Fibrin Clot Retraction by Human Platelets
Correlates with
IIb
3
Integrin-dependent Protein Tyrosine Dephosphorylation*
Sylvie
Osdoit and
Jean-Philippe
Rosa
From U348 INSERM and IFR6 Circulation-Lariboisière,
Hôpital Lariboisière, 41 Boulevard de la Chapelle,
75475 Paris Cedex 10, France
Received for publication, October 2, 2000
 |
ABSTRACT |
We have analyzed tyrosine phosphorylation
associated with retraction of the fibrin clot by washed platelets in
purified fibrinogen. Retraction was dependent on integrin
IIb
3, based on absence of
retraction of
IIb
3-deficient
thrombasthenic platelets. However, only a subset of
IIb
3-blocking antibodies or peptides were
able to inhibit retraction, suggesting a differential engagement of
IIb
3 in fibrin clot retraction
versus aggregation. Immunoblotting demonstrated a
phosphorylated protein pattern comparable with aggregation at early
time points. However, as opposed to aggregation, tyrosine
phosphorylation decreased rapidly in parallel to retraction (up to 60 min). Dephosphorylation was
IIb
3-dependent, since it was
blocked by
IIb
3-specific inhibitors and
was absent in thrombasthenic platelets. Inhibition of platelet clot
retraction by phenyl-arsine oxide and peroxovanadate, suggested
a role for tyrosine phosphatases. Cytochalasin D and E (5 µM) blocked fibrin clot retraction and tyrosine
dephosphorylation, suggesting regulation by actin cytoskeleton
assembly. Tyrosine phosphatase activities were found associated with
clot retraction using the "in-gel" tyrosine phosphatase assay;
however, none were
IIb
3-dependent. An 85-kDa
protein and to a lesser degree "Src" showed the closest dose-dependent correlation between inhibition of tyrosine
dephosphorylation and inhibition of retraction. We thus postulate that
IIb
3 engagement in fibrin clot retraction
drives, in an actin cytoskeleton-dependent manner, the
interaction of tyrosine phosphatases and of the tyrosine-phosphorylated substrates 85-kDa protein and Src, the dephosphorylation of
which regulates the force generation and/or transmission required for full contraction of the fibrin matrix.
 |
INTRODUCTION |
Matrix retraction (or contraction) is a cellular event subsequent
to cell matrix adhesion that is biologically significant, since it is
involved in such relevant phenomena as morphogenesis during
embryogenesis, wound healing, or the final steps of hemostasis (1, 2).
Cellular matrix retraction corresponds macroscopically to the reduction
of the matrix volume, due to the activity of cells that actively
reorganize the extracellular matrix by shortening and thickening matrix
fibers (3). Matrix contraction is exhibited by numerous cell types,
including smooth muscle cells, fibroblasts, monocytes, endothelial
cells, or platelets. Although the underlying mechanisms involve
integrin engagement, cytoskeletal reorganization, and force generation
by molecular motors (1, 2), the exact molecular requirements for each
of these steps as well as their coordination are still poorly understood.
Platelets provide a particularly attractive model with
which to address the issue of matrix retraction, since 1) platelets are
easy to isolate and to analyze, 2) they develop a contractile activity
in fibrin clot that is easy to assess, and 3) retraction activity is
linked to engagement of a well characterized integrin,
IIb
3 (also termed glycoprotein IIb-IIIa,
or gpIIb-IIIa).
IIb
3 is the most abundant
integrin at the platelet surface (4) and acts as a fibrinogen receptor
in platelet aggregation. Both structure-function relationships and the
signaling pathways triggered by
IIb
3
engagement have been defined in detail for aggregation and adhesion
(5). In contrast, engagement of
IIb
3 in
fibrin clot retraction has been the subject of only a limited number of
studies and remains poorly understood. The first evidence for a role of
IIb
3 in retraction has been deduced from
studies of a human hereditary hemorrhagic condition termed Glanzmann
thrombasthenia, which is characterized by a quantitative or a
qualitative defect in
IIb
3 (6). In this
recessive disorder, bleeding tendency is associated with the inability
of the patient's platelets to aggregate and to retract a fibrin clot.
More direct evidence for a role of
IIb
3 in fibrin clot retraction was gained from studies using
IIb
3-specific monoclonal antibodies and
IIb
3 antagonist peptides (7-9). In addition, a differential engagement of
IIb
3 in fibrin clot retraction versus aggregation has been suggested (10, 11). Altogether, these studies have demonstrated that
IIb
3
is an important component of fibrin clot retraction, but the presence
of plasma in most studies has precluded more refined biochemical
characterization as well as the study of retraction-associated platelet signaling.
Signaling associated with platelet aggregation, with
adhesion to immobilized adhesive proteins (fibrinogen, fibrin, von
Willebrand factor), or with spreading has been extensively studied (for
a review, see Ref. 5). In these experimental settings, signaling as
mediated by
IIb
3 was shown to involve
tyrosine kinases, including Syk, Fak, and Src, as well as tyrosine
phosphatases, such as PTP1B (5, 12, 13). In contrast, signaling
associated with clot retraction by platelets has been the subject of
only a few reports. These include the observation that tyrosine kinase
inhibitors inhibit fibrin clot retraction (14), a study of calpain
engagement in clot relaxation, in which partial inhibition of
retraction was restored by the calpain inhibitor calpeptin (15), and a study on Rho-A, an actin cytoskeleton regulator of the Rho family of
small G-proteins, for which no involvement in clot retraction could be
demonstrated (16). Finally, a report demonstrated that knock-in mice
expressing the conservative mutations Tyr
Phe747 and
Phe759 of the
3 cytoplasmic tail exhibited
platelets with altered clot retraction ability (17), suggesting a role
for
3 cytoplasmic tail tyrosine phosphorylation in clot
retraction. However signaling events associated with clot retraction
were not examined. To date, no thorough analysis of signaling and
particularly protein tyrosine phosphorylation during clot retraction
has been reported.
In the present paper, we analyze retraction of a fibrin clot by washed
platelets in a plasma-free system. We confirm that like in
platelet-rich plasma,
IIb
3 is central to
retraction, based on experiments using
IIb
3-specific antagonists or
IIb
3-deficient thrombasthenic platelets.
Next, we demonstrate that clot retraction is associated with a strong
and short initial wave of tyrosine phosphorylation, followed by a
sustained
IIb
3-dependent
tyrosine dephosphorylation of several polypeptides. Dephosphorylation
parallels retraction, is specifically blocked by
IIb
3 blockers, and is absent in
thrombasthenic platelets. Dephosphorylation is specific for retraction,
since it is not observed with thrombin alone. In turn, tyrosine
phosphatase inhibitors block clot retraction, thus suggesting a
functional role for tyrosine phosphatases in retraction. Tyrosine
dephosphorylation was inhibited by actin polymerization inhibitors.
Tyrosine phosphatase activities were found associated with clot
retraction using the "in-gel" tyrosine phosphatase assay, but none
were
IIb
3-dependent. Thus,
IIb
3 engagement in retraction does not
induce specific sets of tyrosine phosphatases but rather may drive the
specific interaction of preexisiting tyrosine phosphatases with
tyrosine-phosphorylated substrates. Among the latter, an 85-kDa protein
exhibited the highest sensitivity to phenyl-arsine oxide
(PAO).1 We conclude that
IIb
3 engagement in fibrin clot retraction drives, in an actin cytoskeleton-dependent manner, the
interaction of tyrosine phosphatases and of specific substrates and
particularly an 85-kDa protein, the dephosphorylation of which may be
involved in force generation and/or force transmission required for
full contraction of the fibrin matrix.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Human fibrinogen was purchased from Stago
(Courbevoie, France) or Kordia (Leiden, The Netherlands). Sodium
orthovanadate (NaVO4), H2O2,
PAO, genistein, erbstatin A, RGDS, RGES, the
-400-411
dodecapeptide, cytochalasin D and E, and the monoclonal anti-actin IgG
were from Sigma-Aldrich (Meylan, France).
IIb
3-specific monoclonal antibodies were
kindly provided by Dr B. S. Coller for 10E5 (18) and 7E3 (19)
(Mount Sinai Hospital, New York), Dr. T. S. Kunicki for AP2 (20)
(Scripps Clinic, La Jolla, CA), Dr. P. J. Newman for AP3 (21) (The
Blood Center, Milwaukee, WI), and Dr. R. P. McEver for Tab (22)
(OMRF, Oklahoma City, OK). Horseradish peroxidase-labeled polyclonal
anti-mouse or -rabbit IgGs were from Amersham Pharmacia Biotech.
The phosphotyrosine-specific monoclonal antibody 4G10 was from UBI
(EuroMedex; Illkirch, France). The
IIb
3
antagonist cyclic heptapeptide Integrilin® (23) was kindly provided by Dr. D. R. Phillips (COR Therapeutics, Palo Alto, CA), and the peptidomimetics Ro44-9883 (24) and Ro43-5054 (25) were provided by
Dr. B. Steiner (Hoffman-LaRoche, Basel, Switzerland). The ECL chemiluminescent kit for Western blotting detection was from Pierce.
Fibrin Clot Retraction Assay--
Whole blood was collected with
informed consent from healthy volunteers, or from a thrombasthenic
patient (C. B.) by venipuncture and anticoagulated in 1:9 (v/v) ACD-C
(130 mM citric acid, 124 mM sodium
citrate, 10 mM glucose, pH 4.0; Ref. 26). Washed platelets were then isolated as previously described (25) and resuspended in
resuspension buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 3 mM KCl, 0.5 mM
MgCl2, 5 mM NaHCO3, 10 mM glucose). CaCl2 was added extemporaneously,
at a final concentration of 1 mM. Fibrinogen was purchased
purified from contaminating fibronectin, von Willebrand factor, and
plasminogen and dialyzed in platelet resuspension buffer (see above) to
eliminate sodium citrate. Purity and absence of degradation was
checked by SDS-PAGE and Western blotting. Glass tubes designed for
aggregation were used for retraction assays. First, a 6% (w/v)
polyacrylamide cushion was polymerized at the bottom of the tubes to
avoid clot adherence. Tubes were then rinsed extensively in distilled
water. Washed platelets resuspended at 600,000/µl in resuspension
buffer containing fibrinogen (4 mg/ml) were dispensed in 0.25-ml
aliquots, and clot retraction was initiated by quickly pipetting down
0.25 ml of bovine thrombin (usually 2.0 units/ml) in resuspension
buffer. The reaction was developed at 37 °C. Pictures were taken at
time intervals using a digital camera. Quantification of retraction was
performed by assessment of clot area by use of the NIH Image 1.67e
software, and data were processed using Excel 4.0. Data were expressed
as follows: percentage of retraction = ((area
t0
area t)/area
t0) × 100.
Preliminary experiments have shown that optimal calcium concentrations
in our in vitro system were between 1 and 10 mM.
All experiments were therefore conducted at 1 mM
Ca2+. Similarly, no significant differences in speed and
final extent of retraction were obtained from 0.1 unit/ml to 1.0 unit/ml thrombin, indicating that at all concentrations tested,
thrombin was not limiting, and a concentration of 1 unit/ml thrombin
was chosen for all subsequent experiments. A fibrinogen concentration
of 2 mg/ml was chosen to reproduce physiological conditions. Whenever inhibitors were to be tested in the retraction assay, they were included in the platelet suspension buffer and usually preincubated at
room temperature prior to retraction induction by
thrombin.CaCl2 was added to Integrilin® to compensate for
sodium citrate present in the stock solution.
Western Blotting--
When clots were to be analyzed by Western
blotting, they were solubilized by adding 125 µl of a 5× stock
solubilization buffer (50 mM Hepes, pH 6.8, 10% SDS, 100 mM dithiothreitol, 25 mM EDTA, 50% glycerol,
0.025% bromphenol blue) and heated at 90 °C for 20 min. 20-50 µg
of platelet proteins were separated on 6% SDS-PAGE gels and
electrotransferred to nitrocellulose membrane using a Hoeffer
electroblotter appliance at 200 mA for 90 min. Processing of
blotted membranes was carried out as described previously (26), and
bound IgGs were detected by chemiluminescence using the manufacturer's instructions. Quantitation was achieved by digitizing Western blot
autoluminograms and assessing band area and gray scale pixel values
using the SigmaGel 1.0 computer program. Variation in band intensities
due to the Western blotting procedure was corrected using actin as an
internal standard for each sample, and then intensity values were
normalized using the corresponding band as reference in the 5-min
retraction control run in each gel.
In-gel Analysis of Tyrosine Phosphatase
Activity--
Determination of tyrosine phosphatase activity was
achieved by the in-gel method of K. Burridge and Nelson (27). A
poly(Glu-Tyr) peptide was 32P-labeled as described except
that c-Src (Sigma) was used for labeling. Labeled poly(Glu-Tyr)
(~1 × 108 cpm/mg) was included in a 13%
SDS-polyacrylamide gel (0.1% bisacrylamide (w/v)) at 100,000 cpm/ml
before polymerization. After electrophoresis, the SDS-PAGE gel was
processed exactly as described to renature tyrosine phosphatase
activity. After Coomassie staining and drying, the gel was subjected to
autoradiography. Bands corresponding to tyrosine phosphatase proteins
appeared clear on a dark background of phosphorylated peptide. Negative
pictures of autoradiographs were generated after scanning and image processing.
 |
RESULTS |
Plasma-free Clot Retraction Assay Is Dependent upon
IIb
3 Integrin Engagement--
We
have developed a plasma-free retraction assay using purified fibrinogen
and washed platelets. Photographs of a typical control clot retraction
are shown in Fig. 1A
(upper panel) from 0 to 60 min. The extent of
retraction was assessed from quantitation of clot surface area (see
"Experimental Procedures") and was expressed as percentage of total
clot surface versus time, as illustrated in Fig.
1A, lower panel, control
curve. In preliminary experiments (not shown), we have
tested varying conditions including thrombin concentrations from 0.1 to
4 units/ml and Ca2+ concentrations from 1 to 10 mM, at the physiological fibrinogen concentration of 2 mg/ml. No difference was found in retraction kinetics (nor in protein
tyrosine phosphorylation patterns). We therefore considered our
conditions nonlimiting and chose arbitrarily to perform experiments at
concentrations of 1 mM Ca2+ and 1 unit/ml
thrombin.

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Fig. 1.
Fibrin clot retraction in plasma-free
conditions is mediated by integrin
IIb 3
engagement. A, kinetics of fibrin clot retraction in buffer
and washed platelets and its inhibition by
IIb 3-specific monoclonal antibodies.
Platelets were washed and resuspended at 3 × 108/ml
in buffer (see "Experimental Procedures") in the presence of 2 mg/ml human fibrinogen, and fibrin clot was initiated by 1.0 unit/ml
bovine thrombin at 37 °C. Upper panel, time
frames of a retracting clot at the indicated time points.
Lower panel, clot retraction kinetics curves in
the absence or the presence of anti- IIb 3
monoclonal antibodies. Clot surface areas were assessed by digital
processing and plotted as percentage of maximal retraction
(i.e. volume of platelet suspension) (see "Experimental Procedures"). Values are
plotted as a function of time. Control platelets are shown by
open diamonds (continuous
line). Monoclonal antibodies used were 10E5
(anti- IIb 3, open
diamonds, dashed line), Tab
(anti- IIb, open circles,
dashed line), AP3 (anti- 3,
open squares, dotted line),
7E3 (anti- 3, closed squares,
dashed line), or mixes: 10E5 + AP3
(closed triangles, continuous
line), 10E5 + Tab (closed triangles,
dotted line), 7E3 + Tab (closed
squares, dotted line), or Tab + AP3
(closed circles, continuous
line). Data correspond to means ± S.E. from 5-9
experiments. B, inhibition of retraction by
IIb 3-specific peptide antagonists.
Platelets were preincubated with antagonists at 37 °C for 10 min
prior to the addition of fibrinogen and clot initiation by thrombin.
Antagonists were RGDS (striped bar), the
-400-411 dodecapeptide from fibrinogen (horizontal
striped bar), and Integrilin® (closed
bars). Values are maximal retraction obtained at 60 min and
are expressed as means ± S.E. from three experiments for RGDS and
-400-411 dodecapeptide and nine experiments for Integrilin®.
C, defective clot retraction in plasma-free conditions with
IIb 3-deficient thrombasthenic platelets.
Platelets from thrombasthenic patient CB (open
circles) were washed and subjected to clot retraction in
buffer, comparatively with control platelets (closed
diamonds) or control platelets added with Tab + AP3 mix (20 µg/ml each) (closed triangles). Values are
mean ± S.E. from one experiment in triplicate.
|
|
Previous studies have demonstrated
IIb
3 integrin involvement in retraction
of fibrin clot in plasma (7-10). To verify the engagement of
IIb
3 in our plasma-free conditions, we
used various
IIb
3-specific inhibitors
(Fig. 1). Among several
IIb
3-blocking monoclonal antibodies (Fig. 1A, lower
panel), the
3-specific monoclonal antibody
7E3 inhibited retraction efficiently (80%), confirming previous
observations (7). In addition, combinations of monoclonals, ineffective
when used alone, such as AP3 (
3-specific) and Tab
(
IIb-specific), or either one in combination with 10E5 (
IIb
3-specific) also blocked retraction
efficiently. Surprisingly, the aggregation-blocking 10E5 (as well as
AP2, not shown) was unable to block retraction when used alone. This
suggests a differential engagement of
IIb
3 with fibrin compared with fibrinogen
and/or in retraction compared with aggregation.
In Fig. 1B, a comparative analysis was conducted with the
IIb
3 antagonists RGDS and
-400-411
dodecapeptide and the highly specific peptide Integrilin®.
Integrilin® inhibited retraction efficiently (82 ± 11%
inhibition at 25 µM), confirming the specific engagement
of
IIb
3 in fibrin clot retraction. Of
note, neither RGDS nor the
-400-411 dodecapeptide had any effect;
this result together with the higher concentration of Integrilin®
required for full inhibition of retraction (25 µM)
versus aggregation (2 µM; Ref. 23 and data not
shown) suggests higher affinity/avidity of fibrin for
IIb
3 compared with fibrinogen, possibly
due to the polymeric state of the former.
In Fig. 1C,
IIb
3 engagement in
fibrin clot in our assay conditions was confirmed by the low extent of
retraction of platelets from a type I thrombasthenic patient exhibiting
no detectable
IIb
3 by Western blotting
(see Refs. 26 and 28; data not shown). This result is comparable with
that obtained with the monoclonal antibody mix Tab + AP3.
Altogether, our data 1) confirm the involvement of
IIb
3 integrin in fibrin clot retraction
in plasma-free conditions, and thereby validate our assay and 2)
suggest differential engagement of
IIb
3
in retraction versus aggregation.
Fibrin Clot Retraction Kinetics Correlates with Protein
Tyrosine Dephosphorylation--
We then asked whether differential
engagement of
IIb
3 corresponded to
differential
IIb
3 signaling; we thus
assessed tyrosine phosphorylation associated with clot retraction,
since it is a prominent signaling pathway triggered by
IIb
3 (5). Fig.
2 shows phosphotyrosine protein patterns
during retraction, as assessed by Western blotting and the
corresponding band intensity quantitation. In Fig. 2A, a
time course of phosphotyrosine protein phosphorylation during fibrin
retraction with normal platelets showed an initial pattern comparable
with platelet aggregation (26), including a 125/130- and a 100/105-kDa
doublet (the latter slightly altered in migration by a 100-kDa
nonphosphorylated protein identified as the cross-linked
-
fibrinogen/fibrin chain dimer; data not shown), a faint 85-kDa band,
cortactin as a 77-80-kDa doublet, a 64-kDa band, Src as a 60-kDa band,
and a 48-kDa band. Cortactin and Src have been identified previously by
immunoprecipitation (26). This pattern evolved with time, exhibiting a
short initial wave of phosphorylation culminating at 5 min, followed by
a slower wave of dephosphorylation, which paralleled most of the
retraction extent (Fig. 2B). This phenomenon was not due to
protein degradation, as ascertained by even Ponceau S patterns (not
shown) and reprobing of the blot with anti-actin (bottom
panel, actin). Dephosphorylation was not due to
thrombin stimulation per se, since platelets stimulated with
thrombin in the absence of added fibrinogen yielded a tyrosine phosphorylation pattern that remained steady with time, as opposed to
platelets in conditions of retraction (Fig. 2C). Thus, the pattern of tyrosine phosphorylation, characterized by a short initial
wave of tyrosine phosphorylation followed by a slower dephosphorylation
pattern, is specific to fibrin clot retraction.

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Fig. 2.
Kinetics of tyrosine phosphorylation of
platelet proteins during clot retraction. A, platelet
protein tyrosine phosphorylation during clot retraction kinetics by
Western blotting. Platelets were subjected to retraction for different
times and immediately solubilized. Proteins were separated by SDS-PAGE,
electroblotted, and reacted with a phosphotyrosine-specific monoclonal
antibody (upper panel), or actin-specific
monoclonal antibody (lower panel). Bound
antibodies were then detected by luminol-based chemiluminescence and
film exposure (autoluminogram). A typical experiment is shown.
Identification of migrating bands is indicated on the right.
B, quantitative kinetics of protein tyrosine phosphorylation
during clot retraction. Autoluminograms were digitized, and bands were
quantitated by digital processing. Relative quantitation was achieved
using the 5-min time point arbitrarily chosen as 1, in arbitrary units
(A.U.) and using the actin signal in each lane to normalize
data. Open circles, continuous
line, Src; closed diamonds,
continuous line, the 125/130-kDa doublet;
open squares, dashed line,
the 100/105-kDa doublet; open triangles,
continuous line, cortactin; closed
squares, dotted line, p64. Plotted
data are means ± S.E. from four experiments. Quantitative clot
retraction curve from corresponding experiments is shown as
closed circles and dashed
line (error bars are omitted for the
sake of clarity). C, comparative analysis of tyrosine
phosphorylation of platelet proteins by Western blotting, in retraction
and thrombin stimulation. Control platelets were subjected to Western
blotting and tyrosine-phosphorylated protein detection after clot
retraction (retraction, right panel)
or thrombin stimulation (1.0 units/ml), i.e. in the absence
of added fibrinogen (thrombin, right
panel). These are representative autoluminograms from four
separate experiments. Bands are identified between the
retraction and thrombin panels.
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|
Tyrosine Dephosphorylation Is Dependent on
IIb
3 Integrin Engagement--
Fig.
3 shows that when retraction was
inhibited by the retraction-blocking association of
anti-
IIb
3 (Tab, AP-3) monoclonal antibodies (middle panel), initial tyrosine
phosphorylation occurred in all conditions but was not followed by
dephosphorylation, as opposed to control platelets (left
panel). The same absence of dephosphorylation was noted when
retraction was blocked by the synthetic inhibitor Integrilin® (not
shown) or when thrombasthenic platelets lacking
IIb
3 were used (right
panel). The correlation in kinetics between retraction and
protein tyrosine dephosphorylation and the absence of dephosphorylation
in retraction-blocking conditions both argue strongly in favor of a
protein tyrosine dephosphorylation activity specifically triggered by
IIb
3 engagement in retraction. Interestingly, the initial wave of tyrosine phosphorylation appears
IIb
3-independent, since it is preserved
in
IIb
3-blocking conditions or in
thrombasthenic platelets.

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Fig. 3.
Immunodetection of tyrosine-phosphorylated
platelet proteins by Western blotting during clot retraction in the
presence of
IIb 3-specific
monoclonal antibodies or of
IIb 3-defective
thrombasthenic platelets. Platelets were subjected to retraction
for different times and immediately solubilized. Proteins were then
separated by SDS-PAGE and electroblotted, and tyrosine-phosphorylated
proteins were detected by a phosphotyrosine-specific monoclonal
antibody, luminol-based chemiluminescence, and autoluminogram.
Left panel, control platelets; medium
panel, platelets in the presence of a mix of
anti- IIb and anti- 3 monoclonal antibodies
(20 µg/ml of Tab and AP3, respectively); right
panel, platelets from a thrombasthenic patient (no
IIb 3 integrin detectable by Western
blot). Shown are representative autoluminograms from four separate
experiments for each. Percentage of retraction values (% retr.) are indicated at the top of each blot at
corresponding times. Bands are identified between
Control and
anti- IIb 3
panels.
|
|
Tyrosine Phosphatase Inhibitors Block Clot Retraction--
To test
whether tyrosine phosphatases played any role in retraction, we
performed clot retraction in the presence of various concentrations of
PAO or of peroxovanadate (H2O2 + NaVO4), two distinct inhibitors of tyrosine phosphatases
(29, 30). Fig. 4, A and
B, demonstrates that both inhibitors totally inhibited clot
retraction, with IC50 of 0.4 µM for PAO and
4-800 µM for peroxovanadate, while enhanced tyrosine
phosphorylation was demonstrated by Western blotting for the major
phosphorylated substrates p48, Src, p64, cortactin, p85, 100/105, and
125/130 (not shown). Altogether, our results suggest that tyrosine
phosphatase activity is directly or indirectly involved in clot
retraction.

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Fig. 4.
Effect of the tyrosine phosphatase inhibitors
PAO and peroxovanadate on platelet clot retraction. Various
concentrations of the tyrosine inhibitors PAO (A, control
(closed diamonds); 0.25 µM
(closed squares); 0.30 µM
(closed triangles); 0.50 µM
(closed circles); 0.75 µM
(open triangles); 1 µM
(open circles)) or peroxovanadate (1 mM H2O2 + NaVO4)
(B, control (closed diamonds); 0.1 mM (closed squares); 0.4 mM (closed triangles); 1 mM (closed circles); 2 mM
(open circles); 4 mM (open
diamonds)) were preincubated for 10 min at room temperature
with platelets prior to thrombin induction of clot retraction.
Quantitation was as in Fig. 1. Data represent the means ± S.E. of
nine experiments for PAO and eight experiments for
peroxovanadate.
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Cytochalasin D, an Inhibitor of Actin Polymerization, Inhibits Clot
Retraction and
IIb
3-dependent
Protein Tyrosine Dephosphorylation--
Cytochalasins are known to
inhibit clot retraction (3). To check if inhibition of actin
cytoskeleton affected
IIb
3-dependent tyrosine
dephosphorylation, we preincubated platelets with
cytochalasin D, a well studied inhibitor of actin polymerization. We
obtained a dose-dependent inhibition of clot retraction
(Fig. 5A), with full
inhibition at 2-5 µM. The dose-dependent
inhibition of retraction was comparable between cytochalasin D and E
(not shown). Analysis of protein tyrosine phosphorylation by Western
blotting showed that inhibition of actin polymerization completely
inhibited protein tyrosine dephosphorylation (Fig. 5B).
These results thus suggest that
IIb
3
regulates protein tyrosine dephosphorylation via actin cytoskeleton
assembly.

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Fig. 5.
Effect of cytochalasin D on clot retraction
and on tyrosine phosphorylation of platelet proteins.
A, control platelets (closed diamonds)
or platelets preincubated with cytochalasin D at various concentrations
(0.5 µM (closed squares); 1 µM (closed circles); 5 µM (open circles)) were subjected
to clot retraction, and data were expressed as percentage of complete
retraction. Data are the means ± S.E. of four experiments.
B, the kinetics of tyrosine phosphorylation of platelet
proteins in the course of clot retraction in the presence of 5 µM cytochalasin D was assessed by Western blotting.
Left panel, control platelets; right
panel, cytochalasin D-treated platelets. Shown is a
representative autoluminogram from three experiments. Bands are
identified on the right.
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Platelet Tyrosine Phosphatase Patterns during Clot
Retraction by in-gel Analysis--
One interpretation of our results
is that dephosphorylation during retraction is due to induction by
IIb
3 of a de novo tyrosine phosphatase activity. Alternatively,
IIb
3
may regulate tyrosine phosphatase/tyrosine-phosphorylated substrate
interaction. To distinguish between these possibilities, we looked for
correlations between retraction blockade by Integrilin® or
cytochalasin D and variations in the SDS-PAGE pattern of tyrosine
phosphatase activities of platelets, obtained by the in-gel method (see
"Experimental Procedures"). Fig. 6
shows a negative autoradiograph after in-gel phosphatase activity
analysis of platelets after retraction for 5 and 60 min, compared with
retraction inhibited by the
IIb
3-blocker Integrilin® or by cytochalasin D. Resting platelets (lane
1) exhibit two major bands at 130 and 32 kDa, as well as
several minor bands at 100, 85, 49, and 22 kDa. After 5-min retraction,
the 130-kDa and the 32-kDa bands were strongly diminished, while a new
26-kDa band appeared, the other bands remaining unchanged
(lanes 2 and 3). After 60-min
retraction, all bands diminished except for the unchanged 26-kDa
band. Thus, retraction correlated with a specific pattern of tyrosine
phosphatases, which evolved with time. However, the same pattern was
obtained when clot retraction was inhibited by the
IIb
3 blocker Integrilin®
(lanes 4 and 5) or by cytochalasin D
(lanes 6 and 7). This result thus
suggests that tyrosine phosphatases induced in conditions of fibrin
clot retraction are independent from
IIb
3
engagement or from actin polymerization. Although we cannot rule out a
possible "technical" artifact, this result suggests that
dephosphorylation during retraction is not due to the de
novo induction of a new tyrosine phosphatase by
IIb
3. It thus follows that
IIb
3-dependent
dephosphorylation is due to the regulation by
IIb
3 engagement of a specific interaction of tyrosine phosphatases and of tyrosine-phosphorylated substrates, possibly through actin cytoskeleton assembly.

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|
Fig. 6.
Comparative in-gel analysis of platelet
protein-tyrosine phosphatase patterns during clot retraction.
SDS-PAGE where 32P-labeled poly(Glu-Tyr) peptide
(105 cpm/ml) was included in the gel was carried out on
extracts of clots containing control platelets before (0,
lane 1) or after retraction was induced by
thrombin (lanes 2-7), in control buffer
(Rct, lanes 2 and 3), in
the presence of 25 µM Integrilin® (Int,
lanes 4 and 5) or of 10 µM cytochalasin D (CytoD, lanes
6 and 7) for 5 min (lanes
2, 4, and 6) or 60 min
(lanes 3, 5, and 7). After
renaturation (see "Experimental Procedures"), the gel was dried and
exposed to autoradiography. A negative digitized picture is presented,
which is a representative of four independent similar experiments.
Molecular weights of prominent bands are indicated on the
right.
|
|
Inhibition of Clot Retraction by Integrilin®, PAO, and
Cytochalasin D Correlate with Dose-dependent Patterns of
Protein Tyrosine Dephosphorylation--
We thus reasoned that if
relevant to retraction, tyrosine phosphatase substrates should exhibit
dose-dependent inhibition of dephosphorylation with
preferably all retraction blockers. The ratios of phosphorylation
levels at 60 min versus 5 min of clot retraction, in the
absence or the presence of the inhibitors Integrilin®, PAO, and
cytochalasin D were calculated for each substrate determined by Western
blotting and compared. A ratio of 1 or more indicates absence of
dephosphorylation or rephosphorylation, respectively. In Fig.
7A, 60:5 ratios were plotted
as a function of Integrilin® concentration. The 100-105-kDa, 85-kDa,
cortactin, Src, and 48-kDa substrates reached a ratio of 1 (i.e. without dephosphorylation from 5 until 60 min) at 10 and 25 µM, efficient inhibitory concentrations (see
retraction dose-response curve, upper right
inset). 85-kDa substrate exhibited the largest
difference in ratio between 0 and 25 µM, indicating the
highest sensitivity to Integrilin®. Quantitation of dephosphorylation
in the presence of PAO (Fig. 7B) showed high and
dose-dependent ratios for 85-kDa, 48-kDa, and 64-kDa
substrates and Src. Of note, 85-kDa ratios were
dose-dependent throughout the whole range of PAO
concentrations tested (0.3-0.6 µM), best fitting the
retraction dose-response curve (upper right
inset). In addition, 85-kDa protein demonstrated the largest
difference between ratios at 0.6 µM (r = 2.0) and 0 µM (r = 0.2), indicating again
the highest sensitivity to PAO. Thus 85-kDa phosphorylation appears
particularly sensitive to both an
IIb
3
block by Integrilin® and to tyrosine phosphatase inhibition and
correlates dose-dependently with clot retraction inhibition. Fig. 7C analyzes the phosphorylation ratio of
platelet proteins during retraction in the presence of the actin
polymerization inhibitor, cytochalasin D. Several substrates showed a
high 60:5 ratio, particularly 64-kDa protein, Src, and 85-kDa protein,
suggesting that regulation of their phosphorylation state is highly
dependent on tyrosine phosphatase activities dependent on actin
cytoskeleton assembly. Altogether, these results suggest that the
85-kDa protein, as a substrate for tyrosine phosphatase activity, and
its
IIb
3- and actin
polymerization-dependent dephosphorylation are functionally related to retraction.

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|
Fig. 7.
Comparative quantitation of the
dephosphorylation level of platelet phosphotyrosyl proteins during clot
retraction blocked by Integrilin®, PAO, or cytochalasin D. Western blots carried out in conditions of inhibition of clot
retraction by Integrilin®, PAO, or cytochalasin D were digitized, and
relative quantitation of the phosphorylation state of each substrate
was assessed as indicated under "Experimental Procedures." After
standardization (see "Experimental Procedures"), values were
normalized relative to the 5-min control of the experiment. Ratios of
phosphorylation at 60 min versus 5 min of retraction were
then calculated, with the 5-min value arbitrarily chosen as 1. Mean
values are expressed as mean ± S.E., of 3-5 experiments; no
error bars are indicated where only the mean of two experiments is
expressed. A value above 1 (marked by the dashed
line) indicates that tyrosine phosphorylation proceeded,
while a value below 1 shows dephosphorylation activity. A,
the 60 min/5 min ratios were calculated and plotted for Integrilin® at
0 (open bars), 5 (light
gray bars), 10 (dark gray
bars), and 25 µM (black
bars). B, 60 min/5 min ratios for PAO at 0 (open bars), 0.3 (light
gray bars), 0.4 (dark gray
bars), 0.5 (black bars), and 0.6 µM (dashed bars). C, 60 min/5 min ratios in control platelets (open bars)
or in the presence of 5 µM cytochalasin D
(black bars). The upper
right panels in A and B
represent the average dose-dependent inhibition curves
obtained with the corresponding inhibitor with the samples used for
Western blotting. Error bars are not indicated for sake of
clarity.
|
|
 |
DISCUSSION |
In the present study, we have addressed the questions
of adhesive and signaling determinants of retraction of the fibrin clot by platelets. To this end, we have developed an assay with washed platelets and pure fibrinogen to analyze platelet proteins by Western
blotting. Since most previous studies on platelet retraction were
carried out in plasma (8, 9), we sought to validate our newly designed
plasma-free assay. Because of the minimal composition of our system
compared with plasma, we wished to check the characteristics of
IIb
3 integrin engagement. The efficient
blocking of clot retraction by the
3-specific monoclonal
antibody 7E3 and the
IIb
3-specific
antagonist peptide Integrilin® confirmed the engagement of
IIb
3. The inability of several
IIb
3-specific aggregation-blocking agents
to alter retraction, including RGDS and the
-400-411 dodecapeptide, as well as the monoclonals 10E5 and AP2 (not shown), is suggestive of a
differential engagement of
IIb
3 in
retraction versus aggregation. This is consistent with the
work by Cohen et al. (7) who first showed that RGDS and 10E5
did not inhibit (but instead increased) isometric clot tension. Our
results are also consistent with the work of Rooney et al.
(10), who showed that recombinant fibrinogen in which the
408-411
region was deleted did support platelet retraction and not aggregation.
The molecular mechanism underlying differential engagement of
IIb
3 with fibrin in retraction
versus fibrinogen in aggregation remains to be elucidated,
although differences in avidity of polymeric(fibrin) versus
monomeric (fibrinogen) ligands for
IIb
3
may be at play.
Protein tyrosine phosphorylation is one of the major "outside-in"
signaling events triggered by
IIb
3
engagement and has been thoroughly studied in platelet aggregation and
spreading (5). We found that retraction was characterized by a brief initial phase of protein tyrosine phosphorylation followed by a
dephosphorylation wave, affecting the same substrates previously found
in aggregation or platelet adhesion: 125/130- and 100/105-kDa doublets,
cortactin, 64-kDa protein, and the Src tyrosine kinase (Refs. 5 and 26
and Fig. 2). Tyrosine dephosphorylation paralleled retraction kinetics
during most of its extent and was not due to thrombin activation of
platelets. In contrast, the initial tyrosine phosphorylation was
clearly
IIb
3-independent,
since neither its intensity nor its pattern were affected when
IIb
3 was blocked by Integrilin® or
monoclonal antibodies or when
IIb
3-defective thrombasthenic platelets
were used (Fig. 3). A corollary to this observation is that tyrosine
phosphorylation in platelet fibrin clot retraction is under the control
of an
IIb
3-independent system, which
remains to be determined. In conclusion, as opposed to aggregation,
where
IIb
3 engagement clearly controls
successive waves of tyrosine phosphorylation and dephosphorylation (5, 31, 32), our data are consistent with
IIb
3 engagement in the fibrin clot
controlling essentially activation of tyrosine dephosphorylation.
Two hypotheses, not necessarily mutually exclusive, could account for
IIb
3-dependent tyrosine
dephosphorylation: 1)
IIb
3 specifically
activates one or several tyrosine phosphatase activity(ies), or 2)
IIb
3 mediates the specific interaction of
tyrosine phosphatases with their tyrosine-phosphorylated substrates. To
distinguish between these two possibilities, we used the in-gel
technique developed by Burridge and Nelson (27), the only technique
among several tested that preserved tyrosine phosphatase activity after the denaturation conditions required to solubilize the fibrin clot
(i.e. proteins boiled in SDS for 20 min, in the presence of
a disulfide reducing agent). Using that elegant method, we found a
pattern of tyrosine phosphatase activities specifically induced during
retraction. However, we found that this pattern was
IIb
3-independent, since it was
unaffected during retraction inhibition by Integrilin® or cytochalasin
D. In addition, it was similar to that described previously by Pasquet
et al. (33) in platelets activated by thrombin and collagen
and neither subjected to aggregation or retraction and is thus probably
not specific to retraction. Thus, within the limits of this technique,
we cannot conclude as to the induction of a specific tyrosine
phosphatase activity by
IIb
3 engagement
in clot retraction. Altogether, our results are in favor of the idea
that
IIb
3-dependent
dephosphorylation during clot retraction is the consequence of an
IIb
3-driven specific tyrosine
phosphatases/tyrosine-phosphorylated substrates interaction rather than
activation of tyrosine phosphatases.
An important finding in the present work is that tyrosine
phosphatase inhibitors, PAO and peroxovanadate, which act through different mechanisms, inhibited retraction. This clearly suggests a
functional role for tyrosine phosphatases and thus for
dephosphorylation in retraction. We have also found that the strong
inhibitory effect of cytochalasins on retraction correlated with
inhibition of protein tyrosine dephosphorylation (Fig. 5). Altogether,
our results are thus consistent with a model in which
IIb
3 drives actin cytoskeleton assembly,
where the interaction of tyrosine phosphatases with phosphoprotein
substrates may take place. Dephosphorylation of these substrates may
then be involved directly or indirectly in force generation and/or
force transmission, leading to reorganization of the fibrin
matrix and clot retraction. Ezumi et al. (32) have
also reported tyrosine dephosphorylation in the course of thrombin-induced platelet aggregation. This dephosphorylation was
thought to be the consequence of the translocation of the tyrosine
phosphatases PTP1B and PTP1C (also known as SHP1) to the actin
cytoskeleton in an
IIb
3-dependent manner (32).
From these studies, it was difficult to conclude as to the exact
significance of dephosphorylation in aggregation, in particular since
no attempt at inhibiting phosphatase activity was made and also because
it only correlated with the late phase of aggregation. We propose that
tyrosine dephosphorylation in aggregation corresponds to reinforcement
of aggregates, due to either force generation and/or reorganization of
the actin cytoskeleton allowing force transmission and leading
to the irreversible phase of aggregation. Thus secondary aggregation
may be equivalent to retraction. This postulate is the subject of
current investigations.
Tyrosine dephosphorylation has been shown to correlate with platelet
microparticle release (33). Interestingly, microparticles were elicited
by the synergistic activation of platelets by collagen and thrombin,
(or the A23187 Ca2+ ionophore), thrombin alone being
ineffective. However, only a subpopulation of stimulated platelets
bound annexin V, which was used to probe for phosphatidylserine surface
exposure, a marker of microparticle release. This platelet
subpopulation exhibited extensive protein tyrosine dephosphorylation.
These results led the authors to conclude that there is a strong
correlation between microparticle release and protein tyrosine
dephosphorylation. Other investigators have demonstrated that platelet
microparticle release involves cortical actin membrane skeleton
reorganization, possibly controlling membrane phospholipid
redistribution (34, 35). It is therefore tempting to speculate that
tyrosine dephosphorylation has a common role in both platelet
microparticle release and fibrin retraction, possibly via actin
cytoskeleton reorganization.
We have sought to determine whether pathways other than tyrosine
phosphorylation and known to be dependent upon
IIb
3 engagement were involved in fibrin
clot retraction. The signaling and
IIb
3-dependent protease
µ-calpain may positively regulate aggregation (36). Interestingly,
one of its targets is PTP1B (37), thus making µ-calpain an attractive
candidate for activating
IIb
3-dependent tyrosine
phosphatase activity in clot retraction. However, calpeptin (as well as
other calpain inhibitors) was without effect on retraction (data not
shown). This strongly suggested that calpains are not predominant
positive regulators of fibrin clot retraction, at least in our
conditions. This is consistent with the work of Schoenwaelder et
al. (15), who clearly demonstrated that calpains regulate clot
retraction negatively, since the calpain inhibitor calpeptin suppressed
partial inhibition of retraction as induced by platelet stirring. We
were also unable to demonstrate any role in clot retraction for the
phosphatidyl 3-kinase or mitogen-activated protein kinases, based on
the absence of inhibitory effect of the phosphatidyl 3-kinase
inhibitors wortmannin and Ly-294002 and the mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase inhibitor
PD98059 (data not shown).
Among the four tyrosine phosphatases described in platelets
(PTP1B, SHP1, SHP2, and HePTP; Refs. 12, 13, 31, 32, and 37), PTP1B and
SHP1 have been shown to be translocated to the actin cytoskeleton (32,
37-39). PTP1B appears as the most likely candidate, because of its
strict
IIb
3-dependent and
complete cytoskeletal translocation upon platelet thrombin aggregation (32). However, we have not been able to test this hypothesis, because
the denaturing conditions required for disrupting the fibrin clot were
not compatible with isolation of intact cytoskeleton. Nonetheless,
information concerning tyrosine-phosphorylated substrates that undergo
dephosphorylation, most likely after they are targeted to the actin
cytoskeleton along with tyrosine phosphatases, is equally relevant to
understanding the molecular dynamics of retraction. We found that
inhibition of clot retraction by the
IIb
3
integrin-specific inhibitor Integrilin® and by the tyrosine
phosphatase inhibitor PAO correlated in a dose-dependent
manner with patterns of "rephosphorylation" by dephosphorylation
inhibition of a panel of substrates. Both patterns shared consistently
a marked dose-dependent inhibition of dephosphorylation of
85-kDa protein and to a lesser extent of 64-kDa protein, Src, and
48-kDa protein. While upon inhibition of actin polymerization by
cytochalasin D, 64-kDa protein and Src yielded the strongest
superphosphorylation signal, the 85-kDa protein demonstrated
rephosphorylation in correlation with clot retraction inhibition.
Altogether, these results lead us to postulate that tyrosine
dephosphorylation of the 85-kDa protein (and possibly others) by a
tyrosine phosphatase activity is involved in clot retraction, possibly
at a convergent step between
IIb
3-dependent signaling,
actin cytoskeleton reorganization, and force
transmission/generation.
What is the role of tyrosine dephosphorylation in clot
retraction? Our study demonstrates a close relationship between
retraction and tyrosine dephosphorylation of several substrates,
including an unidentified 85-kDa protein. In cell migration, it has
been proposed that tyrosine dephosphorylation was a general mechanism for the release of focal adhesion plaques from the rear of the cell to
allow forward motion of the cell body (40). It is possible that an
equivalent mechanism is at play in retraction of the fibrin matrix by
platelets and that tyrosine dephosphorylation is relevant to
reorganization of adhesion plaques and/or of the platelet cytoskeleton. For example, it is possible that force generation by myosin and its
transmission to the fibrin matrix through
IIb
3 requires a specific organization of
an actin-myosin contractile cytoskeleton capable of supporting the
"strong" force transmission developed against increasing resistance
of the fibrin network. This organization may require remodeling of the
cytoskeleton by tyrosine phosphatases, including disruption of bonds
between actin bundles and focal adhesion plaques or actin cross-links.
In this respect, Choquet et al. (41) have shown that the
movement of beads coated with fibronectin on fibroblasts and restrained
with an optical trap responded by a localized, proportional
strengthening of the cytoskeleton linkages, allowing stronger force to
be exerted on the integrins. This integrin-dependent
strengthening of cytoskeleton linkages was clearly dependent upon
tyrosine phosphatase activity, since it was inhibited by PAO. It
is possible that in retraction, increasing rigidity of the fibrin
matrix induces a strengthening of cytoskeleton linkages, as a
consequence of a cytoskeletal rearrangement dependent on tyrosine
dephosphorylation of p85. An alternative hypothesis, not necessarily
exclusive from the first, is that tyrosine dephosphorylation may act
positively on the molecular motor(s) in retraction.
 |
ACKNOWLEDGEMENTS |
We thank F. Grelac for skilled technical
assistance during the first steps of the experimental design of the
platelet retraction assay and Drs. B. S. Coller, D. R. Phillips, R. P. McEver, and P. J. Newman for providing 10E5
and 7E3, Integrilin®, Tab, and AP-3, respectively. We
express special thanks to Sylviane Levy-Toledano and Marijke Bryckaert
for continuous support throughout this study as well as for useful
critical advice during manuscript preparation. We owe much to Leslie V
Parise for critical reading and editing of the manuscript. Finally, we are indebted to Jacques P. Caen, who gave us access to thrombasthenic patient C. B., who voluntarily and generously donated blood for this study.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 33-153203788;
Fax: 33-149958579; E-mail:
jean-philippe.rosa@inserm.lrb.ap-hop-paris.fr or
jprosa{at}infobiogen.fr.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M008945200
 |
ABBREVIATIONS |
The abbreviations used are:
PAO, phenyl-arsine
oxide;
PAGE, polyacrylamide gel electrophoresis;
NaVO4, sodium orthovanadate.
 |
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