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Review article
Molecular mechanisms of platelet exocytosis: insights into the “secrete” life
of thrombocytes
Guy L. Reed, Michael L. Fitzgerald, and János Polgár
The critical role played by platelets in hemostasis, thrombosis,
vascular remodeling, and healing is related to their function as
exocytotic cells that secrete important effector molecules at the site
of vascular injury. Recent insights into molecular mechanisms of
secretion indicate that platelet granule secretion is homologous to
exocytosis in neurons and other cells but involves a plateletselective machinery that is uniquely coupled to cell activation. This
review summarizes these recent insights in the context of the
burgeoning literature on mechanisms of exocytosis in neurons and
other cells—illustrating the fundamental homologies and the
unique characteristics of platelet secretion.
Overview of platelet secretion
Physiologic role of exocytosis
Platelets are anuclear secretory cells that respond to substances
produced by vascular injury (eg, thrombin, adenosine diphosphate
[ADP], collagen, and so forth).1 Activation causes platelets to
change shape, secrete their intracellular granules, and aggregate
with each other.1 Platelets normally contain at least 3 types of large
intracellular granules known as alpha, dense, and lysosomal
granules.2 Although it was previously believed that only the alpha
and dense granules were released in vivo, recent evidence indicates
that lysosomal granules may also be secreted under physiologic
circumstances.3,4 Platelet exocytosis provides the local, concentrated delivery of key effector molecules at the sites of vascular
injury. These effector molecules amplify cell activation, initiate
thrombus production, mediate intracellular adhesion, and trigger
both cell proliferation and migration. Platelet secretion plays an
important role in hemostasis because humans with defective
platelet exocytosis suffer from a moderate bleeding tendency
(see below).
Platelet granule development and contents
Platelet alpha granules contain polypeptides such as coagulation
proteins (eg, fibrinogen, factor V), soluble adhesion molecules (eg,
von Willebrand factor, vitronectin), growth factors (eg, plateletderived growth factor, epidermal growth factor), protease inhibitors (eg, plasminogen activator inhibitor-1, ␣2-antiplasmin), and
membrane adhesion molecules (eg, P-selectin, ␣IIb␤3). Platelet
dense granules contain small molecules (eg, ADP, calcium, magnesium) that are important for activating cells.3
When platelets originate by segmentation from megakaryo-
From the Cardiovascular Biology Laboratory, Harvard School of Public Health,
Boston, MA; Cardiology Division, Massachusetts General Hospital, Boston,
MA.
cytes, they contain a complex network of intact membrane
structures that includes the plasmalemma, the granules, the surfaceconnected canalicular system (SCCS) and the dense tubular
system. The development of platelet granules requires the activation of a platelet-selective genetic program because deletion of a
transcription factor, NF-E2, blocks granule formation, as well as
platelet release from the megakaryocyte.5 Membranes of the alpha
granules contain proteins found on the plasma membrane (eg,
␣IIb␤36,7 CD36,8 CD9, Rap1b9). Indeed as much as 10% of the
glycoprotein (GP)Ib-IX-V complex can be found associated with
the alpha granule membrane.7 This is consistent with the fusion of
endocytic vesicles with alpha granules. Alpha granules also contain
a mixture of proteins synthesized by the megakaryocyte (eg,
␤-thromboglobulin) as well as proteins endocytosed from the blood
(eg, fibrinogen) by both the megakaryocyte and platelet.10-21
Platelets contain coated pits and coated vesicles21-25 and take up
proteins by the process of receptor-mediated endocytosis; for
example, fibrinogen is taken up by the integrin ␣IIb␤3.15,16,21,26,27
Thus the alpha granules appear to develop from the homotypic
fusion of trans-Golgi vesicles in megakaryocytes as well as from
heterotypic fusion of these vesicles with endocytic vesicles.10,28
The dense granules contain small molecules such as serotonin
that are taken up and concentrated through specific transporting
and storage mechanisms.29-31 Although the contents of the alpha
and dense granules differ from each other, recent studies have
shown the presence of the alpha granule protein P-selectin and the
plasma membrane proteins ␣IIb␤3 and GPIb in the dense granule
membrane.32-35 This suggests that dense granules, like alpha
granules, arise from both endogenous syntheses in the megakaryocyte as well as from heterotypic fusion with endocytic vesicles
budding from the plasma membrane.35
Ultrastructural features of secretion
Platelet activation causes dramatic cytoskeletal rearrangements
that are important for shape change, adhesion, aggregation, exocytosis, and retraction.2,36,37 When platelet exocytosis occurs, granules fuse with the target plasmalemma or SCCS membranes to
secrete their cargo into the extracellular space. Some experts favor
a model in which granules individually fuse with the SCCS
membrane,38,39 whereas others suggest that platelet secretion
involves compound fusion of the granules with each other and then
with the plasmalemma membrane.40-43 In bovine platelets, which
lack an SCCS, the dense granules anchor in close physical
apposition to the plasma membrane (in a manner similar to vesicle
Public Health, II-127, 677 Huntington Ave, Boston, MA 02115; e-mail:
[email protected].
Supported in part by National Institutes of Health grant HL-64057.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: Guy L. Reed, Cardiovascular Biology Laboratory, Harvard School of
© 2000 by The American Society of Hematology
Submitted February 11, 2000; accepted May 23, 2000.
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BLOOD, 15 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 10
docking in neurons, see below). These anchored vesicles then fuse with
the plasmalemma in response to increased intracellular Ca ⫹⫹.44
At least 3 systems of cytoskeletal fibers are present in the
platelet: the membrane skeleton, microtubules, and microfilaments
(long actin filaments). When platelets are activated, the percentage
of filamentous actin rapidly increases.36,45-47 The membrane skeleton is associated with the network of cytoplasmic actin filaments,
various actin-binding proteins, and surface membrane glycoproteins (reviewed in Fox36 and Hartwig and Kwiatkowski48) and is
preserved in Triton X-100 lysates of platelets as a continuous layer
at the periphery of the cytoplasmic filaments.47 In resting platelets,
circumferential bundles of microtubules play an important role in
maintaining the discoid shape of platelets. Microtubule depolymerization leads to the loss of the discoid shape.49 Microtubules also
appear to play an important role in platelet secretion because
colchicine (a tubulin ligand)50 and monoclonal antibodies to alpha
and beta subunits of tubulin51 inhibit platelet secretion (when
introduced into permeabilized cells). Microtubule-associated proteins regulate the stability and phosphorylation of the microtubules;
these proteins may also play a role in microtubule reorganization.52
The contractile ring and stress fibers of platelets may also be
involved in exocytosis and retraction.37 Cytoskeletal reorganization
is mediated through actin polymerization and depolymerization
regulated by phosphatidylinositol-4,5-bisphospate (PIP2), which
binds to actin regulatory proteins such as scinderin and gelsolin.53
Consistent with these observations, recombinant scinderin potentiated Ca⫹⫹-induced serotonin release in permeabilized platelets,
whereas PIP2, or 2 peptides that interfere with scinderin-actin
interactions, blocked exocytosis.54
In summary, changes in the platelet cytoskeleton are linked to
the process of cell activation and appear to play a critical role in
granule movement and exocytosis. Unfortunately the mechanisms
through which cytoskeletal alterations moderate platelet secretion
remain poorly understood.
Platelet secretory disorders: storage
pool diseases
There are several recent reviews of platelet secretory defects.40-42,55,56
Congenital defects in platelet secretion are uncommon, and, the
storage pool diseases (SPDs) are the best characterized abnormalities. Patients with the SPDs may have abnormal bleeding times and
platelet aggregation defects.57,58 The SPDs have been categorized
into 3 groups by electron microscopy: decreased or abnormal alpha
granules (␣-SPD), dense granules (␦-SPD), or both (␣␦-SPD).
Patients with the ␦-SPDs have diminished or absent platelet
dense granules and frequently have vesicular defects in other cells
besides platelets. For example, ␦-SPD is found in patients with
Chediak-Higashi syndrome (CHS) and Hermansky-Pudlak syndrome (HPS). CHS is characterized by immunologic deficiency,
neutropenia, neurologic abnormalities, and large cellular inclusion
bodies.59 In addition to abnormal secretion of ADP and platelet
aggregation related to missing platelet dense granules, patients with
CHS also have abnormalities of other storage organelles including
melanosomes, cytolytic granules, and lysosomes.58 HPS (oculocutaneous albinism, platelet dysfunction, and lysosomal lipofuscin
ceroid deposition) is another type of ␦-SPD.60 HPS platelets lack
dense granules, and there is abnormal lysosome morphology and
function in other cells.61 A type of SPD has been found also in
strains of minks, cattle, and mice (eg, the gunmetal, sandy, cocoa,
muted, and mocha mouse).62-64
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3335
The ␦-SPDs may represent the effect of different gene mutations at distinct steps in lysosomal vesicular trafficking. Studies of
B- and T-cell lines in CHS have associated abnormal lysosomalendosomal protein sorting with the immune abnormalities seen in
patients with CHS.65,66 Defects in the LYST (lysosomal trafficking
regulator) gene have been identified in patients with CHS and in
beige mice.67-69 Introduction of a yeast artificial chromosome
containing the LYST gene corrects the giant lysosome morphology
of beige mouse fibroblasts.67 On the basis of these results and the
homology of the carboxy terminal domain of LYST with VPS15, a
kinase implicated in sorting of vacuolar proteins in yeast, it has
been suggested that the LYST gene regulates some aspect of
lysosomal trafficking.69
Mutations in different genetic loci have been identified in
patients with HPS and in strains of animals demonstrating HPSlike storage pool defects such as the mocha and pale-ear mice.70 A
duplication has been identified in a novel gene (HPS1) in 22
patients of Puerto Rican descent with HPS that was not found in
normal controls of Puerto Rican descent or HPS patients who were
not Puerto Rican.71 The HPS1 gene is predicted to encode a 700
amino acid polypeptide of unknown function.72 Pale-ear mice have
defects in platelet dense granules and melanosomes and carry an
insertional mutation in an exon of a 3⬘ coding sequence in the
murine counterpart of the HPS locus.73 The mocha mouse has
HPS-like storage pool defects involving platelet dense granules,
melanosomes, and lysosomes. In these mice the gene for the
␦-subunit of the AP-3 complex is mutated.74 AP-3 complex
function appears critical for normal dense granule maturation
because mutations have been found in the ␤-3A subunit of
non-Puerto Rican HPS patients and the Pearl mouse.75,76
Although the relationships between the genetic defects of CHS,
HPS, and the molecular pathogenesis of abnormal vesicle trafficking and function are not yet fully defined, these insights suggest
that normal development of platelet dense granules is related to
lysosomal vesicle trafficking. Defects in vesicle formation and
trafficking markedly affect downstream processes such as exocytosis, even if the exocytotic machinery is intact.77
The ␣-SPD or gray platelet syndrome is characterized by
enlarged platelets devoid of normal alpha granule staining as
visualized by the gray color of a Wright-stained blood smear.78
These platelets have normal serotonin uptake and normal (to
increased) numbers of dense granules and release lower amounts of
serotonin in response to thrombin stimulation.79 The ␣-SPD
platelets contain small abnormal vesicles that resemble alpha
granule precursors associated with the Golgi apparatus in
megakaryocytes.78,80 Although these platelets lack platelet factor 4
(a secreted protein), they have detectable amounts of the alpha
granule protein P-selectin on the membranes of their internal
vesicles and can translocate it to the plasma membrane in response
to thrombin.81,82 These findings suggest that the defect in ␣-SPD is
not in exocytosis itself but in the targeting of secretory proteins to
the alpha granule.81 It is interesting to note that this defect was not
seen in the targeting of von Willebrand factor or P-selectin to
the Weibel-Palade bodies of endothelial cells in 2 patients with
this disorder.83
The ␣␦-SPDs are deficient in both granule types and display a
loss of primary and secondary aggregation responses to most
common agonists.84,85 There is heterogeneity in P-selectin expression with most patients having little or no platelet P-selectin,
whereas a small number have normal levels. The incomplete
penetrance of the defect was confirmed by electron microscopy.82
The empty sack syndrome is another secretory disorder in
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BLOOD, 15 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 10
which there is a reduced level of stored nucleotides and serotonin,
but granule membrane proteins are present in normal numbers as
measured by granulophysin content.86 Other patients with defective
secretion have been identified with disorders that appear to involve
impaired aggregation and cell signaling.87-89
Comparison of the physiology of platelet
and neuronal secretion
Studies of vesicle trafficking and fusion in yeast, neurons, and
neuroendocrine cells have provided a conceptual framework for
elucidating regulated exocytosis in platelets. Regulated exocytosis
is “triggered” by intracellular signal(s) produced by cell activation
or membrane depolarization and is a subset of the vesicle trafficking and fusion processes that move molecules in and out of cells.
The triggered secretion of neurotransmitters is intensively
studied and has been the subject of several recent reviews.90-97
There are also recent reviews of exocytosis in neuroendocrine98,99
and pancreatic cells.100 It is generally agreed that neuronal exocytosis proceeds through a sequence of vesicle docking, priming,
triggering, and fusion that leads to release of neurotransmitters into
the synaptic cleft (Figure 1). Electron microscopy has demonstrated the docking or close apposition of vesicles to the plasma
membrane at the active zone of neurons.101 Although docking is
necessary for exocytosis, only a subset of the vesicles present can
be released rapidly indicating that additional “priming” or activation of the docked vesicle is necessary. The release of vesicles is
“triggered” by increases in the intracellular Ca⫹⫹ concentration.
Recent studies of platelet secretion (see below) have indicated
that it bears important similarities to neuronal exocytosis despite
the fact that these cells have different origins (mesoderm versus
ectoderm, respectively). Figure 2 highlights important similarities
and differences between the 2 processes. Neurons contain small
synaptic vesicles (SSV) loaded with neurotransmitters such as
␥-aminobutyric acid (GABA) and acetylcholine. The SSV take up
their neurotransmitters in the cell body and appear to recycle
contents after exocytosis through endocytosis. Neurons also have
small and large dense core vesicles that contain catecholamines and
neuropeptides, respectively. Platelet dense granules resemble the
neuronal small dense core vesicles because they contain small
molecules such as serotonin that are taken up and concentrated
from extracellular pools. Indeed, platelet dense granules have been
used as a model system for understanding the uptake and transport
of serotonin in amine-containing neurons.102,103 Similarly, the alpha
Figure 1. Steps in neuronal exocytosis. Synaptic vesicles containing neurotransmitters dock with the plasma membrane. The docked vesicles are primed for
secretion in an ATP/Mg⫹⫹-dependent process that readies them for immediate
release in response to local increases in Ca⫹⫹ secondary to membrane depolarization. Subsequently the vesicle and plasma membranes fuse (through a SNAREdependent mechanism) and the vesicle contents are released into the extracellular space.
Figure 2. Physiology of regulated exocytosis in neurons and platelets. The size
and contents of vesicles, kinetics of exocytosis, secretory signals, Ca⫹⫹ levels,
magnitude of exocytosis, and role played by endocytosis in both cell types are shown.
LDCV indicates large dense core vesicles; SSV, small synaptic vesicles.
granules resemble the large dense core vesicles of neurons because
they contain a number of proteins that are synthesized in the
megakaryocyte before platelet segmentation.
There is a marked difference in the kinetics of exocytosis and
signal transduction between these 2 types of cells. Neurotransmitters are released from SSV within 200 ␮s, whereas platelet granule
release typically takes 2 to 5 seconds—a 10 000-fold difference in
rates. Although increases in Ca⫹⫹
trigger both platelet and neuronal
i
exocytosis, there are significant differences between these cells in
the magnitude of the calcium signal and how it is generated. In
neurons membrane depolarization down the axon elicits Ca⫹⫹
influx via Ca⫹⫹ channels. This leads to local increases in Ca⫹⫹
concentration of ⬃200 ␮mol/L, which triggers the exocytosis of
vesicles in close proximity. Protein phosphorylation and other
signaling mechanisms may also have modulating effects on
neuronal exocytosis. In contrast, platelet secretion is triggered
when extracellular receptors are activated by agonists (such as
thrombin and ADP). This increases cytosolic intracellular Ca⫹⫹ to
2 to 10 ␮mol/L and activates protein kinase C, a kinase whose
activity is known to be important for platelet secretion (see below
and Figure 3). In response to potent agonists, secretion occurs after
Figure 3. Platelet activation, intracellular signaling, and secretion. When a
ligand or agonist such as thrombin interacts with its cognate receptor on the platelet
membrane, phospholipase C is activated through a G-protein–dependent mechanism. Alternatively, when collagen interacts with platelets, phospholipase C is also
activated but through other mechanisms (not shown). Phospholipase C cleaves PIP2
to DAG, which activates protein kinase C. Phospholipase C also produces IP3, which
releases Ca⫹⫹ from the platelet dense tubular system. Increased intracellular Ca⫹⫹
and protein kinase C work synergistically to induce platelet exocytosis. Protein kinase
C phosphorylates secretory molecules such as PSP.
BLOOD, 15 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 10
a lag phase of ⬃1.5 seconds and is nearly complete within 5
seconds.104,105 Finally, notable differences are apparent between
these cells in the magnitude of exocytosis in response to a signal.
The magnitude and rate of platelet exocytosis is related to the
potency of the stimulus and strong agonists appear to release nearly
all granules. In contrast, vesicles in neurons are released in a
quantal fashion.
Conserved molecular mechanisms
of exocytosis
SNARE machinery
The SNARE (soluble NSF attachment protein receptor) hypothesis
has been an influential model of general vesicular fusion.106 The
SNARE hypothesis states that the vesicle fusion apparatus contains
3 components: t-SNAREs, v-SNAREs, and a soluble SNAP/NSF
component (Figure 4). The t-SNAREs are target receptors such as
the syntaxins and SNAP-25 that are found on the target membrane
(the plasma membrane in the case of exocytosis). The v-SNAREs
are vesicle-associated membrane receptors (VAMP or synaptobrevins). The soluble components consist of NSF (N-ethylmaleimide-sensitive factor) and the soluble NSF-attachment proteins
(SNAPs; ␣, ␤, ␥ isoforms). In yeast cells, vesicle membrane fusion
requires 3 sequential steps of priming, docking, and fusion.
Priming is thought to involve dissociation of the SNARE complex,
“activation” of a t-SNARE, and adenosine triphosphate (ATP)mediated release of SNAP catalyzed by NSF.107,108 Docking, the
stable association between vesicles and membranes (or between
vacuoles), requires a low-molecular-weight GTPase or Rab-like
molecule.109 Fusion between membranes requires the formation of
ternary core complexes between the t-SNAREs and a v-SNARE,
but not NSF.109 The formation of a ternary complex between the
t-SNAREs (SNAP-25 and syntaxin) and the v-SNARE (VAMP)
may drive membrane fusion by overcoming the energy barrier that
normally blocks it (see below).110 It is important to note that
priming has a different meaning for neuroendocrine cells than it
does for yeast. For neuroendocrine cells priming is a Mg-ATP–
dependent step that follows docking that readies vesicles for rapid
exocytosis in response to Ca⫹⫹ signals (Figure 1), and probably
involves the SNAREs.97,111,112
In neurons, syntaxin 1, SNAP-25, and VAMP 2 form a stable
trimolecular SNARE complex.90-97,106 Syntaxin 1 belongs to a large
MOLECULAR MECHANISMS OF PLATELET EXOCYTOSIS
3337
family (at least 16 members) of type 1 integral membrane proteins
that are classically found on the plasmalemma or acceptor membrane.97 SNAP-25 in neurons (or related molecules SNAP-23 and
SNAP-29 in other cells) attaches to target membranes (ie, plasmalemma) via palmitoylated residues in the middle half of the
molecule, making its NH2 and COOH terminal ends free to interact
with syntaxin and VAMP. VAMPs 1 and 2 are also type I membrane
proteins found in vesicle membranes. The VAMP family includes at
least 8 known members.97 It has been postulated that the combinatorial complexity of the syntaxin–SNAP-25–VAMP system may
play a role in directing vesicles to the correct target membranes for
fusion, but in vitro experiments have not confirmed this hypothesis.113 Although the SNARE complex is sufficient in some systems
for mediating vesicle-membrane mixing, the kinetics and regulation of this process in neurons, platelets, and other cells argues that
other molecules are involved.97,114,115 The SNARE proteins may
participate in the initial formation of a hemifusion stalk between
membranes with subsequent generation of the fusion pore, lipid
mixing, and exocytosis requiring other, as yet unidentified, molecular interactions.97
Sec1/Munc 18
Members of the Sec1/Munc 18 gene family regulate interactions
between the SNARE proteins. The Sec1/Munc 18 proteins bind to
the syntaxins and through this binding prevent formation of the
core SNARE complex. At the same time the Sec1/Munc 18
molecules have been shown to be required for exocytosis. Sec 1,
one of the genes found necessary for the final phase of exocytosis in
yeast,116 has a homologue in worms (unc-18) that is required for
normal acetylcholine release at the nerve terminal117 and for
neurotransmitter release in flies.118 The murine homologue of
Sec1/unc-18 (Munc-18-1) binds to syntaxin 1 in neurons and
through this binding prevents formation of the SNARE core
complex between syntaxin 1, SNAP-25, and VAMP119 that is
required for fusion.
Deletion of the Munc 18-1 gene in mice is reported to be lethal
because it prevents vesicle exocytosis even though it does not
interfere with attachment of vesicles to the plasma membrane.97
The 3-dimensional structure of the neuronal Sec1-syntaxin complex has recently been solved.120 This structure has suggested that
the Sec1 proteins may provide specificity for the pairing of vesicles
with the correct target membranes in addition to modulating
SNARE complex formation.
Excitation and secretion coupling molecules
Figure 4. A hypothetical model for interactions of the SNARE machinery in
platelet secretion.90-97,106,120,158-162 Platelets contain the SNARE molecules syntaxin
2 and 4, SNAP-23, and VAMP. PSP (platelet Sec1 protein) binds to syntaxin 4 and
can inhibit SNARE complex formation. Platelet granule membranes contain Rabs
that may interact with PSP through effector molecules to play a role in vesicle docking
and targeting. PSP is phosphorylated in a protein kinase C-dependent manner when
platelets are activated by thrombin. This relieves the inhibitory effects of PSP on
SNARE complex formation by blocking its binding with syntaxin. Alternatively,
conformational changes induced in PSP by other molecules (potentially homologues
of Doc2, Munc13, and so forth) may release it from syntaxin, permitting SNARE
complex formation and the initiation of fusion and exocytosis.
Although yeast models are useful for understanding constitutive
vesicular secretion, they do not account for the regulated exocytosis “triggered” in specialized secretory cells, such as neurons or
neuroendocrine cells, by second messengers such as Ca⫹⫹ transients (reviewed in Calakos and Scheller91). The Ca⫹⫹ sensors
involved in regulated exocytosis are likely to differ between cell
types and secretory processes.98 Potential Ca⫹⫹ sensor/effector
molecules identified in neurons include synaptotagmin 1, Munc-13,
and Doc2.121 These proteins contain protein kinase C-C2 or
potential Ca⫹⫹-binding domains. Synaptotagmin 1 knockout mice
show impaired fast Ca⫹⫹-dependent vesicle exocytosis in neurons121,122 arguing that this protein plays a critical role in Ca⫹⫹triggered secretion in these cells. Munc13 (the murine homologue
of unc13) contains a protein kinase C-C1 or diacylglycerol
(DAG)-phorbol ester-binding domain, translocates to the plasma
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membrane, and enhances phorbol ester-induced release of neurotransmitters at neuromuscular junctions.123,124 Unc-13 is necessary
for normal synaptic function in Caenorhabditis elegans.125,126
Munc 13 can displace Sec1 (unc18) from syntaxin and deletion of
the Munc13-1 gene in mice causes impaired synaptic vesicle
release in response to action potentials.127,128 However, because
Munc-13 does not appear to bind Ca⫹⫹, its role as a direct Ca⫹⫹
sensor has been challenged.127 The C2 domains of Doc2 interact
with Ca⫹⫹ and phospholipid, which may target it to synaptic
vesicles and other membranes.129 One domain of Doc2 binds the
murine Sec1 protein (Munc18) and competes with Munc18syntaxin binding, whereas another domain of Doc2 interacts with
Munc13 in a manner that is enhanced by Ca⫹⫹ and phorbol
ester.130,131 In genetic experiments, overexpression of Doc2 increases secretion in PC12 cells, suggesting that this protein plays a
critical role in exocytosis.132 Several other potential Ca⫹⫹ sensors
have been proposed including the annexins, calcyclin, calmodulin,
unc31, frequenin, rim, and scinderin.133 Although these molecules
may prove to modulate other Ca⫹⫹-dependent steps in the exocytotic pathway, the late-acting or triggering Ca⫹⫹ sensor is likely to
interact directly with the SNARE proteins because of their critical
role in the initiation of vesicle fusion.
Other modulators of exocytosis
Abundant evidence implicates the Rab proteins, a large family of
Ras-related low-molecular-weight GTPases in vesicle trafficking
and fusion. At present the precise role played by the Rab proteins in
exocytosis remains unclear despite evidence that these molecules
interact with the SNAREs in yeast 134-136 and are involved in vesicle
docking, in a step between NSF/SNAP action and fusion.107 The
Rab proteins may exert their effects through effector molecules that
specifically interact with the guanosine triphosphate (GTP)- or
guanosine diphosphate (GDP)-bound form of the molecule. Deletion of the rab3a gene in mice causes a mild phenotype with an
increase in the number of vesicle fusion events elicited by an action
potential137; this suggests that Rab3A may play an inhibitory role in
neuronal exocytosis.
Molecular mechanisms of platelet exocytosis
Activation-secretion coupling
The platelet is activated (Figure 3) when specific physiologic
ligands (eg, ADP, thrombin, thromboxane A2, platelet activating
factor, collagen, epinephrine) interact with cognate receptors on the
plasma membrane. Receptor engagement activates phospholipase
C either via G-protein–coupled mechanisms (eg, thrombin, reviewed in Brass et al138) or via other signaling mechanisms (eg,
collagen reviewed in Barnes et al139). Phospholipase C cleaves PIP2
to inositol-1,4,5-trisphosphate (IP3) and DAG. In turn IP3 increases
cytosolic Ca⫹⫹ from 40 to 100 nmol/L to 2 to 10 ␮mol/L, which
initiates granule secretion.140-145 There is strong evidence in
platelets that protein kinase C also plays an important role in
inducing secretion. Platelets contains several isoforms of protein
kinase C (␣,␤⌱,␤⌱⌱,␦,␨,␩,␪), many of which are activated by DAG
and Ca⫹⫹.146 After cellular activation by thrombin, protein kinase
C phosphorylates pleckstrin, myristoylated-alanine rich C kinase
substrate (MARCKS), and several other substrates within seconds,
with kinetics that parallel the kinetics of platelet secretion and
aggregation.147-149 Phorbol esters mimic DAG in their stimulation
BLOOD, 15 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 10
of protein kinase C; they induce platelet secretion and aggregation
and potentiate the effects of calcium ionophores or agents that
150-152 Inhibitors of protein kinase C block dense
increase Ca⫹⫹
i .
granule secretion153 and have been used to demonstrate synergism
between Ca⫹⫹
and protein kinase C activity in platelet secretion.154
i
Although the protein kinase C isoform(s) required for platelet
secretion is not known, protein kinase C␤ has been shown recently
to be necessary for exocytosis in mast cells.155
A potential role for low-molecular-weight GTP-binding proteins (eg, the Rabs and other GTPases) in exocytosis has been
suggested because the exocytotic effects of Ca⫹⫹ are potentiated by
GTP-␥-S (which stimulates both heterotrimeric and low-molecularweight G-proteins), whereas AlF4 (which only activates heterotrimeric G-proteins) does not induce secretion.156,157
Current knowledge
Recent studies have revealed that the secretory machinery in
platelets has important homologies to the machinery found in
neurons and other cells. Platelets can form core SNARE complexes
in vitro that support SNAP-dependent NSF-ATPase activity158 and
this ␣SNAP-dependent NSF ATPase activity has been shown to be
critical for exocytosis of both alpha and dense granules.159 Platelet
membranes contain syntaxins 2 and 4, which play distinctive roles
in granule exocytosis; syntaxin 2 is involved in dense granule
release, whereas syntaxin 4 is necessary for platelet alpha granule
secretion.158,160-162 Platelets contain abundant amounts of SNAP23, and a VAMP, which interact and form SNARE complexes that
reportedly dissociate after cell activation (Figure 4).160,161 SNAP-23
is required for dense granule exocytosis, but it is not yet clear
which VAMP is functionally involved in secretion.162,163 A platelet
Sec1 protein (PSP) has been cloned, which is orthologous to
Munc-18c.161 PSP forms a tight complex with syntaxin 2 and
syntaxin 4 that can prevent the formation of the SNARE complex
(Figure 4)161,164; antibodies against PSP inhibit dense granule
secretion in permeabilized cells (J. Polgar and G. L. Reed, 1998,
unpublished observations). PSP is phosphorylated in platelets
activated by thrombin through a protein kinase C-dependent
mechanism. Protein kinase C-phosphorylation of PSP inhibits
syntaxin 4 binding, which relieves the inhibitory effect of this
molecule on SNARE complex formation.161 Alternatively, other as
yet uncharacterized platelet molecules, such as homologues of
Doc2, Munc 13, and so on, may induce a conformational change
that releases PSP from syntaxin (Figure 4).120
Platelets also contain a number of different Rab molecules
including Rab 3b, 6, 8, 11, 31.77,165 Rabs 3b, 6, and 8 are
phosphorylated when cells are activated by thrombin. Rab 6
phosphorylation proceeds through protein kinase C mechanism and
is associated with alterations in the GTP/GDP binding affinities and
cell localization.165,166 To date, no direct functional role has been
established for the Rabs in platelet secretion.
Mechanisms of exocytosis in other
blood cells
At present very little information is available about the mechanisms of secretion in leukocytes or endothelial cells. It has been
argued that leukocytes use lysosomes for both storage and exocytosis, unlike typical secretory cells, which use separate organelles for
storage and release of their secretory products.167 SNARE proteins
have been found in neutrophils, and phagocytosis appears to occur
BLOOD, 15 NOVEMBER 2000 䡠 VOLUME 96, NUMBER 10
through a SNARE-dependent mechanism.168-172 In mast cells,
SNAP-23 relocation from the plasmalemma to the granule membrane is required for compound granule-granule, granule-plasmalemma fusion typical of these cells.173 Overexpression of Rab 3d in
rat basophilic leukemic cells (RBL-2H3 cells) inhibits exocytosis
of hexosaminidase stimulated by interactions with the high-affinity
IgE receptors.174 Evidence indicates that endothelial cell secretion
also occurs through an NSF–SNARE-dependent mechanism175
though NSF-dependence has not been found by other
investigators.176
Summary and future directions
Despite their unique characteristics, platelets use a molecular
machinery for exocytosis that is homologous to that used in other
types of secretory cells (eg, neurons) and different organisms (yeast
to humans). However, many of the important physiologic differences between platelets and neurons in the regulation and kinetics
of exocytosis are due to the fact that platelet secretory molecules
arise from different genes and have distinctive features.
The core SNARE-related molecules responsible for platelet
secretion have recently been identified. Now the regulatory linkages or signaling mechanisms that link these molecules and the
process of platelet activation need to be elucidated more completely. Because the coupling of cell activation to exocytosis differs
significantly between platelets and neurons, it is likely that the
molecules that transduce these processes in platelets will be quite
different from their counterparts in neurons. Unlike neuronal
exocytosis, secretion in platelets is triggered by extracellular
ligand-receptor interactions that lead to cell activation. These
receptors are coupled to second messengers, such as Ca⫹⫹, DAG,
and protein kinase C, that signal to the exocytotic machinery.
Recent studies have demonstrated that thrombin activation of
platelets induces protein kinase C-mediated phosphorylation of
PSP, providing important linkages between thrombin activation
and the secretory machinery.161 Increased Ca⫹⫹
is a critical
i
determinant of secretion in platelets but the molecular sensor(s) in
platelets that transduces or couples the Ca⫹⫹
signal to secretion
i
must still be discovered.
Platelets represent a unique opportunity for examining exocytosis in a cellular system that is not confounded by the membrane
trafficking events found in actively synthetic cells. Beyond studies
MOLECULAR MECHANISMS OF PLATELET EXOCYTOSIS
3339
of the exocytotic machinery, important insights into secretion have
come from physiologic, pharmacologic, ultrastructural, and clinical
investigations. It is clear from ultrastructural analyses and from
studies of patients with secretory deficiencies, that platelet exocytosis is the net result of several molecular and cellular processes
acting in sequence. These processes include the formation, packaging, “differentiation,” and maintenance of secretory granules. The
molecular mechanisms underlying these processes are important
areas for future research. Valuable insights into this process are
likely to come from identification of the genes responsible for
secretory pool disorders in humans and animals. A particularly
striking difference between platelets and nonhematopoietic cells is
the strong functional association between changes in platelet shape
(mediated by the cytoskeleton) and the exocytotic process. Further
studies are necessary to define these linkages at the molecular level.
Although many of the signaling pathways involved in platelet
activation have been identified, the linkages of these signals to the
secretory machinery are poorly understood, even though patients
with impaired platelet activation-secretion coupling mechanisms
have been described. The fact that some of the patients with platelet
storage pool disorders do not appear to have functional defects in
their other secretory cells suggests that there may be a megakaryocyte- and platelet-selective molecular machinery for granule formation, packaging, maintenance, and exocytosis that is responsible for
the special function of the alpha and dense granules.
Insights into the molecular mechanisms responsible for platelet
exocytosis will help define the causes of human platelet secretory
disorders. These discoveries will provide general clues for understanding exocytosis in other vascular cells. Insights into these
mechanisms may also be useful for designing a new class of
therapeutic molecules to reduce the role played by platelet secretion in thrombosis and vascular remodeling—processes that contribute to cardiovascular and cerebrovascular disease.
Acknowledgments
The authors are grateful to the other members of the laboratory,
particularly Sul-Hee Chung, Aiilyan Houng, and Lin Liu. Given the
scope of this review, it has not been possible to discuss many
important contributions to the field of platelet secretion, and the
authors apologize to researchers whose work has not been cited.
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