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Glycoprotein IIIa Is Phosphorylated in Intact Human Platelets
By Leslie V. Parise, Anne B. Criss, Lisa Nannizzi, and Mark R. Wardell
The glycoprotein Ilb-llla complex (GP Ilb-llla) is a multifunctional transmembrane protein on platelets. Its most completely described function is as a fibrinogen receptor that
mediates platelet aggregation, but it is also involved in clot
retraction, signal transduction, calcium transport, and
other events. However, the mechanisms that regulate the
functions of GP Ilb-llla during platelet activation are largely
unknown. One possible mechanism is phosphorylation,
since several other receptors are regulated by this process.
We found that GP Illa, but not GP Ilb, was phosphorylated
in 32P-labeled platelets, predominantly on threonine residues. Furthermore, GP llla phosphorylation increased four-
fold in platelets activated with thrombin or phorbol 12myristate 13-acetate, but not at all in platelets treated with
prostacyclin, an inhibitor of platelet activation. The thrombin-induced increase in phosphorylation was inhibited by
pretreating platelets with prostacyclin or with staurosporin, a specific protein kinase C inhibitor. Thus, there is
an increase in the level or turnover of phosphate on GP llla
during platelet activation, most likely involving protein
kinase C. This phosphorylation may regulate some aspect(s) of GP Ilb-llla function.
0 1990 by The American S o c i e t y of Hematology.
G
mechanism by which activation-induced changes in G P
IIb-IIIa function could be regulated.
LYCOPROTEINS IIb A N D IIIa are abundant integral membrane proteins on platelets that exist as
Ca2+-dependent heterodimer complexes, termed glycoprotein (GP) IIb-IIIa.'~*Glycoprotein IIb, the a-subunit (molecular weight [mol wt] 140,000), consists of a heavy chain
(mol wt 120,000) disulfide-linked to a light chain (mol
wt = 25,000); G P IIIa, the @-subunit,is a single polypeptide
of mol wt i~ 105,000.2Recent cloning and sequencing studies
have established that G P IIb-IIIa is a member of a large
family of adhesive receptors, the integrins (reviewed in
reference 3). These studies also predict that G P IIIa and the
light chain of G P IIb each contain a membrane-spanning
region near its carboxy t e r m i n ~ s , ~a . ~short cytoplasmic
domain (20 and 41 amino acids, respectively), and a much
larger extracellular component.
The G P IIb-IIIa complex contributes to several platelet
functions, most of which are acquired during platelet activation. For example, it acquires the ability to bind fibrinogen,6>'
thereby mediating platelet aggregation during thrombus
formation. In addition, G P IIb-IIIa is believed to mediate
subsequent clot retraction by bridging the extracellular fibrin
network to the contractile apparatus of the latel let.^,^ The
complex also plays a role in signal transduction in stimulated
platelets; for example, in tyrosine-specific protein phosphorylation" and in N a + / H + exchange across the plasma
membrane." Furthermore, G P IIb-IIIa contributes to platelet adhesion to the subendothelium" and acts as a passive
Ca2+tran~p0rter.I~
Although G P IIb-IIIa has multiple functions, the mechanisms that regulate them are largely unknown. Since phosphorylation regulates the functions of other receptors, such
as the @-adrenergic, insulin, and epidermal growth factor
receptors (reviewed in reference 14), we investigated whether
any components of the G P IIb-IIIa complex are phosphorylated. The present study shows that G P IIIa is phosphorylated, primarily on threonine residues, and that the phosphorylation or turnover of phosphate increases dramatically in
thrombin- or phorbol 12-myristate 13-acetate (PMA)activated platelets. The agonist-induced phosphorylation is
inhibited by prostacyclin (PGI,), an inhibitor of the intracellular events leading to platelet a ~ t i v a t i o n . 'In
~ addition,
evidence is provided that G P IIIa phosphorylation in platelets is catalyzed by protein kinase C (PKC). This study
clearly implicates phosphorylation of G P IIIa as a potential
-
-
Blood, Vol 75,No 12 (June 15). 1990:pp 2363-2368
MATERIALS AND METHODS
Platelet preparation. Human platelets were obtained on the day
of use and washed as previously described16 but in the absence of
PGI, and EDTA, and were resuspended to 1 to 2 x lo9platelets/mL
in buffer A (12 mmol/L NaHCO,, 138 mmol/L NaCl, 5.5 mmol/L
glucose, 2.9 mmol/L KCI, 10 mmol/L HEPES, pH 7.4). The
platelet suspension was incubated with 1 mCi of H,[32P]04(ICN
Biomedicals, Inc, Irvine, CA)/mL of platelets at room temperature
for 30 minutes, sedimented at 730g for 10 minutes, and resuspended
to 0.7 to 0.8 x 10' platelets/mL in buffer A with 0.36 mmol/L
NaH,P04 H,O. The platelets were then incubated at 37OC without
stirring for the times and at the concentrations of agents indicated in
the figure legends. The concentrations of agents were chosen on the
basis of their ability to induce either maximal aggregative or
inhibitory responses. The concentrations of PGI, were chosen
according to their ability to inhibit platelet aggregation induced by
0.1 U of thrombin/mL. These concentrations varied with platelets
from different donors and ranged from 60 to 370 nmol/L. The actual
phosphorylation experiments with thrombin and PMA were done
with activated, aggregation-competent platelets but without stirring
and, therefore, without aggregation per se, so that GP IIb-IIIa could
be quantitatively and reproducibly immunoprecipitated.
Immunoafinity chromatography. Approximately 1.2 x 1O9
platelets were incubated at 37OC with platelet agonists, antagonists,
or appropriate buffers, as indicated in the figure legends and text.
The incubations were terminated by diluting the sample with an
From the Gladstone Foundation Laboratories for Cardiovascular
Disease, Cardiovascular Research Institute. University of California at San Francisco, San Francisco, CA; and ihe Department of
Pharmacology and Centerfor Thrombosis and Hemostasis, School
of Medicine, University of North Carolina, Chapel Hill, NC.
Submitted November 8, 1989; accepted February 27. 1990.
Supported by FIRST Award No. R29HL38405 and Grant No.
1989-91-A-07 from the American Heart Association-NC aflliate
(L.V.P.).
Address reprint requests io Leslie V. Parise. PhD. Department of
Pharmacology, CB #7365, Faculty Laboratory Ofice Building,
University of North Carolina, Chapel Hill. NC 27599-7365.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7512-0027$3.00/0
2363
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PARSE ET&
2364
q u a l volume of icecold lysis buffer (2% Triton X-100.20 mmol/L
Tris. 2 mmol/L sodium metavanadatc. 40 mmol/L molybdic acid.
80 mmol/L sodium pyrophosphate. 4 mmol/L EGTA. 0.2 mmol/L
trifluoperazine. 0.2 mmol/L leupeptin. 2 mmol/L phcnylmethylsulfonyl fluoride. 40 mmol/L KH,PO,. pH 7.2). For platelets treated
with thrombin. 0.2 U of hirudin/mL was also included in the lysis
buffer.
The lysate was antrifuged for I5 minutes a t 31.OOOg to sediment
the platelet cytoskeleton. Glycoprotein Ilb-llla was purified from
the supernatant by preclearing on a 0.2-mL glycine-Sepharose
column (glycine 1 I mg/mL] covalently coupled to CNRr-activated
Sepharose ISigma. SI Louis. MO])followed by adsorption onto a
0.4-mL column of A,A.-Scpharosc (AIAI is a specific anti-GP
Ilb-ll la monoclonal antibody" supplied by Dr Joel Bennett. and was
coupled at I mg/mL of CNBr-activated Sepharox). The column
was washed with 23 column volumes of buffer R ( I50 mmol/L NaCI.
O.I%Triton X-100.0.02%NaN,. ZOmmol/LTris.pH 7.4).TheGP
Ilb-llla was eluted with a buffer of 0.1%Triton X-100. 2 mmol/L
CaCI,. 0. I mol/L glycine, pH I I .3. and each fraction was collected
into 0.5 volume of neutralization buffer (0.1% Triton X-100. 2
mmol/L CaCI,. I mol/L Tris. pH 6.6). Glycoprotein Ilb-llla was
precipitated from I mL of each fraction with trichloroacetic acid
(I2'X-).resuspendedinZIOpLofbufferR.andmixcdwith95pLofa
modified Laemmli sample buffer (32.3% glyarol Ivol/vol]. 6.5%
sodium dodecyl sulfate (SDS) [wt/vol]. 0.01% bromphenol blue
Iwt/vol]. 0.04 N NaOH. 0.2 mol/L Tris [final concentration. using
I mol/L stock Tris. pti 6.8)) with or without 16%8-mercaptoethano1 (vol/vol). and heated a t loooC for 5 minutes before being
subjected to electrophoresis.'' Gels for autoradiography were dried
immediately; the proteins were neither fixed nor stained.
Imnrunopncipirarion. For immunoprecipitation. I mL of lysate was centrifuged at 12.800g for 15 minutes at 4%. and the
supernatant was added to 50 pL of glycine-Sepharose (resuspended
I : I in buffer B) and nutated at 4% for I hour. The lysate was again
antrifuged at 1 2 . 8 0 0 ~for I minute to sediment the glycineSepharose. The lysate supernatant was then added to 50 p L of the
anti-GP Ilb-llla monoclonal antibody. A,&, coupled to Sepharose.
and nutated at 4% for 3 hours. The h,A&pharosc
was xdimented
as described above. and the supernatant was discarded. The A,&Sephanrse was washed with an additional 1.25 mL of buffer R and
rescdimented. The CP Ilb-llla was released by muspending the
-
-1GPllb-
2
3
r
A,A,-Scpharosc in 0.25 mL of the pH 11.3 elution buffer and
recovering the supernatant.
Phmphwmino acid analysis. Glycoprotein Ilb-llla was ilated by immunoaffinity chromatography a s described above. exthat I .5 to 2 x IO' lyscd platelets and a 0.8 to I mL A,A,-Sepharosc
column were used. Approximately 2.4 mL of the immunoafinitypurified GP Ilb-llla was precipitated with trichloroacetic acid
( 1 2%). redissolved with 400pL of a modified Laemmli sample buffer
( 10.9% glycerol [vol/vol]. 2.2% SDS Iwt/vol]. 5.5% 8-mercaptoethanol [vol/voll. 0.002% bromphenol blue [wt/vol]. 0.14 N NaOH.
and 0.07 mol/L Tris [final concentration. using I mol/L stock Tris.
pH 6.81). and heated to loooC for 5 minutes.'" After electrophoresis
on a preparative 7.5% gel. GP llla was electrocluted at 100 V for 3
hours into IS mmol/L ammonium carbonate and 0.1% SDS (wt/
vol). pH 8.2. To remove the SDS. the electroeluted protein was
precipitated with IS volumes of I% (vol/vol) HCI in acetone at
-20% for 2 hours. and the precipitated GP llla was pellcted by
low-speed antrifugation. The pellet was washed in HCl/acetone
and ramtrifuged. and the acetone-supernatant was discarded. The
washed pellet was dissolved in 6 N HCI. sealed in vacuo. and
hydrolytnd for 2 hours at I IOOC. The hydrolysates were dried over
solid NaOH in vacuo. dissolved in 80 pL of electrophoresis buffer
( I % pyridine Ivol/vol]. 10% glacial a a t i c acid [vol/vol]. pH 3.5).
and then electrophoresed along with phosphoserine. phosphothnw
nine. and phosphotymine standards on Whatman / I chromatography paper for 1.25 hours at 3 kV in the electrophoresis buffer
described above. The p i t i o n s of the standards were determined
after staining with 0.2% ninhydrin (wt/vol) in acetone. and the
"P-labeled amino acids were identified after autoradiography.
RESULTS
To determine whether GP Ilb or GP llla was phosphorylated in intact human platelets, washed platelets were labeled
with H,['*PJO,
and lyscd with an icc-cold bufTcr containing
Triton X-I00 and inhibitors of phosphatase and kinase
activity. After removal of the cytoskeleton. GP Ilb-llla was
purified from the lysate by immunoafinity chromatography
with A2A, a monoclonal antibody that recognizes the GP
Ilb-llla complex." The purified material was examined on
4
5
'
I
.
L
Fbl. M " t h o f p b @ o q h d
II
I
I
Caomassie 32p
I
1251
3ip
I
1251
QPnbtrom t"
I-WWMQ+
p
w
w OP nb-llb from "P-(.kkd plat.kn
was olutrophorosod am 7.6% SDS-PO)Y.cry(.mido gds run undu rodudng llanos 1
through 3)or nonrodudng conditions llama
4 and 6). Lano 1. g d s t a i d with Coocruuk
Brilliant Bluo; b m2 and 4. autoradiograms
showing that OP Ilk. but not GP Ilb. m s
phosphorylatod; lanos 3 and 6, autoradiograms of '%I.a(od t3P Ilb and OP Ilb
standards. imolatad as proviously dastxibod." -0th
Itb was oborvod t o
bo phor9hwy(.tod
rogmrdloas of whothor it
m s i h t o d from control platdots or plat+
lots t r u t o d with thrombin, M A , or PO4
(Fig 2): tho aramplos in knos 2 and 4 aro
from P M A - t r u t o d pbtolots.
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2365
G P l l l ~PHOSPHORYLATION IN HUMAN PLATELETS
SDS gels by Coomassie staining and autoradiography (Fig
1). The purified material was essentially homogeneous (Fig
1, lane 1). Only one component of the complex was phosphorylated. This was identified as G P IIIa (lanes 2 and 4) on the
basis of its co-electrophoresis with iodinated G P IIIa (lanes 3
and 5) from the purified G P IIb-IIIa complex.
The possibility that G P IIIa was phosphorylated only as a
consequence of detergent lysis of platelets was eliminated by
two experiments. First, "P-labeled platelets were lysed in the
presence of excess unlabeled adenosine triphosphate ( A T P
0.5 mg/mL); the unlabeled ATP would be expected to
inhibit phosphorylation of G P IIIa that might occur after
lysis and the release of cytoplasmic [32P]ATP.No inhibition
of G P IIIa phosphorylation by excess unlabeled ATP was
observed (data not shown). Second, when unlabeled platelets
were lysed in the presence of [32P]ATP,no phosphorylation
of G P IIIa was observed (data not shown). Thus, phosphorylation occurs before lysis, most likely on the cytoplasmic
domain of G P IIIa.
To determine whether G P IIIa underwent increased phosphorylation when platelets were activated, "P-labeled platelets were exposed to the agonists thrombin or PMA. Thrombin caused a 4.1-fold increase in G P IIIa phosphorylation
within 3 minutes, followed by a steady decrease (Fig 2A).
The time course of G P IIIa phosphorylation was similar to
other events that occur under similar conditions during
platelet activation, eg, the expression of platelet fibrinogenbinding activity." Further, the fold-increase was comparable
with that of other platelet proteins whose functions are
regulated by phosphorylation (see Discussion). As shown in
Fig 2B, PMA, an agent that directly activates PKC," caused
an increase in G P IIIa phosphorylation similar to that
induced by thrombin (4.3-fold over the control value a t 5
minutes). This was also followed by a decrease. The decrease
in phosphorylation shown in Fig 2A and B was specific for
G P IIIa; it did not occur with total precipitated phosphoproteins (data not shown). Therefore, the decrease probably
represents a loss of phosphate from G P IIIa, rather than an
exchange with unlabeled phosphate.
The changes in G P IIIa phosphorylation shown in Fig 2
were also apparent when G P IIIa phosphorylation was
measured in separate experiments as the amount of '*P
incorporated per milligram of G P IIIa. In those experiments,
32P-labeled platelets were treated for 3 minutes with thrombin or 5 minutes with PMA. After lysis, relatively large
amounts of G P Ilb-IIIa were isolated from these platelets by
immunoaffinity chromatography. The milligram amount of
GP IIIa was quantitated from Coomassie-stained gels versus
a standard curve of G P IIIa on the same gels, and the amount
of 32P was quantitated by densitometry of G P IIIa on
autoradiograms. When the results were expressed as the
amount of 32P/mg of G P IIIa, we found that thrombin
induced a 2.5-fold increase and PMA induced a 4.5-fold
increase in G P IIIa phosphorylation relative to the control.
The responses of platelets to agonists are inhibited by
agents such as PGI, that elevate cyclic adenosine monophosphate (AMP) levels within the cytosol. The basis for the
inhibition is presumably related to the phosphorylation of
certain platelet proteins by cyclic AMP-dependent protein
Thrombin
80
401
>-
m
L, ,
c
0.5 1
r
Control
#
3
5
10
3
6
Control
401
9)
""1
-0.5 1
3
5
1'0
TIME (minutes)
Fig 2. Time course of "P incorporation into GP llla in intact
platelets. Washed, "P-labeled platelets were incubated at 37°C
with either (A) human thrombin (0.1 UlmL: 50 U/mL stock in 0.75
mol/L NaCI, 8.3 mmol/L polyethylene glycol BOOO, 10 mmol/L Tris.
pH 7.4). ( 6 )PMA (10 pmol/L; 5 mmol/L stock in dimethylsulfoxide), or (C) PGI, (60nmol/L; 0.14 mmol/L stock in 0.1 mol/L NaCI.
50 mmol/L Tris, pH 12) for the indicated times. Control samples
for each treatment were incubated with the buffer alone. Upon
completion of each incubation, the platelets were lysed with
ice-cold lysis buffer and GP Ilb-llla was isolated by immunoprecipitation (see Materials and Methods). The relative amount of "P
incorporated into GP llla was quantitated by densitometry of
autoradiograms of 7.5% polyacrylamide gels containing the immunoprecipitated and reduced glycoproteins. Densitometrywas done
under conditions of linear exposure of the autoradiogram. The
data shown are representative of two (A) or three (B and C)
experiments per treatment. Each data point is en average of
duplicate samples.
kinase.,' When resting platelets were treated with PGI,, G P
IIIa phosphorylation was not induced (Fig 2C), suggesting
that cyclic AMP-dependent protein kinase was not responsible for phosphorylating G P IIIa. In a separate experiment,
PGI, did inhibit the thrombin-induced phosphorylation of
GP IIIa, indicating that the agonist-induced phosphorylation
is on the stimulus-response pathway of platelets. (In this
experiment, pretreatment of platelets with PGI, (370 nmol/
L) for 1 minute before the addition of thrombin (0.1 U/mL)
7.5% (SE, n = 3) inhibition of the
resulted in a 72%
thrombin-induced increase.)
To determine which amino acid(s) was phosphorylated,
32P-labeled GP IIIa was electroeluted from a preparative
SDS-gel, partially hydrolyzed with acid, and subjected to
high-voltage paper electrophoresis under conditions that
separated P-serine, P-threonine, and P-tyrosine. Regardless
of whether platelets were treated with PMA, thrombin, or
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PARIS ET AL
2366
b u l k (ie. control). threonine was observed to be the major
"P-labcled amino acid residue (Fig 3). A low level of serine
phosphorylation was alsoobserved (Fig 3. lanes I and 3). but
no tyrosine phosphorylation was apparent.
Rccause PMA directly activates PKC and induces GP l l l a
phosphorylation. we investigated whether this enzyme participated in the thrombin-induced phosphorylation of GP I 1 la.
since thrombin is a physiologic platelet activator.'? I n thcsc
experiments. platelets were pretreated with staurosprin. an
inhibitor of PKC." The concentration of staurosprin ( I
pmol/l.) used was a minimum value. selected because i t was
just high enough to maximally inhibit the thrombin-induced
phosphorylation of P47. a known substrate of PKC in
platclcts" (Fig 4A). Phosphorylation of P47 was inhibited
e,- -.'
1
2
-
Thrombin
PMA
3
P-serP-TtU-
P-Tyt-
Control
Fig 3.
odd analpis of t3P ))(. hd.1.dtrom
" P - ~ a k k d piatekts. a m o t o i n IIIJ trom = ~ - t a k t e dp ~ ~ ~ o
was subjected to partial amino acid hydrolysis followed by highvoltage paper electrophoresis u n d a conditions that separated
P-serine I P - S r ) . P-threonine IP-Thr), and P-tyroshe (P-Tyr). The
positions of those amino acids were determined by ninhydrin
staining of unlabeled phosphory(.ted standards that had been
added to each sample before elmxrophoresis. Autoradiography of
the papor electrophoretogram showed that threonine was the
major phosphorylated amino add from contrd. thrombin-treated.
or PMA-treated platelots. P, inorganic phosphate.
under conditions in which many other platelet proteins
remained phosphorylated. even at relatively high concentrations of staurosprin (Fig 4A. inset). supporting the relative
specificity of this inhibitor. This finding contrasts with results
obtained with sphingosine. another inhibitor of PKC": at a
concentration that minimally inhibited P47 phosphorylation.
sphingosinecauscd the disappearance of many other phosphorylated bands in a total platelet lysate (data not shown).
When '-'P-labeled platelets were pretreated with staurosporin. the thrombin-induced phosphorylation of GP I 1 la
was completely inhibited (Fig 4R)-cvidencc that PKC
phosphorylatesGP I1l a in thrombin-stimulated platelets.
DISCUSSION
This study shows that GP l l l a of the GP I l b - l l l a complex
is phosphorylated. primarily on threonine residues. in intact
human platelets. Activation of platelets with the physiologic
agonist thrombin dramatically increases this phosphorylation. whereas pretreatment of platelets with PGI,. an inhibitor of platelet activation. inhibits this increase. The agonistinduced increase in GP l l l a phosphorylation appears to be
mediated by PKC. Thus. the activation-induced phosphorylation or turnover of phosphate on GP l l l a is a potential
regulatory mechanism for one or more of the many functions
associated with the GP I l b - l l l a complex in platelets.
Four lines of evidence suggest that PKC is the likely
mediator of GP l l l a phosphorylation. ( 1 ) Thrombin and
PMA. which inducc GP l l l a phosphorylation. are known to
activate PKC in platelets." (2) The relatively specific inhibitor of PKC. staurosprin (which has been used to identify
PKC-mediated events in
completely inhibits the
thrombin-induced phosphorylation of GP I l l a . (3) PGI,.
which activates the cyclic AMP-dependent protein kinase.
docs not induce GP l l l a phosphorylation. eliminating this
enzyme as a possible candidate. (4) The amino acid residues
that become phosphorylated are threonine (and. to a lesser
extent. serine). and PKC is a serine-threonine-specific
entyme.-" The putative cytoplasmic domain of GP l l l a does
contain two threonine residues that are especially likely
targets for PKC-mediated phosphorylation: residua 758 and
762 (see reference 4 for the complete amino acid sequence of
GP llla).""' Precisely which residuc(s) is phosphorylated
remains to be determined.
Several lines of evidence suggest that the agonist-induced
phosphorylation of GP l l l a has the potential to regulate GP
I l b - l l l a function in platelets. First. the time course of the
increased phosphorylation is comparable with the time course
of certain changes in function related to GP I l b - l l l a : for
example, fibrinogen binding to platelets.'' tyrosine-specific
protein phosphorylation in thrombin-stimulated pla~clets.'~
and the redistribution of GP I l b - l l l a on the surface of
t s
activated platelets." Second. the magnitude of the change in
GP l l l a phosphorylation is roughly comparable with that of
other platelet proteins whose functions are affected by
phosphorylation. Some examples are the 2.5-fold increase in
myosin light chain phosphorylation in thrombin-treated
platelets." which enables actin to cause myosin-ATPase
activation": the twofold to threefold increase in phosphorylation of actin-binding protein in prostaglandin E,-treated
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GPIIIA PHOSPHORYLATION IN HUMAN
PLATELETS
2367
Fig 4. Inhibition of thrombin-stimulated P47 and GP IIla phosphorylation by staurosporin. (AI UP-labeled platelets w e r e treated for 10
minutes a t 37°C with increasing concentrations of staurosporin to determine the minimum concentration that maximelly inhibited P47
phosphorylation. The inset shows t h e phosphorylation pattern of whole platelet lysates from UP-labeled platelets. N o t e the number of
proteins that remain phosphorylated a t concentrations of staurosporin that maximally inhibit P47 phosphorylation. The graph showa the
amounts of P47 phosphorylation in t h e presence of increasing concentrations of staurosporin, as determined by densitometry. Based on
this experiment, a concentration of 1 pmol/L staurosporin was selected for further study. (B) UP-labeled platelets were pretreated for 10
minutes a t 37°C with 1 pmollL staurosporin or solvent alone (dimethylsulfoxide) followed by thrombin (0.1 U/mLl for 3 minutes. The
broken line indicates the basal level of phosphorylation in untreated platelets (23.7%). Staurosporin inhibited the thrombin-induced
phosphorylation by 104.6% 1 6.6% (SE, n = 4).
platelets t h a t is reported t o stabilize this protein against
calpain hydrolysis": a n d t h e 1.4-fold increase in G P Ib,
phosphorylation t h a t inhibits actin polymerization.'' Third.
agonist-induced phosphorylation w a s inhibited by PGI,. a n
agent t h a t also inhibits agonist-induced changes in platelet
function.
W h e n platelets a r e activated. GP I l b - l l l a is converted
from a form that c a n n o t bind extracellular fibrinogen into a
form t h a t can." In addition, undefined activation-induced
events cause t h e intracellular d o m a i n of t h e G P Ilb-llla
complex lo bind lo the cytoske1eton.x The critical imporlance
of these changes in t h e function of GP I l b - l l l a is exemplified
by Glanzmann's thrombasthenia. a hereditary bleeding disorder. PhtClCtS from these patients have either lOW amOUntS Of
or functionally a b n o r m a l G P Ilb-llla,'x~'" resulting in t h e
absence of agonist-induced platelet aggregation o r clot r e
traction. Despite t h e importance of agonist-induced changes
in G P I l b - l l l a , very little is known a b o u t t h e intracellular
mechanisms t h a t regulate them. The present study shows an
activation-induced chemical modification of G P l l b l l l a .
Further studies are necessary t o assess t h e consequence of
G P llla phosphorylation on t h e numerous functions associated with G P llb-llla.
ACKNOWLEDGMENT
We thank Dr Joan Fox for many hclprul suggalions: Kerry
Humphrey for manuscript preparation; A] Averbach and Sally
Gullat1 Seehafer for editorial assistance; and Charles Benedict for
graphics. wc also thank Dr Joel Bcnnett (University of Pennsylvania) and Dr John Fenton I ] (New York State Department o f H a ] t h .
Albany, NY) for their generous gifts ofA:A,and human thrombin.
rcspectivcly.
REFERENCES
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Ca . '-dependent complexa of platelet membrane glycoproteins Ilb
and Ilia in solution as determined by crossed immunoelectrophorcsis. Blood 58:268. I98 I
2. Jcnnings LK. Phillips DR: Purification ofglycoprotcins Ilband
I I I from human platelet plasma membranes and characteri7ation of
a calcium-dependent glycoprotein Ilb-Ill complex. J Biol Chcm
257:10458. 1982
3. Phillips DR, Charo IF, Parise LV. Fitzgerald LA: The platelet
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4. Fitzgcrald LA. Steincr B, Rall S C Jr. Lo S-S, Phillips DR:
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5 . Poncz M. Eisman R. Hcidcnrcich R. Silver SM. Vilaire G.
Surrey S. Schwartz E. Bennett JS: Structure of the platelet
membrane glycoprotein Ilb. Homology to the Q subunits of the
vitronectin and fibronectin membrane receptors. J Biol Chem
26223476. 1987
6. Bennett JS. Vilaire G:Exposure of platelet fibrinogen receptors
by ADP and epinephrine. J Clin Invest 64:1393. 1979
7. Parise LV, Phillips DR: Reconstitution of the purified platelet
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8. Phillips DR. Jennings LK, Edwards HH: ldcntification of
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Cell Biol86:77. 1980
9. Cohen I. Gerrard JM. White JG: Ultrastructureofclots during
isometriccontraction. J Cell Biol93:775. 1982
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1 1 . Banga HS. Simons ER. Brass LF. Rittenhouse SE: Activation of phospholipasa A and C in human platelets exposed to
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1990 75: 2363-2368
Glycoprotein IIIa is phosphorylated in intact human platelets
LV Parise, AB Criss, L Nannizzi and MR Wardell
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