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1822 Part XII: Hemostasis and Thrombosis Chapter 111: Megakaryopoiesis and Thrombopoiesis 1823

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Flt-3 Ligand and the reduced polyploidy of the remaining megakaryocytes. A
The flt-3 ligand initially was identified as a ligand for a novel member similar result occurs in humans. Patients with congenital amegakary-
of the protein tyrosine kinase family of receptors. This growth factor ocytic thrombocytopenia (CAMT) display numerous homozygous or
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also affects megakaryocyte formation. Like stem cell factor, to which it compound heterozygous nonsense or severe missense mutations of the
is most closely related, flt-3 ligand is found in both soluble and mem- thrombopoietin receptor c-Mpl (Chap. 117). 131,132 The effect of throm-
brane-bound forms, is a noncovalently linked dimer, and affects pri- bopoietin on hematopoietic stem cells is particularly revealed by con-
marily primitive hematopoietic cells. Although several studies have sideration of children with CAMT. Within 5 years of birth, nearly every
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shown that flt-3 ligand used alone does not support megakaryocyte patient with CAMT develops aplastic anemia as a result of stem cell
colony formation, some studies suggest it works in synergy with other exhaustion.
megakaryocyte stimulatory agents to augment the proliferation of The thrombopoietin gene displays an unusual 5′ flanking struc-
megakaryocytic progenitor cells in culture. 114,115 Administration of flt-3 ture. Unlike the majority of genes that initiate translation of the encoded
ligand to mice expands the number of marrow and splenic progenitor polypeptide with the first ATG codon present in the mRNA, throm-
cells that can give rise to megakaryocytes in vitro. However, genetic bopoietin translation initiates at the eighth ATG codon located within
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elimination of either flt-3 ligand or its receptor does not produce a the third exon of a full-length transcript. However, because the eighth
platelet phenotype. ATG of thrombopoietin mRNA is embedded in the short, open read-
ing frame of the seventh ATG, its translation is particularly inefficient
Thrombopoietin because of the mechanism of ribosomal initiation. As such, little
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The term thrombopoietin was first coined in 1958 to describe the primary thrombopoietin protein is produced for any given amount of mRNA.
regulator of platelet production. A major impetus to the discovery of Although this molecular arrangement has no known physiologic conse-
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thrombopoietin in 1986 was the identification of the myeloproliferative quences, it forms the basis for an unusual form of disease, a disorder of
leukemia virus (MPLV), which induces a vast expansion of hematopoi- translation efficiency. Four cases of autosomal dominant familial throm-
etic cells. The responsible viral oncogene was characterized in 1990, bocytosis have been linked to mutations in the region surrounding the
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and its cellular homologue c-Mpl was cloned in 1992. Based on the initiation codon. In two families, a single mutation in different nucleo-
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presence of two copies of the hematopoietic cytokine receptor motif tides of the intron 3 splice donor sequence results in alternate splicing
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and the ability of a fusion of c-Mpl and the IL-4 receptor to signal in fac- of the primary thrombopoietin transcript, eliminating the seventh and
tor dependent cells, c-Mpl clearly encoded a growth factor receptor, eighth ATG codons, creating a new aminoterminus by fusing of the fifth
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but its ligand was not known. Using three distinct strategies, four sepa- open reading frame with the thrombopoietin coding sequence. This
rate groups were able to clone complementary DNA for the correspond- novel thrombopoietin mRNA is efficiently translated, resulting in sup-
ing hormone and report their results in 1994 (reviewed in Ref. 75). The raphysiologic levels of hormone production and nonclonal expansion of
gene for thrombopoietin encodes a 36-kDa polypeptide, which also is thrombopoiesis. 135,136 In another mutant thrombopoietin allele, deletion
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predicted to be extensively posttranslationally modified, resulting in an of a single nucleotide within the seventh open reading frame leads to
approximately 50- to 70-kDa protein. its fusion with the thrombopoietin coding sequence and now enhanced
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Thrombopoietin bears striking homology to erythropoietin, the translation of thrombopoietin from the seventh ATG codon. A fourth
primary regulator of erythropoiesis, within the aminoterminal half of mutation has been described within the seventh open reading frame,
the predicted polypeptide. The two proteins are more closely related leading to premature termination of that short peptide, preventing its
than any other two cytokines within the hematopoietic cytokine fam- interference with translation initiation from the usual eighth initiation
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ily, sharing 20 percent identical amino acids, an additional 25 per- codon, again enhancing thrombopoietin production (reviewed in
cent conservative substitutions, and identical positions of three of the Ref. 139). Of note, while reactive thrombocytosis is not thought to lead
four cysteine residues. Unlike any of the other cytokines in the family, to hypercoagulability (Chap. 119), several patients in these pedigrees
thrombopoietin contains a 181-residue carboxyl-terminal extension, developed thromboses, raising the physiologic question of why should
which bears homology to no known proteins. Two functions have been chronic stimulation of platelets with enhanced levels of thrombopoietin
assigned to this region: it prolongs the circulatory half-life of the hor- lead to hypercoagulability.
mone, and it aids in its secretion from the cells that normally synthesize The physiologic regulation of thrombopoietin production has
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the hormone. 124 received much attention. Experimental induction of immune-mediated
The biologic activities of thrombopoietin have been demonstrated thrombocytopenia results in relatively rapid restoration of platelet lev-
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in vitro and in vivo, in mice, rats, dogs, nonhuman primates, and man. els, followed by a brief period of rebound thrombocytosis. In these
Incubation of marrow cells with thrombopoietin stimulates megakary- experimental cases and in most naturally occurring cases of thrombo-
ocyte survival and proliferation, alone and in combination with other cytopenia, plasma hormone concentrations vary inversely with platelet
cytokines. In vivo, thrombopoietin stimulates platelet production in counts, rising to maximal levels within 24 hours of onset of profound
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a log-linear manner to levels 10-fold higher than baseline 3,61,125 without thrombocytopenia. Two non–mutually exclusive models have been
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affecting the blood red or white cell counts. In addition, because of its advanced to explain these findings. In the first model, thrombopoie-
effect on hematopoietic stem cells (Chap. 16), the number of erythroid tin production is constitutive, but its consumption, and hence the level
and myeloid progenitors and mixed myeloid progenitors in marrow and remaining in the blood to affect megakaryopoiesis, is determined by
spleen also are increased, 126,127 an effect that is particularly impressive the mass of c-Mpl receptors present on platelets and megakaryocytes
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when the hormone is administered following myelosuppressive ther- accessible to the plasma. In this way, states of thrombocytosis result in
apy. 126,128,129 This effect likely results from the synergy between thrombo- increased thrombopoietin consumption (by the expanded platelet mass
poietin and the other hematopoietic cytokines circulating at high levels of c-Mpl receptors), reducing megakaryopoiesis. Conversely, throm-
in this condition. bocytopenia reduces blood thrombopoietin destruction, resulting in
Based on genetic studies, thrombopoietin clearly is the primary elevated blood levels of the hormone that drive megakaryopoiesis and
regulator of thrombopoiesis. Elimination of either the c-Mpl or Tpo platelet recovery. This model is based on one of the mechanisms reg-
gene leads to profound thrombocytopenia in mice as a result of a greatly ulating macrophage colony-stimulating factor levels. The invariable
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reduced number of megakaryocyte progenitors, mature megakaryocytes, levels of thrombopoietin-specific mRNA present in the liver and kidney

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