor  c-kit  leads to a 50 percent reduction in circulating platelet levels. The cytokine acts in synergy with thrombopoietin to enhance megakaryocyte production in semisolid and suspension culture systems.  Evidence that IL-3 contributes to normal or accelerated megakaryopoiesis   in vivo  is weak. Genetic elimination of the IL-3 gene fails to affect platelet counts, even when combined with thrombopoietin receptor deficiency, but the cytokine can induce growth of marrow progenitors into colonies containing immature megakaryocytes  in vitro  in the absence of thrombopoietin . The chemokine SDF-1 appears to play a role in megakaryocyte proliferation.  In vitro , SDF-1 acts in synergy with thrombopoietin to support the survival and proliferation of megakaryocyte progenitors. 

21. Megakaryoblast : Signal Transduction The survival and proliferation of megakaryoblasts depends on at least two thrombopoietin -induced signaling pathways: PI3K and mitogen-activated protein kinase ( MAPK). In the presence of chemical inhibitors of PI3K, the favorable effects of thrombopoietin on megakaryocyte progenitor survival and proliferation are eliminated,  although constitutively activating this pathway is not sufficient for thrombopoietin -induced growth. MAPK is another important signaling pathway stimulated by thrombopoietin . Using purified marrow megakaryocytic progenitors and model cell lines, several groups showed that inhibition of MAPK blocks megakaryoblast maturation  because of its effect of activating Ets transcription factors.

22. Stage II Megakaryocytes Stage II megakaryocytes contain a lobulated nucleus and more abundant, but less intensely basophilic, cytoplasm. Ultrastructurally , the cytoplasm contains more abundant α granules and organelles. The demarcation membrane system begins to expand at this stage of development. Stage II megakaryocytes measure up to 30 μm in diameter, constitute approximately 25 percent of marrow megakaryocytes, and are the stage of development during which endomitosis is most prominent , generating cells displaying ploidy values of 8N to 64N.

23. Stage II Megakaryocyte: Cytoplasmic Development Early in megakaryocyte development, the cytoplasm acquires a rich network of microfilaments and microtubules. Toward stages III and IV, the proteins accumulate in the cell periphery, creating an organelle poor peripheral zone. Biochemically , the megakaryocyte cytoskeleton is composed of actin, α- actinin , filamin , non-muscle myosin (including the product of the  MYH9  gene), mutated in several giant platelet thrombocytopenic syndromes, β 1 - tubulin, talin , and several other actin-binding proteins. Like platelets, megakaryocytes can respond to external stimuli by changing shape, transporting organelles around the cytoplasm, and secreting granules. These functions are dependent on the microfilament and microtubule systems of the cell. In addition, microtubules play a vital role during the later stages of platelet formation.

24. Stage II Megakaryocyte: Platelet Granule Formation Although more prominent in later stages of differentiation, platelet-specific α granules first begin to form adjacent to the Golgi apparatus as 300- to 500-nm round or oval organelles in stage II megakaryocytes. Three distinct compartments are recognized in α granules: (1) a central, electron-dense nucleoid, containing fibrinogen, platelet factor-4, β- thromboglobulin , transforming growth factor (TGF)- β 1 , vitronectin , and tissue plasminogen activator-like plasminogen activator; (2) a peripheral zone, containing tubules and von Willebrand factor (arranged much like that seen in endothelial cell Weibel -Palade bodies); and (3) the granule membrane, containing many of the critical platelet receptors for cell rolling (P- selectin ), firm adhesion ( GPIb -V-IX), and aggregation (integrin α IIb β 3 ).

25. Stage II Megakaryocyte: Platelet Granule Formation Proteins present in α granules arise from  de novo  megakaryocyte synthesis (e.g., GPIb -V-IX, GPIV, integrin α IIb β 3 , von Willebrand factor, P- selectin , β- thromboglobulin , platelet-derived growth factor), nonspecific pinocytosis of environmental proteins (albumin and immunoglobulin G), or cell surface membrane receptor-mediated uptake from the environment (e.g., fibrinogen, fibronectin, factor V). Insights into platelet granule formation have come from a molecular understanding of Hermansky-Pudlak Syndrome (HPS). In this disorder, characterized by oculocutaneous albinism and a qualitative platelet bleeding disorder, a complex of at least eight proteins form in various granule associated complexes such as the biogenesis of lysosome-related organelles complexes, which affect δ granule formation.  These complexes are thought to be involved in cargo transport of a number of subcellular granules, such as lysosomes, melanosomes , and platelet δ granules.

26. Stage III/IV Megakaryocytes Continued cytoplasmic maturation characterizes stage III/IV megakaryocyte development. Cells are extremely large (40–60 μm in diameter) and display a low nuclear-to-cytoplasmic ratio. Cytoplasmic basophilia disappears as cells progress from stage III to IV. The demarcation membrane system gradually replaces the endoplasmic reticulum and Golgi apparatus during the final stages of maturation. The nucleus usually is eccentrically placed.

27. Stage III/IV Megakaryocytes The nucleus remains highly lobulated but single at all stages of megakaryocyte development. In occasional marrow sections, neutrophils or other marrow cells are seen transiting through the cytoplasm of the mature megakaryocyte, a process termed emperipolesis , and is of no pathologic significance.

28. Date of download: 12/21/2014 Copyright © 2014 McGraw-Hill Education. All rights reserved. Megakaryocyte morphology. A. Normal human marrow biopsy. Two megakaryocytes are evident. In one case the section is through the cell at the level of the nuclei (horizontal arrow), and in the other it is through the cytoplasm above or below the nucleus (vertical arrow). B. Normal human marrow aspirate. Mature (stage III) megakaryocyte with a multilobated nucleus and abundant cytoplasm. C. Normal human marrow aspirate. Mature megakaryocyte with a neutrophil embedded in the cytoplasm. Many ultrastructural studies have confirmed that this appearance represents marrow cells entering the canalicular system of megakaryocyte cytoplasm through its opening to the exterior of the cell (emperipolesis).
(Used with permission from Lichtman’s Atlas of Hematology, www.accessmedicine.com.) Legend : From: Chapter 113. Megakaryopoiesis and Thrombopoiesis Williams Hematology, 8e, 2010

29. Date of download: 12/21/2014 Copyright © 2014 McGraw-Hill Education. All rights reserved. Megakaryocyte morphology. A. Normal human marrow biopsy. Two megakaryocytes are evident. In one case the section is through the cell at the level of the nuclei (horizontal arrow), and in the other it is through the cytoplasm above or below the nucleus (vertical arrow). B. Normal human marrow aspirate. Mature (stage III) megakaryocyte with a multilobated nucleus and abundant cytoplasm. C. Normal human marrow aspirate. Mature megakaryocyte with a neutrophil embedded in the cytoplasm. Many ultrastructural studies have confirmed that this appearance represents marrow cells entering the canalicular system of megakaryocyte cytoplasm through its opening to the exterior of the cell (emperipolesis).
(Used with permission from Lichtman’s Atlas of Hematology, www.accessmedicine.com.) Legend : From: Chapter 113. Megakaryopoiesis and Thrombopoiesis Williams Hematology, 8e, 2010

30. Date of download: 12/21/2014 Copyright © 2014 McGraw-Hill Education. All rights reserved. Megakaryocyte morphology. A. Normal human marrow biopsy. Two megakaryocytes are evident. In one case the section is through the cell at the level of the nuclei (horizontal arrow), and in the other it is through the cytoplasm above or below the nucleus (vertical arrow). B. Normal human marrow aspirate. Mature (stage III) megakaryocyte with a multilobated nucleus and abundant cytoplasm. C. Normal human marrow aspirate. Mature megakaryocyte with a neutrophil embedded in the cytoplasm. Many ultrastructural studies have confirmed that this appearance represents marrow cells entering the canalicular system of megakaryocyte cytoplasm through its opening to the exterior of the cell (emperipolesis).
(Used with permission from Lichtman’s Atlas of Hematology, www.accessmedicine.com.) Legend : From: Chapter 113. Megakaryopoiesis and Thrombopoiesis Williams Hematology, 8e, 2010

31. Stage III/IV Megakaryocytes: Proplatelet formation Careful microscopic studies have localized marrow megakaryocytes to the abluminal surface of sinusoidal endothelial cells. In specially prepared specimens, the megakaryocytes can be seen issuing long, slender cytoplasmic processes between endothelial cells and into the sinusoidal lumen, structures termed  proplatelet processes . The processes have been reproduced  in vitro  and  in vivo . The processes consist of a β- tubulin cytoskeleton and highway, transporting organelles and platelet constituents from the megakaryocyte to the terminal projection, the nascent platelet.

32. Date of download: 12/23/2014 Copyright © 2014 McGraw-Hill Education. All rights reserved. Megakaryocyte proplatelet processes in the marrow sinusoid. Scanning electron micrograph showing the luminal view of the confluence of two marrow sinusoids with two proplatelet processes protruding through the lining endothelial cells. One of the processes has intermittent constrictions (arrows), indicating potential sites for platelet formation. Other cells depicted include lymphocytes and erythrocytes (magnification ×3000).
(From Becker RP, De Bruyn P,54 with permission.) Legend : From: Chapter 113. Megakaryopoiesis and Thrombopoiesis Williams Hematology, 8e, 2010

33. Stage III/IV Megakaryocytes: Membrane Composition Most of the specific characteristics of platelet membranes are present at stages III and IV of megakaryocyte development. Megakaryocyte membrane lipid composition progressively changes through development, achieving approximately four times the content of phospholipids and cholesterol as found in immature cells. Megakaryocytes contain about the same amounts of membrane neutral and phospholipid as platelets, but contain relatively more phosphatidylinositol and less phosphatidylserine and arachidonic acid.

34. Platelet Formation Platelet formation involves massive reorganization of megakaryocyte cytoskeletal components, including actin and tubulin, during a highly active, motile process in which the termini of the process branch and issue platelets. The size of the individual platelets formed is of interest. Unfortunately , little is known about this aspect of platelet formation except that tubulin is proposed to  act  as a measuring device for the proper site to pinch off platelets from proplatelet processes. The mechanism of platelet formation clearly must be affected in some way by the transcription factor GATA-1, the glycoprotein Ib -IX complex, the Wiskott -Aldrich syndrome protein, and platelet myosin, as defects in each of these genes leads to unusually large or small platelets. Finally , localized cytoplasmic membrane proteolysis, a sublethal form of apoptosis, likely plays a role in initiating the final stages of platelet formation

35. Extrinsic Regulation of Megakaryocyte Production: Thrombopoietin The term  thrombopoietin  was first coined in 1958 to describe the primary regulator of platelet production. A major impetus to the discovery of thrombopoietin in 1986 was the identification of the myeloproliferative leukemia virus (MPLV), which induces a vast expansion of hematopoietic cells. The responsible viral oncogene was characterized in 1990, and its cellular homologue c - Mpl  was cloned in 1992. The gene for thrombopoietin encodes a 36-kDa polypeptide, which also is predicted to be extensively posttranslationally modified, resulting in an approximately 50- to 70-kDa protein.

36. Extrinsic Regulation of Megakaryocyte Production: Thrombopoietin Thrombopoietin bears striking homology to erythropoietin, the primary regulator of erythropoiesis, within the amino-terminal half of the predicted polypeptide. The two proteins are more closely related than any other two cytokines within the hematopoietic cytokine family, sharing 20 percent identical amino acids, an additional 25 percent conservative substitutions, and identical positions of three of the fourcysteine residues . Unlike any of the other cytokines in the family, thrombopoietin contains a 181-residue carboxyl-terminal extension, which bears homology to no known proteins. Two functions have been assigned to this region: it prolongs the circulatory half-life of the hormone, and it aids in its secretion from the cells that normally synthesize the hormone.

37. Extrinsic Regulation of Megakaryocyte Production: Thrombopoietin Incubation of marrow cells with thrombopoietin stimulates megakaryocyte survival and proliferation, alone and in combination with other cytokines. In vivo , thrombopoietin stimulates platelet production in a log-linear manner to levels 10-fold higher than baseline without affecting the blood red or white cell counts. In addition, because of its affect on hematopoietic stem cells, the number of erythroid and myeloid progenitors and mixed myeloid progenitors in marrow and spleen also are increased , an effect that is particularly impressive when the hormone is administered following myelosuppressive therapy . This effect likely results from the synergy between thrombopoietin and the other hematopoietic cytokines circulating at high levels in this condition . MPL gene is located on chromosome 1p34.

38. c-MPL Receptor

39. Extrinsic Regulation of Megakaryocyte Production: Thrombopoietin Based on genetic studies, thrombopoietin clearly is the primary regulator of thrombopoiesis . Elimination of either the c - Mpl  or  Tpo  gene leads to profound thrombocytopenia in mice as a result of a greatly reduced number of megakaryocyte progenitors, mature megakaryocytes, and the reduced polyploidy of the remaining megakaryocytes. A similar result occurs in humans. Patients with congenital amegakaryocytic thrombocytopenia (CAMT) display numerous homozygous or mixed heterozygous nonsense or severe missense mutations of the thrombopoietin receptor  c- Mpl .  The effect of thrombopoietin on hematopoietic stem cells is particularly revealed by consideration of children with CAMT. Within 5 years of birth, nearly every patient with CAMT develops aplastic anemia as a result of stem cell exhaustion.

40. Extrinsic Regulation of Megakaryocyte Production: Thrombopoietin The thrombopoietin gene displays an unusual 5′ flanking structure. Unlike the majority of genes that initiate translation of the encoded polypeptide with the first ATG codon present in the messenger ribonucleic acid (mRNA), thrombopoietin translation initiates at the eighth ATG codon located within the third exon of a full-length transcript. However , since the eighth ATG of thrombopoietin mRNA is embedded in the short, open reading frame of the seventh ATG, its translation is particularly inefficient because of the mechanism of ribosomal initiation. As such, little thrombopoietin protein is produced for any given amount of mRNA. Although this molecular arrangement has no known physiologic consequences, it forms the basis for an unusual form of disease, a disorder of translation efficiency. Four cases of autosomal dominant familial thrombocytosis have been linked to mutations in the region surrounding the initiation codon.

41. Extrinsic Regulation of Megakaryocyte Production: Thrombopoietin It has been seen that plasma  hormone concentrations vary inversely with platelet counts, rising to maximal levels within 24 hours of onset of profound thrombocytopenia. Two nonmutually exclusive models have been advanced to explain these findings. In the first model, thrombopoietin production is constitutive, but its consumption, and hence the level remaining in the blood to affect megakaryopoiesis , is determined by the mass of c- Mpl receptors present on platelets and megakaryocytes accessible to the plasma. In this way, states of thrombocytosis result in increased thrombopoietin consumption (by the expanded platelet mass of c- Mpl receptors), reducing megakaryopoiesis . Conversely , thrombocytopenia reduces blood thrombopoietin destruction, resulting in elevated blood levels of the hormone that drive megakaryopoiesis and platelet recovery.

42. Extrinsic Regulation of Megakaryocyte Production: Thrombopoietin A second model suggests thrombopoietin expression is a regulated event. Very low platelet levels can induce thrombopoietin -specific mRNA production. Several studies show that thrombopoietin mRNA levels are modulated in response to moderate to severe thrombocytopenia, at least in the marrow. The signal(s) responsible for this form of thrombopoietin regulation is being uncovered, but is, at least in part, mediated by transcriptional enhancement . CD40 ligand, platelet-derived growth factor, fibroblast growth factor, TGF-β, platelet factor-4, and thrombospondin modulate thrombopoietin production from marrow stromal cells.

43. Thrombopoietin Mimetics : Romiplostim It has been approved for use in refractory ITP. In immune thrombocytopenia, immune dysregulation leads to the production of autoantibodies or immune complexes that accelerate peripheral platelet destruction by binding to platelets, causing platelet phagocytosis, along with T-cell and possibly complement-mediated lysis. The production of new platelets is also suppressed; antiplatelet antibodies have been shown to bind to megakaryocytes in the bone marrow, causing both a decrease in the number of megakaryocytes and the inhibition of megakaryocyte maturation.

44. Thrombopoietin Mimetics : Romiplostim  Plasma levels of endogenous thrombopoietin are typically high in patients who have thrombocytopenia associated with bone marrow failure syndromes. However, thrombopoietin levels are usually normal or only slightly increased in patients with immune thrombocytopenia, for reasons that remain unclear. The fact that thrombopoietin levels in immune thrombocytopenia are lower than anticipated led to the concept of treating the disorder by means of exogenous stimulation of thrombopoietin receptors.

45. Thrombopoietin Mimetics : Romiplostim Initial trials involved the use of recombinant forms of thrombopoietin . However, clinical trials were stopped when thrombocytopenia — resulting from the development of autoantibodies against endogenous thrombopoietin — developed in healthy volunteers receiving these agents. Thus, development began on a second generation of thrombopoietin mimetics or agonists that are structurally dissimilar to thrombopoietin and thus do not lead to the formation of autoantibodies . Romiplostim ( Nplate , Amgen) is a synthetic fusion protein that has four peptides consisting of 14 amino acid residues connected to an IgG Fc fragment, producing a “ peptibody ”. Romiplostim binds to the thrombopoietin receptor and activates intracellular signaling pathways (the JAK–STAT and MAP kinase pathways), which stimulates platelet production.

46. Thrombopoietin Mimetics : Romiplostim Eltrombopag ( Promacta , GlaxoSmithKline) is a small nonpeptide molecule that binds to the transmembrane region of the thrombopoietin receptor, which activates the same intracellular pathways that are activated by romiplostim .  In initial pharmacodynamic studies, a single intravenous or subcutaneous dose of romiplostim resulted in an increase in the platelet count after 5 to 8 days in a dose-dependent fashion. The peak platelet count was reached between days 12 and 16, and by day 28 the platelet count had fallen to baseline.  Eltrombopag has shown a similar pattern of response in platelet count.

47. AMG 531 Unique platform “peptibody” Made in E. coli Molecular weight = 60,000 D 4 Mpl binding sites Bussel JB et al. N Engl J Med. 2006;355:1672. No sequence homology with TPO Cleared endothelial FcRn Recycled Cleared RES Fc Carrier Domain TPO Agonist Peptides Fc Carrier Domain TPO Agonist Peptides

48. The romiplostim trials included two 24-week placebo-controlled phase 3 studies, one enrolling 63 splenectomized patients and the other enrolling 62 non splenectomized patients. The primary efficacy end point was a durable response, which was defined as a platelet count of at least 50,000 per cubic millimeter for at least 6 of the final 8 weeks.

49. Thrombopoietin Mimetics : Romiplostim This end point was achieved in 38% of splenectomized patients and in 61% of nonsplenectomized patients in the romiplostim groups, as compared with no splenectomized patients and 5% of nonsplenectomized patients in the placebo groups (P<0.001 for both comparisons). Patients receiving romiplostim were more likely to reduce or discontinue concurrent medications (primarily glucocorticoids) and to require less rescue medication than patients in the placebo group.  In an open-label extension study involving 292 patients, 94.5% achieved a platelet count of at least 50,000 per cubic millimeter during the study, and more than 50% had a platelet count of at least 50,000 per cubic millimeter during at least 90% of all visits at a median of 78 weeks. 

50. Romiplostim : 38% Durable Response, 79% Overall Response Durable Response Overall Response Number of Weeks Platelet Response Platelet response: platelet count ≥ 50 x 10 9 /L Durable platelet response: platelet response for ≥ 6 weeks of final 8 weeks, in the absence of rescue medications during 24 week trial Overall response: either durable or transient platelet response ( ≥ 4 weekly platelet responses) Error bars represent standard deviation of the mean 0.0 38.1 ( P = 0.0013) 0.0 78.6 20 40 60 80 100 ( P < 0.0001) Durable Platelet Response (%) Overall Platelet Response (%) Mean (SE) Number of Weeks With Platelet Response 0.2 (0.1) 12.3 (1.2) 5 10 15 20 ( P < 0.0001) 20 40 60 80 100 Placebo Romiplostim

51. Lancet 2008;371:395

52. Romiplostim (AMG 531): Summary In splenectomized patients: 38% durable response, 79% overall response Increased and maintained platelet counts over 24 weeks Significantly decreased the use of rescue medications All romiplostim patients discontinued or reduced concurrent ITP therapy (corticosteroids, azathioprine, danazol ) Romiplostim appeared to be well tolerated

53. Romiplostim : Summary of Long-term Dosing Efficacy Data Summary The majority of patients achieved long-term platelet counts > 50 x 10 9 /L and double the baseline value Mean platelet count maintained between 50 and 250 x 10 9 /L over 2 years Use of concomitant and rescue medications was substantially reduced over time No trend in this study for adverse events to increase in frequency with longer drug exposure One patient had neutralizing antibodies to AMG 531; negative on retesting.

54. Small molecule, non-peptide thrombopoietin receptor (TPO-R) agonist Does not compete with TPO for binding to TPO-R Low immunogenic potential Active only in humans, chimps Stimulates megakaryocyte proliferation and differentiation Orally bioavailable Increases platelet counts in normal volunteers Thrombopoietin MW 64,000 Eltrombopag MW 442 Eltrombopag : Oral Platelet Growth Factor

55. Eltrombopag was approved by the Food and Drug Administration (FDA) on the basis of the results of two 6-week, placebo-controlled clinical trials and the initial results of an open-label extension study. The primary efficacy measurement in both randomized studies was the proportion of patients achieving a platelet count of at least 50,000 per cubic millimeter on day 43. In a dose-adjustment study involving 118 patients, this platelet count was achieved more often in the groups who received daily oral eltrombopag (at a dose of either 50 mg or 75 mg) than in those receiving placebo.

56. Primary Endpoint: Percentage of Patients With Platelets ≥50,000/µL at Day 43 Visit † Responders (%) Placebo § Eltrombopag P <0.001 ‡ OR = 9.61 (3.31, 27.86) † Last observation carried forward. ‡ Indicates significance at 5% (2-sided) level of significance. § 1 patient received IVIg on Day 1. Logistic regression analysis adjusted for randomization stratification variables.

57. Conclusions The EXTEND data suggest that oral eltrombopag was well tolerated and safe Eltrombopag up to 75 mg/day increased and sustained platelet counts >50,000/ μ L in the majority of patients Eltrombopag reduced the incidence and severity of bleeding

58. Thrombopoietin Mimetics : Eltrombopag In a subsequent phase 3 placebo-controlled trial involving 114 patients who were treated with 50 mg of eltrombopag per day, a platelet count of at least 50,000 per cubic millimeter was achieved by day 43 in 59% of patients in the eltrombopag group, as compared with 16% of those in the placebo group (P<0.001). In an open-label extension study involving 299 patients who completed a previous eltrombopag study, 87% of patients achieved a platelet count of at least 50,000 per cubic millimeter during treatment. 

59. Thrombopoietin Mimetics Both romiplostim and eltrombopag have been found to reduce bleeding complications. In the romiplostim extension study, the rate of moderate-to-severe bleeding decreased from 23% to 12% within the first 24 weeks and to 6% during weeks 24 through 48. In the eltrombopag extension study, the incidence of any bleeding symptoms declined from 56% at baseline to 16% by week 52 and to 20% by week 104.

60. Thrombopoietin Mimetics Romiplostim and eltrombopag are approved by the FDA for patients with chronic immune thrombocytopenia who have an insufficient response to glucocorticoids, intravenous immune globulin, or splenectomy. Clinically , these agents are typically used in patients who have persistent or chronic immune thrombocytopenia and ongoing bleeding, with or without previous splenectomy and one or more courses of rituximab. Since thrombopoietin -receptor agonists cross the placenta, their safety in pregnancy has not been shown, so their use in such cases is not recommended.

61. Thrombopoietin Mimetics The recommended initial dose of romiplostim is 1 μg per kilogram of body weight, administered subcutaneously once weekly, with subsequent dose adjustment on the basis of the platelet count. The mean therapeutic dose is 3 to 4 μg per kilogram, with a maximum dose of 10 μg per kilogram. Romiplostim is available in 250-μg and 500-μg vials as a lyophilized powder. O nly providers who are enrolled in a regulated prescriber program called NEXUS (Network of Experts Understanding and Supporting Nplate and Patients) may prescribe romiplostim . The NEXUS program requires providers to enroll all patients receiving romiplostim in a registry and to enter baseline data for patients as well as periodic safety information. Details regarding the NEXUS program are available at  www.nplatenexus.com . These restrictions do not apply to the use of romiplostim outside the United States. In most other countries, the drug can be self-administered by the patient at home.

62. Thrombopoietin Mimetics The recommended initial dose of eltrombopag for most patients is 50 mg daily given orally, with subsequent dose adjustment on the basis of the platelet count (to a maximum of 75 mg daily or a minimum of 25 mg daily ). Patients with hepatic dysfunction and patients of Asian ethnic background (in whom plasma concentrations of the drug are higher than in white patients) should initiate treatment at a dose of 25 mg once daily. Eltrombopag should be taken 1 to 2 hours after a meal because of interactions with food. It should not be taken within 4 hours after taking antacids, dairy products, or supplements that contain polyvalent cations , such as calcium, magnesium, and aluminum. Like romiplostim , eltrombopag was approved by the FDA under restricted terms. In the United States, the drug can be prescribed only by participants in a regulated prescriber program called Promacta Cares. Details regarding the program are available at  www.promactacares.com .

63. Thrombopoietin Mimetics When either romiplostim or eltrombopag is given, the platelet count should be measured weekly until a stable count (>50,000 per cubic millimeter for at least 4 weeks without dose adjustment) has been achieved and monthly thereafter. Treatment should be withheld temporarily when the platelet count is 200,000 to 400,000 per cubic millimeter; it is not a goal of therapy to achieve and maintain a normal platelet count.

64. Thrombopoietin Mimetics Eltrombopag therapy can cause hepatic injury. Thus , patients receiving eltrombopag should have levels of serum aspartate aminotransferase, alanine aminotransferase, and bilirubin checked every 2 weeks during the dose-adjustment phase of therapy and monthly after the establishment of a stable dose.

65. Thrombopoietin Mimetics : Adverse Effects The most common adverse effects of thrombopoietin -receptor agonists in clinical trials included headache, nausea, vomiting, fatigue, diarrhea, arthralgia, and nasopharyngitis . Worsened thrombocytopenia after the discontinuation of the thrombopoietin -receptor agonist occurs in 8 to 10% of patients, with an increased risk of bleeding during the first 4 weeks. Tapering of the agent or reinitiation of other treatment for immune thrombocytopenia is recommended if severe thrombocytopenia supervenes. The platelet count typically recovers to pretreatment levels after several weeks.

66. Thrombopoietin Mimetics In prospective studies, patients receiving eltrombopag had hepatobiliary laboratory abnormalities; 11% of patients receiving eltrombopag and 7% receiving placebo had aminotransferase values at least 3 times the upper limit of the normal range and alkaline phosphatase or total bilirubin values at least 1.6 times the upper limit of the normal range. These abnormalities may resolve despite continued therapy. However, the package insert specifies that eltrombopag therapy should be discontinued if alanine aminotransferase levels increase to 3 times the upper limit of the normal range or higher and are progressive, are persistent for 4 weeks or more, are accompanied by an increase in the direct bilirubin level, or are accompanied by clinical symptoms of liver injury or evidence of hepatic decompensation. No similar effects have been seen with romiplostim .

67. Thrombopoietin Mimetics In a study of extended romiplostim treatment involving 291 patients, 25 venous or arterial thromboembolic events occurred in 17 patients. In an eltrombopag extension study involving 299 patients, 16 patients had 21 thromboembolic events. The frequency of thromboembolic events did not increase with the duration of treatment in either study. Most thromboembolic events that have been associated with the use of thrombopoietin -receptor agonists have been observed in patients with at least one additional risk factor for thrombosis.

68. Thrombopoietin Mimetics The American Society of Hematology Guidelines: T he use of thrombopoietin -receptor agonists is recommended for adult patients at risk for bleeding who have a relapse after splenectomy or who have a contraindication to splenectomy and do not have a response to at least one other therapy. It was also suggested that these agents could be considered for adult patients at risk for bleeding who have not had a response to one line of therapy and have not undergone splenectomy.

69. c- mpl  mutations are the cause of congenital amegakaryocytic thrombocytopenia (CAMT) CAMT is a rare autosomal recessive bone marrow failure syndrome that presents with severe thrombocytopenia which can evolve into aplastic anemia and leukemia. The disorder is expressed in infancy with or without physical anomalies. It is often recognized on day 1 of life or at least within the first month. It is often initially confused with fetal and neonatal alloimmune thrombocytopenia, but the neonate fails to improve and responds only to platelet transfusion.

70. CAMT A recent classification was proposed in 2005. Type I—early onset of severe pancytopenia, decreased bone marrow activity and very low platelet counts. In this group, there is complete loss of functional c- Mpl . Median platelet count is usually 21 × 10 9 /L or below . Type II—milder form with transient increases of platelet counts up to nearly normal values during the first year of life and an onset of bone marrow failure at age 3 to 6 years or later. In this group, there are partially functional receptors for the c- Mpl gene. Median platelet count is usually 35 × 10 9 /L to 132 × 10 9 /L. Type III—there is ineffective megakaryopoeisis with no defects in the c- Mpl gene.

71. CAMT Differential diagnosis for severe CAMT includes thrombocytopenia with absent radii (TAR) and Wiskott -Aldrich syndrome (WAS). The primary treatment for CAMT is bone marrow transplantation. Newer modalities are on the way, such as TPO- mimetics for binding towards partially functioning c- Mpl receptors and gene therapy. Prognosis of CAMT patients is poor, because all develop in childhood a tri-linear marrow aplasia that is always fatal when untreated. Thirty percent of patients with CAMT die due to bleeding complications and 20% -due to HSCT if it has been done.

72. GATA1 Mutation leading to X linked Thrombocytopenia X-linked thrombocytopenia is a well-known clinical condition, found most often in the context of the Wiskott -Aldrich syndrome (WAS) and consisting of thrombocytopenia, defective humoral and cellular immunity, and eczema. Mutations in the WASP gene lead either to the full-blown WAS picture or to isolated X-linked thrombocytopenia. The platelets in this syndrome are typically small sized. However, hereditary macrothrombocytopenia with or without associated thrombopathy has been identified in a variety of syndromes, such as the May- Hegglin anomaly, Bernard- Soulier syndrome, Fechtner syndrome, or Epstein syndrome.

73. GATA1 Mutation leading to X linked Thrombocytopenia Very recently, the May- Hegglin anomaly and Fechtner syndrome have been linked to mutations in the nonmuscle myosin heavy chain 9 gene on chromosome 22. GATA1 is the founding member of the GATA-binding family of transcription factors and has been shown to be an essential protein for normal erythropoiesis and megakaryocyte differentiation. The human gene encoding GATA1 has been mapped to Xp11.23. Shivdasani et al developed a lineage-selective knockout mouse of GATA1, leading to megakaryocyte-specific loss of GATA1 expression and established the critical role of this transcription factor for megakaryocyte growth and platelet development.

74. GATA1 Mutation leading to X linked Thrombocytopenia Very recently Nichols et al described for the first time a mutation in the GATA1 gene in a family with X-linked dyserythropoietic anemia and macrothrombocytopenia . This missense mutation (V205M) leads to a reduced interaction of the N-terminal zinc finger of GATA1 with its essential cofactor FOG1 (for Friend of GATA1).

75. MYH9-Related Platelet Disorders Myosin heavy chain 9 (MYH9)-related platelet disorders belong to the group of inherited thrombocytopenias . The MYH9 gene encodes the nonmuscle myosin heavy chain IIA (NMMHC-IIA), a cytoskeletal contractile protein. Several mutations in the MYH9 gene lead to premature release of platelets from the bone marrow, macrothrombocytopenia,and cytoplasmic inclusion bodies within leukocytes. Four overlapping syndromes, known as May- Hegglin anomaly, Epstein syndrome, Fechtner syndrome, and Sebastian platelet syndrome, describe different clinical manifestations of MYH9 gene mutations.

76. MYH9-Related Platelet Disorders Macrothrombocytopenia is present in all affected individuals, whereas only some develop additional clinical manifestations such as renal failure, hearing loss, and presenile cataracts. The bleeding tendency is usually moderate, with menorrhagia and easy bruising being most frequent. The biggest risk for the individual is inappropriate treatment due to misdiagnosis of chronic autoimmune thrombocytopenia. To date, 31 mutations of the MYH9 gene leading to macro-thrombocytopenia have been identified, of which the upstream mutations up to amino acid 1400 are more likely associated with syndromic manifestations than the downstream mutations.

77. MYH9-Related Platelet Disorders In 1909, May described a family in which several members had enlarged platelets but minor if any bleeding symptoms. In 1945, Hegglin found Do¨hle body like inclusions within the leukocytes of affected individuals with a dominantly-inherited giant platelet disorder. This led to the term May- Hegglin anomaly (MHA) to describe the triad of thrombocytopenia, giant platelets, and leukocyte inclusion bodies . These inclusion bodies are spindle shaped and appear bright blue in standard blood films.

78. MYH9-Related Platelet Disorders In 1972, Epstein et al described Epstein syndrome (EPS) as the first macro-thrombocytopenic syndrome characterized by giant platelets, associated with deafness and nephritis; however, in contrast with MHA leukocyte inclusion bodies were absent. In 1985, Peterson et al characterized another dominantly inherited macro-thrombocytopenic syndrome, characterized by interstitial nephritis, cataract, deafness (i.e., a syndrome complex resembling Alport syndrome), and leukocyte inclusions. These inclusion bodies were much smaller than those observed in the MHA and typically round rather than spindle shaped. These authors called the disorder Fechtner syndrome (FS).

79. MYH9-Related Platelet Disorders In 1990, Greinacher et al described a milder variant macrothrombocytopenia , Sebastian platelet syndrome (SPS), with small inclusion bodies in leukocytes.Individuals were recognized to have developed cataracts at a young age (45 to 50 years), and all affected family members that were >50 years of age had developed high-tone hearing impairment. In 1999, the inheritance of these giant platelet disorders was linked to a 5.5-Mb region on the short arm of chromosome 22q.2,15 The syndromes MHA, EPS, FS, and SPS are now recognized to be related disorders, caused by mutations in the MYH9 gene, with phenotypic differences related to the presence or absence of the additional features of cataracts, nephritis, and sensorineural hearing loss.

80. MYH9-Related Platelet Disorders An MYH9-related macrothrombocytopenia should be suspected in individuals that have large platelets, a high MPV, a broad platelet histogram, and a peak preceding the leukocyte histogram.

81. MYH9-Related Platelet Disorders

82. MYH9-Related Platelet Disorders Platelet aggregometry and platelet function studies using the PFA-100 do not show major defects in MYH9 disorders. Because of the altered composition of the platelet cytoskeleton, the shape change in the aggregation curve is typically absent.

83. MYH9-Related Platelet Disorders

84. Hypothesis on the Mechanism of Bleeding with MYH9-Related Disorders The most important reason for bleeding is the reduced clot stability due to impaired clot retraction by platelets with a disturbed cytoskeleton. This is aggravated in case of iron-deficiency anemia.

85. Bernard- Soulier syndrome (BSS) Bernard- Soulier syndrome (BSS) is an autosomal recessive giant platelet disorder that is usually caused by reduced or absent expression of the glycoprotein (GP) Ib -IX-V receptor complex. Platelets in BSS are as large as in the MYH9 disorders and appear similar to MYH9 platelets by light microscopy and electron microscopy ultrastructural analysis. Aggregation studies can help to distinguish a MYH9 disorder from the rare, dominant Bolzano type of BSS, in which the GP IbIX -V complex is expressed but functionally impaired. In BSS, there is typically absent agglutination with ristocetin , despite aggregation responses to other agonists. The diagnosis of BSS is typically confirmed by quantitative analysis of GP Ib -IX complex expression on the platelets or genetic testing.

86. Paris-Trousseau syndrome/Jacobson syndrome This is a disorder with large platelets that contain giant a-granules. This syndrome is distinguished from MYH9 disorders by its features of mental retardation, facial and cardiac abnormalities, and its genetic cause, which is a heterozygous deletion of one part of chromosome 11q23.

87. X-linked macrothrombocytopenia X-linked macrothrombocytopenia can be caused by a mutation in the GATA-1 gene Xp11–12, a transcription factor important for megakaryopoiesis and erythropoiesis. The disorder is associated with splenomegaly and red cell abnormalities ( reticulocytosis and anemia) and thrombocytopenia with platelet function defects (decreased agglutination with ristocetin , weak aggregation with collagen).

88. Gray platelet syndrome (GPS) It is an autosomal recessive bleeding disorder associated with giant platelets containing empty a-granules and moderate thrombocytopenia—the appearance of pale and gray platelets in the blood smear is an important diagnostic clue to distinguish this condition from MYH9 disorders. The genetic defect is unknown.

89. Thank You