0022-3565/02/3003-952–957$3.
00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 300:952–957, 2002
Close Relationship between the Platelet Activation Marker
CD62 and the Granular Release of Platelet-Derived Growth
Factor
JOCHEN GRAFF,1 UTE KLINKHARDT,1 VALÉRIE B. SCHINI-KERTH, SEBASTIAN HARDER, NICOLE FRANZ,
STEFFEN BASSUS, and CARL M. KIRCHMAIER
Institute of Clinical Pharmacology, University Hospital, Frankfurt/Main, Germany
Received August 29, 2001; accepted November 6, 2001
ABSTRACT
The expression of CD62 on the surface of platelets is consid-
ered to be an indicator of platelet degranulation and secretion.
We characterized the relationship between CD62 expression
and platelet-derived growth factor (PDGF)AB and PDGFBB se-
cretion in response to thrombin-receptor activating peptide
(TRAP). The principal findings were 1) expression of CD62 as a
constituent of platelet !-granule membrane and secretion of
PDGF, an important ingredient of !-granules, can be stimulated
by TRAP-induced activation in a dose-dependent fashion; 2)
the activation marker and secretion product are closely corre-
lated with each other; and 3) changes in the CD62 expression
Platelet secretion of vasoactive factors plays an important
role in the development of atherosclerosis (Ross, 1999) and
restenosis after coronary interventions (Chandrasekar and
Tanguay, 2000). A major mitogenic compound released by
aggregating platelets is platelet-derived growth factor
(PDGF), which markedly stimulates smooth muscle cell pro-
liferation and migration (Heldin and Westermark, 1999).
Neutralizing antibodies to PDGF, and competitive PDGF
receptor blocking agents have been shown to inhibit neointi-
mal formation in animal and human studies (Serruys et al.,
1997; Bilder et al., 1999; Waltenberger et al., 1999). PDGF
exists in three different and biologically active isoforms (AA,
BB, AB). Platelets contain mainly PDGFAB and small
amounts of PDGFBB, which are stored in !-granules (Hart et
al., 1990; Heldin and Westermark, 1999). Activation of plate-
lets, e.g., by thrombin or ADP, is associated with the trans-
location of CD62 (P-selectin) from the !-granule membrane
to the outer surface (Leytin et al., 2000a, b). Once exposed at
activated platelets, CD62 allows the interaction of leukocytes
with platelets by interacting with leukocyte PSGL-1, thereby
Supported by the Paul und Cilli-Weil Stiftung, Frankfurt/Main, Germany
1
Contributed equally to this work.
ABBREVIATIONS: PDGF, platelet-derived growth factor; TRAP, thrombin-receptor activating peptide; FITC, fluorescein isothiocyanate; FACS,
fluorescence-activated cell sorter; PE, phycoerythrin; PLT, platelet; PRP, platelet rich plasma.
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Vol. 300, No. 3
4496/965635
Printed in U.S.A.
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induced by a drug, namely clopidogrel, or by a disease, namely
diabetes, are paralleled by changes in PDGF secretion. Al-
though CD62 is perceived as an activation marker of platelets
indicating enhanced aggregability and secretion of !-granular
content, the proof that the CD62 status and its modifications
reflect directly the actual secretion of the most important plate-
let mitogen, PDGF, has so far not been given. This ex vivo-in
vitro study shows that at least for the activation pathway pro-
vided by the PAR-1 receptor for which TRAP is the selective
agonist, CD62 expression on platelets could be a surrogate for
their secretory activity.
triggering inflammatory responses (Furie and Furie, 1995;
Evangelista et al., 1996; Zahler et al., 1999).
Flow cytometric determination of CD62 is commonly used
to quantify platelet activation status (Hagberg and Lyberg,
2000; Leytin et al., 2000b; Zeiger et al., 2000). The expression
of CD62 on the surface of platelets is considered to be an
indicator for platelet degranulation and secretion (Gawaz et
al., 1996; Michelson et al., 1996; Neumann et al., 1997) and a
predictor of acute coronary events (Hollander et al., 1999). It
has been shown that upon in vitro activation of platelets,
CD62 is detected at the platelet surface, and both !-granule-
derived products, like "-thromboglobulin, and dense granule-
derived products, like serotonin, are released (Rand et al.,
1996). However, the correlation between the surface expres-
sion of CD62 and granule content released is still unclear.
Moreover, it remains to be determined whether changes in
CD62 expression induced by either drugs or diseases are
paralleled by changes in the secretion of granule-derived
products. Therefore, the aim of the present study was to
characterize the relationship between platelet activation sta-
tus (translocation of CD62) and PDGF secretion from !-gran-
ules in response to different concentrations of thrombin-re-
ceptor activating peptide (TRAP), and to determine whether
such a relationship is maintained under antiplatelet drugs
 Relationship of CD62 Expression PDGF Release in Platelets
for which it is known that they either reduce CD62 expres-
sion (namely, the thienopyridine clopidogrel) (Rupprecht et
al., 1998; Klinkhardt et al., 2000) or do not influence CD62
expression (namely the GPIIb/IIIa-inhibitor abciximab)
(Fredrickson et al., 2000; Graff et al., 2001), or in clinical
conditions that are associated with platelet hyper-reactivity
(e.g., diabetes).
Materials and Methods
Chemicals
TRAP (H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-OH) was obtained from
Bachem (Heidelberg, Germany). FITC-anti-CD62 antibody, PE-anti
CD42b antibody (IgG1-mouse) and FACS solution for analysis in a
FACScan cytometer was obtained from Becton Dickinson (Heidel-
berg, Germany). PDGFAB was determined by an immunoassay (hu-
man PDGFAB; Quantikine, R & D Systems, Wiesbaden, Germany),
which has a 10% cross-reactivity with PDGFAA and a 2% cross-
reactivity with PDGF BB and a limit of sensitivity of 8.4 pg/ml. In
several samples, we also determined PDGFBB with a newly available
immunoassay (human PDGFBB; Quantikine, R & D Systems), which
has a 0.1% cross-reactivity with PDGFAB and a limit of sensitivity of
15 pg/ml. Clopidogrel (Plavix) was obtained from Sanofi-Synthelabo
(Berlin, Germany), and abciximab (Reopro) was a gift from Eli Lilly
Inc. (Indianapolis, IN).
Experimental Protocols
The following experimental protocols were performed during this
study.
Protocol 1. Relationship between platelet secretion of PDGFAB
and CD62 expression in healthy subjects. Blood was drawn from the
antebrachial vein of male volunteers (n ! 6, age 25– 41 years) into
3.18% sodium citrate.
Protocol 2. Effect of ex vivo treatment in whole blood with the
GPIIb/IIIa-antagonist abciximab on platelet secretion of PDGFAB
and expression of CD62. Blood was drawn from healthy subjects (n !
6, age 22–38 years) as indicated above and spiked with abciximab (5
#g/ml final concentration). This concentration of abciximab has been
proven to confer "80% inhibition of platelet aggregation and GPIIb/
IIIa-receptor activation (Klinkhardt et al., 2000).
Protocol 3. Effect of in vivo treatment with clopidogrel on platelet
secretion of PDGFAB and expression of CD62 in healthy subjects (n !
8, age 25– 41 years). Platelet secretion of PDGFAB and expression of
CD62 was determined before and after oral administration of clopi-
dogrel (loading dose of 2 # 75 mg/day, followed by 75 mg/day for 6
days). Blood was drawn as indicated above.
Procotol 4. Relationship between platelet secretion of PDGFAB
and CD62 expression in diabetic patients and age-related healthy
control subjects. Patients with noninsulin-dependent diabetes mel-
litus (n ! 8, age 56 –71 years) were selected from the diabetes day
clinic of the Deutsche Klinik of Diagnostik (Wiesbaden, Germany)
and compared with a group of healthy control patients (n ! 8, age
59 – 69 years). Blood was drawn as indicated above.
All clinical protocols have been approved by the Institutional
Review Board of the University Clinic of Frankfurt/Main, and writ-
ten informed consent was obtained. No antiplatelet agent such as
aspirin or clopidogrel was taken by the study subjects during the last
4 weeks before the study.
Flow Cytometry Analysis
Citrated whole blood (250 #l) was diluted 1:1 in Hepes buffer
(20 mM Hepes, 137 nM NaCl, 2.7 mM KCl, 1 mM MgCl2, 5.6 mM
glucose and 1 g l$1 bovine serum albumin, pH 7.4) and carefully
mixed. TRAP at a final concentration of 0 (i.e., unactivated), 1, 2,
5, 10, 20, or 30 #M in the case of protocol 1, and 5 #M for all other
protocols, was added to blood samples to activate platelets. There-
953
after, the sample (30 #l) was washed with Hepes buffer by cen-
trifugation for 5 min at 750g. Platelet sediment was resuspended
in Hepes buffer (200 #l) and incubated with saturating concen-
trations of FITC-anti-CD62 (10 #l) at room temperature for 30
min in darkness. Subsequently samples were incubated with sat-
urating concentration of PE-anti-CD42b antibody (20 #l), which is
used to set a gate for platelet events during the analysis. After
incubation with labeled antibodies, samples were diluted with 1
ml of sodium citrate solution (3.8%) in Dulbecco’s phosphate-
buffered saline, and centrifuged at 750g for 5 min. The labeled
platelet pellets were resuspended in 300 to 400 #l of the FACS
solution. Acquisition and processing of data from 5,000 platelets
were carried out with CONSORT software (Becton Dickinson).
Fluorescence channels were set on logarithmic scales. Expression
of CD62 was quantified either as mean fluorescence intensity
(MFI, given in arbitrary units) or as CD62 positive platelets (%%)
identified by binding of FITC-labeled CD62 antibody to the sur-
face of platelets (Evangelista et al., 1996; Hagberg and Lyberg,
2000).
Determination of PDGF (Enzyme-Linked Immunosorbent
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Assay). Platelet rich plasma (PRP) was prepared by centrifugation
of citrated blood at 754g for 5 min at 24°C. Subsequently, 1 ml of PRP
from the upper part of the tube content was transferred into a new
tube, and platelet count was obtained. These conditions have been
proven to confer an optimal yield of platelets (400 –500 ! 104 per #l)
with a minimum contamination by leukocytes (0.01– 0.03 ! 104 per
#l). The platelet suspension was incubated at 37°C for 2 min and for
protocol 1 subsequently stimulated with TRAP at concentrations of 0
(i.e., unactivated), 1, 2, 5, 10, 20, and 30 #M and incubated for 20 min
at room temperature. In samples obtained during protocols 2 to 4, 0
and 5 #M TRAP was used. Thereafter, platelet aggregates were
removed by centrifugation at 754g for 5 min, and 500 #l of the
supernatant were transferred into new tubes, which were centri-
fuged for another 30 min with 48,000g. The supernatant was kept
and stored at $70°C before use. Furthermore, one sample of platelet
poor plasma was obtained from each subject by centrifugation of
citrated whole blood with 2500g for 15 min. For the determination of
the total amount of PDGF in PRP, platelets were lysated according to
the following methods (Hart et al., 1990): 1 ml of PRP was mixed
with 1 ml of lysate buffer (0.5% Triton X-100, 50 nM triethanol-
amine, diluted in 10 ml of Dulbecco’s phosphate-buffered saline),
then frozen and thawed three times before centrifuged as described
above. The amount of PDGFAB (and in some samples also of PDG-
FBB) in the aliquots was determined by the use of a commercially
available immunoassay. The concentration of PDGF in each PRP
aliquot was adjusted to the actual platelet count of each sample and
is quoted as nanograms per 109 platelets.
Statistical Evaluation. Results are presented as mean values &
S.D. Concentration-response curves for the increase in CD62 expres-
sion or secretion of PDGF in response to TRAP (protocol 1) were
established on the basis of a sigmoidal Emax model [E ! (Emax !
C$)/(EC50$ % C$)]. The minimization of model parameters by least
squares based on the Newton-Gauss algorithm was carried out using
a computer program (Scientist, MicroMath Inc., Salt Lake City, UT).
The correlation between CD62 expression and PDGFAB release fol-
lowing activation by increasing TRAP concentration has been deter-
mined using a second order polynomial function, and alternative
curve functions were rejected on the basis of statistical comparison of
the curve fit according to the Akaike criterion. The relationship
between PDGFAB and PDGFBB release in the same sample was
assessed by linear regression analysis and Spearman’s rank order
correlation coefficient. Differences between observations in samples
spiked or not spiked with abciximab (protocol 2) and in diabetic
patients and their controls (protocol 4) were assessed by the U test
(Mann-Whitney). The differences before and after clopidogrel treat-
ment (protocol 3) were assessed by paired t test.
954 Graff et al.
Results
PDGF Response to Activation with TRAP (Protocol
1). Platelets activated by TRAP release PDGF in a concen-
tration-dependent manner, and the concentration-response
curve for TRAP yielded an EC50 of 7.9 & 2.0 #M. Lower
concentrations of TRAP (1 and 2 #M) only slightly provoked
PDGFAB release, compared with unactivated platelets. Max-
imal PDGFAB release was seen at 20 #M TRAP (Fig. 1A). The
maximal PDGFAB content as determined from platelet lysate
(131 ng/109 PLT) was only less above the maximum of the
estimated TRAP concentration-response curve. In platelet
poor plasma, the amount of PDGF was 4 & 1 ng/ml. In a
subset of samples of protocol 1 and 3, PDGFBB release was
also investigated. The ratio between the BB and the AB
isoform, calculated from all samples was 0.09 & 0.04. Linear
regression analysis showed that the ratio was constant over
Fig. 1. PDGFAB-release (ng/109 platelets) in PRP and CD62 expression
(MFI) in whole blood after activation with TRAP (protocol 1). Mean
values & S.D. (n ! 6).
all concentration levels of PDGF, and therefore seems not to
depend on the strength of TRAP as inducer of PDGF release
(Fig. 2). Furthermore, the ratio in those samples obtained in
protocol 3 under clopidogrel were not different from the sam-
ples obtained during protocol 1.
CD62 Expression to Activation with TRAP and Cor-
relation to PDGF Release (Protocol 1). CD62 expression
was determined in all samples that were stimulated with
TRAP. Differences in the shape of the concentration-response
curve were dependent from the analysis of the flow cytomet-
ric data. After stimulation with 5 #M TRAP, the expression
of CD62 quantified as CD62 positive (%%) platelets showed
76%% platelets, suggesting a larger effect as when the re-
sponse is given as MFI, where the increase in MFI from
baseline approximated 50% of the maximal response. The
EC50 of TRAP was 2.4 & 1.0 #M for the CD62 response in %%
and 4.3 & 0.7 for MFI, in closer agreement to the EC50 for
release of PDGF (Fig. 1B). Furthermore, since the concentra-
tion-response curve for MFI showed a better correlation to
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the PDGFAB release than expression of CD62%% platelets
after various TRAP concentrations (r2 ! 0.82 versus 0.74)
(Fig. 3), the MFI of CD62 expression has been regarded for
reporting the subsequent experiments. From protocol 1, we
chose a TRAP concentration of 5 #M as stimulus for platelet
activation in the subsequent experiments. At this concentra-
tion effects in CD62 expression or PDGF release induced by
disease or drugs might be detected in either direction.
Effect of Platelets Spiked in Vitro with Abciximab
(Protocol 2). CD62 expression (MFI) was not affected by
spiking the blood with abciximab (5 #M/ml) both in unacti-
vated as well as in TRAP-activated samples. PDGFAB secre-
tion even showed a slight but not significant increase under
abciximab after TRAP activation (29 to 42 ng/109 PLT) (Table
1).
Effect of Clopidogrel on Activation of Platelets (Pro-
tocol 3). Baseline values of CD62 expression and PDGFAB
release were similar at days 1 and 6. After activation with 5
#M of TRAP, a significant decrease (p ' 0.02) in CD62
Fig. 2. Linear regression analysis (dashed line, 95% confidence interval)
of PDGFAB release and corresponding PDGFBB release in a subgroup of
subjects from protocol 1.
 Relationship of CD62 Expression PDGF Release in Platelets
Fig. 3. Correlation between CD62 expression (MFI) and PDGFAB release.
Data have been fitted to a second order polynomial function, and the
mean curve (solid line) and 95% confidence interval (dashed lines) are
given. The inset shows the curve based on determination of the fraction
of CD62 positive platelets in percentage (%).
expression (238 to 158 MFI), as well as PDGFAB (23 to 12
ng/109 PLT), from day 1 to day 6 could be demonstrated (Fig.
4). When the CD62-PDGF data pairs were superimposed to
the curve obtained during protocol 1 from a different group of
subjects, almost all data were within the 80% prediction
interval (Fig. 5).
Platelet Activation in PRP from Patients with Dia-
betes Mellitus (Protocol 4). Compared with age-matched
controls, platelets from diabetic patients showed a signifi-
cantly elevated release of PDGFAB in unactivated PRP (16.4
ng/109 PLT versus 9.4 ng/109 PLT, p ' 0.01) as well as after
stimulation with 5 #M of TRAP (45.8 versus 28.7 ng/109 PLT,
p ' 0.03) (Table 1). CD62 expression in unactivated samples
was similar in both groups but was significantly enhanced
after TRAP stimulation in the diabetes group (276 to 217
MIF, p ' 0.03). When the CD62-PDGF data pairs were
superimposed to the data obtained from protocol 1, only a few
data pairs fell of the 80% prediction interval of the reference
curve (Fig. 5).
Discussion
The principal findings presented in this paper are: 1) ex-
pression of CD62 as a constituent of platelet !-granular
membrane and secretion of PDGF, an important ingredient
of !-granules, can be stimulated by TRAP-induced activation
in a dose-dependent fashion; 2) the activation marker and the
secretion product are closely correlated with each other; and
3) changes in the CD62 expression induced by a drug, namely
clopidogrel, or a disease, namely diabetes, are paralleled by
changes in PDGF secretion. Although CD62 is perceived as
an activation marker of platelets indicating enhanced aggre-
gability and secretion of !-granular content, the proof that
the CD62 status and its modifications reflect directly the
actual secretion of the most important platelet mitogen,
PDGF, has not been given so far. This ex vivo-in vitro study
shows that at least for the activation pathway provided by
955
the PAR-1 thrombin receptor, for which TRAP is the selective
agonist (Kahn et al., 1999), CD62 expression on platelets is a
surrogate for their secretory activity. From the stimulus-
response curves, we established a concentration of 5 #M
TRAP that allows detection of drug- or disease-induced mod-
ifications in both directions. On the other hand, patients or
treatment groups in our study were not distinguishable on
the basis of their baseline secretion of PDGF or their baseline
expression of CD62. Samples collected independently from
the TRAP dose-response curve obtained from protocol 1 were
mostly within the prediction interval (Fig. 5) and suggested a
robust and reproducible relationship.
Flow cytometric detection of CD62 expressed on the plate-
let surface after !-granule release is a common method used
to characterize platelet activation in various experimental
and clinical conditions (Evangelista et al., 1996; Leytin et al.,
2000b). Parameters widely used to describe the activation
status are either percentage of CD62 positive platelets in the
total platelet population (%%) or the MFI of CD62 positive
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cells expressed in arbitrary units. Assaying %% platelets will
quantify the proportion of activated cells but will disregard
the quantity of expressed platelet surface antibody CD62
(Michelson et al., 2000). MFI represents the mean epitope
density of CD62 molecules on the average platelet surface
and therefore reflects the activity of the single platelet but
not their quantity (Evangelista et al., 1996). In our study, we
found a better correlation to PDGF secretion when using the
MFI to describe CD62 expression instead %% platelets. The
curve describing the relationship between CD62%% platelets
and PDGF release (Fig. 3, inset) starts extremely shallow,
which implies that even upon mild activation a large propor-
tion of the platelet population is detected as %%, but the
overall secretion levels remains relatively low. In contrast, at
levels of CD62%% over 80%, PDGF secretion increases 2-fold
despite only minor variations in CD62 expression.
PDGF is one of the major mediators for vascular smooth
muscle cell proliferation and migration that occur in initial
hyperplasia in the process of restenosis and atherosclerosis
(Ross 1999). Platelet granules are reported to contain mainly
PDGFAB and PDGFBB (Hammacher et al., 1988; Hart et al.,
1990). Animal experiments confirmed the role of the B-chain
in intimal hyperplasia, especially in preventing vascular
smooth muscle cells from apoptosis (Leppanen et al., 2000).
Accordingly, the growth activating potency of PDGFBB is
reported to be about 4-fold larger than that reported for
PDGFAB (Hart et al., 1990). From our data, based on enzyme-
linked immunosorbent assay technique, the ratio of the
PDGF isoforms AB and BB was approximately 1:10 and not
1:3 as quoted from a recent report employing reversed phase
HPLC technique for differential PDGF assay (Hart et al.,
1990).
We tried to prove whether the relationship between CD62
expression and secretion of PDGF is maintained under mod-
ifications induced by pharmacological or clinical conditions.
The thienopyridine P2Y12 receptor antagonists are the only
antiplatelet drugs that are known to reduce CD62 expression
(Rupprecht et al., 1998; Klinkhardt et al., 2000), whereas
treatment with GPIIb/IIIa inhibitors (Fredrickson et al.,
2000; Klinkhardt et al., 2000), or aspirin (Rinder et al., 1993;
Fredrickson et al., 2000; Klinkhardt et al., 2000) did not. We
could demonstrate a significant decrease in the platelet acti-
vation marker CD62 and the secretion product PDGF after 6
956 Graff et al.

TABLE 1
Protocols 2 to 4: CD62 expression and corresponding release of PDGFAB
Data are expressed as mean & S.D.

PDGF (ng/109 Platelets) CD62 (MFI)

Unactivated 5 #M TRAP Unactivated 5 #M TRAP

Protocol 2: drug treatment in vitro: abciximab (n ! 6)

Control 8.7 & 4.8 29.4 & 14.0 74 & 9 199 & 34
5 #g/ml abciximab 8.7 & 4.2 41.8 & 23.0 71 & 6 227 & 35
Protocol 3: drug treatment in vivo (healthy subjects:
clopidogrel (n ! 8))
Day 1: before clopidogrel 8.0 & 2.5 22.7 & 8.8 98 & 17 238 & 25
Day 6: under clopidogrel 6.3 & 3.8 11.6 & 3.0b 95 & 18 158 & 23b
Protocol 4: diabetic patients and age-adjusted controls (n ! 8)
Control 9.4 & 2.4 28.7 & 14.9 113 & 24 218 & 30
Diabetics 16.4 & 4.0a 45.8 & 8.0a 119 & 6 276 & 46a
a
p ' 0.05 for difference between groups (U test).
b
p ' 0.05 for difference between day 1 and day 6 (paired t test, two-sided probability).

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Fig. 4. CD62 expression (MFI) (top) and PDGFAB release (ng/109 plate-
lets) (bottom) in healthy volunteers (n ! 8) before (day 1) and under (day
6) clopidogrel intake at baseline (BL) and after TRAP (5 #M) stimulation
(protocol 3). Results are presented as means & S.D.

days of clopidogrel treatment. Since it has been observed that

via stimulation of phosphatidylinositol-3 kinase the P2Y12
receptor is a necessary constituent for sustained aggregation
induced by TRAP (Trumel et al., 1999; Storey et al., 2000), a
cross talk between signaling pathways of the PAR-1 throm-
bin receptor and purinergic receptors is likely and might
explain why clopidogrel effects TRAP-induced degranulation.
In diabetic patients, platelet hyper-reagibility is impli-
cated as a risk factor for both microvascular and macrovas-
cular disease (Bern 1978; Barnett 1991) and is associated
with an enhanced platelet aggregation response at sites of
vascular injury (Winocour 1992) and enhanced CD62 expres-
sion on the platelet surface (Tschoepe et al., 1991; Rauch et
al., 1999). However, it has not been determined whether
platelets from diabetic subjects release more of the content of
their granules in response to activation. Although our group
of diabetic patients (noninsulin-dependent diabetes mellitus)
was small, we observed a significantly higher PDGF-release
from platelets after stimulation with TRAP than in age-
matched controls. However, CD62 expression and PDGF re-
lease did not match in unactivated platelets, which might
indicate that a separate mechanism must be taken into
account to explain the dissociation between the parameters,
at least in patients with noninsulin-dependent diabetes
mellitus.
Thrombus-bound platelets at sites of vascular injury could
Fig. 5. Baseline and TRAP-induced CD62 expression and PDGFAB re-
lease obtained in subgroups (diabetics, elderly controls, and healthy
volunteers [before and under clopidogrel intake]) superimposed on the
curve and 80% prediction interval (dashed line) of protocol 1 from a
different subgroup of healthy subjects.
act as a depot for !-granule release of factors like PDGF with
sustained effects on the remodeling process (Schini-Kerth et
al., 1997). Therefore investigation of platelets activated by a
defined stimulus, mimicking the local thrombin response
might be closer to the situation of platelet depots in a throm-
bus than the determination of systemic serum growth factor
levels. However, some limitations of our study results need
attention. For platelet activation, we used TRAP (SFLL-
RNP), which is a synthetic peptide and stimulates the PAR-1
receptor without proteolytic cleavage like the physiological
activator thrombin, which also activates PAR-4 and GPIb-
receptors (Coughlin 1999; Kahn et al., 1999; Ofosu and
Nyarko, 2000). On the other hand, it has been demonstrated
that PAR-1 is the primary binding site for human thrombin
at platelets, and PAR-1 binding contributes to almost all
platelet-activating effects mediated by thrombin (Ofosu and
Nyarko, 2000). PDGF itself interferes with platelet activa-
tion, and it has been shown in heparinized blood that
PDGFBB in concentrations of 100 ng/ml reduced TRAP-in-
duced platelet aggregation and platelet microparticle forma-
 Relationship of CD62 Expression PDGF Release in Platelets
tion by approximately 20% (Selheim et al., 1999). From our
study, the steep shape of the curve describing the relation-
ship between PDGF release and CD62 is determined by data
pairs obtained after strong TRAP stimulation. In this part of
the curve, the increase in the MFI seems to be terminated
already despite a further increase in PDGF, which indeed
might indicate a negative feedback of PDGF on platelet ac-
tivation. Nevertheless, in the above-mentioned study (Sel-
heim et al., 1999), PDGF although reducing platelet aggre-
gation did not affect the expression of CD62.
In conclusion, our data support the utility of flow cytomet-
ric determination of CD62 as a target parameter during
clinical studies with antiplatelet agents and in acute or
chronic vascular diseases. The use of a defined stimulus, as
used in common platelet aggregation tests, might allow for
detection of drug- or disease-induced modifications and is
also closely related to the secretion of the potent mitogen
PDGF. However, marked discrepancies in the level of CD62
expression based on the flow cytometric protocol or device
used have been described (Serebruany et al., 1999), and the
need for standardization of the methodology must be empha-
sized.
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Address correspondence to: Prof. Dr. Sebastian Harder, Institute of Clinical
Pharmacology, at the Pharmazentrum Frankfurt, University Hospital, Theodor-
Stern-Kai-7, D-60590 Frankfurt/Main, Germany. E-mail: harder@em.uni-
frankfurt.de