3348? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
The biogenesis of platelets
from megakaryocyte proplatelets
Sunita R. Patel, John H. Hartwig, and Joseph E. Italiano Jr.
Hematology Division, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts, USA.
Megakaryocyte development. Megakaryocytes are rare myeloid cells
(constituting less than 1% of these cells) that reside primarily in
the bone marrow (1) but are also found in the lung and periph-
eral blood. In early development, before the marrow cavities have
enlarged sufficiently to support blood cell development, mega-
karyopoiesis occurs within the fetal liver and yolk sac. Megakary-
ocytes arise from pluripotent HSCs that develop into 2 types of
precursors, burst-forming cells and colony-forming cells, both
of which express the CD34 antigen (2). Development of both cell
types continues along an increasingly restricted lineage culminat-
ing in the formation of megakaryocyte precursors that develop
into megakaryocytes (1). Thrombopoietin (TPO), the primary
regulator of thrombopoiesis, is currently the only known cytokine
required for megakaryocytes to maintain a constant platelet mass
(3). TPO is thought to act in conjunction with other factors,
including IL-3, IL-6, and IL-11, although these cytokines are not
essential for megakaryocyte maturation (4).
Megakaryocytes tailor their cytoplasm and membrane systems
for platelet biogenesis. Before a megakaryocyte has the capacity to
release platelets, it enlarges considerably to an approximate diam-
eter of 100 µm and fills with high concentrations of ribosomes
that facilitate the production of platelet-specific proteins (5). Cel-
lular enlargement is mediated by multiple rounds of endomito-
sis, a process that amplifies the DNA by as much as 64-fold (6–9).
TPO, which binds to the c-Mpl receptor, promotes megakaryocyte
endomitosis. During endomitosis, chromosomes replicate and the
nuclear envelope breaks down. Although interconnected mitotic
spindles assemble, the normal mitotic cycle is arrested during ana-
phase B. The spindles fail to separate, and both telophase and cyto-
kinesis are bypassed. Nuclear envelope reformation (10, 11) results
in a polyploid, multilobed nucleus with DNA contents ranging
from 4N up to 128N within each megakaryocyte (12).
In addition to expansion of DNA, megakaryocytes experience sig-
nificant maturation as internal membrane systems, granules, and
organelles are assembled in bulk during their development. In par-
ticular, there is the formation of an expansive and interconnected
membranous network of cisternae and tubules, called the demar-
cation membrane system (DMS), which was originally thought to
divide the megakaryocyte cytoplasm into small fields where indi-
vidual platelets would assemble and subsequently release (13). DMS
membranes have continuity with the plasma membrane (14, 15) and
are now thought to function primarily as a membrane reservoir for
the formation of proplatelets, the precursors of platelets. A dense
tubular network (16) and the open canalicular system, a channeled
system for granule release, are also formed before the assembly of
proplatelets begins. Specific proteins associated with platelets, such
as vWF and fibrinogen receptors, are synthesized and sent to the
megakaryocyte surface, while others are packaged into secretory
granules with such factors as vWF, which is loaded into α-gran-
ules (17). Still other proteins, such as fibrinogen, are collected from
plasma through endocytosis and/or pinocytosis by megakaryocytes
and are selectively placed in platelet-specific granules (17, 18). Also
assembled during megakaryocyte maturation are mitochondria and
dense granules, which, like α-granules, derive from Golgi complexes.
Thus, as terminally differentiated megakaryocytes complete matu-
ration, they are fully equipped with the elements and machinery
required for the major task of platelet biogenesis.
The flow model of platelet formation. Despite the identification of
platelets over 120 years ago, there is still little consensus on many
of the mechanisms involved in platelet biogenesis. However, recent
evidence supports a modified flow model of platelet assembly. In
this model, platelets are assembled along essential intermediate
pseudopodial extensions, called proplatelets, generated by the out-
flow and evagination of the extensive internal membrane system
of the mature megakaryocyte (19). In 1906, Wright introduced the
initial concept that platelets arise from megakaryocyte extensions
when he described the detachment of platelets from megakaryocyte
pseudopods (20). Almost a century later, studies on megakaryocytes
producing platelets in vitro have revealed the details of platelet
assembly and have led us back to the classical proplatelet theory of
platelet release in which platelets fragment from the ends of mega-
karyocyte extensions (21–23). The discovery and cloning of TPO in
1994 and its receptor, c-Mpl, have allowed major advances in the
study of thrombopoiesis (24). TPO has facilitated the development
of in vitro megakaryocyte culture systems through which the pro-
Nonstandard?abbreviations?used: DMS, demarcation membrane system; GP, glyco-
protein; PKCα, protein kinase Cα; TPO, thrombopoietin.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 115:3348–3354 (2005).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
cess of platelet formation can be directly visualized and analyzed
(25–29). These systems have successfully reconstituted the transi-
tion of terminally differentiated megakaryocytes into fully func-
tional platelets. Megakaryocytes cultured in the presence of TPO
extend numerous proplatelets, consistent with the flow model. The
proplatelets generated in the in vitro systems are structurally simi-
lar to those seen in vivo extending into bone marrow sinusoids and
within the bloodstream (30–32). Significantly, platelets released
from megakaryocytes in vitro are structurally and functionally
similar to those found in vivo (28, 29). They are discs, 2–3 µm in
diameter, that contain a marginal microtubule band, and change
shape in response to platelet agonists, including thrombin.
Transcriptional control of platelet formation. A complete understand-
ing of platelet formation will rely heavily on the identification
of cellular controls active at each step of the elaborate process
described above. To date, only a few transcription and signaling
factors have been implicated in platelet generation, in part because
of the rarity of megakaryocytes in bone marrow.
GATA-1 and FOG (friend of GATA) are 2 transcription factors
with major roles in thrombopoiesis. GATA-1 acts early in mega-
karyocyte development, where it is involved in lineage commitment
of megakaryocytes, as well as erythrocytes, from their respective
progenitor cells (33). GATA-1 also functions later in megakaryocyte
development, controlling proliferation. Mutant mice that fail to
accumulate GATA-1 within their megakaryocytes exhibit throm-
bocytopenia and possess an increased number of immature mega-
karyocytes within their bone marrow. These megakaryocytes exhibit
small size, underdeveloped DMSs, reduced platelet-specific granule
content, decreased polyploidization, and an excess of rough endo-
plasmic reticulum (34). In humans, missense mutations in GATA-1
that disrupt its interaction with FOG-1 lead to thrombocytopenia
and abnormal bone marrow megakaryocytes. A truncated version
of GATA-1, expressed in transient myeloproliferative disorder and
acute megakaryoblastic leukemia, is able to interact with FOG-1
but lacks an N-terminal activation domain, resulting in decreased
transcriptional activation (35). Thus GATA-1 and FOG-1 appear to
play critical roles in megakaryocyte maturation.
The transcription factor NF-E2 has been identified as a major
regulator of platelet biosynthesis. NF-E2 is a heterodimer of p45
and p18 subunits that assumes the basic leucine zipper motif
(36). NF-E2 null mice experience lethal thrombocytopenia and
die from hemorrhage since they lack circulating platelets (37).
Megakaryocytes from NF-E2–null mice fail to undergo pro-
platelet formation, although megakaryocyte maturation appears
intact (38). Interestingly, megakaryocytes from NF-E2 knockout
mice lack β1-tubulin, the major β-tubulin isoform expressed in
megakaryocytes and platelets (39). NF-E2 has also been shown to
interact with the promoter for Rab27b, a small GTPase identified
in platelets. Rab27b expression is high in terminally differenti-
ated megakaryocytes, and its inhibition in megakaryocytes results
in attenuated proplatelet production, which suggests a role for
Rab27b in proplatelet formation (40). NF-E2 may also affect
thromboxane synthase (41) and caspase-12 (42), both of which
are reduced in NF-E2–null megakaryocytes.
Overview of proplatelet formation. The assembly of platelets from
megakaryocytes involves an elaborate dance that converts the cyto-
plasm into 100- to 500-µm-long branched proplatelets on which
the individual platelets develop. The proplatelet and platelet for-
mation process generally commences from a single site on the
megakaryocyte where 1 or more broad pseudopodia form. Over
a period of 4–10 hours, the pseudopodial processes continue to
elongate and become tapered into proplatelets with an average
diameter of 2–4 µm. Proplatelets are randomly decorated with
multiple bulges or swellings, each similar in size to a platelet, which
gives them the appearance of beads connected by thin cytoplas-
mic strings (Figure 1). The generation of additional proplatelets
continues at or near the original site of proplatelet formation and
spreads in a wavelike fashion throughout the remainder of the cell
until the megakaryocyte cytoplasm is entirely transformed into
an extensive and complex network of interconnected proplatelets
(27, 36). The multilobed nucleus of the megakaryocyte cell body is
compressed into a central mass with little cytoplasm and is eventu-
ally extruded and degraded. Platelet-sized swellings also develop at
the proplatelet ends and are the primary sites of platelet assembly
and release, as opposed to the swellings along the length of the
proplatelet shaft (Figure 1). The precise events involved in platelet
release from proplatelet ends have not been identified.
Microtubule organization in proplatelets. Microtubules, hollow poly-
mers assembled from αβ-tubulin dimers, are the major structural
component of the engine that drives the elongation of proplate-
lets. It is well known that microtubule bundles assembled by the
megakaryocytes fill proplatelet processes (27, 43, 44), as shown in
Figure 2. Likewise, when megakaryocytes are retrovirally directed to
express GFP-tagged β1-tubulin, fluorescent microtubules densely
fill their proplatelets (45). The microtubules arrays are essential
for proplatelet formation (26). Proplatelets fail to form in mega-
karyocytes treated with agents that inhibit microtubule assem-
bly (1–10 µm nocodazole) (26, 27, 46). Transgenic mice lacking
β1-tubulin, the most abundant platelet β-tubulin isoform, assemble
microtubules poorly, develop thrombocytopenia (15–30% reduction
in platelet count), and have spherocytic circulating platelets, a con-
sequence of defective marginal band formation (44). The marginal
bands of β1-tubulin–null platelets are composed of a reduced micro-
tubule mass and have only 2–3 coilings instead of the normal 8–12.
The importance of β1-tubulin in platelet-shape maintenance is sup-
ported by a recent study that identified the first β1-tubulin varia-
tion in humans, where a double-nucleotide mutation results in the
substitution of a highly conserved glutamine with a proline (Q43P)
(47). In heterozygous individuals carrying the Q43P mutation,
Anatomy of a proplatelet. Differential interference contrast image of
proplatelets on a mouse megakaryocyte in vitro. Some of the hall-
mark features of proplatelets, including the tip, swellings, shafts, and a
branch point, are indicated. Scale bar, 5 µm.
3350? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
β1-tubulin expression was reduced in platelets, which were enlarged
and spherocytic because of defects in the microtubule marginal
band. Additionally, the Q43P mutation is thought to occur quite
frequently in the normal population (∼10%) and impart a protec-
tive effect against cardiovascular disease in men (47).
Electron microscopy studies of megakaryocytes undergoing
proplatelet formation have provided insights into how microtu-
bule reorganization powers proplatelet growth (27). Just before
proplatelet formation, microtubules consolidate in a mass just
beneath the cortical plasma membrane. These microtubules align
into bundles and fill the cortex of the first blunt process extended
by megakaryocytes, signaling the beginning of proplatelet develop-
ment. The microtubules merge into thick linear bundles that fill
the proplatelet shafts when the proplatelets lengthen and taper.
At the free proplatelet end, the microtubule bundles form loops,
which reenter the proplatelet shaft. This process gives rise to the
bulbous tips of the proplatelets, each measuring 3–5 µm in diam-
eter. These studies also were the first to recognize that the platelet-
sized swellings that occur along proplatelet shafts are not nascent
platelets but are instead points where the microtubule bundles of
the shaft diverge for a short distance and then reconvene to locally
thicken the proplatelet shaft. Coiling of microtubules, the signa-
ture of circulating platelets, occurs only at the proplatelet ends
and not within the platelet-sized swelling positioned along the
proplatelet shaft. Therefore, the primary site of platelet assembly
is at the end of each proplatelet.
Proplatelet elongation. Proplatelets grow from the megakaryocyte
cell body at an average rate of 0.85 µm/min, in good agreement
with the 4–10 hours required to convert the entire megakaryocyte
cytoplasm into proplatelets with average lengths of 250–500 µm
(48). Microtubule assembly dynamics within megakaryocytes and
proplatelet formation are complex, and their exact relationship
to growth, other than supplying microtubule mass, is unclear. In
recent studies, EB3, a protein that binds the plus end of micro-
tubules, fused to GFP was expressed in murine megakaryocytes
and used as a marker of microtubule plus-end dynamics. Imma-
ture megakaryocytes without proplatelets employ a centrosomal-
coupled microtubule nucleation/assembly reaction, which appears
as a prominent starburst pattern when visualized with EB3-GFP.
Microtubules assemble only from the centrosomes and grow out-
ward to the cell cortex, where they turn and run in parallel with the
cell edges (49). However, just before proplatelet production, cen-
trosomal assembly ceases and microtubules begin to collect in the
cell cortex. Once proplatelet extension begins, microtubule nucle-
ation and growth occur continuously throughout the entire pro-
platelet, including the shaft, swellings, and tip. The EB3-GFP stud-
ies also revealed that microtubules polymerize in both directions
in proplatelets, e.g., toward both the tips and the cell body (49).
This demonstrates that the microtubules composing the bundles
have a mixed polarity. The rates of microtubule polymerization are
approximately 10-fold faster than the proplatelet growth rate.
Although microtubules are continuously polymerizing in pro-
platelets, polymerization per se does not provide the forces for
elongation. Proplatelets continue to elongate at normal rates even
when microtubule polymerization is inhibited by drugs that block
net microtubule assembly, which suggests another mechanism for
proplatelet elongation (49). Consistent with this idea, proplatelets
possess an inherent microtubule sliding mechanism, similar to the
extension of a fire engine ladder. Dynein, a minus-end microtubule
molecular motor protein, localizes along the microtubules of the
proplatelet and appears to directly contribute to microtubule slid-
ing, since inhibition of dynein, through disassembly of the dynac-
tin complex, prevents proplatelet formation (49). Microtubule slid-
ing can also be reactivated in detergent-permeabilized proplatelets.
ATP, known to support the enzymatic activity of microtubule-based
molecular motors, activates proplatelet elongation in permeabi-
lized proplatelets (48) that contain both dynein and its regulatory
complex, dynactin. Thus, dynein-facilitated microtubule sliding
appears to be the key event in driving proplatelet elongation.
Platelet amplification. Each megakaryocyte has been estimated to
generate and release thousands of platelets (50–52). If platelet for-
mation is restricted to a relatively limited number of proplatelet
ends, platelets would have to form and release on a minute time
scale. (The average megakaryocyte has approximately 5–10 origi-
nal proplatelets. If 1,000 platelets are constructed, then each end
would have to produce 100–200 platelets over a 4-hour time course,
equivalent to 25–50 platelets per hour.) Analysis of time-lapsed
video microscopy of proplatelet development from megakaryo-
cytes grown in vitro, however, has revealed that ends are amplified
in an elaborate process that repeatedly bends and bifurcates the
proplatelet shaft to form new ends (27). End amplification initiates
when a proplatelet shaft is bent into a sharp kink, which then folds
back on itself, forming a loop in the microtubule bundle. The new
loop eventually elongates, forming a new proplatelet shaft branch-
ing from the side of the original proplatelet. Loops lead the pro-
platelet tip and define the site where nascent platelets will assemble
and where platelet-specific contents are trafficked (Figure 3). In
marked contrast to the microtubule-based motor that elongates
proplatelets, actin-based force is used to bend the proplatelet in
end amplification. Megakaryocytes treated with one of the actin
toxins cytochalasin and latrunculin can extend long proplatelets
but fail to branch and are decorated with few swellings along their
length (27). Despite extensive characterization of actin filament
dynamics during platelet activation, how actin participates in this
reaction and the cytoplasmic signals that regulate bending have yet
to be determined. Immunofluorescence and electron microscopy
of megakaryocytes undergoing proplatelet formation indicate that
actin filaments are distributed throughout the proplatelet and are
particularly abundant within swellings and at proplatelet branch
Localization of microtubules within proplatelets. (A) Immunofluorescence
studies on murine megakaryocytes grown in culture and labeled with
β1-tubulin antibodies indicate that microtubules line the entire length
of proplatelets, including shafts and the tip. (B) Immunofluorescence
studies further show that microtubule coils similar to those seen in
mature platelets occur in both proplatelets and released platelet-sized
particles (arrow). Scale bar, 5 µm.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
points (27, 53). Studies also indicate that protein kinase Cα (PKCα)
associates with aggregated actin filaments in megakaryocytes
undergoing proplatelet formation, and inhibition of PKCα or inte-
grin signaling pathways prevents actin filament aggregation and
proplatelet formation in megakaryocytes (53). However, the role of
actin filament dynamics in platelet biogenesis remains unclear.
Organelle transport in proplatelets. Nascent platelets forming at the
proplatelet tips must be loaded with their contents of organelles
and platelet-specific granules. This process occurs along the shafts
of proplatelets, as organelles and granules travel in a discontinu-
ous fashion from cell body to proplatelet. Despite bidirectional
movement along shafts, the particles are eventually captured at the
proplatelet tip (54). Immunofluorescence and electron microscopy
studies indicate that organelles are in direct contact with microtu-
bules (48), and their movement appears to be independent of actin.
Of the 2 major microtubule motors, kinesin and dynein, only the
plus end–directed kinesin is situated in a pattern similar to that of
organelles and granules and is likely responsible for transporting
these elements along microtubules (54). It appears that a 2-fold
mechanism of organelle and granule movement occurs in platelet
assembly. First, organelles and granules travel along microtubules,
and second, the microtubules themselves can slide bidirectionally
in relation to other motile filaments to indirectly move organelles
along proplatelets in a “piggyback” fashion.
Proplatelet release. In vivo, proplatelets extend into bone mar-
row vascular sinusoids, where they may be released and enter the
bloodstream. The actual events surrounding platelet release in vivo
have not been identified because of the rarity of megakaryocytes
within the bone marrow. The events leading up to platelet release
within cultured murine megakaryocytes have been documented.
After complete conversion of the megakaryocyte cytoplasm into a
network of proplatelets, a retraction event occurs, which releases
individual proplatelets from the proplatelet mass (27). Proplate-
lets are released as chains of platelet-sized particles, with the most
commonly released structure resembling a barbell, a narrow shaft
connecting 2 teardrop-shaped tips. Electron microscopic analysis
of these barbell structures illustrates linear microtubule bundles
within the shafts and a coil of microtubules, reminiscent of the
mature platelet marginal band, within the ends. Individual plate-
let-sized particles are also released from proplatelet-producing
megakaryocytes or released proplatelets. Although the actual
release event has yet to be captured, the platelet-sized particle is
thought be liberated as the proplatelet shaft increasingly narrows.
See Figure 4 for an overview of platelet formation.
Apoptosis in platelet biogenesis. The process of platelet assembly
in megakaryocytes exhibits some characteristics associated with
apoptosis, including cytoskeletal reorganization, membrane con-
densation, and ruffling. These similarities have led to further
investigations aimed at determining whether apoptosis is a major
force driving proplatelet formation and platelet release. Apopto-
sis, programmed cell death, is responsible for destruction of the
nucleus in senescent megakaryocytes (55). However, it is thought
that a specialized apoptotic process may lead to platelet assembly
and release. Apoptosis has been described in megakaryocytes (56)
and found to be more prominent in mature megakaryocytes as
opposed to immature cells (57, 58). A number of apoptotic factors,
both proapoptotic and antiapoptotic, have been identified in mega-
karyocytes (reviewed in ref. 59). Apoptosis-inhibitory proteins such
as Bcl-2 and Bcl-xL are expressed in early megakaryocytes. When
overexpressed in megakaryocytes, both factors inhibit proplatelet
formation (60, 61). Bcl-2 is absent in mature blood platelets, and
Bcl-xL is absent from senescent megakaryocytes (62), consistent
with a role for apoptosis in mature megakaryocytes. Proapoptotic
factors, including caspases and NO, are also expressed in megakary-
ocytes. Evidence indicating a role for caspases in platelet assembly
is strong. Caspase activation has been established as a requirement
for proplatelet formation. Caspase-3 and caspase-9 are active in
mature megakaryocytes, and inhibition of these caspases blocks
proplatelet formation (60). NO has been implicated in the release
of platelet-sized particles from the megakaryocytic cell line Meg-01
and may work in conjunction with TPO to augment platelet release
(63, 64). Other proapoptotic factors expressed in megakaryocytes
and thought to be involved in platelet production include TGF-β1
and SMAD proteins (65). Of interest is the distinct accumulation of
apoptotic factors in mature megakaryocytes and mature platelets
(66). For instance, caspase-3 and caspase-9 are active in terminally
differentiated megakaryocytes. However, only caspase-3 is abun-
dant in platelets (67), while caspase-9 is absent (66). Similarly, cas-
pase-12, found in megakaryocytes, is absent in platelets (42). These
data support differential mechanisms for programmed death in
platelets and megakaryocytes and suggest the selective delivery
and restriction of apoptotic factors to nascent platelets during
proplatelet-based platelet assembly.
Disorders of platelet production
A diversity of factors can contribute to anomalous platelet counts;
one of these is inappropriate platelet production. Disorders of
inappropriate platelet production are grouped into 2 major cat-
egories, inherited and noninherited disorders. Inherited platelet
disorders occur in individuals harboring genetic mutations with-
in genes that are active during the process of platelet biogenesis.
Identification of the precise genetic lesions that give rise to such
disorders gives further insight into the mechanisms of platelet
formation. These rare disorders, including the 3 major disor-
ders described below, can result in severe thrombocytopenia and
increased bleeding times.
Bernard-Soulier syndrome. Bernard-Soulier syndrome is an auto-
somal dominant disorder characterized by macrothrombocytope-
nia (thrombocytopenia with increased platelet volume), increased
bleeding time, and impaired platelet agglutination (68). The
underlying cause is absent or deficient expression of the glycopro-
tein (GP) Ib/IX/V complex, which forms the vWF receptor on the
platelet surface (69). Binding of vWF to the GPIb/IX/V complex
is an essential step in hemostasis. A host of mutations within the
genes that encode GPIbα, GPIbβ, or GPIX have been identified
and linked to Bernard-Soulier syndrome. How these mutations
translate into macrothrombocytopenia, however, is still unknown.
Proplatelet amplification. Megakaryocytes increase their proplatelet
number through formation of branched extensions off of existing pro-
platelets. Initially, the shaft of the parent proplatelet (A) is sharply bent
(B). This bend then folds back on itself to form a loop (C). The loop
elongates to form a new proplatelet with a novel tip (D).
3352? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
Results from studies on GPIbα knockout mice, a model of Ber-
nard-Soulier syndrome, indicate that the megakaryocyte DMS
is disordered (70). These megakaryocytes went on to produce
aberrantly large megakaryocyte fragments and proplatelets with
reduced internal membrane content (71). Since the GPIb/IX/V
complex is linked to the membrane skeleton, it is thought that
the genetic defects of Bernard-Soulier syndrome may alter normal
cytoskeletal dynamics during platelet formation (69).
MYH9-related disorders. MYH9-related disorders are characterized
by macrothrombocytopenia. Patients with these disorders, includ-
ing May-Hegglin anomaly, Sebastian syndrome, Fechtner syn-
drome, Alport syndrome, and Epstein syndrome, may also experi-
ence hearing loss, cataract, nephritis, and/or granulocyte inclusion
body formation (69). The genetic defects in each of these illnesses
occur within the MYH9 gene, which encodes nonmuscle myosin
heavy chain IIA, the sole myosin isoform expressed in platelets and
neutrophils. Myosin molecules are hexameric protein complexes
composed of 2 heavy chains that dimerize and 2 pairs of myosin
light chains. Each heavy chain consists of a globular head contain-
ing ATPase activity and actin-binding sites, and an α-helical tail or
rod. Although numerous mutations have been identified through-
out the MYH9 gene, mutations are most commonly seen to occur
in exons encoding the α-helical tail, a region that promotes myosin
dimerization and filament formation. Studies examining the 4 most
common myosin rod mutations identified in MYH9-related disor-
ders indicate that the mutations result in diminished myosin dimer
and filament formation in vitro (72). Moreover, studies completed
on 2 MYH9 mutations that reside within the myosin head domain
reveal that the defects dramatically decrease both the MgATPase
activity and actin filament translocation of myosin in vitro (73).
The thrombocytopenia that occurs in MYH9-related disorders
is thought to be a result of defective platelet production, since
Overview of megakaryocyte production of platelets. As megakaryocytes transition from immature cells (A) to released platelets (E), a systematic
series of events occurs. (B) The cells first undergo nuclear endomitosis, organelle synthesis, and dramatic cytoplasmic maturation and expan-
sion, while a microtubule array, emanating from centrosomes, is established. (C) Prior to the onset of proplatelet formation, centrosomes disas-
semble and microtubules translocate to the cell cortex. Proplatelet formation commences with the development of thick pseudopods. (D) Sliding
of overlapping microtubules drives proplatelet elongation as organelles are tracked into proplatelet ends, where nascent platelets assemble.
Proplatelet formation continues to expand throughout the cell while bending and branching amplify existing proplatelet ends. (E) The entire
megakaryocyte cytoplasm is converted into a mass of proplatelets, which are released from the cell. The nucleus is eventually extruded from the
mass of proplatelets, and individual platelets are released from proplatelet ends.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
both megakaryocyte numbers and platelet clearance are normal
(74). Myosin has been theorized to function in platelet formation,
specifically during the bending and branching process, where an
actomyosin interaction could possibly provide the forces required
to contort the proplatelet shaft (27). Also, recent studies on plate-
lets carrying MYH9 defects show that a greater amount of myo-
sin is associated with the actin cytoskeleton in resting platelets
and that, upon activation, MYH9-mutated platelets have altered
cytoskeletal dynamics (75). Defects in surface expression of the
GPIb/IX/V complex in platelets carrying MYH9 mutations have
also been reported (76).
Gray platelet syndrome. Gray platelet syndrome is another autoso-
mal dominant disease that presents with macrothrombocytopenia.
These large platelets appear gray due to a reduction in α-granule
content (77). α-granules normally contain a number of proteins
including von Willebrand factor, and fibrinogen. In Gray platelet
syndrome, platelets inadequately package these proteins within
α-granules (78). As a consequence, a number of clot promoting fac-
tors fail to be released upon platelet activation, which increases the
risk of bleeding. Although the precise genetic defects responsible
for gray platelet syndrome are unknown, evidence indicates that
cytoskeletal defects can result in poor α-granule packaging (79).
The transition from megakaryocyte to platelets is a complex pro-
cess. Although the basic mechanisms of platelet production have
been investigated, elucidating the specific molecular controls
and cellular events involved in platelet formation and release is
an unfinished task. Major issues still to be addressed include (a)
determination of which factors induce proplatelet formation in
mature megakaryocytes, (b) identification of the mechanism of
platelet release from proplatelets, and (c) understanding of how
the cytoskeleton drives the events that result in proplatelet pro-
duction. Further examination of genetic defects that result in
platelet disorders, in addition to continued molecular, cellular,
and biochemical studies of megakaryocytes as they transition into
platelets, will provide a clearer understanding of these processes.
This knowledge may aid in the future ex vivo expansion of plate-
lets or in vivo therapies aimed at enhancing platelet production in
patients with thrombocytopenia.
This work was supported by NIH grant HL68130 (to J.E. Italiano Jr.).
J.E. Italiano Jr. is an American Society of Hematology Scholar.
S.R. Patel was supported by NIH training grant HL066978-04 and
an NIH/National Heart, Lung, and Blood Institute postdoctoral
fellowship award (HL082133).
Address correspondence to: Joseph E. Italiano Jr., Brigham and
Women’s Hospital, Division of Hematology, 1 Blackfan Circle, 6th
Floor, Boston, Massachusetts 02115, USA. Phone: (617) 355-9007;
Fax: (617) 355-9016; E-mail: firstname.lastname@example.org.
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