Page 1842 - Williams Hematology ( PDFDrive )

1816 Part XII: Hemostasis and Thrombosis Chapter 111: Megakaryopoiesis and Thrombopoiesis 1817

to expand the number of megakaryocytic precursor cells and is com- Cytokine Dependency
pleted by the end of stage II megakaryocyte development. During the The cytokines, hormones, and chemokines that affect the survival and
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endomitotic phase, each cycle of DNA synthesis produces an exact dou- proliferation of megakaryoblasts include thrombopoietin, interleukin
bling of all the chromosomes, resulting in cells containing DNA con- (IL)-3, stem cell factor (also termed mast cell growth factor, steel factor,
tent from eight to 128 times the normal chromosomal complement in and c-kit ligand), and the chemokine CXCL12 (previously termed stro-
a single, highly lobated nucleus. Although poorly understood for many mal cell-derived factor [SDF]-1). Thrombopoietin is the most critical
years, the ability to produce large numbers of normal megakaryocytes (for additional details, see the more extensive discussion in “Hormones
in culture has started to shed light on this enigmatic process. Endomi- and Cytokines” below), as genetic elimination of the TPO gene in mice
tosis is not simply the absence of mitosis but rather consists of recurrent leads to circulating platelet levels approximately 10 percent of normal.
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cycles of aborted mitoses. Cell-cycle kinetics in endomitotic cells also Homozygous or complex heterozygous mutation of the gene encoding
are unusual, characterized by a short G phase, a relatively normal DNA the thrombopoietin receptor cMPL leads to congenital amegakaryo-
1
synthesis phase, a short G phase, and a very short endomitosis phase. cytic thrombocytopenia, in which platelet levels are approximately
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2
During the endomitosis phase, megakaryocytic chromosomes con- 10 percent of normal because of a near absence of megakaryocytic pro-
dense, the nuclear membrane breaks down, and multiple (at advanced genitors and megakaryoblasts (Chap. 117). The importance of stem cell
stages) mitotic spindles form upon which the replicated chromosomes factor to megakaryoblast development is revealed by experimental find-
assemble. However, following initial chromosomal separation, individ- ings both in vitro and in vivo. Genetic reduction in expression of stem
ual chromosomes fail to complete their normal migration to opposite cell factor or its receptor c-kit leads to a 50 percent reduction in circulat-
poles of the cell, the spindle dissociates, the nuclear membrane reforms ing platelet levels. The cytokine acts in synergy with thrombopoietin
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around the entire chromosomal complement, and the cell again enters to enhance megakaryocyte production in semisolid and suspension cul-
G phase. ture systems. Evidence that IL-3 contributes to normal or accelerated
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1
megakaryopoiesis in vivo is weak. Genetic elimination of the IL-3 gene
Regulation of Gene Expression fails to affect platelet counts, even when combined with thrombopoie-
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The promoters for integrin α , GPIb, GPVI, GPIX, and platelet tin receptor deficiency, but the cytokine can induce growth of marrow
IIb
factor-4 genes have been the focus of several studies and are active at progenitors into colonies containing immature megakaryocytes in vitro
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the megakaryoblast stage of development. Consensus sequences for in the absence of thrombopoietin. The chemokine CXCL12 appears
both GATA-1 and members of the Ets family of transcription factors to play a role in megakaryocyte proliferation. In vitro, the chemokine
(e.g., Fli-1) are present in the 5′ flanking regions of these genes, dele- acts in synergy with thrombopoietin to support the survival and prolif-
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tion of which reduces or eliminates reporter gene expression, 21–24 at least eration of megakaryocyte progenitors. The combination of fibroblast
in mature hematopoietic cells. MafB also enhances GATA-1 and Ets growth factor (FGF)-4 and CXCL12 restores megakaryopoiesis in TPO
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activity during megakaryoblast differentiation, induced by activation and c-mpl null mice. 38
of ERK1/2, one of the primary downstream events of thrombopoietin
stimulation. 26 Signal Transduction
Another target of GATA-1 in megakaryocytes is polyphosphate-4- The survival and proliferation of megakaryoblasts depends on at least
phosphatase (P4P), which was first identified by subtraction cloning two thrombopoietin-induced signaling pathways: PI3K and mito-
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between normal and GATA-1 knockdown megakaryocytes. One of gen-activated protein kinase (MAPK; Chap. 17). In the presence of
the unexplained features of megakaryocytes in GATA-1 knockdown chemical inhibitors of PI3K, the favorable effects of thrombopoietin
mice is that, rather than massive cell death as seen in GATA-1–deficient on megakaryocyte progenitor survival and proliferation are elimi-
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erythroid progenitors, the aberrantly developing megakaryoblasts in nated, although constitutively activating this pathway is not sufficient
GATA-1 knockdown marrow are highly abundant and proliferate in for thrombopoietin-induced growth. MAPK is another important sig-
vitro far more than control cells. P4P catalyzes hydrolysis of the D-4 naling pathway stimulated by thrombopoietin. Using purified marrow
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position phosphate of PI P and PI 3,4,5 P. These membrane phospholipids megakaryocytic progenitors and model cell lines, several groups showed
3,4
are products of phosphoinositol 3′-kinase (PI3K) action on membrane that inhibition of MAPK blocks megakaryoblast maturation 26,40–42
phospholipids, and they play an important role in the proliferative and because of its effect of activating Ets transcription factors.
survival response to megakaryocyte growth factors. When reintroduced
into the knockdown mice, P4P diminishes the exuberant growth char-
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acteristic of the knockdown cells. These findings are similar to the STAGE II MEGAKARYOCYTES
phenotype of cells from PTEN or SHIP knockout mice, enzymes that Stage II megakaryocytes contain a lobulated nucleus and more abun-
hydrolyze the D-3 and D-5 positions of PI 3,4,5 P. dant, but less intensely basophilic, cytoplasm. Ultrastructurally, the
Another transcription factor vital for megakaryoblast differentia- cytoplasm contains more abundant α granules and organelles. The
tion is RUNX1 (also termed CBFA2 and AML1), the gene responsible demarcation membrane system begins to expand at this stage of devel-
for thrombocytopenia seen in familial platelet disorder/predisposition opment. Stage II megakaryocytes measure up to 30 μm in diameter,
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to acute myelogenous leukemia (Chap. 119). In this disorder, hap- constitute approximately 25 percent of marrow megakaryocytes, and
loinsufficiency of RUNX1 is associated with thrombocytopenia. As are the stage of development during which endomitosis is most promi-
its genetic elimination in mice leads to significant maturation defects nent, generating cells displaying ploidy values of 8N to 64N.
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in the megakaryocyte lineage, the human disorder almost certainly
results from this genetic alteration. During normal megakaryoblast Endomitosis
differentiation, RUNX1 levels rise and, conversely, fall during ery- Whereas megakaryoblasts are generally thought to be able to expand
throid differentiation. In response to phosphorylation by ERK1/2, by cell division, at an early stage of their maturation, the cells begin
RUNX1, in complex with CBFβ and together with GATA-1, induces to undergo endomitosis, in which cells diverge from the normal cell
integrin α and integrin α expression in megakaryoblast-like cells, cycle during mid- to late anaphase. Like normally mitotic cells, endomi-
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IIb
2
providing the beginnings of a molecular explanation for megakaryo- totic megakaryocytes condense their chromatin into chromosomes,
cyte development. form a spindle, dissolve the nuclear membrane, and assemble the

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