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Membrane-Associated Forms of the PA4 Amyloid Protein Precursor of
Alzheimer’s Disease in Human Platelet and Brain: Surface Expression
on the Activated Human Platelet
By Qiao-Xin Li, Michael C. Berndt, Ashley I. Bush, Baden Rumble, Ian Mackenzie, Anna Friedhuber,
Konrad Beyreuther, and Colin L. Masters
The amyloid proteinprecursor (APP) of Alzheimer’s disease
(AD) is abundantly expressed in platelets, where its primary
function remains undetermined. As an integral transmembrane protein, the release of APP from the membrane may
be a critical event in AD. We examined the association of
APP with human platelet membranes using a combination
of alkali treatment and immunoprecipitationof the carboxylterminus ofAPP. Most of theplatelet membrane-associated
with molecular mass of 100 to 130 kD is reAPP A
(P
)P
,,
moved with alkali treatment and is also truncated at the
carboxyl-terminus. APPM,.
is present at least in part on the
surface of the platelet. The full-length transmembrane form
of APP, as a 140- t o 150-kD minor species, is alkali resistant
and is also present on the plasma membrane. In contrast,
from brain is full-length
(possessingthe
most of the APP”
carboxyl-terminus) with a molecular mass of 105 t o 130 kD
and isresistant t o alkali treatment. lmmunoelectronmicros-
copy shows platelet APP t o be localized t o the a-granule.
Activation of platelets results in a threefold increase in surface APP detectability. In plasma, the 130-kD APP-reactive
band is increased in AD. We find that in the gray platelet
syndrome, platelets contain reduced amounts of APP, with
a corresponding reduction in plasma APP levels, suggesting
that platelets are the major source of plasma APP. Our studies also identify an interaction of APP with platelet membranes which differs from that foundin the brain, and raise
the possibility of a receptor for APP in platelet membranes.
in plateQuantitative differences in the amounts of APP”
lets compared with brain also indicate regulation of the
pathways that determine the cleavage of APP near its transmembrane domain. These pathways are a therapeutic target
in AD, and maybe easily amenable t o investigation in platelets.
0 1994 by The American Society of Hematology.
I
particular interest because it might function in an autoregulaN ALZHEIMER’S DISEASE (AD), the proteolysis of the
tory manner by controlling proteolytic events on the cell
amyloid protein precursor (APP), leads to the extracellusurface.
the prototypic isoform,’
is
predominantly
lar and intracellular accumulation of a 40- or 42-residue PA4
amyloid protein.’S2 The full-length forms of APP (APPFL) expressed in the brain, and lacks the KPI domain.
The circulating forms of APP in blood plasma mayderive
are integral membrane glycoprotein^^.^ in which the transfrom plateletsz3or activated lymphocytesfmonocytes.” The
membrane domain contains part of the amyloidogenic PA4
exclusion of exon 15 through alternate splicing generates a
sequence.’ A closely related family of membrane proteins
series of APP molecules restricted to adherent cells, particu(APLP-l and APPWAPLP-2)627do not contain this amylarly of the lymphocytehnonocyte lineage.25 Although unloidogenic PA4 sequence. The amino-terminus of PA4 is
likely to be the source of the cerebral deposits of PA4,’ the
derived from the cognate exoplasmic region of APP, and
platelet-associated forms of APP provide a readily available
could be generated by a variety of proteases acting close to
source to study the normal structure and function of APP.
the exoplasmic surface. In contrast, the carboxyl-terminus of
Studies by
have focused the
on
released forms
the PA4 domain is derived from the cognate transmembrane
of platelet APP. In this study, we characterize the APPM,,
domain of APP,’ thereby creating a problem in understandof platelet and brain, and examine the expression of surface
ing the mechanism of its release from the membrane before
APP of platelets in both the resting and activated state. We
proteolytic cleavage. Like many integral membrane recepalso find that APP localizes to the a-granules by immunotors, secreted forms of APP occur: principally through a
process of carboxyl-terminal truncation involving proteolytic
cleavage of the full-length protein close to the membrane
From the Department of pathology, The University ofMelbourne,
surface. This action of an APP endoprotease/secretase activParkville, Victoria; The Mental Health Research Institute of Victoria, RoyalPark Hospital, Parkville, Victoria; The Baker Medical
ity normally should prevent the generation of PA4, because
Research Institute, Alfred Hospital, Prahran, Victoria, Australia;
the major pathway of constitutive cleavage appears to occur
and the Center for Molecular Biology, The University of Heidelberg,
atLys16 within the PA4 ect~domain.’.~However, a minor
Heidelberg, Germany.
alternative pathway may exist in which soluble forms of
Submitted June 9, 1993; accepted February 28, 1994.
PA4,- are generated.’&I2Point mutations within or close to
Supported by grants from the National Health and Medical Rethe PA4 region of APP cause AD in some fa mi lie^,'^"^ and
search Council of Australia, the Aluminium Development Council
therefore subtle changes in this region of APP are likely to
of Australia, and the Victorian Health Promotion Foundation. K.B.
affect processing events. Clearly, the amount, topology, and
is supported by the Deutsche Forschungsgemeinschafiand the Bunstability of the membrane-associated forms of APP (APPMem) desministerium f i r Forschung und Technologie.
Address reprint requests to Colin L. Masters, MD, Department
could determine to a large degree whether extracellular or
of Pathology, The University of Melbourne, Parkville, Victoria, 3052
intracellular forms of PA4 are generated. Therefore, the naAustralia.
ture of the association of APP with cell membranes needs
The publication costs of this article were defrayed in parr by page
to be characterized in some detail.
charge payment. This article must therefore be hereby marked
The family of APP glycoproteins now extends to more
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
than 10 isoforms generated by alternate splicing of the APP
indicate this fact.
gene, principally of exons 7, 8, and 15. Exon 7 encodes a
0 1994 by The American Society of Hematology.
Kunitz-type protease inhibitor (KPI) domain2@”and is of
0006-4971/94/8401-0024$3.00/0
Blood, Vol 84,No 1 (July l), 1994:pp 133-142
133
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134
LI ET AL
electron microscopy, strongly substantiating previous reports
using sucrose density gradient centrifugation and immunofluorescence m i c r o ~ c o p y . ~In~ addition,
-~~
our studies of a
patient with the gray platelet syndrome (GPS) suggest that
most of the APP found in normal plasma is derived from
platelets.
MATERIALS AND METHODS
Materials. Electrophoretic Rainbow Standards andI4C-methylated protein molecular weight protein markers and Na[’ZSI]were
purchased from Amersham (Aylesbury, UK). Electrophoresis grade
acrylamide, N,N’-methylene-bis-acrylamide,
N,N,N’,N‘-tetramethylenediamine, ammonium persulphate, and London White Resin (LR
White) were obtained from Bio-Rad (Richmond, CA). Prostaglandin
E, (PGE,) and sodium deoxycholate were from Sigma (St Louis,
MO). Lactoperoxidase and thrombin were from Boehringer-Mannheim (Mannheim, Germany). Protein A-Sepbarose CL-4B was purchased from Pbarmacia (Uppsala, Sweden). Protein concentrations
were determined by the BCAmethod (Pierce, Rockford, IL). All
other chemicals were of analytical reagent grade.
The primary antibodies used were: monoclonal antibody (MoAb)
22C11 (Boehringer Mannheim), raised against a recombinant APP,,,
bacterial fusion protein,’which recognizes an epitope nearthe
amino-terminus of APP; a rabbit antiserum raised against nondenatured (“native”) APP purifiedfrom the membrane fraction of human
brain3”; anti- “carboxyl-terminus” is a rabbit antiserum raised
against a synthetic peptide (residues 653 through 695 ofAPP,,,)
representing the complete cytoplasmic domain of APP.3’ Another
polyclonal rabbit antibody to the cytoplasmic domain (Ab 369),”
was kindly provided by Dr Sam Gandy (Rockefeller University,
New York, NY). MoAb 7H5 to the KPI region of APP33was a kind
gift from Dr Dale Scbenk (Athena Neurosciences, San Francisco,
CA). Affinity-purified antimouse or antirabbit alkaline phosphataseconjugated secondary antibodies (absorbed with human serum proteins) were obtained from Sigma. Antibodies to P-selectin, CD13,
and GPIb have been previously ~haracterized.’~”~
Goat antimouse
and goat antirabbit IgG conjugated to 10 nm gold were from BioCell (Cardiff, UK),and 10 nm streptavidin goldwas a gift from
E.C. Bell (St Vincent’s Institute of Medical Research, Melbourne,
Australia). Biotinylated rabbit antimouse antibody was from Promega.
Platelet and brain membrane preparation. Platelets were obtained
from fresh blood of healthy volunteers using the method described
by Bush et al?’ Platelets were washed twice and sonicated Tyrode’s
in
buffer laclang calcium and magnesium (136.9 mmol/L NaCl, 2.68
mmom KCI,11.9 mmol/L NaHCO,, 0.42 m m o K NaHzP04, pH
6.4) with S mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride
(PMSF), 0.2 mg/mL leupeptin, and 3 pmol/L PGE, . The lysate was
then centrifuged at 100,ooOg for 2 hours to separate the membrane
pellet fraction from the soluble supernatant fraction. Brain membrane
wasobtained from temporal lobe biopsytissuethatwascut
into
pieces, followed by homogenization and centrifugationas above. The
membrane fractions werethenwashedusingthesodium
carbonate
procedure described by Fujiki et al.37 Briefly, the membranes were
resuspended by sonication in 0. l mol/L sodium carbonate at pH 11.5
andincubated for 30 minuteson ice followed by centrifugation
(-233,000g. 1 hour). The treated membranes were used for Westem
blotting and immunoprecipitation as described below.
Tissue culture and rransfection. HeLa cells were grown in Dulbecco’s modified Eagle’s medium (Flow Laboratories, Irvine, CA)
containing penicillin (50 pg/mL), streptomycin (50 pg/mL), fungizone (2.5 pg/mL), and 10% (vol/vol) fetal calf serum (FCS; Commonwealth Serum Laboratories, Melbourne, Australia). The same
medium was used for the rat pheochromocytoma line (PC12) except
10% FCS + 5% horse serum wereused. Twenty-four hours after
subculture, the cells at 1.5 X 10‘ cells/lO-cm flask were transfected
with 25 pg plasmid DNA carrying either APPh9,or APP,,, cDNA
by Ca3(P04)2coprecipitation.3 Cells were harvested 24 hours after
transfection and lysed with buffer containing 50 mmol/L Tris-HCI,
pH 7.5, 150 mmol/L NaC1, 2 mmol/L EDTA, 0.2% Nonidet P-40
(NP-40), and2% Triton X-100. The samples were centrifuged at
10,OOOg for 5 minutes. and the supernatants were used for the Western blot analysis.
To isolate membranes from phaeochromocytoma (PC12) cells,
the cells were harvested in hypotonic buffer containing S mmol/L
Tris-HC1, pH 7.5. After a low-speed spin to remove the cell debris,
membranes were obtained by centrifugation of the lysate at 100,OOOg
for 1 hour and treated with sodium carbonate as described above.
Sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDSPAGE) and Western blotting. Samples (50 pg total protein) were
dissolved in sample buffer containing 2% SDS and 5% P-mercaptoethanol, boiled, and electrophoresed on 8.5% discontinuous polyacrylamide gels in the presence of SDS. The proteins were then
transferred electrophoretically to nitrocellulose as described previously.z’ Nonspecific binding of primary antibody was blocked with
3% bovine serum albumin (BSA) and the nitrocellulose was incubated with MoAb 22C11 ascites (1:7,500) overnight at 4°C. Affinitypurified antimouse IgG conjugated to alkaline phosphatase (1 :2,000)
was used for immunodetection of the bound primary antibody.
Immunoprecipitation ofAPP. Membranes were solubilized with
2% sodium deoxycholate and the extracts were diluted with 5 vol
STE buffer (S0 mmol/L Tris-HC1,pH 7.5, 150 mmol/L NaC1, 2
mmol/L EDTA, I % Triton X- 100, 2 mmol/L PMSF, and 5 pg/mL
leupeptin) followed by preclearing with 5 mg of protein A-Sepharose
in STE buffer containing 0.2% NP-40 for 1 hour at 4°C. The supematantswere then incubated overnight at 4°Cwith 5 pL of either
preimmune serum, or antibodies to native APP or to the carboxylterminus of APP. The antigen-antibody complexes were precipitated
by 5 mg of protein A-Sepharose for 1 hour at 4°C. The immunoprecipitates were resuspended in sample buffer (containing SDS and p mercaptoethanol) and analyzed by SDS-PAGE and Western blotting.
MoAb binding to the surface of human platelets. Purified 22C11
IgG was radioiodinated to a specific radioactivity of 1,900 c p d n g
using chloramine T.” The radioiodinated 22C1 1 was comparable in
activity with unlabeled 22C11 as evaluated by Western blot analysis
of SDS-polyacrylamide gel of platelet APP. Platelets were prepared
from venous blood into acid citrate dextrose (ACD) as anticoagulant
as described by Dunlop et al.3xPlatelets were washed in the presence
of 3 pmol/L PGE, and then either treated with PGE, (3 pmoVL) or
activated with thrombin ( 1 U/mL, 1 minute). Based on the method
described by Berndt et a1’: binding was assayed by incubating various amounts of iodinated MoAb 22C11 with 375 pL of 5 X IOx/
mL platelets for 30 minutes in Tyrode’s buffer (containing 1 mmol/
L EDTA, without calcium or magnesium) at 20°C. Duplicate samples
of the incubation mixtures (100 pL) were overlayed onto 0.75 mL
of 17% sucrose in Tyrode’s buffer containing 1 mmollL EDTA and
microfuged for 2 minutes. The platelet pellets were counted for
radioactivity in a gamma counter. A third 100-pL sample was
counted to determine the total count. Nonspecific binding was determined by incubating platelets with ‘251-labeledMoAb 22C11 in the
presence of 100-fold excess of the unlabeled antibody. Specifically
bound radioactivity was calculated by subtracting the nonspecific
binding from the total bound radioactivity.
Surfiace labeling of platelets. Iodination of resting platelets was
performed essentially according to the method described by Booth
et al.3yWashed platelets isolated in the presence of 3 pmoVL PGEI”
were resuspended to 1 X 1OY/mL inbuffer A (10 mmol/L HEPES,
pH 7.4, 150 mmoVLNaCI, 1 mmolL EDTA). The platelets were
labeled in the presence of Na[lZ51]( I mCi/mL), lactoperoxidase (2.5
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135
MEMBRANE-ASSOCIATED FORMS OF PLATELET APP
pg/mL). and hydrogen peroxide (35 pmolk). After a IO-minute
incubation, the platelets were pelleted by centrifugation as described
above. The platelet pellet was lysed in buffer A (with S mmoll
L EDTA and 1% Triton X-IOO) for immunoprecipitation analysis
followed by autoradiography. Under these conditions, platelet activation is minimal because released proteins known to associate with the
activated platelet surface, such as thrombospondin. are not labeled."'
b~~rn~mocyrc~chemic~~i
ioculix~~ion
ofAPP by electron micrr,.sco~py.
Platelets were isolated from fresh blood and fixed in 4% paraformaldehyde followed by embedding in LR White resin. Ultrathin sections
were prepared on nickel grids, blocked in I % BSA, and immunostained withpurified MoAb 22C1 I ( 1 : l O O ) for 2 hours atroom
temperature. Bound antibody was detected by biotinylated rabbit
antimouse antibody (1:2O) and subsequent IO nm streptavidin-gold
conjugate (l:lO()). Sections were fixed in 2% glutaraldehyde and
counterstained with uranyl acetate and viewed in a Siemens 102
electron microscope (Berlin, Germany) at60 kV. Staining of plateletsurface APP was performed according to the method described by
Kieffer et al."" Briefly, platelet-rich plasma obtained from blood in
ACD" or whole blood was dropped on a formvar-coated nickel
grids. Platelets were allowed to adhere and spread for 15 minutes
at 37°C in a moist chamber. After washing off nonadherent cells,
the grids were blocked with 0. I % BSA and 20% normal serum and
further processed for immunolabeling with different antibodies at
4°C. The primary antibodies were detected with goat antimouse or
goat antirabbit IO nm gold conjugate ( I :S). Finally, platelets were
tixed in 2% glutaraldehyde, dehydrated progressively. stained with
uranyl acetate, and viewed as described above.
Rcr~if~immunoo.s.suy.The radioimmunoassay for APP was performed according to the method described by Rumble et al"' with
the following modification. Rabbit antiserum to native APP"' was
used at 1:8OO dilution and a radioactive tracer was prepared by the
iodination of native APP using the chloramine T method."'
RESULTS
Full-length APP (APPFL) is present inhumanplatelets
anddiffers from thehuman brain isojbrm. APPFI. is of
particular interest because it contains an intact /?A4 sequence
capable of participating in the formation of amyloid. This
form could interact with membranes by being bound to a
surface structure or receptor, by being covalently linked, or
by being an intrinsic transmembrane protein. This question
was approached by using a method of alkali stripping of
membranes that is expected to remove any peripheral/extrinsic membrane protein." Total membranes from fresh platelets prepared in buffer containing 5 mmol/L EDTA were
treated with sodium carbonate at pH 11.5 and examined by
Western blotting using MoAb 22C1 1 (Fig l ) . In a previous
study," we determined the N-terminalsequence of APP from
platelet membranes recognized by MoAb 22C1I , confirming
its identity as APP and not APLPI or APPH/APLP-2. Similar amounts of proteinfromtotal platelet lysates, soluble
fractions, and membrane fractions (before or after alkali
treatment) were also analyzed. As shown in Fig I , there were
prominent 1 0 0 - to 1 IO-kD and 120- to 130-kD bands in the
platelet lysate, soluble fractions, and membrane fractions
(Fig I , lanes 1 through 3). Alkali treatment removed most
of the APP signal in the platelet membrane (Fig I , lane 4)
and only 10% of the APP immunoreactive material remained
with the treatedmembrane as quantitated by radioimmunoassay (data not shown). Treatment with buffer alone as control
didnot affect the APP membrane association (data not
Platelet
Brain
"
Kd
200 -
1 2 3 4 5
-~
6
-l
97.4 -
69 Fig1.Comparisonof
APP immunoreactivities in alkali-treated
platelet and brain membranesby Western blot. The membraneswere
isolated and treated as described in Materials and Methods. Fifty
micrograms of protein was reduced,boiled, and separated on an
8.546 SDS-PAGE andthe APP immunoreactivespecies were detected
using MoAb 22Cll. Lane 1, platelet lysate; lane 2, 100,OOOg soluble
fraction of platelet; lane 3, 100,OOOg pellet fraction of platelet (membrane); lane 4, alkali-treated platelet membrane; lane 5, membrane
from brain biopsy material; lane 6, alkali-treated brain membrane.
The relative molecular mass of standardprotein markers are shown
on the left. APP immunoreactive bands are indicated on the right.
shown). Alkali treatment did not disrupt the membrane association of other platelet membrane proteins P-selectin, GPlb,
and GPIIla (data not shown). These results suggest that most
of the platelet membrane-associated APPis not integral/
intrinsic to the membrane.
There wereweak bands of140 to 150 kD in thetotal
platelet lysate (Fig I , lane 1) and platelet membrane fraction
before or after alkali treatment (Fig I , lanes 3 and 4, respectively). To further confirm the identityof these bands, immunoprecipitation with polyclonal antibodies raised to purified
human brain native APPor to a synthetic peptide corresponding to thecomplete cytoplasmic domain were performed. The
immunoprecipitatesweredetected
withMoAb22C11on
Western blots (Fig 2, A and B). The 1 0 0 - to 130-kD and 140to 150-kDformsofAPP
in thealkali-strippedmembranes
were enrichedby immunoprecipitation with antibody to native
APP (Fig 2A, lane I and Fig 2B, lane l), but only the prominent band at 140 to 150 kD was immunoprecipitated by the
cytoplasmicdomainantibody (Fig 2A,lane 4 andFig2B,
lane 2).Sometimes trace amounts of a band at 130 kD were
also immunoprecipitated by the cytoplasmic domain antibody
(Fig 2B, lane 2). Because the immunoprecipitates were
assessed with an antibody to an amino-terminal epitope (MoAb
22C1 I), these data indicate that only the 140- to 150-kD and
a trace 130-kD APP species in platelet are APPkl,. In fact,
the140-to150-kD
species consists ofatleast threebands
(Fig 2B,lane 2). The 140- to 150-kDimmunoprecipitates
frombothnativeAPPandcytoplasmicdomainantibodies
-
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LI ET AL
136
Anti-native Anti-carboxyl Prebleed
APP terminus
control
/
Kd
200-
1 2 3 4 5
1
7
2
~
II140-150 -C
M
It- 120-130 -C
+100-110
97.4 -
69 L
1
I
B
2
7- 120-130
3100-110
C
Fig 2. Full-length APP in platelet and brain. (A) Alkali-treated platelet membrane (lanes 1 and 4) and brain membrane (lanes 2, 3, and 5)
were immunoprecipitated with an antibody t o native APP (lanes l and 2) or to the carboxyl-terminus of APP (lanes 3 and 4) followed by
Western blotting using MoAb 22Cll.Samples were reduced and boiled before applicationt o the SDS-PAGE. A preimmune serum was used
in lane 5. Molecular-weight markers are shown on theleft. APP immunoreactive bands are indicated on the right.
(B) Immunoprecipitation of
APP from alkali-washed platelet membranes with thesame two antibodies as (A) in a separate experiment. This shows multiplebands in the
140- t o 150-kD region using the antibodyto thecarboxyl-terminus. (C) APP immunoreactivity of materialreleased from alkali-treated platelet
membrane. Lane 1, immunoprecipitates from antibody t o native APP. Lane 2, immunoprecipitates from antibody t o carboxyl-terminus. The
immunoprecipitates weredetected by MoAb22C11 on Western blot. APP immunoreactive bands are indicated on the right.
Each lane represents
the immunoprecipitate fromreleased material of 2 x lo9 total platelets.
were also detected with another polyclonal antibody to the
carboxyl-terminus of APP (Ab 369) (data not shown).
The material released by alkali treatment of platelet membranes c m be immunoprecipitated by native APP antibody
as a group of 100- to 130-kD bands, but is not immunoprecipitated by cytoplasmic domain antibodies (Fig 2C, lanes
I and 2). Each lane represents material derived from 2 X
10" platelets. Therefore. these 1 0 0 - to 130-kD species are
not integralhntrinsic to the membrane, and are best referred
to as "membrane-associated" APP (APP,,,,,). We also observed that protease inhibitors. especially leupeptin, were
required in the lysate buffer to permit detection of APP,:,..
None of the APP immunoreactivities described were immunoprecipitated by the preimmune serum (Fig 2A, lane S).
When a lysate of APP7>,cDNA transfected HeLa cells
was analyzed in this system, the same set of four bands
detected by MoAb 22CI I as reported by Weidemann et al'
were observed (data not shown). Two of these four bands
at 140 kD and I I O kD were detected byan antibody to the
KPI region (7HS)(Fig 3. lane 2). Both the platelet 140-150
kDand the 130 kD APP species were also recognized by
this antibody (Fig 3. lanes 3 and S). This further indicates
that these bands are KPI containing APP species.
To further compare APPMc,,,of brainand platelet APP.
human brain membranes were obtained from biopsy tissue
andtreatedwith
alkali. Figure I (lane S) shows thatthe
major brain APP immunoreactive bands detected by MoAb
22CI 1 are in the 105- to 130-kD range. In contrast to platelets, alkali treatment of brain membranes did not release any
appreciable amount of APP (Fig 1. lane 6 ) , indicating that
most of the material is integral or intrinsic to the membrane.
This is further confirmed by the immunoprecipitationof APP
from alkali-treated brain membranes with both native APP
and cytoplasmic-domain antibodies. The cytoplasmic-domain antibody immunoprecipitated the same APP species as
nativeAPP antibody (Fig 2A,lanes 2 and 3): hence,the
majority of the brain membrane APP species are expected
to be full-length APP. The APP in human brain also consists
of several discernible bands of 105. I I O , I 18, 120, and I30
kD. Among these species only the 130- and I 18-kD bands
wererecognized by the anti-KPI antibody (Fig 3, lanes 4
and 6 ) . Membranes obtained from PC I2 cells showed a similar lack of alkali-releasable APP species (data not shown).
Because the APP,:,,may be 21 potential substrate for PA4
amyloidogenesis. we analyzed platelet samples to detect any
differences of this species between control and AD subjects.
Immunoprecipitations of platelet membranes from AD ( n =
3) and control ( n = 3) were performed using antibodies to
bothnative APP and cytoplasmic domain. comparison of
obvious
the APP in both control and AD did not show any
qualitative or quantitative differences (data not shown). This
is consistent with our previous observations that differences
do not exist in the content of APP in whole platelets between
AD and controls."
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MEMBRANE-ASSOCIATEDFORMS
OF
PLATELETAPP
Cell
Anti-native
APP
lysate immunoprecipitates
/"7"-7
Kd
1
69-
Western
blot:
2
3
4
5
6
"
200-
I
1
I
U
-
Anti-"KPI" 22C11
Fig 3. Identification of APP isoforms containing KPI region from
membranes of platelets and brain. Extracts from alkali-treated membranes of either platelets
(lanes 3 and 5) or brain (lanes 4 and 6) were
immunoprecipitated by an antibody
t o native APP. The products
were reduced, boiled, and thenanalyzed by 8.50/0 SDS-PAGE followed
by Western blot using antisera as indicated below the figure. Cell
lysates from APPBS (lane 1) or APP751(lane 2)transfected HeLa cells
were also analyzed under thesame conditions. The relative molecular mass of standard protein markers is shown on the
left.
APP
immunoreactive bands are indicated on the right.
At least two forms cf APP, the carbox~,l-terminal-trunccrted and full-length APP, crre present on platelet surjcrce.
The above studies indicate that two different forms of APP
are associated with platelet membranes that comprise both
the internal membrane as well as the plasma membrane. To
show the presence of surface APP. washed resting platelets
isolated in the presence of 3 pmol/L PGE, were surfacelabeled with Na ["'l] and lactoperoxidase. The labeled proteins were immunoprecipitated with either native APP or
cytoplasmic domain antibodies. The native APP antibody
(Fig 4, lane 2) precipitated a broad band from 100 to 150
kD consisting probably of both the I W- to 130-kD and 140to 150-kD APP species, because the cytoplasmic domain
antibody (Fig 4, lane 3 ) only precipitated a 140- to 150-kD
protein. This is consistent with the data that the 140- to 150kD band is an APPFI.species present on the platelet surface.
In addition, the 1 0 0 - to 130-kD carboxyl-terminal-truncated
APP was also present, at least in part, on the platelet surface.
Expression of APP on the platelet surfrrce i s increcrsed
during crctivation. Although it cannot be absolutely excluded that no platelet activation occurs during platelet isolation and labeling, the available data are most consistent with
a small degree of surface expression of APP in normal testing
platelets because PGE, was included throughout the procedure and because released proteins known to be associated
with the activated platelet surface, such as thrombospondin,
are not labeled by this procedure.3" Activation of human
blood platelets is associated with changes in platelet morphology, biochemistry, and membrane composition, and results in increased surface expression of several endogenous
platelet proteins. To confirm expression of APP on the plate-
137
let surface and the effect of activation, washed platelets isolated in the presence of 3 ymollL PGE, wereincubated
with increasing amounts of '2'I-labeled MoAb 22CI I under
resting (incubation with PGE,) or stimulated (incubation
with I U/mL thrombin) conditions. A binding curve of
pooled data from four normal donors is shown in Fig S .
The binding of 22C1 I to resting and activated platelets was
evaluated by analysis of variance. Across all 22C 1 I concentrations, binding was greater to activated platelets ( P <
.001). Analysis of the data by Scatchard analysis gave 250
binding sites on resting platelets (kd -20 nmollL) and 730
binding sites on thrombin-activated platelets (kd "30 nmol/
L), with the implied assumption that the 22C1 1 IgG bound
monovalently per measured bindingsite. These low numbers
of binding sites are consistent with our previous radioimmunoassay that measured about 200 copies of full-length APP
(2.06 X IO"" pmol of APP) by the carboxyl-terminus antibody and "2,000 copies of total APP by the amino-terminus
antibody per resting platelet.'3 Under equivalent assay conditions, P-selectin surface expression evaluated using the anti-
Kd
1 2 3
200 -
97.4-
+l 40-150
7100-130
6946Fig 4. Surface labeling of APP in platelets. Washed platelets isolated in the presence of 3 pmol/L PG€, were surface radioiodinated,
lysed, and immunoprecipitatedwith an antibody t o native APP (lane
2). an antibody to thecarboxyl-terminus (lane 3). or preimmune serum (lane 1). The precipitates were reduced, boiled, and separated
on 8.5% SDS-PAGE, followed by autoradiography. The relative molecular mass of "C-labeled protein markers are shown on the left.
The APP bands are indicated on the right. Unidentified radioactive
material migrated with thedye front.
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LI ET AL
138
Ultrrrstructural localization cf APP in the hutncrn platelet.
Previously published.data have shown that platelet APP is
releasedfrom the a-granule by stimuli such as thrombin,
collagen, or calcium ionophore that
induce
degranulation.2~.26.27.44,'15
To localize APP epitopes within the platelet,
/-
"
1
0-
3
4
[22C1l] added, pg/mL
2
5
6
Fig 5. Binding of MoAb 22Cll to human platelets. Washed platelets isolated in the presence of 3 pmollL PG€, were incubated with
increasing amounts of '251-labeledMoAb 22Cll under resting (PGE,,
0)
or activated (thrombin, 0 )conditions. Specificbinding was evaluated as described in Materials and Methods. Binding for each MoAb
22Cll concentration was determined in duplicate and the data as
shown are the pool of separate experiments with four different normal donors.
P-selectin MoAb AK6 increased from -300 binding sites
on resting platelets to greater than 4,000 binding sites on
thrombin-activated platelets (data not shown).
We also measured the ['*'I]-MoAb 22C11 binding sites
on platelets from an individual with the GPS, a congenital
disorder where there are greatly diminished contents in the
platelet a-granule.J3 The number of binding sites of APP
measured from the GPS were -390 for resting and 1,020
for thrombin-stimulated platelets, similar to the numbers
measured for the normal control subjects and taking into
account that GPS platelets are larger than normal platelets.
In contrast to the normal plasma membrane surface expression, the GPS platelets had reduced amounts of soluble APP.
Compared with an age-matched control (Fig 6, lanes I , 3,
and 4), the GPS platelets had very little soluble APP in the
100- to I IO-kD and 120- to 130-kD regions (lane 2) and
hadless membrane-associated APP in the 120-to130-kD
regions (lanes S and 6). Furthermore, Western blot analysis
of plasma APP from both control and GPS cases showed
that the level of APP in GPS plasma was markedly reduced
(data not shown). Radioimmunoassay also showed that control platelets had 7.4-fold more soluble APP than GPS platelets, and a similar reduction was found in the levels of GPS
plasma. After alkali treatment, the control membrane (Fig
6, lane 4) lost the 120- to 130-kD APP species (see Fig I )
and showed a similar pattern to that of GPS platelet membrane APP (Fig 6, lane S). These data suggest that the 120to 130-kD APPMcn,species maybean
extrinsically bound
form of APP, stored in the a-granule. The 140- to 150-kD
APP species described in Fig I were also present in both
the control and GPS platelet membranes (Fig 6, lanes 3
through 6).
immunoelectron microscopy was performed on paraformaldehyde-fixed platelets. APP was mainly localized to the agranule (Fig 7A). This localization of APP substantiates previous biochemical data whichindicatethat
approximately
50% of platelet APP is recovered in the granule fraction
during differential centrifugation of platelet lysate^.'^ It is
also consistent with immunofluorescence microscopy
Localization of APP on the surface by immunogold
electron microscopy was performed on unfixed platelets adhering to grids. These platelets had been activatedby contact.
Scattered gold particles were observed using MoAb 22CI I
(Fig 7R) or rabbit antinative APP (Fig 7C). As controls, an
antibody to GPlb showed a similar but much stronger diffuse
labeling (Fig 7D). That the platelets had been activated by
adherence wasconfirmedwithan
antibody to P-selectin,
which showed prominent clusters of gold particles on the
platelet surface (Fig 7E). Further controls in which the primary antibody was omitted or an irrelevant antibody (antiCD13 [WMIS],'" an IgG, MoAb of the same subclass as
MoAb 22C11) wasused,showedminimal
attachment of
gold particles (Fig 7F).
DISCUSSION
Our studies indicate that the platelet has evolved several
distinct pools of APP. The major form is soluble, carboxylterminus truncated, stored in the a-granule (confirmed by
-
C GPS
Kd
1
2
3
G PS
4
5
6
200 -
97.4 -
I 140-150
I120-1 30
1100-110
69 -
Fig 6. Comparison ofAPP in platelets of GPSand control subjects.
Washed platelets were separated into soluble and membrane fractions. The same amount of protein 150 pgl from control (lanes 1, 3,
and 4) or GPS platelets (lanes 2, 5, and 6) was reduced, boiled, and
resolvedon 8.5% SDS-PAGE followed by Western blotting with
MoAb 22Cll. Lanes 1 and 2, soluble fraction; lanes 3 and 5, membrane fraction; lanes 4 and 6, alkali-treated membranes.The relative
molecular mass of standard protein markers is shown on the left.
APP immunoreactive bands are indicated on the right.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
FORMS
MEMBRANE-ASSOCIATED
Fig 7. lmmunoelectron
microscopic localization of human
platelets with antibodies t o APP,
GPlb, and GMP140. (A) Localization of APP within platelet. Platelets were fixedwith paraformaldehyde and embedded sections
were exposed t o MoAb 2 2 C l l
(l:lOO), then t o biotinylated antimouse IgG, followed by streptavidin-gold conjugate. There is
extensive labeling of the m-granules. (B) Surface immunolabeling of APP onunfixedwhole
platelets adherent t o grids.
Platelets were activated by adhesion. Platelets were reacted
withMoAb22Cll
(1:200) followed by a secondary antibody
conjugated t o 10 nm gold. Gold
particles above background levels were observed diffusely over
theplatelet surface. (C) Same
source of platelets as in (Bl incubated with a polyclonal antibody
t o native APP(1:ZOO). (D) Same
source of plateletsas in (B) incubated with an antibody t o GPlb
(1:ZOOl.There is extensive labeling diffusely over the surface of
platelets. (El Same source of
platelets as in (B) localized with
an antibody t o P-selectin (GMP140,1:ZOO). The label is present
in discrete clusters over the
platelet surface.
(F)
Negative
control with an irrelevant MoAb
t o CD13. Bar, 0.5 pm.
OF PLATELET APP
139
"-l
t.
immunoelectron microscopy. Fig 721) and contains APP species with the KPI don~ain."f'JX This
pool o f APP is available
for rapid release on activation of pl:ttclctsby ;I variety of
a g o n i s t s . ~ ' . ~ f , . ~ 7 . . ' . ' . . l ~ A smaller proportion of the total platelet
pool is APP\,,,,,. which may be divided into two forms o n
the basis of its association with membrnncs under alkaline
conditions.
About 90% of APP,,,.,,,. with molecular mass of 1 0 0 t o
I30 kD. is carboxyl-terminus truncated and can be released
by alkalitreatment. Theseformssharesimilarmolecular
masses with the soluble pool of APP. and appear t o localize
.. .-...
... . .. ....
.;L.:
y
.
.. ...
at least in part on the surface of the platelet. a s suggested
by thesurface-labeling experiments(Fig 4). Theseforms
could be derived from an external source, or more likely are
derived from the truncated
species in a-granules that have
become associated with the plasma membrane after release.
as seen for other a-granule constituents
like thrombospondin.
fibrinogen. and von Willebrand factor.J"-5' The latter possibility is further supportedby the results obtained from platelets
of GPS. where the soluble pool of APP is markedly reduced.
with correspondingly low amounts of APPM,,,,(Fig 6).
Interestingly, the relatively abundant APP\,,,,, 120- to 130-
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140
kD species does not appear to be recognized by MoAb
22C11 when bound to the surface of intact platelets. In this
situation, MoAb 22Cll may only detect APPpL.This is inferred from the finding that the copy number of APP estimated from binding experiments (Fig 5 ) is similar toan
estimate based on a carboxyl-terminus radioimmunoassay
Moreover, the copy number obtained from GPS
platelets that lack appreciable 120-to 130-kD APPM,, is
comparable with that of controls, although this result needs
to be verified infurther GPS subjects. The paucity of MoAb
22Cll immunoreactivity on the platelet surface as judged
by immunogold labeling is also consistent with this concept
(Fig 7). Gardella et alZ9also failed to detect any surface
APP by a less sensitive method of flow cytometry using a
polyclonal antibody against the amino-terminal region close
to the epitope recognized by MoAb 22C11. The masking of
the MoAb 22C 11 epitope could be further explored by using
antibodies to other domains of APP.
Less than 10% of APPM,, in platelets is resistant to alkali
treatment and is immunoprecipitable with antisera raised
against the carboxyl-terminus. This is inferred to be APPFL,
integral to the membrane, with a molecular mass of 140 to
150 M), and responsible for the increased MoAb 22C11
binding sites on the surface of the activated platelet. As
judged by its comigration with APP7s1,the 140- to 150-kD
species is N- and 0-glycosylated, contains a KPI domain,
and is larger than all forms of APPn found in human brain.
These differences in mobility could be caused by differential
glycosylation or an as-yet-undefined additional splicing pattern. There are also trace amounts of APPFL with molecular
mass of 130 kD in the platelet that comigrate with the immature (N-glycosylated) APPT5,. Others2R,29,52
have reported
full-length APP as 140 kD in platelets.
The biologic significance of increased expression of surface APP during activation is uncertain. P-selectin, an integral membrane protein of the a-granule, also undergoes increased expression on the plasma membrane after activation
to a level of about 10,000 copies per platelet.s3 P-selectin
is involved in mediating adhesion of activated platelets to
neutrophils and monocyte^.'^ Although APPFL is present in
low copy number (about 730 copies per activated platelet),
it may also serve as a receptor for intercellular or cell-matrix
interactions. However, the low copy number of surface APP
would make it unlikely to play a major role in cell-cell
adhesion, when compared with the copy number of major
adhesion molecules such as GPIb, where 21,000 copies are
present on the platelet surface.35Another possible function
for the 140- to 150-kDAPPFLmay involve the regulation of
hemostatic protease inhibitory activity on the platelet surface, because it carries the KPI domain. Smith et a145showed
thatthe soluble form of APP can inhibit the activity of
coagulation factor XIa. Studies by others have shown that
the soluble forms of APP with functional KPI domains (also
known as nexin 11), when complexed with a protease, may
be capable of binding to the cell surface and being internali ~ e dThe
. ~ mechanisms
~
involved have not yet been defined.
It is possible that platelet APPMe, may participate in platelet
aggregation. Other studies have shown that APP can bind
to heparan sulfate prote~glycan,~'.~~
laminin,58collagen type
LI ET AL
IV?' and integrin-like receptors,6"and therefore it is likely
that APP is involved in cell-cell or cell-matrix interactions.
However, wehave been unable to showany blocking of
platelet aggregation using a variety of antibodies toAPP
(unpublished data, 1992,1993). Therefore, in this context
surface APP on platelets may subserve some other function
required after the aggregation event.
In contrast to platelets, APPM,, in the brain are predominantly alkali-resistant species that represent integral APPI:L
with molecular mass of 105 to 130 kD. Similar results were
obtained for APPM,, from a pheochromocytoma (PC 12)cell
line. Therefore, the presence of the carboxyl-terminus-truncated APPMem may be characteristic of the platelet. Human
brain lacks readily detectable 140- to 150-kD APPFL species,
but contains boththe
full-length mature and immature
APP751and APP69s(Fig 3). We and others have been unable
to detect APP695species in platelets even though mRNA for
this species has been found to be present.28329
These differences between platelet andbrain APP mayberelevantto
the brain-restricted occurrence of PA4 amyloidosis.
There are circulating forms of APP in p l a ~ m a . * ' ~ The
~'~"~
platelet origin of these forms has been suspected, and the
GPS data presented in this report are consistent with this
concept, where a 7.4-fold decrease in plasma APP was
found. Although activated T cells and macrophages release
APP,24they are an unlikely source of a major proportion of
plasma APP undernormal conditions. InAD plasma, an
increase in the 130-kD APP immunoreactive band has been
identified,h2yetthetotal
amount of plasma APP appears
unchanged. Previously, we found no difference in total APP
immunoreactivities in platelets of AD patients and
In this study, we also found no difference in the amounts of
platelet membrane-associated APP between AD patients and
controls, although subtle differences could easily be missed
by the immunoprecipitation techniques used in the present
study. Further investigations are requiredto elucidate the
altered plasma APP profile in AD.
ACKNOWLEDGMENT
We are indebted to Dr Lindsay Dunlop (Baker Medical Research
Institute, Melbourne, Australia) and Linda Bendall (Department of
Medicine, Westmead Hospital, New South Wales, Australia) for
their valuable technical assistance in the binding and surface-labeling
experiments; to Dr Peter Castaldi (Department of Medicine, Westmead Hospital) for organizing the gray platelet syndrome patient;
and to Dr Renate Kalnins (Austin Hospital, Melbourne, Australia)
for providing brain tissue samples. We are grateful to Dr Jon Currie
andValcy Malone (Mental Health Research Institute of Victoria,
Melbourne, Australia) for coordination of volunteers; to Stephanie
Fuller (Department of Pathology, University of Melbourne, Melbourne, Australia) for preparing the transfected cells; and to Tina
Cardamone for assistance in photography.
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1994 84: 133-142
Membrane-associated forms of the beta A4 amyloid protein precursor
of Alzheimer's disease in human platelet and brain: surface
expression on the activated human platelet
QX Li, MC Berndt, AI Bush, B Rumble, I Mackenzie, A Friedhuber, K Beyreuther and CL Masters
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