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).
3354? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 115 Number 12 December 2005
38. Lecine, P., et al. 1998. Mice lacking transcription
factor NF-E2 provide in vivo validation of the
proplatelet model of thrombopoiesis and show a
platelet production defect that is intrinsic to mega-
karyocytes. Blood. 92:1608–1616.
39. Lecine, P., Italiano, J.E., Kim, S., Villeval, J., and
Shivdasani, R. 2000. Hematopoietic-specific beta1
tubulin participates in a pathway of platelet bio-
genesis dependent on the transcription factor
NF-E2. Blood. 96:1366–1373.
40. Tiwari, S., et al. 2003. A role for Rab27b in NF-E2-
dependent pathways of platelet formation. Blood.
41. Deveaux, S., et al. 1997. p45 NF-E2 regulates expres-
sion of thromboxane synthase in megakaryocytes.
EMBO J. 16:5654–5661.
42. Kerrigan, S.W., Gaur, M., Murphy, R.P., Shattil, S.J.,
and Leavitt, A.D. 2004. Caspase 12: a developmental
link between G-protein-coupled receptors and inte-
grin alphaIIbeta3 activation. Blood. 104:1327–1334.
43. Topp, K.S., Tablin, F., and Levin, J. 1990. Culture of
isolated bovine megakaryocytes on reconstituted
basement membrane matrix leads to proplatelet
process formation. Blood. 76:912–924.
44. Schwer, H.P., Lecine, P., Tiwari, S., Italiano, J.E., and
Hartwig, J. 2001. A lineage-restricted and divergent
beta-tubulin isoform is essential for the biogenesis,
structure and function of blood platelets. Curr. Biol.
45. Schulze, H., et al. 2004. Interactions between the
megakaryocyte/platelet-specific beta 1 tubulin and
the secretory leukocyte protease inhibitor SLPI
suggest a role for regulated proteolysis in platelet
functions. Blood. 104:3949–3957.
46. Handagama, P.J., Feldman, B.F., Jain, N.C., Farver,
T.B., and Kono, C.S. 1987. In vitro platelet release
by rat megakaryocytes: effect of metabolic inhibi-
tors and cytoskeletal disrupting agents. Am. J. Vet.
47. Freson, K., et al. 2005. The TUBB1 Q43P functional
polymorphism reduces the risk of cardiovascular
disease in men by modulating platelet function
and structure. Blood. 106:2356–2362.
48. Italiano, J.E., Shivdasani, R.A., and Hartwig, J.H.
2002. Cytoskeletal mechanics of platelet formation
[abstract]. Blood. 100:132a.
49. Patel, S.R., et al. 2005. Differential roles of micro-
tubule assembly and sliding in proplatelet forma-
tion by megakaryocytes. Blood. doi:10.1182/blood-
50. Trowbridge, E., et al. 1984. The origin of plate-
let count and volume. Clin. Phys. Physiol. Meas.
51. Harker, L., and Finch, C. 1969. Thrombokinetics in
man. J. Clin. Invest. 48:963–974.
52. Kaufman, R., Airo, R., Pollack, S., and Crosby, W.
1965. Circulating megakaryocytes and platelet
release in the lung. Blood. 26:720–731.
53. Rojnuckarin, P., and Kaushansky, K. 2001. Actin
reorganization and proplatelet formation in
murine megakaryocytes: the role of protein kinase C
alpha. Blood. 97:154–161.
54. Richardson, J., Shivdasani, R., Boers, C., Hartwig,
J., and Italiano, J.E. 2005. Mechanisms of organelle
transport and capture along proplatelets during
platelet production. Blood. doi:10.1182/blood-
55. Gordge, M.P. 2005. Megakaryocyte apoptosis: sort-
ing out the signals. Br. J. Pharmacol. 145:271–273.
56. Radley, J.M., and Haller, J.C. 1983. Fate of senescent
megakaryocytes in the bone marrow. Br. J. Haema-
57. Falcieri, E., et al. 2000. Ultrastructural characteriza-
tion of maturation, platelet release and senescence
of human cultured megakaryocytes. Anat. Rec.
58. Zauli, G., et al. 1997. In vitro senescence and apop-
totic cell death of human megakaryocytes. Blood.
59. Kaluzhny, Y., and Ravid, K. 2004. Role of apoptotic
processes in platelet biogenesis. Acta Haematol.
60. De Botton, S., et al. 2002. Platelet formation is the
consequence of caspase activation within mega-
karyocytes. Blood. 100:1310–1317.
61. Kaluzhny, Y., et al. 2002. BclxL overexpression in
megakaryocytes leads to impaired platelet frag-
mentation. Blood. 100:1670–1678.
62. Sanz, C., et al. 2001. Antiapoptotic protein Bcl-xL is
up-regulated during megakaryocytic differentiation
of CD34(+) progenitors but is absent from senes-
cent megakaryocytes. Exp. Hematol. 29:728–735.
63. Battinelli, E., and Loscalzo, J. 2000. Nitric oxide
induces apoptosis in megakaryocytic cell lines.
64. Battinelli, E., Willoughby, S.R., Foxall, T., Valeri,
C.R., and Loscalzo, J. 2001. Induction of platelet
formation from megakaryocytoid cells by nitric
oxide. Proc. Natl. Acad. Sci. U. S. A. 98:14458–14463.
65. Kim, J.A., et al. 2002. Gene expression profile of
megakaryocytes from human cord blood CD34+
cells ex vivo expanded by thrombopoietin. Stem
66. Clarke, M.C., Savill, J., Jones, D.B., Nobel, B.S.,
and Brown, S.B. 2003. Compartmentalized mega-
karyocyte death generates function platelets com-
mitted to caspase-independent death. J. Cell Biol.
67. Brown, S.B., Clarke, M.C., Magowan, L., Sander-
son, H., and Savill, J. 2000. Constitutive death of
platelets leading to scavenger receptor-mediated
phagocytosis. A caspase-independent cell clearance
program. J. Biol. Chem. 275:5987–5996.
68. Bernard, J., and Soulier, J.P. 1948. Sur une nouvelle
variet e de dystrophie thrombocytarie-hemorragi-
pare congenitale. Sem. Hop. Paris. 24:3217–3223.
69. Balduini, C.L., and Savoia, A. 2002. Inherited
thrombocytopenias: from genes to therapy. Hae-
70. Ware, J., Russel, S., and Ruggeri, Z.M. 2000. Gen-
eration and rescue of a murine model of platelet
dysfunction: the Bernard-Soulier syndrome. Proc.
Natl. Acad. Sci. U. S. A. 97:2803–2808.
71. Poujol, C., Ware, J., Nieswandt, B., Nurden, A.T.,
and Nurden, P. 2002. Absence of GPIbalpha is
responsible for aberrant membrane development
during megakaryocyte maturation: ultrastruc-
tural study using a transgenic model. Exp. Hematol.
72. Franke, J.D., Dong, F., Rickoll, W.L., Kelley, M.J.,
and Kiehart, D.P. 2004. Rod mutations associated
with MYH9-related disorders disrupt non-muscle
myosin-IIA assembly. Blood. 105:161–169.
73. Hu, A., Wang, F., and Sellers, J.R. 2002. Mutations
in human nonmuscle myosin IIA found in patients
with May-Hegglin anomaly and Fechtner syndrome
result in impaired enzymatic function. J. Biol. Chem.
74. Hamilton, R.W., et al. 1980. Platelet function,
ultrastructure, and survival in the May-Hegglin
anomaly. Am. J. Clin. Pathol. 74:663–668.
75. Canobbio, I., Noris, P., Pecci, A., Balduini, C.L., and
Torti, M. 2005. Altered cytoskeleton organization
in platelets from patients with MYH9-related dis-
ease. J. Thromb. Haemost. 3:1026–1035.
76. DiPumpo, M., et al. 2002. Defective expression of
GPIb/IX/V complex in platelets from patients with
May-Hegglin anomaly and Sebastian syndrome.
77. Raccuglia, G. 1971. Gray platelet syndrome. Am. J.
78. Oramer, E.M., Vainchenker, W., Vinci, G., Guichard,
J., and Breton-Gorius, J. 1985. Gray platelet syn-
drome: immunoelectron microscopic localisation
of fibrinogen and von Willebrand factor in plate-
lets and megakaryocytes. Blood. 66:1309–1316.
79. Stenberg, P.E., et al. 1998. Prolonged bleeding time
with defective platelet filopodia formation in the
Wistar Furth rat. Blood. 91:1599–1608.