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Dependence of Plasmin-Mediated Degradation of Platelet Adhesive Receptors
on Temperature and Ca2+
By Kenneth J. Winters, Paul R. Eisenberg, Allan S. Jaffe, and Samuel A. Santoro
The effects of activation of plasminogen by streptokinase
and tissue-type-plasminogen activator on platelet activation and the membrane glycoproteins (GPs) that mediate
platelet adhesion and aggregation are not yet fully defined.
To clarify effects on platelets during activation of plasminogen in vitro, w e used monoclonalantibodies (MoAbs), flow
cytometry, and platelets surface-labeled with lZ61 to characterize changes in receptors for fibrinogen (GPllb-llla), von
Willebrand factor (GPlb), and collagen (GPla-Ha). Activation
of plasminogen in plasma with pharmacologic concentrations of plasminogen activators did not degrade GPllb-llla
or GPlb. and caused only a modest decrease in GPla. In
washed platelets GPllb-llla was extensively degraded by
plasmin at 37°C in the absence of exogenous Ca2+, conditions that destabilize the Ilb-llla complex. Degradation of
GPlb in washed platelets displayed a similar although
less-marked dependence on temperature and the absence
of Ca2+. The binding of activation-specific MoAbs did not
increase during activation of plasminogen in plasma. We
conclude that during pharmacologic fibrinolysis, reported
inhibition of platelet function in plasma is not due to
degradation of platelet-adhesive receptors. In addition,
platelet activation observed during thrombolytic therapy
does not appear to be a direct consequence of plasminogen
activation.
0 1990 by The American Society of Hematology.
P
logic activation of plasminogen directly degrades plateletadhesive receptors has direct clinical implications.' In addition, since platelet activation during fibrinolytic therapy may
be due to factors other than the direct action of plasmin on
platelets, it is important to determine whether activation of
plasminogen directly activates platelets.'8-27
Accordingly, the present study was designed to characterize the direct effects of pharmacologic activation of plasminogen on the platelet-surface GP receptors involved in platelet
adhesion and aggregation. This was accomplished by analysis of the binding of monoclonal antibodies (MoAbs) specific
for these receptors with flow cytometry, and by definition of
alterations in platelet-membrane adhesive GPs with platelets
surface-labeled with Iz5I.Specifically, the effects of activation of plasminogen by pharmacologic and suprapharmacologic concentrations of streptokinase (SK) and tissue-type
plasminogen activator (t-PA) were characterized for the
platelet-fibrinogen receptor (GPIIb-IIIa), the vWF receptor
(GPIb), and the collagen receptor (GPIa-IIa).
Our results suggest that minimal plasmin-mediated proteolysis of these platelet-adhesive receptors occurs after pharmacologic activation of plasminogen in plasma.28In addition,
flow cytometry studies performed with activation-specific
MoAbs show no evidence of direct platelet activation during
activation of plasminogen with t-PA or SK. However, in
washed platelet preparations, degradation of GPIIb-IIIa and
GPIb by plasmin is dependent on both temperature and the
absence of added Caz+.
LATELET ADHESION and aggregation are integral
events in the initiation of acute myocardial infarction
(AMI) and modulate both the initial response to fibrinolytic
therapy and the propensity for reocclusion after successful
clot lysis.'-4 Inhibition of platelet aggregation accelerates
initial clot lysis and delays or prevents reocclusion in animal
models of thrombosi~.~*'~
The beneficial effects of adjunctive
therapy with aspirin in clinical studies suggest that similar
effects may occur in patients as we11."~'2Despite evidence
that inhibition of platelet function improves the success of
fibrinolytic therapy, the extent to which activation of plasminogen directly activates platelets or degrades the surface
glycoproteins (GPs) mediating platelet adhesion and aggregation has not been well defined.
Although there is evidence for platelet activation during
thrombolysis in vivo, studies of the effects of plasmin on
platelet function in vitro have yielded conflicting results."-16
Fibrinogen-dependent platelet aggregation and ristocetininduced platelet agglutination can be inhibited by plasmin,
perhaps due to proteolysis of platelet-membrane-adhesive
GPs. However, platelet inhibition in aggregometry studies
may reflect the antiaggregatory effects of fibrinogen and von
Willebrand factor (vWF) degradation products rather than
direct plasmin-mediated proteolysis of platelet-surface
receptors.''-l9 Because platelet dysfunction following thrombolytic therapy may contribute to bleeding complications,
clarification of the circumstances under which pharmacoFrom the Cardiovascular Division and the Division of Laboratory
Medicine, Washington University School of Medicine, St Louis,
MO.
Submitted March 12,1990; accepted June 1 1 , 1990.
Supported by SCOR in Ischemic Heart Disease HL-17646 and
HL-40506. S.A.S. is an Established Investigator of the American
Heart Association.
Address reprint requests to Samuel A . Santoro. MD, PhD,
Division of Laboratory Medicine. Box 8118, 660 S Euclid Ave.
Washington University School of Medicine. St Louis. MO 631 10.
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.
0006-4971/90/7608-0005$3.00/0
1546
MATERIALS AND METHODS
Materials. The plasminogen activators studied were SK (KabiVitrum, Stockholm, Sweden) and single-chain recombinant t-PA
(Genentech, San Francisco, CA). Human plasminogen was purchased from Enzyme Research Labs (South Bend, IN) and '"I was
obtained from Amersham (Arlington Heights, IL). D-phenylalanylL-prolyl-L-arginine chloromethylketone (PPACK) and lactoperoxidase were purchased from Calbiochem (La Jolla, CA). Reagents for
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) were obtained from Biorad Labs (Richmond, CA), and
prestained molecular weight standards were purchased from Bethesda Research Labs (Gaithersburg, MD). Adenosine diphosphate
(ADP) was obtained from Helena Labs (Beaumont, TX). The
and
chromogenic substrate S-2251 (D-Val-Leu-Lys-p-nitroanilide)
Blood, Vol76, N o 8 (October 15). 1990: pp 1546-1557
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DEGRADATION OF PLATELET ADHESIVE RECEPTORS
human plasmin were purchased from KabiVitrum. All other reagents were from Sigma (St Louis, MO).
Preparation of platelets. Platelets were obtained from normal
volunteers who gave written informed consent and were not taking
any medications known to interfere with platelet function. Antecubital venipuncture was performed with a 19-gauge needle after
application of a light tourniquet. Whole blood was anticoagulated
with either 1/10 vol of 3.8% (wt/vol) trisodium citrate, pH 7.4, or
acid citrate dextrose (ACD, 0.038 mol/L citric acid, 0.075 mol/L
trisodium citrate, 0.14 mol/L a-D-glucose), pH 4.5. Heparin sodium
(1 U/mL) or ethylenediaminetetraacetic acid (EDTA, 10 mmol/L)
was used for anticoagulation in several studies. Neither ACD nor
EDTA was used in experiments designed to assess the effects of
activation of plasminogen or platelet activation. Platelet-rich plasma
(PRP) was isolated after centrifugation of anticoagulated whole
blood at l5Og for 15 minutes. For some studies autologous plateletpoor plasma was prepared by centrifuging citrated PRP at 1500g for
10 minutes.
To prepare washed platelets for flow cytometry studies, an
additional 1/10 vol of ACD and PGE, (10 pmol/L) were added to
the PRP, and the platelets were pelleted at 1,500g for 10 minutes.
Platelets were resuspended in 20 mL of washing buffer (0.14 mol/L
NaCI, 2.7 mmol/L KCI, 12 mmol/L NaHCO,, 0.5 mmol/L
Na,P04, 5.5 mmol/L a-D-glucose, 5.0 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid [HEPES], 1/10 vol ACD, IO
pmol/L PGE,, pH 6.5, [Tyrode-HEPES wash buffer]) and centrifuged at 1,500g for 10 minutes. The initial wash was repeated and
the platelets were resuspended in TyrodeHEPES buffer (no ACD or
PGE,, no divalent cations), pH 7.4. For specific experiments 2
mmol/L CaCI, was added as described.
For studies with platelets surface-labeled with "'I, platelets were
pelleted from PRP as described above and washed twice by centrifugation with 20 mL of the following buffer: 0.15 mol/L NaCl, 0.05
mol/L Tris, 1/10 vol ACD, 10 pmol/L PGE,, and 5.5 mmol/L
a-D-glucose, pH 6.5 (TBS wash buffer). Platelets were then resuspended in TBS wash buffer and surface-labeled with "'I by the
lactoperoxidase method." Surfacelabeled platelets were then washed
two more times by centrifugation with TBS washing buffer and
resuspended in autologous citrated plasma or Tyrode-HEPES buffer
(no ACD or PGE,), pH 7.4. Incubations in buffer were performed in
the presence or absence of 2 mmol/L CaCl,.
Plasminogen was activated in citrated PRP with pharmacologic
and suprapharmacologic concentrations of SK (1,000 or 20,000
U/mL) or t-PA (2.5 or 20 pg/mL). For studies of platelets
surface-labeled with Iz5I,the platelets were resuspended in autologous platelet-poor plasma. Washed platelets were incubated with the
same doses of SK or t-PA in Tyrode-HEPES buffer supplemented
with 21 pg/mL of human plasminogen in the presence or absence of
2 mmol/L CaCl,. Control incubations included plasminogen and a
matched volume of t-PA (0.2 mol/L arginine phosphate, pH 7.2) or
SK (0.9% NaCI) buffer. In studies of both washed platelets and
PRP, activation of plasminogen was accomplished at 23O or 37OC for
up to 60 minutes. The platelet count was 1 to 2 x 108/mL during
incubations with plasminogen activators.
In studies of platelets surface-labeled with '"I, incubations were
terminated by the addition of 5 pmol/L PPACK and 200 KIU/mL
aprotinin. After 10 minutes, 1/10 vol ACD was added and the
platelets were pelleted for 2 minutes at 12,OOOg. The supernatant
was removed by aspiration and the platelet pellet was dissolved by
boiling for 10 minutes in electrophoresis gel buffer (0.063 mol/L
Tris, 2% SDS, 10% glycerol, 0.001% wt/vol bromphenol blue) with
or without 1.0% 2-mercaptoethanol. Samples were analyzed on 6%
polyacrylamide gels (SDS-PAGE) by the method of Laemmli.MThe
gels were dried under vacuum and autoradiography was performed
with X-omatic AR-5 film and an imageintensifier screen (Kodak,
1547
Rochester, NY). Gels were developed after 4 to 7 days at -70%.
Major platelet GP bands were analyzed by use of a laser densitometer (LKB 2400, Bromma, Sweden).
Results obtained with '*'I surface-labeled platelets during pharmacologic activation of plasminogen in citrated plasma were compared
to similar autoradiographic studies performed with platelets obtained at hourly intervals from a patient receiving 100 mg of t-PA for
AMI.
Flow cytometry of platelets. Platelet-surface GP degradation
was quantitated by the use of FITC-labeled MoAbs and analysis of
their binding to specific receptors by flow cytometry. MoAb 10E5, a
complex specific antiXPIIb-IIIa antibody, was a gift of B. Coller
(State University of New York, Stonybrook).".'* MoAb 6D1 (B.
Coller) binds to the glycocalicin portion of GPIb." MoAb 12F1
binds to the GPIa subunit of GPIa-IIa (gift of V. Woods, University
of California, San Dieg~).'~.'~
MoAbs specific for platelet activation
were PAC1, which binds to the activated GPIIb-IIIa complex (gift
of S. Shattil, University of Pennsylvania, Philadel~hia),'~"~
and S12,
which recognizes the a granule protein (GMP-140 or PADGEM)
expressed on the platelet surface after platelet secretion (gift of R.
McEver, University of Oklahoma, Tulsa).4042For flow cytometry
MoAbs 10E5,6D1,12Fl,andS12 wereusedata finalconcentration
of 5 to 10 pg/mL. MoAb PACl was used at 9 to 18 pg/mL.
Before Fluorescein isothiocyanate ( FITC) labeling, MoAbs were
dialyzed overnight at 4OC in 0.1 mol/L NaH,PO,, pH 7.4. FITC
stock was prepared immediately before use at 0.8 mg/mL in 0.5
mol/L Na,CO,, pH 9.5. FITC stock was added to MoAb at 1:5
(wt/wt), the pH was readjusted to 9.5, and the incubation was
allowed to proceed at 23OC for up to 2 hours with intermittent
vortexing. The peak corresponding to labeled MoAb was separated
from free FITC on a 30.0 x 0.5 cm Sephadex G-25-300 (Pharmacia,
Uppsala, Sweden) gel filtration column equilibrated with 0.1 mol/L
NaH,P04.
For flow cytometry studies platelets were incubated with t-PA or
SK in citrated plasma or buffer in the presence of plasminogen as
described above. After 60 minutes, 50-pL aliquots of platelets were
removed and added to Tyrode-HEPES buffer, PPACK ( 5 pmol/L),
aprotinin (200 KIU/mL), and FITC-MoAb at 23OC in a final
volume of 200 pL. When calcium had been omitted from washedplatelet incubations it was added to a final concentration of at least
100 pmol/L during incubation with MoAbs before flow cytometry.
After a 30-minute incubation, flow cytometric analysis was performed by use of a Coulter Epics model 753 flow cytometer
(Hialeah, FL) with a 250-mW argon laser emitting at 488 nm.
Fluorescence emission was detected at 525 nm with a band pass
filter. Platelets were distinguished from cellular fragments by their
characteristic forward- and side-light scatter, and a gate was set to
analyze the fluorescence from single platelets. The bound fluorescence from 25,000 platelets was analyzed and plotted as a histogram
of platelet number versus fluorescence intensity per platelet. The
fluorescencescale extends over a 3-decade log scale divided into 256
equal channels. At the gain settings used, 295% of nonspecific
binding fell in the first 15 channels. For the activation-specific
MoAbs PACl and S12, activated platelets were defined as those
whose fluorescence fell above channel 15. For quantitation of
changes in GPs Ib, IIb-IIIa, and Ia-IIa the mean population
fluorescence channel number was calculated with on-line software.
Mean fluorescencechannel number was converted from log to linear
format.
Blood samples were also obtained from patients receiving t-PA
(100 mg) for AMI. Plasmin was inhibited with PPACK ( 5 pmol/L)
and aprotinin (1,000 KIU/mL) and PRP obtained as described
above. Binding of FITC-MoAbs 10E5 (anti-GPIIb-IIIa) and 6D1
(anti-GPIb) to platelets was quantitated by flow cytometry. All
patient samples for flow cytometry and autoradiographic studies
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1548
WlNTERS ET
were obtained in accordance with the guidelines of the Human
Studies Committee at Washington University Medical School.
Plosmin ond rr2-unfiplosmin o~.my.r. Activation of plasminogen was performed in plasma or buffer as described. After 60
minutes aliquots were removed and the platelets were pelleted at
1 2 . 0 0 0 ~for 2 minutes. The s u p m a t a n t was collected and immediately froten at -7OOC. Samples were thawed and diluted 120 with
0.1 I mol/l. NaCI.0.05 mol/l. Tris. pH 7.4. and added to the wells of
a microtiter plate (Costar. RioRad. Richmond. CA). The chromogenic substrate for plasmin activity. S-2251." was added to a final
concentration of 312 pmol/L. Activity of purified human plasmin
was used to develop a standard curve from 0.0025 to 0.02 caseinolytic units (CW) per milliliter. The change in absorbance due to
hydrolysis of the substrate was measured every 20 seconds for 6
minutes with a programmable automated kinetic plate reader at
23% (Molecular Dcvices. Menlo Park. CA). The lower limit of
detectibility for samples assayed with a I:20 dilution was 0.05
CU/mL plasmin activity. The human plasminogen preparation
exhibited no plasmin activity in this assmy.
cr-2-Antiplasmin was measured in P R P with an assay based on the
plasmin-specific chromogenic sub~trateS-2251 a s modified from the
manufacturer's instructions for use with microtiter plates (KabiVitrum). Plasmin was added to samples in e x m c and residual plasmin
activity against the subsrrate S-2251 was measured with the automated kinetic plate reader. cr-2-Antiplasmin levels were derived by
comparison with a standard curve developed with serial dilutions of
pooled human plasma.
Sforisricol mrrhods. Mean channel fluorescence on flow m o m etry was converted to relative mean linear fluorexvnce units a s
described and presented as the mean
standard error (SEM).
Differenm between the means of control and I-PA- or SK-treated
AL
platelets were compared by paired analysis through UK of the
IVilcoxon signed-rank method. and a significant difference was
considered to exist when P < .05.
RESULTS
Ffects on GPs after activation of plasminogen in plasma.
To characterize structural changes in membrane GPs, platelets surface-labeled with "'I wcre suspended in autologouscitrated plasma and incubated with t-PA or S K for up to 60
minutes at 37OC. The migration of platelet GPs Ibn. Ilk.
and 1113 as determined by reduced 6 7 SDS-PAGE was
unchanged aftcr incubation with pharmacologic concentrations of I-PA (2.5 pg/mL) or SK (I.OO0 C/mL. Fig I).
Similar results were obtained with incubations at 23OC (data
not shown). With suprapharmacologic conccntrations of
t-PA (20.0 pg/mL) and SK (20.000 L / m L ) there was some
proteolysis of minor CPs migrating above GP Ibn. but the
migrations of GPs Ibn. Ilbcr. and Illa remained unchanged
(Fig I ) . However. with 1-PA (20 pg/mL) densitometry
demonstrated a mean decrease of 30% in the GPlbn band at
60 minutes (n = 2). The degradation of GPlbn observed
with suprapharmacologic concentrations of t-PA did not
appear to be a function of plasmin activity alone. a s S K
generated higher plasmin acrivity in plasma than t-PA
(Table I ). As expected, cr-2-antiplasmin was depleted during
incubations with I-PA o r SK in PRP (data not shown).
Analysis of the binding of specific MoAbs with flow
cytometry confirmed the apparent lack of degradation of
PRP (37°C)
CONTROL
t-PA 2.5 p g / m l
SK 1,000 IU/ml
211-
69-
5
46-
-
0 15 30 60
0
0 IS 30 60
15 30 60
X
i
CONTROL
211-
107-
?-PA 20 pg/ml
SK 20,000 IlJ/mI
m:b a s : & g
6946-
0 15 30 60
0 15 30 00
I N C U B A T I O N TIME ( m i n )
0 15 30 60
*Dba
Fig 1. Autorodioglowm of CIPm from platw r f a ~ l o b d e dwith '
9 and subjmod to 6%
SDS-PAGE under reducing conditions. Surfoea
labeled platOletS -0
imUb.1.d for 0. 16.30, and
80 minutes in autologous plasma at 37T wtth 1-PA
or SK at the indicated concentrations. Migration of
platekt surface GPs Ibu. I l k . and 111. (arrows) i s
unchaneed aftor activation of plasminogen in
plasma. though GP Ib i s partblty degraded during
acttvatian of plasminogen wtth suprapharmocologic concontrations of t-PA 120 pglmL1 Platelets
incubatedwtthout plosminog.n actrvator are shown
in the first pbnel of each row Icontrot) Migration of
mol w l standards i s indated on tho left side of
oach gel.
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1549
DEGRADATION OF PLATELET ADHESIVE RECEPTORS
Table 1. Plasmin Activity (CU/mL)
Citrated PRP
t-PA 2.5 jtg/mL
t-PA 20.0 pgfmL
SK 1,000 UImL
SK 20,000 UImL
Washed Platelets
(37°C)
m O c-/ Ca2+)
0.19 f .03
0.30 .02
0.30 + .03
0.87 f .08
0.08 f .01
0.27 & .02
0.24 i .02
0.67 i .04
(37o~/+Caz+)
0.08 f .01
0.24 & .03
0.17
.01
0.49 .07
*
*
Wac/- Ca2+)
0.07 & .02
0.22 & .05
0.13 f .01
0.33 & .02
Plasmin activity against the chromogenic substrate $225 1 after activation of plasminogen with t-PA or SK in citrated plasma or buffer. Mean 5 SEM
(n = 8 to 18).
GPs Iba, IIba, and IIIa after activation of plasminogen in
PRP with pharmacologic concentrations of t-PA or SK (Fig
2). Binding of MoAb 10E5 (anti-GPIIb-IIIa) to platelets
did not decrease compared with control after incubation with
both pharmacologic and suprapharmacologic concentrations
of t-PA or SK, as indicated by a lack of change in relative
mean fluorescence (Fig 2A). Similar results were obtained
with MoAb 6D1 (anti-GPIb), although there was a trend
toward decreased MoAb 6D1 binding with 20 pg/mL of
t-PA that failed to achieve significance (P = .07, Fig 2B).
MAb 10E5: PRP 37°C
30
3
30
p
MAb 6D1: PRP 37°C
I"1lm
MAb 12F1: PRP 37°C
n
2.5 ug/ml
1-
20.0 uglml
1,000 IUlml
n
20,000 IU/ml
Ea
Fig 2. Flow cytometric analysis of platelet adhesive receptors.
Each set of bars indicates the mean relative fluorescence (*SEMI
as measured with flow cytometry due to binding of FITC-labeled
MoAbs to platelets. The binding of MoAb 10E5 (anti-GP Ilb-llla),
MoAb 6D1 (anti-GP Ib), and MoAb 12F1 (anti-GP la) to control
platelets (black bars) is compared with the binding of MoAb to
platelets incubated with t-PA or SK in PRP at 37°C at the indicated
concentrations (hatched bars). Aliquots removed at 60 minutes
were incubated with 5 pmol/L PPACK, 200 KIU/mL of aprotinin,
and the specific FITC-MoAb. There was no significant difference in
the binding of each antibody to its respective membrane GP after
incubation with t-PA or SK. except for a modest decrease in the
binding of MoAb 12F1 to GPla after SK. .P < .OB, n = 3 to 6.
Binding of MoAb 12F1, which binds to the Ia subunit of
GPIa-IIa, did not change after incubation with t-PA, although modest decreases in binding of 15% to 20% occurred
after SK (P= .03, Fig 2C). Examination of individual
histograms of platelet number versus fluorescence per platelet did not show any distinct subpopulations of platelets
deficient in the GP receptors characterized with MoAbs
10E5, 6D1, or 12F1. In contrast to the findings at 37OC,
results of flow cytometric studies of platelets incubated in
plasma with pharmacologic and suprapharmacologic concentrations of t-PA or SK at 23OC demonstrated no decrease in
the binding of MoAb 10E5,6D1, or 12F1 (data not shown).
Eyects on GP IIb-IIIa after activation of plasminogen in
buyer. To characterize the effects of plasminogen activation in a purified system, platelets surface-labeled with Iz5I
were incubated at 37OC with 1,000 U/mL of SK in divalent
cation-free Tyrode-HEPES buffer supplemented with 21
pg/mL of human plasminogen. Analysis of autoradiograms
from control platelets incubated in the absence of SK
demonstrated no change in GP IIb-IIIa on either reduced or
nonreduced gels (Fig 3). In contrast, activation of plasminogen with SK produced striking degradation of GPs IIb and
IIIa (Fig 3). Proteolysis was evident by 10 minutes, with the
subsequent generation of three new fragments with apparent
mol wts of 78, 71, and 66 Kd on 6% SDS-PAGE under
nonreducing conditions. Reduced SDS-PAGE confirmed the
proteolysis of GPs IIba and IIIa, with degradation fragments
migrating as a broad band with a mol wt of 65,000 to 70,000
(Fig 3).
Previous studies have demonstrated dissociation of GPIIbIIIa at 37OC in the presence of strong Ca2+-chelating
agent^:^.^^ To determine whether the presence of Ca2+or the
incubation temperature affected the proteolysis of GPIIb or
IIIa in a purified system, incubations of surface-labeled
platelets with 2.5 pg/mL of t-PA were performed under the
following conditions: 23°C without added Ca2+,37OC without added Ca2+,and 37OC with 2 mmol/L Ca2+ (Fig 4).
Control incubations in the absence of plasminogen activators
at 23O and 37OC with or without Ca2+ demonstrated no
change in the migration of GPs IIba and IIIa. At 23OC in the
absence of added Ca2+,GPIIb-IIIa was resistant to plasminmediated proteolysis (Fig 4). At 37OC in the presence of
Ca2+, GPIIIa was resistant to proteolysis while GPIIba
underwent a modest degradation. However, activation of
plasminogen with 2.5 pg/mL of t-PA at 37OC in the absence
of added Ca*+ resulted in almost complete degradation of
both GPs IIba and IIIa (Fig 4). Marked proteolysis was
evident despite generation of only 0.08 0.01 CU/mL of
+_
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WINTERS ET AL
1550
WASHED PLATELETS (37'C)
224
-
-
-SK-1.000 IU/ml (NR)
CONTROL
(NR)
- - _- -
.~
.
c
I
46--------
0
1
5
10 15 3060
*Ib
*IIb
0
X
i
-
CONTROL (R)
1
5
IO
Fig 3. Autorndbgrnma of GPs from plnt.kts
surface-Inbded with '
9 and subjected t o 6%
SDS-PAGE under nonredudng conditions INR. top
r o w ) and after reduetion of disutfide
bonds (R.
bottm row). Wnteleta were incubated with SK in
TyrodsHEPES buffer containing 21 r g / m L of pbsminogen for 60 minutes et 37T in the a b r w e of
added Ca'
Aliquots were removed a t 0. 1, 5. 10.
15, 30. and Bo minutes as indicated. Migration of
platelet surface GPs Ibd. I l k . Illa. and IV are
indicated by the arrows. After a c t b a t h of p l m i nogen w i t h SK in the absence of added Ca'
prottmtysis of GPs lb. Ib. and Itb occurred. indicated by a decrease in the intensity of thew bands
ns well as the appearance of lower mol wt degradation fragments of these GPs. Control platelets
incubated with p1auninog.n alone in the absence
of plaaminogen activator are shown in the first
penel of each row. The migration of m d wt
atandarda is indicated on th. leh side of a h 0.1.
15 30 60
SK 1,000 I W m l (R)
.
224-
46-
I
0
1
' C r r r w - -
5 10 15 30 60
0 1 5 10 15 30 60
INCUBATIOCJ TIME ( m i n )
plasmin activity with 2.5 pg/ml, of I-PA in washed-platelet
prcparations (Table I ) . In addition. activation of plasminogen with t-PA (2.5 pg/mL) or SK (1.ooO U/mL) in plasma
anticoagulated with IO mmol/l, EDTA. a potent chelating
CONTROl
224
X
W A S H E D PLATELETS
?-PA 2.5pg/ml
'
2 s c (-Cd')
-37'C
(+Cd')-
-
--'
37'C ( - c d + )
-rT--
L-"
I
c?
@
agent. also resulted in marked degradation of GP Ilb-llla at
37°C (data not shown).
Binding of Mohb 1OE5 to the Ilb-llla complex was
examincd by flow-cytometric analysis of washed platelets
109-4-
-
I*
7
-
i n46-
0
15 30 60
--e&
0 15 30 60
-q
0 15 30 60
INCUBATION TIME (min)
0 15 30 60
Fig 4. AvtorwJIogrnma of GPa from phdm surface-Inbeled with 'q nnd wbj0ct.d to 6% SDS-PAGE under r.dudng eondlcion8.
Surface-Inbeled washed plntdots were incubated in TyrodsHEPES buff.r with t-PA (2.5pglmL) and 21 pg/mL of pbunin0g.n for 0.16,
30, and 60 minutes under the fdkwing conditiom: 231: without added C.", 3 R with 2 m d / L CS7' in the butter. and 3 m witbout
added CS" A repronntative imubstionof washed platelets w i t h plasmin0g.n alone nt 371: in the nbsmce of Wed Cd is shown in the
first penel (control). Platelet surface GPs Iba, Ilbn, and Ills ere indiested by the arrows. Marked degradation of platelet GPS nba and 111.. and
t o a lesser degree GPlbn. occurred when plaeleta were incubated w i t h (-PA and plasminogen at 37% without added C a 7 ' . The migraion
of mol wt atandards is indicated on the left side of each gel.
.
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DEGRADATION OF PLATELET ADHESIVE RECEPTORS
1551
incubated with t-PA or SK in Tyrode-HEPES buffer supplemented with 21 pg/mL of plasminogen at 23O or 37OC with
or without added Ca2+.Platelets incubated at 37OC for 60
minutes in buffer containing no added Ca2+demonstrated an
80% loss of MoAb lOE5 binding, as indicated by a marked
decrease in relative fluorescence even in the absence of t-PA
or SK (Figs 5 and 6C). Loss of MoAb 10E5 binding in the
absence of t-PA or SK at 37OC without the presence of added
Ca2+ was detectable as early as 15 minutes, which corresponded to the time course of plasmin-mediated degradation
of GPIIb-IIIa when analyzed by SDS-PAGE and autoradiography (Figs 3 and 4). Because the migration of GPs IIb and
IIIa as detected by SDS-PAGE was unchanged in the
absence of t-PA or SK at 37OC without added Ca2+(Figs 3
and 4), the decrease in MoAb 10E5 binding with flow
cytometry suggested a conformational change in GPIIb-IIIa,
leading to loss of the complex specific epitope recognized by
this antibody. Indeed, activation of plasminogen by t-PA or
SK at 37OC in the absence of exogenous Ca2+produced only
an additional 5% to 10% decrease in MoAb 10E5 binding,
suggesting that the small number of GP IIb-IIIa complexes
that retain native conformation under these conditions are
relatively resistant to degradation (Fig 6C). In contrast,
activation of plasminogen at 23OC in the absence of Ca2+or
at 37OC in the presence of added Ca2+, conditions that
maintain the integrity of the IIb-IIIa complex, produced only
small decreases in MoAb 10E5 binding (Fig 6A and B).
Effects on GPs Ib and l a after activation of plasminogen
in buffer. To determine whether GPIb displayed a similar
temperature- and Ca2+-dependentsusceptibility to plasminmediated proteolysis, washed platelets surface-labeled with
"'I were incubated with t-PA or SK in Tyrode-HEPES
buffer in the presence of plasminogen. Densitometric analysis of autoradiograms from platelets incubated with either
t-PA (2.5 pg/mL) or SK (1,000 U/mL) at 37OC in the
absence of Ca2+demonstrated a 50% decrease in GPIb by 60
minutes (Figs 3 and 4). At 23OC in the absence of Ca2+,
activation of plasminogen with 2.5 pg/mL of t-PA produced
no change in GPIba (Fig 4), while incubation at 37OC in the
presenceof Ca2+resulted in a 30% loss of GPIba (Fig 4). The
temperature and Ca2+-dependent proteolysis of GPIb was
most striking with 20 pg/mL of t-PA (Fig 7). Activation of
plasminogen with suprapharmacologicconcentrationsof t-PA
produced a 60% loss of GPIba at 23OC in the absence of
Ca2+,a 75% loss at 37OC in the presence of Ca2+,and a 90%
decrease at 37OC in absence of added Ca2+(Fig 7). A similar
though less marked temperature- and Ca2+-dependentproteolysis of GPIb occurred with 20,000 U/mL of SK (Fig 7).
Binding of MoAb 6D1 (anti-GPIb) as analyzed by flow
cytometry remained stable when platelets were incubated for
up to 60 minutes in Tyrode-HEPES buffer with plasminogen
(21 pg/mL) in the absence of t-PA or SK, regardless of
temperature or Ca2+conditions (Fig 8). Activation of plasminogen with pharmacologic concentrations of t-PA produced no change in the binding of MoAb 6D1, regardless of
temperature or Ca2+conditions (Fig 8). Incubations with SK
(1,000 U/mL) at 37OC in the absence of Ca2+produced a
30% decrease in MoAb 6D1 binding, but this change was not
significant by paired analysis (P= .07,Fig 8C). Incubations
of washed platelets with 20 pg/mL of t-PA resulted in a 50%
decrease in MoAb 6D1 binding at 37OC in the absence of
added Ca2+(P = .04). The results of autoradiography and
flow cytometry suggest that GPIb may undergo partial
proteolysis in washed platelets while binding of MoAb 6D1,
an antibody that binds close to the ligand-binding domain of
GPIb, is preserved. Another explanation for the discrepancy
between the magnitude of GPIb degradation on autoradiography and the amount of MoAb 6D1 binding on flow
cytometry may be the replacement of degraded receptors
from the internal platelet pool of GPIb.so*5'However, both
autoradiographic and flow-cytometric studies demonstrate
the temperature- and Ca2+-dependenceof GPIb degradation
by plasmin (Figs 4,7, and 8).
In contrast to the findings with GPs IIb-IIIa and Ib, there
was no evidence of a decrease in MoAb 12F1 (anti-GP Ia)
binding to washed platelets during activation of plasminogen
with t-PA or SK at 23OC or 37OC in the presence or absence
of Ca2+(data not shown).
Patient studies. Although our observations in citrated
plasma demonstrate that platelet-adhesive GP degradation
does not occur following activation of plasminogen with
pharmacologic concentrations of t-PA or SK (Figs 1 and 2),
it is conceivable that prolonged platelet exposure to activation of plasminogen in vivo may result in proteolysis of
platelet receptors. To examine this possibility, platelets were
obtained from patients receiving a 3-hour infusion of t-PA
WASHED PLATELETS (37'C) :MAb 10E5
Fig 5. Histograms of fluorescence intensity
(x-axis, log scale) against cell number (y-axis) of
the binding of FITC-labeled MoAb 10E5 (anti-GPllbIlla) to washed platelets. Washed platelets were
incubated in fyrode-HEPES buffer containing 21
fig/mL of plasminogen at 37°C without added Ca2+
in the presence or absence of t-PA (top row) or SK
(bottom row). Aliquots were removed at 0 and 60
minutes and incubated with 5 pmol/L PPACK, 200
KIU/mL of aprotinin, and the FITC-labeled MoAb
10E5. Compared with 0 minutes (left panels), after
a 6O-minute incubation in the absence of plasminogen activator (middle panels) there was an 80%
decrease in FITC-MoAb 10E5 binding at 37°C
without added Cazt. When plasminogen was activated with t-PA or SK under identical conditions
there was only a small additional (5% to 10%) loss
in MoAb 10E5 binding (right panels).
t-PA
E
m
LLI
5
0
FLUORESCENCE CHANNEL NUMBER
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1552
WINTERS ET AL
n
MAb 10E5: WP 23°C (-Ca++)
NS
NS
n
n
NS
n
30
20
10
,a
s
'E
3
0
.^lm
-
MAb 10E5: WP 37°C (2mM Ca++)
MAb 10E5: WP 37°C (-Ca++)
n
n
n
30
20
10
MoAbs 10E5 (anti-GPIIb-IIIa) and 6Dl (anti-GPIb) following t-PA therapy for AMI.
Activation-specijc MoAbs during activation of plasminogen in plasma. Platelets were incubated in citrated plasma
at 37OC with pharmacologic and suprapharmacologic concentrations of t-PA or SK for periods of up to 1 hour. The
binding of PACl, an MoAb that binds to the GPIIb-IIIa
fibrinogen receptor on platelets after activation, did not
increase when platelets were incubated with either t-PA or
SK, consistent with a lack of platelet activation in response to
activation of plasminogen (Fig 10). Even after incubation
with 20,000 U/mL of SK there was no evidence of platelet
activation or aggregation (P> .3, n = 9,despite the generation of substantial plasmin activity of 0.87 f 0.08 CU/mL,
a level comparable with that reported to induce platelet
activation by others." Platelets incubated with t-PA or SK in
plasma could still be activated with 10 pmol/L ADP, as
evidenced by an ADP-induced increase in P A C l binding
comparable to that observed with control platelets (Fig 10).
The absence of platelet activation in citrated plasma during
activation of plasminogen was confirmed by the lack of
increased binding of MoAb SI2 documented with flow
cytometry. This antibody binds to the a granule protein
(GMP-140), which is expressed on the platelet surface after
platelet secretion.
0
2.5 ugh1
20.0 uglml
1,000 IU/ml 20,000 IU/ml
Ea
pL-1
Fig 6. Flow cytometric analysis of the effect of activation of
plasminogen on the GPllb-llla complex. Each set of bars indicates
the mean relative fluorescence (*SEMI as measured with flow
cytometry due t o binding of FITC-labeled MoAb 10E5 to washed
platelets. Washed platelets were incubated in Tyrode-HEPES
buffer containing 21 pg/mL of plasminogen with or without t-PA
or SK under the following conditions: 23°C without added Ca2+(A),
37°C with 2 mmol/L Ca2+in the buffer (B). and 37°C without added
CaZ+ (C). Aliquots were removed at 0 and 60 minutes and
incubated with 5 gmol/L PPACK, 200 KlUlmL of aprotinin, and the
FITC-labeled MoAb 10E5. MoAb 10E5 binding at 0 minutes (black
bars) is compared with binding after a 60-minute incubation
without (hatched bars) or with (open bars) plasminogen activator.
Incubationsof platelets with plasminogen in the absence of t-PA or
SK at 23°C without added Ca2+ (A), and at 37°C with 2 mmol/L
Cazi (B), showed no change in MoAb 10E5 binding (hatched bars).
Under identical conditions, activation of plasminogen with t-PA or
SK resulted in little change in MoAb 10E5 binding (A and B. open
bars). In contrast, after a 60-minute incubation of washed platelets
with plasminogen in the absence of t-PA or SK at 37°C without
added Ca2+ (C), MoAb 10E5 binding decreased by 80% (hatched
bars). Activation of plasminogen with t-PA or SK at 37'C in the
absence of added Ca2+caused only a modest additional decrease in
MoAb 10E5 binding (C, open bars). * P .05, n = 3 t o 6.
-=
(100 mg) for AMI. Platelets from one patient were surfacelabeled with 12'1 and subjected to SDS-PAGE and autoradiographic analysis. At 2 and 4 hours after initiation of t-PA
there was no change in the amount of platelet"surface GPs Ib,
IIb, and IIIa, compared with platelets obtained before
fibrinolytic therapy, despite substantial proteolysis of platelet
surface-associated fibrinogen (Fig 9). In addition, flowcytometric analysis of platelets from an additional five
patients demonstrated no change in the binding of FITC-
DISCUSSION
Our results indicate that activation of plasminogen in
citrated plasma by pharmacologic concentrations of t-PA or
SK does not result in degradation of platelet adhesive
receptors for fibrinogen and WF. Although some degradation of the collagen receptor occurs in plasma with SK, it is
modest. However, plasmin-mediated proteolysis of GPIlbIIIa in washed platelets is marked a t physiologic temperatures in the absence of added Ca2+. Similarly, plasminmediated degradation of GPIb in washed platelets is increased
a t physiologic temperatures, in the absence of added Ca2+,in
the presence of increased plasmin activity, and with suprapharmacologic concentrations of t-PA. Furthermore, we
could not find any evidence that activation of plasminogen in
citrated plasma by t-PA or SK directly activates platelets.
Consequently, the platelet activation observed during pharmacologic activation of plasminogen does not appear to
result from the direct action of plasmin on platelet^.'^.'^ In
addition, the resistance of platelet-adhesive receptors to
plasmin-mediated degradation in plasma suggests that the
inhibition of platelet aggregation in plasma observed by
others is most likely due to factors other than the proteolysis
of GPs, Ib, IIb, and IIIa.I7-I9
Previous studies of platelet function have indicated that
activation of plasminogen in plasma can induce both antiaggregatory and proaggregatory response^.'^-^^^'^ Antiaggregatory effects may be due to generation of fibrinogendegradation products that compete with fibrinogen for binding
to the platelet GPIIb-IIIa fibrinogen receptor but do not
support platelet aggregatiom2' In addition, proteolysis of W F
by plasmin may explain the inhibition of ristocetin-induced
platelet aggl~tination.'.'~
Although antiaggregatory effects
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DEGRADATION O f PLATELET ADHESIVE RECEPTORS
1553
WASHED PLATELETS
CONTROL
-
I-PA 2 O y o / m I
1
1
2 r c (-Co")
37'C
(*CO")
37.c (-Co"l
Ir
311-
Fig 7.
Autarediogrann of OPs from plotdots
surface-labeled with '
9 and subjected to 6%
SDS-PAGE under reducing conditions. Surfacelabeledwashed platdots were incubatedin Tyroda
HEPES buffer with 21 UglmL of plasminogen and
either (-PA Itop) 01SK (bottom) under the following conditions: 23'C without added Ca". 37°Cwith
2 mmollL Ca' added to the buffer. and 3 7 C
without added Cd . Aliquot. were removed at 0.
15. 30, and 60 minutes. Representative ineubations of platelets with plssminogenin the absence
of t-PA or SK at 3 r C without added Ce' are
shown in the first panels. Platelet surface GPs Iba.
Ilba, and llla are indicated by the arrows. Marked
degradation of platelet GPs Ilb and llla occurred
after activation of plasminogen with e i t k t-PA or
SK.but only at 3 T C in the absence of added Ca'
Degradation of GPlb was greater after activation
of plasminogen by t-PA than after that by SK.
Proteolysis of GPlb with mivation of plasminogen
by (-PA was evident under all three conditions. but
wes most striking at 3 r C without added Ca'
Migration of mol w
t standards is indicated on the
l e h side of each gel.
0
"
-a
.
.
could also be due to proteolysis of platelet-adhesive GPs by
plasmin. resulting in decreased binding of cohesive ligands.
our results suggcst that significant functional impairment of
platelet receptors is unlikely. MoAbs IOES ( a n t i 4 P l l b Illa) and 6D1 (anti-GPlb) both bind close to the ligandbinding domains of their respective receptors."." Preserved
binding of these MoAbs (as characteriied with flow cytomet r y in plasma) and the absence of structural change of these
GP receptors (as shown by SDS-PAGE during activation of
plasminogen in plasma) suggest that the fibrinogen and vWF
receptors arc likely to retain function. The observation of
Loscalzo and Vaughan that platelets disaggregated by suprapharmacologic concentrations of I-PA in plasma could be
reaggregated by ADP if resuspended with intact fibrinogen is
consistent with functional competence of the GPllb-llla
receptor after activation of plasminogen in plasma."
Degradation of platelet surface GPllb-llla has been
reported in washed platelets during activation of plasminogen with t-PA.'"Our finding that GPllb-llla is not degraded
in association with activation of plasminogen in plasma
suggests that the results obtained in washed-platelet systems
ma); not be extrapolated to explain antiaggregatory effects in
plasma. In addition. degradation of GPllb-Illa in washed
platelets in the absencc of Ca" may explain discordant
observations in studies of washed platelets and platelets in
plasma. Irreversible dissociation of GPllb-llla can be induced by incubation of washed platelets at 37OC with strong
Ca' -chelating agents such as EDTA.""" Chelation of Ca' '
at 23°C docs not result in dissociation of GPllb-llla at
physiologic pH." "suggesting that both physiologic temperature and the absence of Ca" are required to induce
instability of the Ilb-llla complex. We have shown that as
few as two washes in ACD-containing buffer are sufficient to
destabilize GPllb-llla when platelets are subsequently incu'
INCUBATIONTIME (min)
bated at 37OC in the absence of added Ca?'. Under these
conditions. even in the absence of plasmin. the results of
SDS-PAGE and autoradiography demonstrate that GPllb
and l l l a polypeptides are still present on the platelet surface
but that VoAb IOE5 will no longer bind to the Ilb-llla
complex. even following restoration of Ca" before incubation with the antibody. In addition. the dissociated GPllbl l l a receptors on platelets in buffer at 37OC in the absenceof
Ca-" are exquisitely sensitive to plasmin-mediated dcgradation. Marked degradation occurred with plasmin activity less
than 0.1 CU/mL. However. at 37OC in the presence of
exogenous Ca". binding of the complex specific MoAb IOES
to the Ilb-llla complex is maintained even when platelets arc
incubated with suprapharmacologic doses of I-PA or S K in
the presence of plasminogen. Recently Beer and Coller have
reported susceptibility of GPllla to proteolysis by plasmin
and other proteases on washed platelets in the presence of
EDTA. although temperature conditions were not specified."
Our observations arc in agreement with theirs and suggest
that plasmin can degrade both GPllb and llla in the abwnce
of Ca-" at 37°C. However. our results also demonstrate that
the GPllb-llla does not undergo significant plasminmediated proteolysis in the presence of Ca-" .
Proteolysis of platelet GPlb has also been reported in
washed platelets incubated with plasmin and with I-PA in
the presence of plasminogen.""' Although the function of
G P Ib does not require the presence of divalent cations." our
observations in washed platelets suggest that it is more
susceptible to plasmin-mediated degradation at 37OC in the
absence of Ca". I n incubations without plasminogen activators the binding of MoAb 6DI is maintained regardless of
temperature or Ca" conditions. This argues against a major
change in theconformationofGPlbat 37OC in theabsenceof
Ca". However. the increased proteolysis of GPlb at 37OC in
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
WINTERS ET AL
1554
the absence of Ca" suggests that subtle changcs in the
conformation of GPlb or changcs in its microenvironment
under these conditions render GPlb more susceptible to
proteolysis by plasmin.
We also observed a discrepancy between the degrcc of
GPlb degradation in washed platelets determined by SDSPAGE and autoradiography and MoAb 6D1 binding with
flow cytometry. For example, activation of plasminogen with
I-PA (2.5 &mL) at 37°C in the absence of addcd Ca"
rcsultcd in a
decrease in GPlb on autoradiography (Fig
4). while similar incubations caused no change in MoAb 6DI
binding on now cytometry (Fig 8 ) . This suggcsts that GPlb
receptors degraded by plasmin may be replaced by intact
receptors from the internal platelet p o l of GPlb dcscribcd
30p
MAb 601: WP 23.C
(-Ca+*)
211 -
-Iba
-IIbu
c3
I
0
c
x
rL
107
-
-m0
69 -
20-
45
MAb 601 : WP 37°C (2mM Ca++)
"1
-
A
Time ( h r ) 0
-
2
4
Fig 9. Autoradiogram of GPs from platelets sudoco-bkled
with '
9 and subjscred to 8% SDS-PAGE under reducing conditions. Platelets were obtained et baseline and hourly from a patient
receiving e 3-hour infuaion of (-PA (100 mg). Denaitometric
analysia demonstrated leas than a 10%decreaae in the amount of
GPs Ib, Ilb, and 11111 detected on the platelet surface at 2 and 4 hours
after the initiation of t-PA. despite marked protbdyria of plateletsurface-associated fibrinogen chain. (mol w t a 47Kd t o 67Kdl.
.
I
2.5 u q m l
.
20 0 uglml
I T ]
1.OOO iUlml
20.000 IUlml
Gia
Fig 8. Flow yrometrk annlysis of the effect of .etivatiOn of
p4nsminog.n on the GP Ib eomplex. E.ch sel of bars indicates the
mean relative fluorescence ( t SEMI as measured with flow cytometry due t o binding of FITC-bbeled MoAb 6 0 1 t o washed platelets.
Washed platelets were incubated in Tyrode-HEWS buffer containing 21 p g l m L of plaaminogen w i t h or without t-PA or SK under the
following condition.: 2 3 T without e d d d Cay (A), 3 X w i t h 2
mmollL Ce' in the buffer (E). and 37'C without added Cd (C).
Aliquot. were removed at 0 and Bo minutes end incubated with 6
pmol/L PPACK. 2 0 0 KlUlmL of aprotinin. and the FITC-labeled
MoAb 6D1. MoAb 6D1 binding at 0 minutes (black b a r d ia
compared with binding after a 80-minute incubation without
(hatched bars) or with (open bars) plasminogen activator. lncubationa of platelets with plaaminogen in the absence of t-PA or SK et
23'C without added Ca' (AI, and at 37'C with 2 mmol/L Ce' (8).
and 3 7 X without added Ca' IC), showed no change in MoAb 6D1
binding (hatched bars). On paired analysis only 1-PA (20 pglmL1 at
3 7 Z in the absence of added Ce'
resulted in a aignificent
decrease in MoAb 6D1 binding IC). * P
.04. n
36.
- -
by Michelson et al and others.'' However. we could detect no
decrease in "'I surface-labeled GPlb during activation of
plasminogen in plasma with pharmacologic concentrations of
t-PA and SK (Fig I ) . Preliminary patient studies during
fibrinolytic therapy for A M I have yielded discordant
results."'" Administration of t-PA raulted in a small increase in platelet surface GPlb. with a concomitant 197
decrease in total platelet GPlb, compatible with replacement
of degraded GPlb rcccptors from the internal platelet pol."
In contrast, SK therapy for A M I produced a 1 9 ~ d e c r ~ iins e
surface GPlb but no change in GPllla." Our autoradiographic- and flow cytometric patient studics suggest that the
amount of platelet surface GPs Ib, Ilb, and llla docs not
change during infusion of I-PA, although the psqibility of
continuous replacement of degraded GPlb receptors in vivo
cannot be excluded by this method.
These discordant results will require further clinical study.
but presently the following conclusions appear warranted
from our observations and previous studies. First. GPlb is
readily hydrolyzed on washed platelets by high doscs of
plasmin.'*,'' Second. proteolysis of GPlb on washed platelets
can be enhanced by physiologic temperatures and chelation
of Ca". Third, degradation of GPlb is modcst in plasma
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DEGRADATION OF PLATELET ADHESIVE RECEPTORS
1555
CITRATE PRP (37°C) : M A b PAC-I
0 vg/ml
Fig 10. Histograms of fluorescence intensity
(x-axis) against cell number (y-axis) of the binding
of FITC-labeled MoAb PACl to platelets in PRP.
MoAb PACl binds to the GPllb-llla complex only
when platelets are activated. Platelets were incubated for 80 minutes at 37°C in citrated PRP with
or without t-PA or SK. Control incubations without
t-PA or SK show fluorescence consistent with
nonspecific binding (first panels, dashed lines).
Activation of plasminogen by t-PA (top) or SK
(bottom) did not result in increased MoAb PACl
binding (second and third panels, dashed lines). For
comparison, the increase in PACl binding due to
platelet activation after the addition of 10 pmol/L
ADP is shown for platelets incubated under identical conditions (solid lines).
t-PA
$
~
0 IU/ml
"
SK
-0
r),
JI
FLUORESCENCE C H A N N E L NUMBER
complex on activated platelets, or MoAb S12, which binds to
during activation of plasminogen by pharmacologic concenthe a granule protein (GMP-140) expressed on the platelet
trations of plasminogen activators and may be offset by
surface after platelet secretion, demonstrating the absence of
replacement of degraded receptors from internal platelet
direct activation of platelets during activation of plasminost~res.'~~''
Finally, in view of the resistance of GPIb to
gen in citrated plasma.
plasmin-mediated proteolysis in plasma, the marked inhibiIn conclusion, while GPIIb-IIIa, and to a lesser extent
tion of ristocetin-induced platelet agglutination described by
GPIb, can be degraded by plasmin in purified systems in the
others following activation of plasminogen in plasma is likely
absence of Ca2+,these receptors are preserved during phardue to factors other than the degradation of GPIb.17-'9
macologic activation of plasminogen in plasma. Our observaAlthough small decreases in MoAb 12F1 binding to GPIa
tions suggest that functional impairment of the major plateletwere seen with SK in plasma, generally GPIa was resistant to
adhesive receptors is unlikely to occur during activation of
degradation in both plasma and washed-platelet studies.
plasminogen in plasma. Thus, platelets in patients treated
Because MoAb 12F1 does not block collagen binding to
with fibrinolytic agents appear to retain the necessary
GPIa-IIa, it is possible that plasmin-mediated degradation of
complement of surface receptors required to mediate corothe collagen binding site of GPIa-IIa may occur. Because
nary reocclusion after successful fibrinolytic therapy.57The
GPIa is difficult to locate consistently on one-dimensional
mechanisms for platelet activation and impaired platelet
SDS-PAGE studies, definitive conclusions concerning the
function during pharmacologic thrombolysis remain undeeffects of plasmin on Mg2+-dependent platelet adhesion to
collagen will require functional assays and perhaps the use of
fined, but they appear to result from effects of activation of
plasminogen on plasma components rather than direct effects
MoAbs that inhibit the function of GPIa-IIa.
Fitzgerald et a1 have demonstrated elevated levels of
on platelets.
thromboxane metabolites indicative of platelet activation
during plasminogen activation with SK and t-PA.43'32'4
HowACKNOWLEDGEMENT
ever, activation of platelets in vivo may result from other
The authors gratefully acknowledge Drs Barry S. Coller, Sanford
factors, such as release of thrombin from clot,2' re-exposure
J. Shattil, Rodger P. McEver, and Virgil L. Woods Jr for their
of collagen in the ruptured atherosclerotic p l a q ~ e , ~ a~ . ' ~ generous gifts of MoAbs. We thank Dr Ken Stone, Chris Easton,
procoagulant surface on the residual
or direct procoagand Cathy Dirnbeck for technical assistance with flow cytometry,
ulant effects attributable to plasmin interactions with the
and Mary Jane Eichenseer for performing the plasmin assays. The
clotting ~ y s t e m . ~ We
~ . ' ~found no increase in the binding of
authors also acknowledge Dr Burton E. Sobel for his encouragement
and critical review of the manuscript.
MoAb PAC 1, which specifically recognizes the GPIIb-IIIa
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1990 76: 1546-1557
Dependence of plasmin-mediated degradation of platelet adhesive
receptors on temperature and Ca2+
KJ Winters, PR Eisenberg, AS Jaffe and SA Santoro
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