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(Circulation. 2000;102:1931.)
© 2000 American Heart Association, Inc.
Clinical Investigation and Reports |
P-Selectin Expression on Platelets Determines Size and Stability of Platelet Aggregates
From the Division of Cardiology (M.M.) and the Division of Hematology (P.T.), Department of Internal Medicine, University of Texas Houston Medical School.
Correspondence to Michael Merten, MD, Division of Cardiology, Department of Internal Medicine, University of Texas Houston Medical School, 6431 Fannin, MSB 5.284, Houston, TX 77030. E-mail michael_merten{at}yahoo.com
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Abstract |
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Background—P-selectin mediates rolling of platelets and leukocytes on activated endothelial cells. After platelet activation, P-selectin is translocated from intracellular granules to the external membrane, whereas fibrinogen aggregates platelets by bridging glycoprotein (GP) IIb/IIIa between adjacent platelets.
Methods and Results—In this study, we define a novel role for P-selectin in platelet aggregation. Expression of P-selectin on the platelet surface correlated strongly with the mean platelet aggregate size. Inhibition of P-selectin binding to its ligand by either monoclonal anti–P-selectin antibodies directed against the lectin domain or soluble human P-selectin reversed platelet aggregation even when added up to 5 minutes after activation; however, fibrinogen binding to platelets was not affected. This deaggregating effect significantly reduced the maximal size and number of platelet aggregates. When added 1 minute after platelet activation, anti–P-selectin antibody achieved 95% to 100% of the deaggregating effect of EDTA, whereas the anti-GP IIb/IIIa antibody abciximab had no effect. Monoclonal antibodies against known P-selectin ligands, such as P-selectin GP ligand-1 (PSGL-1) or GP Ib, had no effect on platelet aggregation, suggesting a different ligand for P-selectin in platelet aggregate stabilization. In kinetic studies, P-selectin was maximally expressed 10 minutes after platelet activation, whereas maximal activation of GP IIb/IIIa occurred within the first 10 seconds, suggesting that P-selectin operates after fibrinogen binding to activated GP IIb/IIIa.
Conclusions—These results indicate that P-selectin interaction with a ligand, different from PSGL-1 or GP Ib, stabilizes initial GP IIb/IIIa–fibrinogen interactions, allowing the formation of large stable platelet aggregates.
Key Words: platelet-derived factors • glycoproteins • fibrinogen
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The glycoprotein (GP) P-selectin, a member of the C-type lectin family,1 is rapidly translocated from
-granules of platelets and Weibel-Palade bodies of
endothelial cells to the cell surface on
stimulation.2 3 It mediates rolling of platelets and
neutrophils on activated endothelial
cells4 5 as well as interactions of activated
platelets with neutrophils and monocytes.6 However,
the role of P-selectin in platelet aggregation remains to be
elucidated. During platelet aggregation, platelet GP IIb/IIIa
undergoes activation-dependent conformational changes and becomes
competent to bind soluble fibrinogen.7 Fibrinogen then
cross-links platelets by bridging GP IIb/IIIa between adjacent
platelets, thereby leading to the formation of platelet
aggregates.7 Subsequently, there is a progressive
stabilization of platelet-fibrinogen interactions.8 9
These post–fibrinogen binding events are influenced by the extent of
platelet secretion.10 11 12 However, the exact mechanism
of these stabilizing interactions has not been elucidated. In this study, we investigated the role of P-selectin in platelet aggregation and found that P-selectin stabilized initial platelet aggregates formed by GP IIb/IIIa-fibrinogen interactions, allowing the formation of large platelet aggregates. This defines not only a novel role for P-selectin in platelet aggregation but also a new mechanism of platelet aggregation.
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Methods |
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Analysis of Platelet Aggregation
Platelet-rich plasma (PRP) was prepared by centrifugation (1000g for 4 minutes) of citrated blood from healthy volunteers, as previously described.13 All human experiments were performed in accordance with institutional and state guidelines. PRP (
2.5x105 platelets/µL) was incubated
with either 5 µg/mL anti–P-selectin antibody G1 (Biosource, and a
kind gift from Dr Rodger McEver, University of Oklahoma), 5 µg/mL
anti–P-selectin antibody CLB-thromb/6 (Accurate Chemical & Scientific
Corp), 70 µg/mL recombinant human P-selectin (R&D Systems Inc), 60
µg/mL anti-human P-selectin GP ligand-1 (PSGL-1) antibody PL-1
(Immunotech), 40 µg/mL anti-human GP Ib antibodies SZ2 and WM23 (a
kind gift from Dr Jose Lopez, Baylor College of Medicine, Houston,
Tex), or 20 µg/mL isotype-matched control antibody (M4.3) for 5
minutes at 37°C followed by the addition of 3 µmol/L ADP,
3 µmol/L thrombin receptor-activating peptide SFLLRNA, or 3
µmol/L thromboxane receptor agonist U46619. All reagents
used in platelet aggregation were dialyzed in Tris-buffered saline
(pH 7.5) before their use. Aggregation was measured in an aggregometer
(Biodata). After 10 minutes of aggregation in the presence of
isotype-matched control antibody (20 µg/mL) or CLB-thromb/6 (5
µg/mL), 5-µL samples were transferred to microscope slides,
air-dried, and stained with Wright-Giemsa stain. We also compared the reversal of platelet aggregation induced by CLB-thromb/6 or the chimeric anti-GP IIb/IIIa antibody abciximab to that induced by 5 mmol/L EDTA, since 5 mmol/L EDTA has been used to measure reversibility of platelet aggregation.10 11 CLB-thromb/6 (5 µg/mL), abciximab (20 µg/mL), or EDTA (5 mmol/L) was added at 1 minute after platelet activation with 3 µmol/L thrombin receptor-activating peptide.
Determination of Size Distribution of Platelet Aggregates
Platelet aggregates were fixed with 1%
paraformaldehyde 3 minutes after addition of ADP
(1 µmol/L) or thrombin-receptor activating peptide (1.6
µmol/L) and diluted 25-fold in Tris-buffered saline. The size
distribution of platelet aggregates was determined with a FACScan
flow cytometer (Becton Dickinson) by a modification of previously
described methods.14 Light scattering was set at
logarithmic gain and events were counted for 5 minutes (forward scatter
was E00 and side scatter was 275). Platelet aggregate size was
expressed in arbitrary forward scatter units (FSU). The maximal
platelet aggregate size, defined as the FSU below which 99.9% of
the events fall, was determined in the presence of isotype-matched
control antibody (5 µg/mL) or CLB-thromb/6 (5 µg/mL). Also
determined was the number of events beyond 300 FSU, corresponding to
aggregates with a diameter >4 µm (as determined by standard
beads). The mean platelet aggregate size, obtained from 10 000
events representing platelet aggregates >300 FSU, was
determined for ADP concentrations ranging from 0.02 to 0.18
µmol/L. To correlate P-selectin surface expression with platelet
aggregate size, parallel unstirred PRP samples were incubated with
saturating concentrations of PE-labeled anti–P-selectin antibody
(Pharmingen) for 30 minutes before the mean fluorescence of
P-selectin–bound antibody per 10 000 events was measured. There was
no significant change (<2%) of the mean fluorescence of bound
control anti-GP Ib antibody (Pharmingen) after activation of the PRP
samples (unstirred).
Measurement of P-Selectin Surface Expression and GP IIb/IIIa
Activation
Simultaneous measurement of P-selectin surface
expression and activation of GP IIb/IIIa was achieved by a modification
of methods previously described by Frojmovic et al.15 PRP
(50 µL) was diluted with Tris-buffered saline and activated
with 20 µmol/L ADP. Saturating concentrations of FITC-labeled,
activation-specific anti-GP IIb/IIIa antibody PAC-1 (Becton Dickinson)
and PE-labeled anti–P-selectin antibody were added to aliquots of PRP
(5 µL) at different time intervals after activation (5, 10, 30, 60,
180, 300, and 600 seconds), gently mixed, and incubated for 60 seconds.
After quench-dilution (60-fold), the samples were analyzed
by flow cytometry within 45 seconds. Unactivated samples of PRP
served as negative controls.
PAC-1 Binding to Platelets and Effect of Anti–P-Selectin
Antibodies
To examine the effect of anti–P-selectin antibodies on
fibrinogen binding to platelets, we used the binding of the
antibody PAC-1 to platelets. PAC-1 recognizes an
activation-dependent epitope on the GP IIb/IIIa complex of
platelets at or near the platelet fibrinogen
receptor16 17 and inhibits fibrinogen-mediated
platelet aggregation.18
Citrated PRP (2.5x105 platelets/µL) was
either activated with 20 µmol/L thrombin
receptor-activating peptide at 37°C for 5 minutes (no shaking to
avoid aggregation) or left untreated. Aliquots (10 µL) of PRP, fixed
with 1% paraformaldehyde, were incubated with
FITC-labeled PAC-1 (20 µg/mL) for 45 minutes, quench-diluted, and
analyzed by flow cytometry. To determine the effect of various
antibodies on PAC-1 binding to activated platelets, PRP was
incubated with the antibodies CLB-thromb/6 (10 µg/mL), G1 (10
µg/mL), or abciximab (20 µg/mL) for 5 minutes before platelet
activation. Unactivated platelets were used as
controls.
Statistical Analysis
All experimental values are represented as mean±SD.
Statistical significance was evaluated by the paired Student’s
t test; the correlation coefficient was tested by the Fisher
test. A value of P<0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Inhibition of P-Selectin Interaction With Its Ligand Inhibits Platelet Aggregation
We examined the effect of the anti–P-selectin antibodies G1 and CLB-thromb/6, both directed against the lectin domain of P-selectin,19 as well as soluble recombinant human P-selectin on human platelet aggregation. None of these agents had a significant effect on the initial slope of platelet aggregation in human PRP after activation with ADP (Figure 1A
) or thrombin receptor-activating
peptide SFLLRNA (Figure 1B). However, all of these agents
inhibited the extent of platelet aggregation significantly by
deaggregating initially formed platelet aggregates. Similar results
were obtained with platelets activated with the
thromboxane receptor agonist U46619 (data not shown). The
different agonists were used at concentrations sufficient to induce
irreversible platelet aggregation. After activation with 3
µmol/L ADP, there was a 67±8.2% and 71±6.9% inhibition of
platelet aggregation with monoclonal anti–P-selectin antibodies G1
or CLB-thromb/6, respectively, and a 41.3±2.9% inhibition with
soluble recombinant human P-selectin at 10 minutes (Figure 1A).
Even when a high ADP concentration of 10 µmol/L was used to
activate platelets, CLB-thromb/6 inhibited platelet
aggregation by 67% to 73% (data not shown). The
inhibitory effect was even more pronounced after
platelet activation with 3 µmol/L thrombin
receptor-activating peptide, in which a 92±3.0% and 95.3±2.1%
inhibition of platelet aggregation was observed with
anti–P-selectin antibodies G1 or CLB-thromb/6, respectively (Figure 1B). Using twice the concentration of thrombin
receptor-activating peptide (6 µmol/L) to activate
platelets, CLB-thromb/6 inhibited platelet aggregation by 66%
to 71% (data not shown). The inhibitory effect of the
anti–P-selectin antibodies was maximal at 5 µg/mL. Higher
concentrations of soluble recombinant human P-selectin were required to
achieve a similar effect in ADP-induced platelet aggregation
(Figure 1A), probably because soluble P-selectin exists in a
monomeric form with an 40-fold lower affinity toward its ligand than
oligomeric membrane P-selectin.20 21 When added 1 minute
after platelet activation, the anti–P-selectin antibody
CLB-thromb/6 achieved 95% to 100% of the deaggregating effect of
EDTA, approximating the effect of EDTA with increasing time after
platelet activation, whereas the chimeric anti-GP IIb/IIIa antibody
abciximab had no effect (Figure 2A). When
CLB-thromb/6 was added 1, 3, or 5 minutes after platelet
activation, it caused deaggregation of initial platelet aggregates
by 47±4.6%, 28.3±9.1%, and 17±6.6% at 14 minutes after
platelet activation, respectively (Figure 2B). The
anti–P-selectin antibody G1 deaggregated platelets by 60%, 45%,
or 29%, respectively, when added 1, 3, or 5 minutes after platelet
activation (data not shown). Anti-human PSGL-1 antibody, PL-1, which
blocks PSGL-1 binding to P-selectin,22 23 or anti-human GP
Ib antibodies SZ2 (not shown) or WM23, which inhibit the binding of
P-selectin expressing CHO cells to glycocalicin,24 had no
significant effect on ADP or thrombin receptor-activating
peptide-induced platelet aggregation (Figure 1, A and B).
This suggests that the ligand for P-selectin in platelet aggregate
stabilization is different from known P-selectin ligands such as PSGL-1
or GP Ib. The possibility of platelet-leukocyte interactions being
responsible for the deaggregating effect of P-selectin inhibition is
unlikely because the leukocyte number was <0.1% of the platelet
number in PRP, as determined by flow cytometric analysis with
an anti-CD45 antibody (Caltag Laboratories).
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Formation of Platelet Aggregates Depends on P-Selectin
We also monitored the effect of anti–P-selectin antibody
CLB-thromb/6 on platelet aggregation by microscopy. Many large
platelet aggregates with a diameter of 200 to 2000 µm,
containing 0.8 to 800x106
platelets,14 were apparent after platelet
aggregation with thrombin receptor-activating peptide (Figure 3A). Prior incubation with CLB-thromb/6
almost completely inhibited formation of these large aggregates
compared with an isotype-matched control antibody (Figure 3A).
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4 µm diameter, 1100 FSU
To quantify the deaggregating effect, we analyzed the size
distribution of aggregates by flow cytometry (Figure 3B
). Flow
cytometric analysis of platelet aggregates has an
20-fold greater sensitivity than the turbidimetric method of
platelet aggregometry, allowing the quantification of platelet
aggregates barely detectable by the aggregometer.14
Incubation of platelets with anti–P-selectin antibody CLB-thromb/6
before activation with thrombin receptor-activating peptide reduced the
maximal size of platelet aggregates by 48% (from 3 309±89 to
1718±201 FSU, n=3, P<0.005) (Figure 3C).
CLB-thromb/6 also significantly reduced the number of aggregates with a
diameter >4 µm by 54% (from 6993±532 to 3317±430 FSU, n=3,
P<0.005) (Figure 3D). Similar results were obtained
with platelets after activation with ADP. Furthermore, the mean
expression of P-selectin on the platelet surface correlated
strongly with the mean platelet aggregate size produced by the same
ADP concentration (r=0.97, n=9, P<0.0001)
(Figure 4).
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Kinetics of P-Selectin Expression and GP IIb/IIIa
Activation
We next investigated the temporal relation between surface
expression of P-selectin and activation of GP IIb/IIIa during
platelet aggregation by using double
immunofluorescence with activation-specific anti-GP
IIb/IIIa antibody (PAC-1) and an anti–P-selectin antibody. Expression
of activated GP IIb/IIIa reached its maximum within the first
10 seconds of activation with ADP and declined afterward whereas
P-selectin expression increased progressively over 600 seconds (Figure 5).
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) were determined by flow cytometry.
Results shown are mean (±SD) of 3 separate experiments.
Effect of Anti–P-Selectin Antibodies on PAC-1 Binding to
Platelets
To examine the effect of anti–P-selectin antibodies on fibrinogen
binding to platelets, we used the binding of FITC-labeled antibody
PAC-1 to platelets, which binds to an activation-dependent epitope
on GP IIb/IIIa17 18 and inhibits fibrinogen-mediated
platelet aggregation.19 The anti–P-selectin
antibodies G1 and CLB-thromb/6 had no effect on the PAC-1 binding to
activated platelets, whereas under similar conditions,
chimeric murine/human anti-GP IIb/IIIa antibody abciximab inhibited
PAC-1 binding (Figure 6).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this study, we examined the role of P-selectin in platelet aggregation. P-selectin expression on the platelet surface correlated strongly with the mean platelet aggregate size. Furthermore, the inhibition of P-selectin binding to its ligand by either the anti–P-selectin antibodies G1 and CLB-thromb/6, both directed against the lectin domain of P-selectin,19 or soluble human P-selectin inhibited the extent of platelet aggregation by deaggregating initial platelet aggregates. This was achieved even up to 5 minutes after platelet activation. The consequence of this deaggregating effect was a reduction in the maximal size and number of platelet aggregates. These findings indicate that platelet aggregate size and stability depend on P-selectin.
Anti–P-selectin antibody achieved 95% to 100% of the
deaggregating effect of EDTA when added 1 minute after platelet
activation, whereas the chimeric anti-GP IIb/IIIa antibody abciximab
had no effect. Anti–P-selectin antibody inhibits P-selectin binding to
its ligand,22 whereas EDTA inhibits both fibrinogen
binding to GP IIb/IIIa10 11 and P-selectin binding to its
ligand.22 The fact that the anti-P-selectin antibody
approximated the effect of EDTA with increasing time after platelet
activation supports a prominent role for P-selectin in later phases of
platelet aggregation. Furthermore, P-selectin was expressed
progressively over a period of 
10 minutes, whereas maximal activation
of GP IIb/IIIa occurred within the first 10 seconds, suggesting that
the stabilizing effect of P-selectin on large aggregate formation
operates after initial platelet aggregation by GP
IIb/IIIa-fibrinogen interactions. Consistent with this,
anti–P-selectin antibodies did not interfere with PAC-1 binding to
activated platelets.
After the formation of platelet aggregates, a variety of post–fibrinogen-binding events are involved in the stability of the platelet aggregates.8 9 These mechanisms are influenced by the extent of platelet granule release.10 11 12 On the basis of our observations, we propose a new paradigm for platelet aggregation involving P-selectin. In this paradigm, the initial event in platelet aggregation is the binding of fibrinogen to activated GP IIb/IIIa, bridging adjacent platelets. Subsequently, P-selectin, which is progressively expressed on the platelet surface, binds by means of the lectin domain to its binding site on adjacent platelets, stabilizing interactions between already-bridged platelets, thereby allowing the formation of large stable platelet aggregates. The initial bridging by GP IIb/IIIa-fibrinogen might be necessary to approximate P-selectin with its binding site because Glanzmann’s thrombasthenic platelets do not aggregate despite the presence of P-selectin.25
PSGL-1 has been identified as the ligand for P-selectin on neutrophils and monocytes.26 27 A recent study reports that PSGL-1 is also present on platelets, even though expressed 25- to 100-fold lower than on leukocytes.28 Furthermore, platelet GP Ib has been shown to act as a ligand for P-selectin.24 Thus, these studies suggest that PSGL-1 or GP Ib could act as the ligand for P-selectin on platelets. However, the anti–PSGL-1 antibody, PL-1, which blocks PSGL-1 binding to P-selectin,22 23 or anti-GP Ib antibodies SZ2 or WM23, which inhibit the binding of P-selectin expressing CHO cells to glycocalicin,24 had no significant effect on ADP- or thrombin-induced platelet aggregation. These results are consistent with an earlier report that showed anti-GP Ib antibody SZ2 had no effect on ADP- or thrombin-induced platelet aggregation.29 Thus, it is likely that the ligand for P-selectin in platelet aggregate stabilization is different from known ligands for P-selectin, such as PSGL-1 or GP Ib. Potential other P-selectin ligands on platelets are gangliosides containing sialyl Lewis X.30 It has been shown that gangliosides on platelets are redistributed after activation with ADP31 or thrombin.32 These gangliosides could serve as ligands for P-selectin in platelet aggregate stabilization.
P-selectin–carbohydrate bonds have been shown to be of high tensile strength33 and may be a major mechanism of platelet aggregate stabilization. With the formation of increasing numbers of these bonds over time, the initial GP IIb-IIIa-fibrinogen complexes can be internalized or moved toward the canalicular system,34 leaving P-selectin as the only bridging molecule. In fact, Isenberg et al35 have shown that P-selectin was the only glycoprotein present in the contact zone between platelet aggregates 15 minutes after activation.
An inhibitory effect of anti–P-selectin antibodies on platelet aggregation has been reported under certain conditions, but no mechanism has been delineated.25 36 Besides, the anti–P-selectin antibodies used in this study are directed against the lectin domain of P-selectin, whereas antibodies in previous studies were directed against complement repeat domains of P-selectin.19
In 1978, Gartner et al37 demonstrated that thrombin-activated platelets expressed a surface-bound lectin, which is involved in platelet aggregation. Expression of this lectin was secretion-dependent,38 and sugars, which inhibited this lectin activity, also inhibited ADP- and thrombin-induced platelet aggregation.37 Our results are consistent with these observations and suggest that the lectin identified by Gartner et al was P-selectin.
Thus, P-selectin may play a central role in platelet interactions not only with endothelial cells and leukocytes but also with other platelets. Since P-selectin determines the size and stability of platelet aggregates, it may be of importance in arterial thrombosis, where platelet aggregation plays a significant role. Indeed, inhibition of P-selectin function accelerated thrombolysis in a primate model of arterial thrombosis and reduced recurrent coronary arterial thrombosis in dogs.39 40 This thrombolytic effect of P-selectin antagonists, which was attributed to the inhibition of platelet-leukocyte interactions, could have been primarily due to the reversal of platelet aggregation, as shown in our study. Consequently, therapeutic interventions directed against P-selectin or its ligand may be beneficial in the treatment of arterial thrombosis.
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Acknowledgments |
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This work was supported by a grant-in-aid from the American Heart Association and a grant from the Charles Slaughter Foundation.
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Footnotes |
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Guest editor for this article was Barry S. Coller, MD, Mount Sinai School of Medicine, New York, NY.
Received March 2, 2000; revision received May 17, 2000; accepted May 24, 2000.
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