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RAPID COMMUNICATION
Platelet a-Granule and Plasma Membrane Share Two New Components:
CD9 and PECAM-l
By Elisabeth Martin Cramer, Gaetan Berger, and Michael C. Berndt
CD9(p241 and PECAMI (CD311 antigens are well-defined
components of the plateletplasma membrane. Both are integral glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigatedtheir subcellular localization
using immunoelectron microscopy. The monospecificity of
immuthe two polyclonal antibodiesused was confirmed by
noblotting. On normal resting
platelets, immunolabeling for
CD9 and PECAMl was found lining the plasma membrane
and the luminalface of the open canalicular system. Some
labeling was also consistently found on thea-granule limiting membrane. This was confirmed by double labeling experiments in which fibrinogen and von Willebrand factor
(vWF) were used as a-granule markers. CD9 and PECAM-1
were found lining the
membrane of thesame granules that
contained fibrinogenand vWF in their matrix. CD9 and
PECAM-1 thus appear t o have an intracellular distribution
identical t o GPllb-llla, a major aggregation platelet receptor.
To rule outa cross-reactivity of the two polyclonal antibodies with GPllb/llla, we studied PECAMI and CD9 expression
on the platelets from a patient with type I Glanzmann's
thrombasthenia whose platelets
are devoid ofGPllb/llla. The
same pattern oflabeling was observed for bothantigens as
for normalplatelets. Normal platelets were furtherobserved
after stimulation byagonists that either fail t o induce (ADP)
or induce granule secretion
(thrombin). After treatment with
ADP, platelets changed shape and centralized their granules;
unthe plasma membrane
immunolabeling
remained
changed; and gold particles were stillfound decorating the
peripheryof the centralized a-granules. After thrombin
treatment, a-granules fused
with the platelet
membrane and
secretion occured. A significant increase of labeling was
then
observed on the platelet
surface. From these results
we conclude that thea-granule membrane containstwo additional
receptors in common with the plasma membrane. This suggests that a-granulemembranereceptors
may originate
from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (fora-granule-specificreceptors
such as P-selectin) or byendocytosis from theplasma membrane (for proteins distributed in thetwo compartments).
0 1994 by The American Society of Hematology.
D
report the topographic distribution of two additional major
platelet surface GPs, PECAM-I and CD9.
CD9 is a major surface GP on platelets with a copy number (-40,000 per platelet) almost equivalent to the major
surface GP, GPIIb-IIIa.9 CD9 has an apparent molecular
weight of 24 kD and belongs to a family of membrane proteins termed tetraspanins because they transverse the membrane four times.'o.'' The role of CD9 in platelet function is
presently unclear. However, it is known to associate with
GPIIb-IIIa after platelet activation,'* suggesting it may play
a rolein regulating the platelet aggregation response.""'
PECAM- 1 (plateletlendothelial cell adhesion molecule- 1) or
CD31 is a 130-kD integral membrane GP expressed on the
surface of platelets, endothelial cells, monocytes, and neutrophils." There are approximately 7,000 copies of surfaceaccessible PECAM-1 on platelets." PECAM-I isbelieved
to be important in mediating endothelial cell-endothelial cell
cohesiveness,'"*" but, like CD9, its function on platelets is
unclear." However, the recent findingthat PECAM-l becomes phosphorylated and associates with the platelet cytoskeleton on platelet activation" suggests that PECAM- 1
may be a major mediator of postactivation events on platelets.
In this study, we have found that both CD9 and PECAM1, like GPIIb-IIIa and CD36, are located notonlyonthe
plasma membrane and luminal surface of the OCS, but also
in the a-granule membrane, with the a-granule content for
each being approximately the same (-30% of total). The
data are consistent with the membrane proteins of the cygranule arising in one of the two ways, ie, by targetted
localization to the a-granule membrane, as occurs with Pselectin, or byan endocytotic mechanism that delivers a granule constituents such as fibrinogen and IgG to the granule as well as platelet plasma membrane GPs.
URING HEMOSTASIS, platelet membrane glycoproteins (GPs) allow the platelet to react to and interact
with its environment. Platelet adhesion is mediated by recognition of subendothelial bound von Willebrand factor (vWF)
by a specific platelet receptor, the GPIb-IX complex.' The
GPIb-IX complex is also a critical component in maintaining
platelet shape because it spans the membrane bilayer and is
linked to a network of submenbraneous actin filaments via
its cytoplasmic association with actin-binding protein.' Topographically, the GPIb-IX complex is found on the external
plasma membrane and on the luminal face of the surfaceconnected open canalicular system (OCS).' In contrast,
P-selectin, which after expression on thrombin-activated
platelets mediates their interaction with neutrophils and
monocytes, is specifically targetted to the a-granule membrane in resting platelets?-6 Other major membrane GPs,
such as the platelet aggregation receptor GPIIb-IIIa and
CD36 (GPIV), are present on both the platelet plasma membrane and on the a-granule membrane.',' In this study, we
From the lnserm U.348, Hapita1 Lariboisiire, Paris, France; and
the Vascular Biology Laboratory,Baker Medical Research Institute,
Prahran, Victoria, Australia.
Submitted May 20, 1994: accepted June 11, 1994.
Supported by the Associcitibn pour la Recherche sur le Cancer
(ARC).
Address reprint requests to Elisabeth Martin Cramer, MD, PhD,
INSERM U . 91, Hapita1 Henri Mondor, 51 avenue du Marichal de
Lattre de Tassigny, 94000 Creteil, France.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8406-0043$3.00/0
1722
Blood, Vol 84, No 6 (September 15), 1994: pp 1722-1730
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1723
CD9 AND PECAM-1 IN PLATELETS
MATERIALS AND METHODS
Platelets
Platelet samples were taken from three normal healthy volunteers
and from two patients with Glanzmann's thrombathenia type
Blood samples collected by venipuncture into plastic tubes containing ACD-C buffer (6.8 mmoVL citric acid, 11.2 mmoVL trisodium citrate, 24 mmom glucose, pH 4.2). The platelet-rich plasma
( P m ) was obtained by centrifugation for 10 minutes at 180g and
22°C. Isolated platelets were obtained by centrifugation of PRP for
10 minutes at 1,100g and 22°C and washed three times with Tyrode's
buffer (36 moYL citric acid, 5 mmom KCl, 2 mmom CaC12, 1
mmoVL MgC12, 103 mmoYL NaCl, 5 mmom glucose, pH7.4)
containing 3.5 mg/mL bovine serum albumin (Sigma Chemical CO,
St Louis, MO) and 7 ndmL prostaglandin E, (Sigma). The washed
platelets were resuspended in Tyrode's buffer containing 2.9 mmoV
L CaClz. When indicated, washed platelet samples were exposed to
either 0.05 U/mL human a-thrombin (Sigma) at 22°C for 5 minutes
or 20 pmoVL adenosine diphosphate (ADP; Sigma) at 37°C for 5
minutes, without stimng. The reaction was stopped by fixation with
1% glutaraldehyde (Ladd Research Industries, Burlington, UK) in
Tyrode's buffer.
Purification of CD9 and PECAM-1
For CD9 purification, human platelet membranes were selectively
extracted with isotonic and hypotonic buffers containing 0.1% Triton
X-100, as previously described in detail.= M e r centrifugation of
the hypotonic extract at 100,OOOg for 1 hour at 2"C, the supernatant
was loaded onto a 10 X 1 cm column of FMC 56-Affigel 10 (2 mg
FMC 56/mL of agarose). FMC 56 is a previously described murine
monoclonal antibody directed against CD9." After thorough washing, CD9 was eluted with 0.1 m o m glycine, pH 2.4 (0.1% wthol),
in Triton X-100. To prevent disulfide-dependent dimerization of the
CD9 on neutralization, the CD9-containing fractions were made 1
mmoVL in N-ethylmaleimide and dialyzed initially against 0.01 mol/
L sodium phosphate buffer, pH 6.0 (containing 1 mM N-ethylmaleimide, 0.15 M sodium chloride, 0.1% [wt/vol] Triton X-100, 0.02%
[wt/vol] NaN3) and then against the same buffer, but without Nethylmaleimide. CD9 was then rechromatographed on a second 10
X 1 cm column of M C 56-Affigel 10 and processed in similar
manner to the first FMC 56-affinity column. The purified CD9 gave
a single 24-kD band on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under both nonreducing and reducing
conditions.
For PECAM- 1 purification, the platelet membranes were extracted
and the membrane extract chromatographed on concanavalin ASepharose 4B as previously described? Although the methyl-a-mannopyranoside eluate contained PECAM-1, the majority of the
PECAM- 1 did not bind to theconcanavalin A-Sepharose 4B column.
The flow through of this column was therefore then loaded onto a
10 X 1 cm column of WM 59-Affigel 10 (2 mg WM59/mL of
agarose). WM 59 is a previously described monoclonal antibody
directed against PECA"1.17 After thorough washing, platelet
PECAM-l was eluted with 0.1 m o m glycine, 0.1% (wt/vol) Triton
X-100, pH 2.4. The eluted fractions were immediately neutralized
by the addition of one-fifth volume of 1 m o m Tris, pH 8.0, and
then dialyzed against 0.01 m o m Tris, 0.15 moVL sodium chloride,
1 mom calcium chloride, 0.1% (wt/vol) Triton X-100, 0.02%
NaN3, pH 7.4, and stored at -70°C. The purified protein on SDSPAGE consisted of a protein band corresponding to PECA"1 and a
proteolytic degradation product of -20 k D lower molecular weight.
Antibodies
Affinity-purified rabbit anti-CD9 and anti-PECAM antibodies
were raised in New Zealand white rabbits and affinity-purifiedon the
respective antigen coupled to Affigel 10/15, as has been previously
described in detail for other platelet membrane GPS?.~.~
Antifibrinogen and anti-VW antibodies were purchased from Cappel Laboratories (Downington, PA) and Dakopatts (Copenhagen, Denmark),
respectively, and used at a U100 dilution.
For immunoelectron microscopy, a U125 and 1/50 dilution of the
affinity-purified polyclonal antibodies directed against PECAM-l
and CD9 were used, respectively. Goat antirabbit antibody (IgG)
coupled to 10-nm colloidal gold particles (GAR-G10) were purchased from Amersham (Les Ulys, France) and used at a 1/50 dilution. Protein-A coupled to 10- and 20-nm gold particles was kindly
provided by H. Heijnen (Cell Biology Department, University Hospital, Utrecht, The Netherlands).
Electron Microscopy
Cells were prepared for immunoelectron microscopy as follows.
They were fixed in 1% glutaraldehyde in 0.1 m o m phosphate buffer,
pH 7.2, for 2 hours at 22°C; washed three times in the same buffer;
and embedded in glycol methacrylate (GMA) as described prev i ~ u s l y .The
~ ~ immunochemical reactions were then performed on
thin sections by the method of Bendayan.26Briefly, the sections were
labeled by a first incubation with the polyclonal rabbit antibodies
for 2 hours at 22°C in a humidified atmosphere. The incubation with
GAR-G or protein A-G was performed for l hour at room temperature and the sections were counterstained with uranyl acetate and
lead citrate. Samples were observed on a Philips EM 300 or 450
CM 10 electron microscope.
To identify the labeled granules, a double-labeling was performed
using the soluble proteins, vW,and fibrinogen as a-granule markers. For the double-immunolabeling experiments, frozen sections
were used as previously described.' Bound anti-CD9 or antiPECA"1 were detected with protein A coupled to 5-nm gold particles. Anti-vWF or antifibrinogen were detected with protein A coupled to 15-nm gold particles.
Quantitative Study
Quantitative data were obtained by counting the gold beads associated with the various structures on an average of 15 platelet equatorial sections in each experimental condition.
Analytical Methods
Western Blot analysis of SDS-PAGE-separated platelet membrane GPs was performed as previously described.'
RESULTS
Specifcity of the Polyclonal Antibodies
Figure l establishes the monospecificity of the affinitypurified polyclonal antibodies to human CD9 and PECAM1 as evaluated by Western Blot analysis. Both antibodies
reacted with asingle major band,consistent with the reported
molecular weight characteristics of CD9 (lane 3)" and
PECA"1 (lane 2)''
CD9
Resting platelets. Immunogold staining for CD9 gave
the following labeling pattern. Consistent labeling of the
plasma membrane and of the luminal face of the O C S was
observed as well as a definiteimmunostaining of the periphery of the a-granules (Fig 2a and b).a-Granules were identified due to their high number,large size, and dark nucleoids.
Either because the reaction was close to the threshold of
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CRAMER,BERGER,
j
-.
ANDBERNDT
-!
B
A
Fig 1. lmmunoblotting of the
polyclonal anti-PECAM-l (A)
and anti-CD9 (B) on a platelet lysate after electrophoresis shows
a single bandfor PECAM-1 at
-130 kD (lane 21 and a single
band forCD9 at -24 kD (lane 3).
Lane 1shows the immunoblot of
anti-PECAM-l against purified
PECAM-1 and its proteolytic
degradation products. Samples
in lanes 1 and 2 were run under
reducing conditions and in lane
3 under nonreducing conditions.
sensitivity of this immunocytochemistry or because a heterogeneous population of a-granules exists as far as CD9content is concerned, only 77% of the granules contained one
or more gold particles (versus 19% of the mitochondria,
which contained rarely more than 1 gold bead, representing
the background staining as control structures). However,
overall, -28% of the total gold particles were found associated with a-granules.
Thrombasthenic platelets. The same qualitative and
quantitative labeling pattern as for normal platelets was
found in thrombasthenic platelets (Fig 2c). This result excludes a cross-reaction between the polyclonal anti-CD9 antibody and GPIIb-IIIa and confirms the monospecificity of
the antibody as determined by the Western blot analysis. In
addition, the data indicate that there is no difference in the
subcellular localization of CD9 in Glanzmann’s thrombasthenia.
ADP-stimulated platelets. After treatment with ADP,
which induced platelet shape change without secretion, immunolabeling for CD9 was found on the same structures as
in resting platelets, eg, plasma membrane (including pseudopods), OCS, and the centralized a-granule limiting membrane (Fig 2d).
Thrombin-stimulated platelets. An increase in plasma
membrane CD9-associated immunolabeling was observed
after thrombin stimulation, coinciding with the fusion of the
a-granules with the cell surface of the activated platelets.
Indeed, the labeling associated to the surface increased of
40% compared with the initial level. No other cell organelle
displayed any significant number of gold particles.
PECAM I
Resting platelets. Normal platelets were examined after
immunogold labeling of PECAM-l.The immunolabeling
was overall weaker than for CD9. Gold particles could be
observed outlining the outer face of the plasma membrane
and the luminal side of the O C S . Moreover, labeling of the
limiting membrane of the a-granules was consistently found
(Fig 3a and b). Like CD9, an average of 27% of the total
number of particles were found labeling the granules,
whereas the remaining particles were associated with the
plasma and OCS membranes. Forty-three percent of the agranules were labeled with one or more gold particles against
14% of the mitochondria taken as control structures. Smaller
granules of other types and the cytoplasmic matrix were not
labeled.
Thrombasthenic platelets. No differences were detected
in PECAM-l immunolabeling between platelets from the
>
Fig 2. lmmunogold localization ofCD9 in human platelets. (a) In resting platelets, gold labeling forCD9 is present on theplasma membrane
(arrowheads), the OCS, and on the a-granule membrane (A and inset). Mitochondria(m) as control structures are devoid of any labeling.
(Original magnification x 55,600.) (b) At a higher magnification, CD9 is detected on theexternal face of the plasma membrane (arrowheads)
and the luminal face of the OCS as well as along the internal face of the a-granule membrane (A). (Original magnification x 53,600.) (cl In
platelets from a patient with Glanzmann’s thrombasthenia, the same labeling pattern isfound. (Original magnification x 57,500.) (dl In ADPtreated platelets that have undergone reversible activation without secretion, the distribution of CD9 remains unchanged. Gold labeling is
found on the
plasma membrane now displaying pseudopodia (arrowheads), as well as on thecentralized a-granules (Al. (Original magnification
x 42,300.) (el After treatmentwith 0.1 UlmL ofa-thrombin, most of the a-granules
have fused andsecreted into theOCS. CD9 immunolabeling
is found along the fewremnant, swollen intracytoplasmic a-granules (A) in the OCS and on the plasma membrane (arrowheads). (Original
magnification x 32,500.)
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CD9 AND PECAM-1 IN PLATELETS
.~
1725
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1726
CRAMER,BERGER,
normal subjects and from the patients with type I thrombasthenia (Fig 3c).
ADP-stimulated platelets. With ADP, platelets undergo
shape change with centralization of a-granules. The peripheral actin-rich zone is deprived of gold labeling, which is
concentrated in the central zone where the a-granules are
located. Both the OCS and plasma membrane pseudopodia
remain labeled (Fig 3d).
Thrombin-stimulated platelets. After thrombin stimulation, the a-granule membranes aggregate and fuse with the
OCS membrane, where they discharge their content, creating
secretion areas. On these platelets, immunolabeling for
PECAM-l was found on the plasma membrane, particularly
on pseudopods; on the OCS membrane; and also in secretion
areas membrane. On thrombin-stimulated platelets, which
had undergone a virtually full degranulation, an average increase of 44% of the initial labeling was observed on the
plasma membrane (Fig 3e).
Control
The primary antibody (anti-PECAM1 or anti-CD9) was
replaced byan irrelevant polyclonal antibody, antihuman
myeloperoxidase, and this led to a complete negativity of
the staining. A similar negative result was obtained when
the primary antibodies were omitted from the reaction.
Double Immunolabeling
To further confirm that both CD9 and PECA“1 were
associated with the a-granules, double-immunolabeling experiments were performed using antibodies against the
known a-granule markers, vWF and fibrinogen. When platelets were double-immunolabeled for the a-granule protein,
vWF, together with CD9, vWF was found eccentrically located in the a-granules, as previously described,*’ and the
same granules displayed labeling for CD9 on their periphery
(Fig 4a). A similar result was obtained with the doubleimmunolabeling for fibrinogen and CD9, withfibrinogen
more randomly distributed on the a-granule matrix than
vWF (Fig 4b). The mitochondria were negative for the three
markers. In a similar approach, labeling for PECA”1 was
present at the internal face of the same granules in which
vWF and fibrinogen were detected, allowing for the identification of these structures as a-granules (Fig 5).
DISCUSSION
The a-granuleof the platelet packages and stores a variety
of proteins that are released on activation. Many of these
AND BERNDT
proteins are endogenously synthesized and packaged within
the megakaryocyte, eg, thrombospondin, P-thromboglobulin, and vWF.” For these proteins, the corresponding
mRNAs are present in megakaryocytes and the proteins can
be immunodetected in transit during traffic in the Golgi sacc ~ l e s . Other
~~~~
proteins
’
in the a-granule, such as fibrinogen,
albumin, and IgG, are stored via a different mechanism involving endocytosis from the surrounding extracellular med i ~ m . ~ ’We
.~~
have previously demonstrated that GPIIb-IIIa
is not only found on the external face of the plasma membrane and the luminal face of the OCS, but also on the inner
face of the a-granule membrane,7and it is probable that the
incorporation of fibrinogen into the a-granule is a receptormediated process dependent on fibrinogenbinding to GPIIbIIIa. Fibrinogen is present in platelet a-granules at higher
concentration than other plasma-derived proteins such as
albumin and IgG,34 andits presence in the granule is dependent on functional GPIIb-IIIa. Thus, fibrinogen is absent
or markedly reduced in a-granule content in patients with
Glanzmann’s thrombasthenia whose platelets lack or have
dysfunctional GPIIb-IIIa.’5 Alternatively, blockage of the capacity of GPIIb-IIIa to bind fibrinogen in vivo inhibits uptake
of fibrinogen into the a - g r a n ~ l eMoreover,
.~~
barbourin, a
specific antagonist of GPIIb-IIIa, also inhibits the endocytic
uptake of fibrinogen into the a - g r a n ~ l e s . ~ ~
The GPIIb-IIIa-mediated endocytosis of fibrinogen into
the a-granule appears to predominate in plasma over other
proteins such as vWF that can also bind to GPIIb-IIIa
through an RGD-dependent mechanism. The endocytosis of
vWF has been ruled out. In an animal model in which a
normal pig was engrafted with the marrow of a pig with
severe von Willebrand’s disease, resulting in vWF-deficient
platelets circulating in vWF-rich plasma, no a-granule vWF
was detected.” In this regard, the mechanism for a-granule
packaging of megakaryocytic-derived proteins appears to be
distinct to that for endocytically derived proteins. Thus, in
the a-storage disease, Gray platelet syndrome, proteins such
as vWF and P-thromboglobulin are essentially absent from
the platelet, whereas the levels of albumin, IgG, and fibrinogen are only moderately reduced.35Furthermore, GPIIb-IIIa
can still be localized to the P-selectin-containing immature
granules in Gray platelet^,^ consistent with a relatively normal endocytic mechanism for plasma protein incorporation
into the a-granule.
Recently, we reported that, in addition to GPIIb-IIIa, another major platelet surface GP, CD36 (GPIV), was also
located in significant quantity on the inner face of the a -
*
Fig 3. lmmunogold localization of PECAM-1 in platelets.(a) In resting platelets,the PECAM-1 a n t i r n is detected onthe plasma membrane
and OCS, although the extant of labeling is leas than for COS. Gold partides are also eruoclated with a-granulea (A and inset). lOriglnal
magnification x 48O
, OO). (b) A better appreciation of PECAM-1 localization can be seen at high magnification with gold partides docorating
the external face of the plaama membrane (arrowheads) andthe internal side ofthe limiting membrane of the a-granules. a4ranuIes [A) can
be identified by their specific morphology,as d i s c u d in the text. Mitochondria (m) are not labeled. (Original magnification x 109,000.) (cl
Platelm from a patient with type I Olanzmann’s thrombasthania expreas PECAM-1 on the plasma membrane (arrowheads) and a-granule
membrane (A) as for normal platelets. (Original magnification x 65,SOO.l (d) When platelets have been activated with ADP, PECAM-l is still
detected along the plasma membrane (arrowheads) and on the gathered m-granules (A). The intermediary actin-rich zone is devoid of gold
labeling. (Original magnification x 48.000.) (e) Thrombin-treated platalets contain large secretion areas(S) whose limiting membrane, made
by fusion of the a-granules and OCS, is labeled. PECAM-1 expression is increased on the plasma membrane and associated pseudopodia
(arrowheads). (Original magnification x 40,OOO.)
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CD9 AND PECAM-1 IN PLATELETS
1727
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BERGER,
1728
1
l,
CRAMER,
AND BERNDT
m
' . ,'!
,
,
I
' '-*A
' ,
* .
,
. ,,,,
,
Fig 4. Normal platelets doubleimmunolabeled
for CD9 (5 nm gold) and either vWF or fibrinogen
as a-granule markers (15 nm gold). (a) a-Granules
contains vWF distributed along one of their poles.
CD9 is detectedalong the periphery of the same
granules(arrowheads) that display a largenucleoid
(n), as well as on the plasmamembrane (pm). (Original magnification x 101,000.) (b) a-Granulesdisplay
labeling for both CD9 lining their membrane (arrowheads)and fibrinogen in their matrix. CD9 isalso
found along the OCS. Mitochondria (m) as control
structures are negative for both antigens. (Original
magnification x 190.000.)
granule membrane.' In the present study, we have extended
this observation to two additional major surface GPs,
PECAM-I and CD9. This finding for PECAM-l, although
novel, is nevertheless consistent with the data of Metzelaar
et al." who showed an increased expression of PECAM-I
on platelets after thrombin activation. The investigators suggest the existence of an intracellular pool of PECAM-I,
without identifying its a-granular origin. Although there are
technical limitations to quantitation in immunogold localization studies and potential differences in antibody accessability with different antigens, the percentage of a-granule localization relative to total distribution is nevertheless in the
range of 20% to 35% for all four surface GPs, GPIIb-IIIa,
CD36, PECAM-I, and CD9 (this study, Berger et a1* and
calculated from the data of Cramer et a]'). Targeting of the agranule-specific GP, P-selectin, to the a-granule membrane
requires a specific signal localized within the cytoplasmic
domain.39" P-selectin is only expressed on the plasma membrane of quiescent cells by default if the cell lacks storage
organelles. In contrast, GPIIb-IIIa appears to be incorporated
into the a-granule membrane as a consequence of the endocytosis of vesicles containing plasmatic fibrinogen (see
above). We would suggest that the presence of three additional surface GPs in a-granules in similar relative amounts
to GPIIb-IIIa argues that, when endocytosis occurs simultaneously to internalization of plasma proteins, plasma membrane may be internalized and many of its components become common to the storage granule membrane. This
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CD9 AND PECAM-1 IN PLATELETS
1729
Fig 5. Normal platelet double immunolabeled for
PECAM-1 (5 nm gold) and either vWF or fibrinogen
as a-granule markers (15 nm gold). (a) The antifibrinogen labels the matrix of the a-granules except
for the dark nucleoid. Anti-PECAM-l is present
along the a-granule membrane (arrowheads). (Original magnification x 138,600.) (bl vWF is located in
the electron-lucent zone of the a-granules next to
the dark nucleoid (nl, whereas PECAM-l is detected
at the inner face of the membrane of the same granules (arrowheads). Mitochondria (m) are not labeled.
(Original magnification x 109,000.)
conclusion is supported by the finding that another major
platelet membrane GP, GPIb-IX, has not been detected in
a - g r a n ~ l e s Regions
.~
of the plasma membrane containing
GPIb-IX would be expected to be structurally ordered due
to the association of GPIb-IX with the membrane network
of actin-filaments associated with the maintenance of platelet
shape’ and therefore less likely to undergo a process of pinocytosis. However, further evidence is clearly required to fully
explain the presence and potential biologic relevance of surface membrane GP distribution between the plasma and agranule membranes.
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From www.bloodjournal.org by guest on August 9, 2017. For personal use only.
1994 84: 1722-1730
Platelet alpha-granule and plasma membrane share two new
components: CD9 and PECAM-1
EM Cramer, G Berger and MC Berndt
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