Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2191-2192
CORRESPONDENCE
The Pathway of Exocytosis in Human Platelets
 |
LETTER |
To the Editor:
In a report titled "Regulated Secretion in Platelets: Identification
of Elements of the Platelet Exocytosis Machinery," recently published by Lemons et al1 in Blood, the authors
have investigated the molecular events involved in membrane fusion in
platelets. Therein, the function of molecules mediating the secretory
events of the exocytosis in platelets was attributed to membranes of structures that might be involved in this process. There are no doubts
or any objection to the biochemical results of the article. On the
other hand, the investigators discuss their original findings on a
critical morphological basis. In the introduction they wrote, "In
one model of platelet activation,2-4 these stimuli
(collagen, thrombin, and ADP) trigger morphological changes in the
platelet resulting in the apparent movement of the secretory granules
to the center of the cell and their subsequent fusion with the surface connected canalicular system (SCCS)." The disadvantage of this model
is that the fusion of membranes of secretory organelles with the
membranes of the SCCS has never been demonstrated.5,6 In
contrast, it was clearly shown in studies, using rapid freezing with a
time resolution in the range of milliseconds to capture fusion
events,7 that the secretory organelles
(
-granules8,9 and dense core bodies9,10)
fuse with the plasma membrane when the platelets were stimulated
before. The fusion of secretory organelles with the plasma membrane in
human platelets is a phenomenon seen in secretory cells in
general.8,11 From morphological observations6,8
and morphometrical measurements,12,13 it was concluded that
the fate of the SCCS during stimulation is to become evaginated within
seconds to allow the surface enlargement necessary for formation of
pseudopodia or spreading. Fully stimulated platelets do not show SCCS
but do show the membranes of degranulating organelles.6,8,10 As a consequence, thrombin-stimulated gray platelets, which lack -granules, do not contain such membrane convolutes.14
Furthermore, the investigators wrote that platelet granule membranes do
not appear to be predocked, in comparison with synaptic vesicles.
Interestingly, -granules and also dense core granules dock before or
at the beginning of stimulation to the plasma membrane. In activated
platelets after organelle centralization by the action of the
contractile cytoskeleton, the membranes of both types of secretory
organelles show docking.8,10 Docking, also named apposition, is longer-lasting than membrane fusion and stable enough to
be demonstrable with chemical fixation.8,15,16 Granules in
apposition with the plasma membrane maintain this position during
subsequent shape change and internal contraction. These reactions
progress in the time range of seconds. An investigation using the
atomic force microscope on living platelets gave support to this view
of platelet exocytosis.17 Secretory organelles that are
apposed to the surface membrane (and arrested there) fuse and induce
sequential fusion and the formation of compound granules. The
constriction of the contractile cytoskeleton in platelets moves
(mobile) platelet organelles to the platelet center and into contact
with each other, supporting further apposition and leading to formation
of compound granules.6,11 It should be noted that the
formation of compound granules during platelet secretion was already
suggested from immunocytochemical and morphological investigations by
Ginsberg et al in 1980.18
From their results, Lemons et al1 suggested that the
molecular mechanism working in platelets for exocytosis is similar to
that one described in other cells, most notably neurosecretory cells. I
would like to add the suggestion that the compound exocytosis of
platelets corresponds to that one of other secretory cells, most likely
mast cells.19,20 The attribution of regulatory molecules
with distinct functions in exocytosis to certain membranes should
reflect the described secretory pathway in
platelets.
Eberhard Morgenstern
Department of Medical
Biology
Saarland University
Homburg/Saar, Germany
| |
REFERENCES |
1.
Lemons PP,
Chen D,
Bernstein AM,
Bennett MK,
Whiteheart SW:
Regulated secretion in platelets: Identification of elements of the platelet exocytosis machinery.
Blood
90:1490,
1997[Abstract/Free Full Text]
2.
Stenberg PE,
Shuman MA,
Levine SP,
Bainton DF:
Redistribution of alpha-granules and their contents in thrombin-stimulated platelets.
J Cell Biol
98:748,
1984[Abstract/Free Full Text]
3.
Escolar G,
White JG:
The platelet open canicular system: A final common pathway.
Blood Cells
17:467,
1991[Medline]
[Order article via Infotrieve]
4.
White JG:
Platelet ultrastructure
, in Bloom AL,
Forbes CD,
Thomas DP,
Tuddenham EGD
(eds):
Haemostasis and Thrombosis.
Edinburgh, UK, Churchill Livingston
, 1994
, p 49
5.
Hols H,
Sixma JJ,
Leunissen-Bijvelt J,
Verkley A:
Freeze-fracture studies of human blood platelets activated by thrombin using rapid freezing.
Thromb Haemost
54:574,
1985[Medline]
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6.
Morgenstern E,
Neumann K,
Patscheke H:
The exocytosis of human blood platelets. A fast freezing and freeze-substitution analysis.
Eur J Cell Biol
43:273,
1987[Medline]
[Order article via Infotrieve]
7.
Monck JR,
Fernandez JM:
The exocytotic fusion pore.
J Cell Biol
119:1395,
1992[Free Full Text]
8. Morgenstern E, Edelmann L: Analysis of dynamic cell processes by
rapid freezing and freeze substitution, in Plattner H (ed.): Electron
Microscopy of Subcellular Dynamics. Boca Raton, FL, CRC, 1989, p 119
9.
O'Toole E,
Wray G,
Kremer J,
McIntosh JR:
High voltage cryomicroscopy of human blood platelets.
J Struct Biol
110:55,
1993[Medline]
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10.
Morgenstern E,
Bastian D,
Dierichs R:
The formation of compound granules from different types of secretory organelles in human platelets (dense granules and alpha-granules). A cryofixation/-substitution study using serial sections.
Eur J Cell Biol
68:183,
1995[Medline]
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11.
Morgenstern E:
Platelets morphology/ultrastructure
, in Bruchhausen Fv,
Walter U
(eds):
Platelets and their Factors. Handbook of Experimental Pharmacology, vol 126.
Heidelberg, Germany, Springer Verl
, 1997
, p 27
12.
Frojmovic MM,
Milton JG:
Human platelet size, shape and related functions in health and disease.
Physiol Rev
62:185,
1982[Free Full Text]
13.
Frojmovic MM,
Milton JG:
Physical, chemical and functional changes following platelet activation in normal and giant platelets.
Blood Cells
9:359,
1983[Medline]
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14.
Morgenstern E,
Patscheke H,
Mathieu G:
The origin of the membrane convolute in degranulating platelets. A comparative study of normal and "gray" platelets.
Blut
60:15,
1990[Medline]
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15.
White JG:
The secretory pathway in bovine platelets.
Blood
69:878,
1987[Abstract/Free Full Text]
16.
Morimoto T,
Ogihara,
Takisawa H:
Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation.
J Cell Biol
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17.
Fritz M,
Radmacher M,
Gaub HE:
Granula motion and membrane spreading during activation of human platelets imaged by atomic force microscopy.
Biophys J
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18.
Ginsberg MH,
Taylor L,
Painter RG:
The mechanism of thrombin-induced platelet factor 4 secretion.
Blood
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19.
Chandler DE,
Heuser JE:
Arrest of membrane fusion events in mast cells by quick freezing.
J Cell Biol
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20.
Chandler DE:
Exocytosis and endocytosis: Membrane fusion events captured in rapidly frozen cells.
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| |
RESPONSE |
The letter by Dr Morgenstern has highlighted the controversy regarding
the nature of the intracellular events leading to platelet exocytosis.
It has been well established that, subsequent to stimulation, human
platelets undergo a series of morphological rearrangements that include
the formation of pseudopodia and the centralization of secretory
granules. This process culminates in the release of granule contents
into the extra-platelet space, but the route of that delivery is
ambiguous. It is unclear whether granules fuse directly to
invaginations of the plasma membrane called the surface-connected
canalicular system (SCCS)1 or whether, after compound
fusion of the granules, the resulting organelle fuses directly with the
plasma membrane.2 Morphological experiments supporting both
models have been presented, yet neither has been definitively proven.
As stated in a commentary on one of these models,3 until
there is a marker to unequivocally distinguish SCCS from other plasma
membrane domains, it will be difficult to resolve this debate using
morphological techniques.
Our long-term goal is to determine the molecular mechanisms involved in
platelet exocytosis. The SNARE hypothesis4 offers a
framework for this endeavor, and neurotransmission has served as an
excellent paradigm, because many of the proteins involved in that
regulated exocytosis event have been identified and
characterized.5 In Lemons et al,6 we
demonstrated that platelets contain the general and specific elements
of the membrane fusion machinery predicted from the SNARE hypothesis.
This justifies our use of neurotransmission as a paradigm
in the study of platelet exocytosis. Presently, we are continuing the
search for additional proteins that facilitate the various events of
platelet exocytosis (and perhaps endocytosis; Bernstein and Whiteheart,
manuscript submitted). Subsequent experiments will focus
on the specific function(s) and the intracellular localization of these
proteins. It is hoped that these experiments will yield a more detailed
picture of the molecular events that lead to the platelet release
reaction and will ultimately resolve the conflict mentioned
above.
Sidney W. Whiteheart
Department of
Biochemistry
University of Kentucky College of Medicine
Lexington,
KY
| |
REFERENCES |
1.
Escolar G,
White JG:
The platelet open canalicular system: A final common pathway.
Blood Cells
17:467,
1991
2.
Morgenstern E,
Neumann K,
Patscheke H:
The exocytosis of human blood platelets. A fast freeze substitution analysis.
Eur J Cell Biol
43:273,
1987
3.
Wencel-Drake JD:
Platelet secretion and receptor cycling.
Blood Cells
17:486,
1991
4.
Rothman JE:
Mechanisms of intracellular protein transport.
Nature
372:55,
1994[Medline]
[Order article via Infotrieve]
5.
Südhof TC:
The synaptic vesicle cycle: A cascade of protein-protein interactions.
Nature
375:645,
1995[Medline]
[Order article via Infotrieve]
6.
Lemons PP,
Chen D,
Bernstein AM,
Bennett MK,
Whiteheart SW:
Regulated secretion in platelets: Identification of elements of the platelet exocytosis machinery.
Blood
90:1490,
1997
