Page 2101 - Hematology_ Basic Principles and Practice ( PDFDrive )

1864 Part XII Hemostasis and Thrombosis

Release of Mature Platelets exhibit a highly disorganized DMS, and fail to generate proplatelets
in vitro, a phenotype indicative of a late block in megakaryocyte
Details of how mature platelets release from the proplatelet tips are maturation. Therefore NF-E2 appears to control the transcription of
beginning to come into focus. In vitro, maturation of proplatelets a limited number of genes involved in cytoplasmic maturation and
ends in a rapid retraction that separates a variable portion of the platelet formation. Shivdasani and colleagues generated a subtracted
proplatelets from the residual cell body, leaving behind a naked, cDNA library enriched in transcripts downregulated in NF-E2
denuded nucleus (see Fig. 124.5C). Activation of apoptotic pathways knockout megakaryocytes. Using this approach, these investigators
in the cell body has been shown to be coincident with this event. have started to identify the downstream targets of NF-E2 and to
Junt and colleagues have used intravital fluorescence microscopy to analyze their role in the terminal stages of megakaryocyte differentia-
visualize proplatelet production in the opened cranial marrow cavity tion. Putative transcriptional targets of NF-E2 include β 1 tubulin,
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of living mice. Yellow fluorescent protein (YFP)–labeled megakaryo- thromboxane synthase, and proteins that regulate inside-out signaling
cytes were seen to protrude proplatelets and release megakaryocyte via α IIb β 3 integrin. The zinc finger protein GATA1 is also a transcrip-
fragments into the marrow sinusoids of living mice. Notably, these tion factor that plays a critical role in driving the expression of genes
anucleate fragments typically exceed platelet dimensions, suggesting essential for megakaryocyte maturation. However, unlike NF-E2,
that platelet morphogenesis continues in the circulation. In line with which appears to drive the later stage of megakaryocyte development,
these observations, we have recently identified a previously unrecog- GATA1 functions at multiple stages of development. Initially, GATA
nized intermediate stage in platelet formation and release, which we proteins were thought to regulate red blood cell maturation because
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termed the preplatelet. Preplatelets are defined as discoid cells genetic disruption of the GATA1 gene in mice results in embryonic
(3–10 µm) that retain the capacity to convert into barbell-shaped lethality secondary to a block in erythropoiesis. However, several
proplatelets and undergo fission into platelets. The conversion of more recent observations also implicate GATA1 as a regulator of
preplatelets to barbell proplatelets is powered by microtubule-based megakaryocyte differentiation. First, forced expression of GATA1 in
forces. It is likely that the microtubule motors that drive proplatelet the early myeloid cell line 416b induces megakaryocyte differentia-
extension are involved in aspects of platelet release, as well as in the tion. Second, Shivdasani and colleagues used targeted mutagenesis of
process of microtubule coiling. Sliding of an uncoiled portion of the regulatory elements within the GATA1 locus to generate mice with
microtubule relative to the rigid microtubule bundle in the proplatelet a selective loss of GATA1 in the megakaryocyte lineage. These
tip would provide a simple mechanism to effect platelet release and knockdown mice expressed sufficient levels of GATA1 in erythroid
would explain the variable morphology of the small but reproducible cells to circumvent the embryonic lethality caused by anemia. GATA1
percentage (<5%) of dumbbell-shaped platelets that are present in deficiency in megakaryocytes leads to severe thrombocytopenia.
blood. Recently, it was demonstrated that individual human platelets Platelet counts are reduced to approximately 15% of normal, and the
have the innate capacity to duplicate and form new cell bodies that small numbers of circulating platelets are round and larger than
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undergo fission into platelets. The morphologic similarities between normal. These mice have increased numbers of small megakaryocytes
platelets that form new cell bodies and preplatelets are striking. that exhibit an accelerated rate of proliferation. The small cytoplasmic
Whether or not newly released platelets exhibit a preplatelet pheno- volume of GATA1-deficient megakaryocytes typically contains an
type, which allows them to form barbell-shapes and divide again, is excess of rough endoplasmic reticulum, very few platelet-specific
not clear. granules, and an underdeveloped or disorganized DMS, suggesting
that maturation of megakaryocytes is arrested in GATA1-deficient
megakaryocytes.
Location of Platelet Release A family with X-linked dyserythropoietic anemia and thrombo-
cytopenia due to a mutation in GATA1 has been described. A
Megakaryocytes are produced in the bone marrow, and some undergo single-nucleotide substitution in the N-terminal zinc finger of
fragmentation into platelets in this location. It has been suggested GATA1 inhibits the interaction of GATA1 with its essential cofactor,
that, by extending into the bone marrow sinusoids, proplatelets friend of GATA1 (FOG). Although megakaryocytes in affected
provide a mechanism for extension into the bone, allowing release of family members are abundant, they are unusually small and exhibit
platelets directly into the circulation. 19,20 Megakaryocytes have been several abnormal features, including an abundance of smooth endo-
identified in intravascular sites within the lung, leading to a theory plasmic reticulum, an underdeveloped DMS, and a lack of granules.
that some platelets are formed from their parent cell in the pulmonary These observations suggest an essential role for the FOG1-GATA1
circulation. interaction in thrombopoiesis. Genetic elimination of FOG in mice
unexpectedly resulted in specific ablation of the megakaryocyte
lineage, suggesting a GATA1-independent role for FOG in the early
Transcriptional Regulation of Platelet Formation stages of megakaryocyte development; therefore GATA1 and FOG
are required for megakaryocyte generation from a common bipoten-
Megakaryocyte development and platelet formation are controlled by tial progenitor.
the coordinated action of transcription factors that specifically turn Several knockout mice also indicate a role for additional transcrip-
on the genes of megakaryocyte precursors or suppress the expression tion factors in megakaryocyte development. Mice carrying a null
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of genes that support other cell types. Gene-targeting studies in mutation in Fli-1, a member of the ETS family of winged helix-turn-
mice have identified several genes that are crucial for megakaryocyte helix transcription factors that bind purine-rich sequences in gene
development and platelet formation. Leading the list of transcription promoters, exhibit defects in megakaryocyte development. Mega-
factors that play an essential role in megakaryocyte maturation and karyocytes cultured from mice lacking Fli-1 contain reduced numbers
platelet biogenesis is the basic leucine zipper heterodimer NF-E2. of α-granules, disorganization of the demarcation membranes, and a
NF-E2 is a protein composed of a ubiquitously expressed 18–20-kDa reduction in size. Mice lacking the hematopoietic zinc finger (Hzf)
small-Maf subunit and a p45 subunit that is restricted to erythroid protein, a transcription factor that is predominantly expressed in
and megakaryocytic lineages. Although NF-E2 was postulated to be megakaryocytes, have reduced numbers of α-granules in megakaryo-
a transcription factor that specifically drove the expression of genes cytes and platelets. Therefore Hzf may regulate the transcription of
essential for erythropoiesis, mice lacking p45 NF-E2 do not exhibit genes involved in the synthesis of α-granule components and/or their
defects in erythropoiesis. Instead, mice deficient in the p45 subunit packaging into α-granules. SCL, a basic helix-loop-helix transcrip-
or two of the small-Maf subunits die of hemorrhage shortly after birth tion factor initially identified in a subset of human T-cell leukemia
because of a complete lack of circulating platelets. Although mega- with multilineage characteristics, also appears to be critical for mega-
karyocytes undergo normal endomitosis and proliferate in response karyopoiesis. Results of deletion of SCL in mice indicate that this
to TPO, mice deficient in p45 NF-E2 produce increased numbers of transcription factor is required for proper erythroid and megakaryo-
megakaryocytes that are larger than normal, contain fewer granules, cyte development.