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Platelet Dense Granule Membranes Contain Both Granulophysin and P-Selectin
(GM P 140)
-
ByS.J. Israels, J.M. Gerrard, Y.V. Jacques, A. McNicol, B. Cham, M. Nishibori, and D.F. Bainton
We recently reported the characterizationof a platelet granule membrane protein of molecular weight (mol wt) 40.000
called granulophysin (Gerrard et al: Blood 77:101, 1991).
identified by a monoclonal antibody (MoAb D545) raised to
purified dense granule membranes. Using immunoelectronmicroscopic techniques on frozen thin sections, this protein
was localizedin restingand thrombin-stimulatedplatelets. In
resting platelets, labeled with antigranulophysin antibodies
and immunogold probes, label was localized to the membranes of one or two clear granules per platelet thin section.
D545 also labeled dense granules in permeabilized whole
platelets and isolated dense granule preparations examined
by whole-mount techniques. Expression of granulophysin on
the platelet surface paralleled dense granule secretion as
measured by ‘%-serotonin release under conditions in which
lysosomal granule release, as measured by B-glucuronidase
secretion, was less than 5%. After thrombin stimulation,
both the surface-connected canalicular system and the plasma
membrane were labeled, demonstrating redistribution of
granulophysin associated with degranulation. Double labeling experiments with D545 and antibodies to the *granule
membrane protein, P-selectin, demonstrated labelingof both
P-selectin and granulophysin on dense granule membranes.
Distribution of both proteins on the plasma membrane after
platelet stimulation was similar. The results demonstrate
that granulophysin is localized to the dense granules of
platelets and is redistributed to the plasma membrane after
platelet activation.
8 1992 by The American Society of Hematology.
P
granulophysin in resting and thrombin-activated human
platelets. We also compared the distribution of granulophysin with that of P-selectin (previously known as GMP140 or PADGEM), a well-characterized a-granule membrane protein10-12that mediates adhesion of activated
platelets with n e ~ t r o p h i l s . ~ ~ J ~
LATELETS CONTAIN a varied assortment of biologically active molecules stored in several types of
granules. Release of these molecules after platelet stimulation results in their interaction with other platelets, blood
cells, and the vessel wall. These secreted components
contribute to promotion of hemostasis, wound healing, and
formation of atherosclerotic plaques. In particular, secretion of dense granule contents plays a pivotal role in
formation of platelet thrombi. These storage organelles
(also called dense bodies or &granules) contain serotonin,
adenine and guanine nucleotides, calcium, magnesium, and
inorganic phosphate.’ The inherent density of the core in
these granules in human platelets when viewed in thin
section or by whole-mount technique results from their
calcium content?
A decrease in the number of dense granules or their
contents has clinical implications because many patients
with this condition have a bleeding diathe~is.3,~
This condition, called storage pool deficiency (SPD), may be acquired
or congenital, and several different congenital types have
been described, including those associated with albinism,
such as the Hermansky-Pudlak
those associated with a-granule deficiency: and those that are associated with an autosomal dominant inheritance pattern but
have no associated abnormalities.*We recently described a
platelet dense granule membrane protein, granulophysin,
identified using monoclonal antibodies (MoAbs) D545 and
D503, raised against purified human platelet dense granule
membranes? As analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the protein has a molecular weight (mol wt) of 40 Kd and was
shown to be deficient in a patient with the HermanslyPudlak syndrome.
Our earlier studies using immunofluorescent techniques
demonstrated that the antigranulophysin antibodies reacted minimally with unstimulated human platelets unless
the platelets were made permeable with saponin. After
thrombin activation, however, nonpermeable platelets
showed staining with the fluorescent-tagged MoAb. In the
present study, we used immunoelectron-microscopytechniques to investigate the ultrastructural localization of
Blood, Vol80, No 1 (July I), 1992:pp 143-152
MATERIALS AND METHODS
Antibodies. MoAbs D545 and D503, both directed against
granulophysin,were prepared after immunization of BAL.B/c mice
with platelet dense granule protein as described in detail previously? D541, an MoAb produced using the same dense granule
protein preparation, was shown to be directed against P-selectin as
analyzed by Western blot using purified P-selectin provided by Dr
R. P. McEver (Oklahoma City, OK), who also provided the
polyclonal rabbit antiserum to P-seIectin.lo Colloidal gold conjugates were obtained from Janssen Pharmaceutica (Beerse, Belgium).
Preparation of platelets. Blood was obtained from aspirin-free
normal adult volunteers by venipuncture and fixed immediately15
or drawn into acid-citrate-dextrose(ACD) anticoagulant. Plateletrich plasma (PRP) was obtained by centrifugation of blood at 8OOg
for 5 minutes. Platelets were washed and resuspended in 0.1 mol/L
phosphate buffer, pH 7.4. Resting platelets were allowed to
incubate at 37°C for 1 hour before fixation. Some platelet samples
were stimulated with 1 U/mL bovine thrombin (Sigma, St Louis,
MO) for 1 or 5 minutes before fixation. Platelets used for
From the Department of Paediatrics and the Manitoba Institute of
Cell Bwlogy, University of Manitoba, Winnipeg, Manitoba, Canada;
and the Department of Pathology, University of Califontia School of
Medicine, San Francisco, CA.
Submitted June 24,1991; accepted March 11, 1992.
Suppored by Grants No. MA7396 from the Medical Research
Council of Canada and No. HLB316lOfrom the National Institutes of
Health.
Address reprint requests to Sara J. Israels MD, Manitoba Institute of
Cell Biology, 100 Olivia St, Winnipeg, Manitoba, Canada, R3E OW.
The publication costs of this article were &frayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1992 by The American Sociey of Hematology.
0006-4971/92/8001-0025$3.00/0
143
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144
ISRAELS ET AL
Fig 1. (a) Tnwmiuion electron micrograph (TEM) of a human p1.t.M to lllu"ta the morphology of typkl organelleawhen emboddod In
Epon (drrctibed Inthe Materialsand Methodsd o n ) . Thh cell contains many trgnnuies (a),
as well aa one seratonln-containing d e w gnnuk
(arrow) and the SCCS. (Originalmagnification x40,OOO.) (b) TEM showing localizationof MoAb 0545 to Mw(y identified dense granule membrane
protein of 40 Kd. Platelets were dripped into 8% paraformaldehyde and prepared for frozen thin-section lmmunocytochemimy. The gold label
(GAR-10) is contained in the dense granule (arrow) and is not evident in the ogranulea (a), SCCS, or on the plasma membrane (pm). The MoAb
was used at a concentretion of 10 FglmL, and a bridge antibody rabbit-anti mouse IgG was used at a dilution of 1:lOO; GAR-10 was then applied.
(Original magnification x50,OOO.)
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PLATELET DENSE GRANULE MEMBRANE PROTEINS
immunocytochemistry were fixed in 4% or 8% paraformaldehyde
in phosphate buffer, infiltrated with 2.1 mollL sucrose, and
embedded in sucrose solution before freezing and storage in liquid
NZ.'~
Platelets prepared for morphology alone were fixed in 1.4%
glutaraldehyde and 1.9% paraformaldehyde in 0.2 mollL sodiumcacodylate buffer with 0.025% CaC12, post fixed in OSOSstained
with uranyl acetate, and embedded in Epon.I6
Immunocytochemical techniques on f i z e n thin sections. Frozen
thin sectionswere cut as previously described.I5Immunocytochemical procedures were performed on frozen thin sections" with the
following antibodies. The primary antibody, D545, was used in a
concentration of 10 &mL. lmmunogold probes, goat anti-mouse
IgG-gold (10 nm, GAMIO) or protein A conjugated to 10 nm gold
(Janssen Pharmaceutica) were used at a dilution of 1:50. For some
experiments, a bridging second antibody, rabbit antimouse, was
used, followed by gold conjugated to goat antirabbit IgG (10 nm,
GARIO). For double-label experiments, D545 was used as above,
with polyclonal antiserum to P-selectin. After washing, the cells
were labeled with goat antirabbit IgG-gold (5 nm, GARS) and goat
anti-mouse IgG-gold (10 mm, GAMIO). Controls for each experiment included substitution of buffer or nonimmune serum for the
specific primary antibody.
145
Reparation of densegmnules. Dense granules were prepared as
described previouslypusing a modification of the method of Rendu
et allRby centrifugation of a whole platelet homogenate through
40% metrizamide at 1lO.ooqS for 30 minutes. The sedimented
pellet was markedly enriched in dense granules. The material
above the metrizamide layer contained the remaining platelet
membranes and was enriched in a-granules. It was used as a dense
granule-depleted membrane fraction for some studies.
Western blors. Westem blots were performed as previously
described! Electrophoresis was performed on a 7.5% polyacrylamide gel with a 4% stacking gel according to the method of
Laemmli,I9 after initial incubation of protein for 30 minutes in a
Weber-Osbome bufferm (three parts sample to one part 6% SDS.
40 mmollL NaP04, pH 7.0,20% glycerol, and 0.01% bromophenol
blue). Proteins were transferred to nitrocellulose at 100 V for 1
hour at 15°C. The nitrocellulose was blocked for 1 hour using 10%
nonfat powdered milk, washed with tris-buffered saline (TBS),and
incubated with monoclonal anti-P-selectin (D541). After washing
with TBS, the nitrocellulose was incubated with biotinylated goat
antimouse IgG from Vectastain ABC Kit (Dimension Laboratories, Mississauga. Ontario, Canada) and then with Vector ABC
peroxidase-conjugate. Blotting was completed by incubating with 6
Flg 2. TEM of thrombin-rtimulated(1 mi-)
platelets 1llUrh.h.mdhMbuHon of gmnuloph@n. Aftof the frozenthin Maion waa Incubated
wkh MoAb D545, GAM-10 gold was added. Label is evident along the plasma membranes (pm) of activated platelets 88 well 88 in intracellular
vacuoles, preaunublyportions of the SCCS. (Original magnification x60,OOO.)
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ISRAELS ET AL
146
mg 4-chloro-I-napthol in 2 mL methanol and 50 FL 30% H202 in
10 mL TBS until color development was achieved.
hpamrion of whole mounts for elecmn micmcopy. PRP or
isolated dense granules were fixed in I% paraformaldehydelO.l%
glutaraldehyde. Platelets or isolated dense granules were made
permeable by incubation in Triton X-100 (Mallinkrodt, Mississauga, Ontario, Canada) (0.1%) for 3 minutes before beingwashed
with TBS. Platelets or dense granules were then allowed to settle
on formvar-coated (J.B. EM Services Inc, St Laurent, Quebec,
Canada) grids before incubation with D545 (or nonimmune ascites
control) and GAMIO.
Differential secretion of semronin and pglucumnidase. PRP was
aliquoted for parallel analysis of serotonin (dense granule) release,
B-glucuronidase (lysosomal granule) release, and flow cytometric
analysis of granulophysin expression on the platelet surface. Each
of these samples was washed and resuspended in a HEPESTyrode’s buffer?’ and aliquots from each were then treated as
follows: ( I ) 0.5 mmollL colchicine for 45 minutes, then 5 U/mL
thrombin for 5 seconds; (2) 0.5 mmollL colchicine for 45 minutes,
then 5 UlmL thrombin for 30 seconds; (3) 0.4 mmol/L RGDSP
peptide and 1 mmol/L CaClz for 1 minute, then 5 U/mL thrombin
for 5 minutes; or (4) no treatment. Colchicine was used to inhibit
lysosomal granule secretion,u and the RGDSP peptide was used to
minimize aggregation under conditions in which secretion was
maximum.l’ Serotonin release was measured from platelets prelabeled with 14C-serotoninas previously
and B-glucuronidase secretion was measured by the method of Hoehn and
Kanfer.2s In each case, values were expressed as the amount
released into the supernatant as a percentage of the total platelet
content.
Flow cyromerry analysis of gmnulophysin dbtriburion. Samples
for analysis were treated as described above, and activation was
terminated by addition of an equal volume of cold ACD. The
platelets were pelleted, resuspended in HEPES-Tyrodes buffer
containing 10% ACD, and incubated for 1 hour on ice in the dark
with D545 (80 pglmL) conjugated directly to FlTC or indirectly
labeled with D545, biotinylated goat anti-mouse IgG (1:IW). and
FITC-avidin (1:3.000). To some samples, unlabeled D545 was
added before the fluorescent labeling was performed. Samples
were fixed with 1% paraformaldehyde and analyzed using an
EPICS model 753 flow cytometer (Coulter Electronics, Hialeah,
FL) equipped with an argon ion laser (500 mW, 488 nm).
Fluorescencewas detected at 525 nm. Forward and 90” light scatter
measurements were used to establish gates for intact viable
platelets. Single-parameter,255channel. log-integralgreen fluorescent histogramswere obtained, each based on 1 x I@ gated events.
RESULTS
Distribution of P-selectin on unstimulated platelets was
previously studied by frozen thin section and shown to be
on the membranes of a-granules.10Although in previous
studies the dense granule population could not be identified because the granules do not retain their dense core
during this type of tissue preparation, some unidentified
clear vesicles were labeled and the possibility that they were
dense granules was considered.In These “dense” granules
are easily identified in platelets prepared by conventional
fine-structural techniques (Fig la). In this study, we used
Fig 3. Preparationsimilar to that d d b o d In legend to Fig 2 but fixed 5 minutus after exporun to thrombin. Again the label h on the plasma
membraneand in SCCS. Bridges (arrow)betweencoils do not contain the label. (Originalmagni&.tion x50,OOO.)
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PLATELET DENSE GRANULE MEMBRANE PROTEINS
the recently developed MoAb D545, raised to purified
dense (serotonincontaining) granule membranes which
identifies a 40-Kd protein, granulophysin, to determine the
ultrastructural localization of granulophysin. To accomplish this, we incubated frozen thin sections of platelets
with D545 followed by a second bridging antibody and a
colloidal goldconjugated third antibody.
Resting platelets examined by electron microscopy showed
that immunoreactive granulophysin was localized in one or
two granules per platelet (Fig lb). Other identifiable
granules, the surfaceconnected canalicular system (SCCS)
and plasma membrane were not labeled. When platelets
were stimulated with thrombin, the immunogold label was
redistributed to the SCCS and plasma membrane (Figs 2
and 3). After 1-minutestimulation, some label could still be
observed in granules, although it had also appeared in the
SCCS and on the plasma membranes. Five minutes after
exposure to thrombin, labeling of granules was rare. Distribution along the membrancs of the SCCS and plasma
membrane appeared to be homogeneous when evaluated
with protein A-gold as the immunoprobe for the primary
147
antibody because protein A-gold does not clump as IgGgold probes may.%There was no apparent concentration of
label on pseudopods. At some sites of cellcell contact no
label was observed (Fig 3).
Double labeling of resting platelets with D545 and
polyclonal antiserum to P-selectin, a protein previously
identified in the membrane of a-granules, showed that
P-selectin was present not only along the a-granule membranes but also along the membranes of granules labeled by
D545 (Fig 4). However, D545 did not label a-granules. The
double-labeled granules often appeared as empty vesicles
on the frozen scctions, with no evidence of a dense core.
Occasionally,small vesicularstructureswere observed within
the granules (Fig 4). These structures have been observed
previously in densecore chromaffin granules, particularly
after freezing.27Five minutes after thrombin stimulation,
double labeling with the two antibodies demonstrated
distribution of both granulophysin and P-selectin to the
SCCS and plasma membrane (Fig 5). Labeling of P-selectin
was more abundant than that of granulophysin, but it is not
possible to draw conclusions from this because it may
Flg4. Frozenthin d o n of a restingplatelet incubatedwith two antibodies: a polydonai antibody against P - w M n iabded wwh OAR4 and
MoAb OS45 against granulophysin labeled with GAM-10. The 5-nm gold (P-seldn) is evident in a-granules and the dense granule, whereas the
10-nm gold (arrows), denoting the presence of granulophysin, is evident In dense granules only. (Original magnification x80,OOO; inset x77,OOO.)
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Fig 5. Double Iabellng of thrombln-stimulatd (5 minute) platelets &om dhhlbution of both anti-P-wldn (5 nm gold) and a d granulophyain(10 nm gold) adbodies tothe plasma membrane (pm), aa wail as in some SCCS. (Odginalmagnf&.tion x65,OOO.)
represent only the difference in binding between polyclonal
antibodies and MoAbs. In addition, P-selectin is distributed
all over the plasma membrane, even at cellcell contact
points.
Western blot analysis showed that P-selectin was present
in the whole platelet homogenate, the dense granuledepleted fraction and the dense granule-enriched preparation (Fig 6), in contrast to granulophysin which has previously been shown to be specific to the dense granuleenriched fraction, with very low amounts in the dense
granule-depleted fractions?
Because the serotonincontaining granules do not retain
their electron-dense core after preparation for cryosectioning, we also examined, by electron microscopy, whole
mounts of permeabilized platelets and isolated dense granules labeled with D545 and colloidal goldconjugated second antibody (Fig 7). Labeling with D545 of identifiable
electron-dense granules could be observed in quiescent
platelets permeabilized with Triton X-100 for 3 minutes
before incubation with the antibody (Fig 7a). Isolated
dense granules prepared from dense granule-enriched
fractions also were labeled with D545 when granules were
made permeable with Triton X-100before incubation with
D545 (Fig 7b-e).
To investigate the possibility that some of the clear
vesicles labeled on cryosections might represent lysosomal
membranes, we evaluated redistribution of granulophysin
to the platelet plasma membrane after stimuli that produced either dense granule secretion only or dense granule
plus lysosomal secretion. Differential secretion was evaluated by quantifying I4C-serotoninrelease from dense granules and p-glucuronidase release from lysosomes. Redistribution of granulophysin to surface membranes of platelets
was evaluated by flow cytometry using FITC-tagged D545
(Fig 8,Table 1): 5.5% to 12.5% of quiescent platelets were
positive after labeling with D545-FITC depending on the
labeling method. After stimulation with thrombin for 5 and
30 seconds (with colchicine and without external calcium),
conditions that produced significant dense granule secretion (71%, 80%) but minimal lysosomal granule secretion
(2.5%. 4.4%), the percentage of fluorescent-positivecells
increased to 66% and 69%. Maximal dense granule secretion (91.1%) as well as release of 31.8% of p-glucuronidase
from lysosomes caused an additional 7.8% of platelets to
express granulophysin on their surface (Fig 8, C through E).
Experiments using indirect labeling of D545 with FlTC
yielded substantial expression of D545 expression under
conditions of selectivc serotonin granule secretion, but a
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PLATELET DENSE GRANULE MEMBRANE PROTEINS
kDa
-205
-116
-77
-46
A B C
Fig 6. Western blot with antCP-..l.cWn MoAb 0541. I
.
"A
contrlns a whole platelot homogenate, lam B contains a dense
granuledepleted fraction (rich in egranu1.r). and lane C contains the
dense gmnule-enrichedfraction. Equivalentamounts of protein (10 pg
per lane) were applied to each lane. Samples were solubilized in
Weber-hbome buffer and separated, nonredud, on a 7.5% hemmli gel as described in the Materials and Methods section.
relatively larger increment in association with lysosomal
granule secretion.
DISCUSSION
Identification of granulophysin by MoAbs as a protein
specific to dense granules in platelets has provided a tool to
further evaluate dense granules at an ultrastructural level.
Although frozen thin sections have been very useful in
examining the a-granule and its fusion with the SCCS and
149
plasma membrane through observation of P-selectin, an
integral granule membrane protein, identifying dense granules has not been possible because they lose their distinctive dense core during the preparati~n'~,''and cannot be
distinguished from other vesicles. Using D545 and immunoelectron microscopy techniques, we were able to identify
granulophysin-containing membrane vesicles specifically.
These vesicles did not contain electron-dense material
(indeed, they usually appeared less electron dense than
a-granules) and lacked other morphologically distinguishing features. Usually one or two were evident per platelet,
which coincides with the mean dense granule count in thin
sections of resting platelets.zB There was no evidence of
granulophysin present in a-granules, SCCS or on the
plasma membrane of resting platelets.
Using whole mount techniques that allow dense granules
to be visualized on transmission electron microscopy by
their inherent electron density, we were able to demonstrate labelingof dense granules both in whole platelets and
isolated dense granule preparations made permeable with
Triton X-100. Neither quiescent platelets nor isolated
dense granules show labeling unless made permeable. The
evidence provided both by the cryosections showing membrane staining of specific vesicles and by the whole mounts
showing specific labeling of dense granules support the
localization of granulophysin to the dense granule membrane.
Double labeling with antibodies to both P-selectin and
granulophysin demonstrated the presence of P-selectin in
the granulophysin-positive granules, as well as the more
numerous granulophysin-negative a-granules. This is supported by the results of Western blot analysis, which
demonstrates the presence of P-selectin in both dense
granule-enriched and dense granule-depleted fractions.
Therefore, P-selectin appears to be common to both types
of granule membranes. This finding is also supported by the
study of Lages et alZPdemonstratingboth decreased expression of P-selectin in a patient with severe a8 SPD and
labeling by anti-P-selectin of both a-granules and unidentified clear vesicles in control platelets.10*29
With the antigranulophysin antibody, the clear vesicles that label with both
anti-P-selectin and antigranulophysin as dense granules
can now be identified. Failure of the a-granules to stain
with D545 also supports our previous finding that granulophysin is not present in the membranes of all granules.
Because the vesicles labeled with D545 on cryosections
could not be specifically identified and because lysosomes
also lack identifying morphologiccharacteristics on cryosections, we investigated the possibility that some of these
vesicles might represent lysosomes, using conditions that
produce differential secretion of dense granules (as measured by 14C-serotoninrelease) and lysosomes (as measured by secretion of p-glucuronidase). With addition of
colchicine, which inhibits lysosomal secretion, brief stimulation with thrombin (5 and 30 seconds) produced significant
dense granule secretion ( > 70% serotonin release) accompanied by redistribution of granulophysin to the platelet
plasma membrane in 69% of cells quantified by flow
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ISRAELS nAL
150
-7.
WMomountsdpbtaktta)orbd.t.d
donla grm(.r (b tlNOl#t 0 ) f l r d wkh 1% pM(*
nukkhyd.IO.1K gMu.ldrhld. and lre8t.d wkh
triton X-100 tor 3 minut- fa. c through 01 Mom
a
l
n
g pl.t.d on gcldr. L.b.Hngwkh Ds45 m n d GAM
10 (0. b, d. 0 ) 01 " u n o
"sand GAM 10 n
control IC).
~OdgIrulNgnlfkdons: lo) x27.OOO. fb)
xS6.OOO. IC)
x 16O.OOO. Id) x66.OOO. lo) x68.OOO.1
1&1.
UmtimuW
Thrombin 5 m+ cokhkine
Thrombin 30 seconds + colchicine
Thrombin 5 minutes
Values am meen L. SE.
-
& e n t l o n d s o " & O ~ m d ~ d O r m ( o O h * J n o n P I . h k ( ~
Saaonln
s.o*m 1%)
In * 81
7.71 :1.73
71.29 t 3.22
80.50 L. 1.37
91.14 L. 1.03
-
SmHOn 1%)
In
S,
0.80 t 0.32
2.48 L. 0.81
4.42 t 0.89
31.82 t 4.87
C.m L.b.cd Wflh DYSflTC1%)
kb*.ccLMml
In * 6)
Di..ccL.b.l
In * I)
6.54 :2.18
23.17 t 7.18
30.69 t 5.95
51.67 t 4.0s
12.5
66.1
69.0
76.8
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PLATELET DENSE GRANULE MEMBRANE PROTEINS
1
I
A\
I
cl
151
B/
membranes and its expression on the platelet surface
paralleling dense granule secretion. However, some granulophysin may also be present on lysosomal membranes and
some of the clear vesicles labeled on the cryosections may
represent lysosomes. In this regard, Nieuwenhuis et a130,3i
demonstrated that lysosomal membranes are redistributed
to the platelet plasma membrane after activation.
After stimulation with thrombin, granulophysin was redistributed to the SCCS and plasma membrane as was previously described for P-selectin.lo Like a-granules, dense
granule membranes apparently fuse with the SCCS on
platelet stimulation and release their contents into the
extracellular space through the SCCS. After degranulation,
granulophysin, like P-selectin, becomes expressed on the
plasma membrane. Distribution of granulophysin on the
plasma membrane did not demonstrate any clustering or
concentration in specific sites that might suggest a specific
function. This homogeneous distribution is similar to that
of P-selectin. P-Selectin, which on the platelet plasma
membrane mediates adhesion of activated platelets to
neutrophils, has been identified as a member of the selectin
family of vascular cell surface re~ept0rs.l~
The function of
granulophysin is unknown, and whether its presence on the
plasma membrane of stimulated platelets plays a functional
role or is simply a marker of dense granule membrane
fusion with external membranes is not clear. The role of
granulophysin, which may be related to granule integrity, is
probably of broader significance than to the platelet alone
because the presence of granulophysin has been identified
in endothelial cells, n e ~ t r o p h i l s ,and
~ ~ lymphokine-activated killer cells.9
LL
’
I
I
Fig 8. Flow cytometric analysis of D545-FITC binding to quiescent
and activated platelets as described in the Materials and Methods
section. Representative histograms of fluorescence recorded after
binding of directly labeled 0545-FITC. Vertical line separates negative
and positive fluorescent populations. Fluorescence intensity is displayed on a three-decade logarithmic scale. (A) Resting platelets and
(B) resting platelets incubated with unlabeled D545 before addition of
D545-FITC. (C) Plateletspretreated with colchicine before exposure to
thrombin for 5 seconds. (D) Plateletspretreated with colchicine before
exposure to thrombin for 30 seconds. (E) Platelets exposed to
thrombin for 5 minutes without colchicine and with external calcium.
(F) Platelets treated as in (E) but incubated with D545-FITC in the
presence of excess unlabeled D545.
cytometry. These conditions produced minimal lysosomal
secretion ( < 5%). When conditions also allowed lysosomal
secretion to occur (thrombin stimulation for 5 minutes),
fluorescence increased by 7.8% more. These results support the presence of granulophysin on the dense granule
ACKNOWLEDGMENT
We thank C. Robertson and E.M. McMillan for technical
assistance, A. Lundy for manuscript preparation, and Dr E. Rector
for expert assistance with flow cytometry analysis.
REFEREiNCES
1. White JG, Gerrard JM: Ultrastructural features of abnormal
blood platelets. Am J Pathol83:590, 1976
2. Gerrard JM, Rao GH, White JG: The influence of reserpine
and ethylenediaminetetraacetic acid (EDTA) on serotonin storage
organelles of blood platelets. Am J Pathol87:633,1977
3. Holmsen H, Weiss HJ: Further evidence for a deficient
storage pool of adenine nucleotides in platelets from some patients
with thrombocytopathia: ‘Storage pool disease.’ Blood 39:197,1972
4. Israels SJ, McNicol A, Robertson C, Gerrard JM: Platelet
storage pool deficiency: Diagnosis in patients with prolonged
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75:118, 1990
5. Hermansky F, Pudlak P: Albinism with hemorrhagic diathesis
and unusual pigmented reticular cells in the bone marrow: Report
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6. Witkop CJ, Krumwiede M, Sedano H, White JG: Reliability
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1992 80: 143-152
Platelet dense granule membranes contain both granulophysin and Pselectin (GMP-140)
SJ Israels, JM Gerrard, YV Jacques, A McNicol, B Cham, M Nishibori and DF Bainton
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