Fibrin Clot Retraction by Human Platelets Correlates with alpha IIbbeta 3 Integrin-dependent Protein Tyrosine Dephosphorylation*

Sylvie Osdoit and Jean-Philippe RosaDagger

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have analyzed tyrosine phosphorylation associated with retraction of the fibrin clot by washed platelets in purified fibrinogen. Retraction was dependent on integrin alpha IIbbeta 3, based on absence of retraction of alpha IIbbeta 3-deficient thrombasthenic platelets. However, only a subset of alpha IIbbeta 3-blocking antibodies or peptides were able to inhibit retraction, suggesting a differential engagement of alpha IIbbeta 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 alpha IIbbeta 3-dependent, since it was blocked by alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha IIbbeta 3 (also termed glycoprotein IIb-IIIa, or gpIIb-IIIa). alpha IIbbeta 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 alpha IIbbeta 3 engagement have been defined in detail for aggregation and adhesion (5). In contrast, engagement of alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 in fibrin clot retraction was gained from studies using alpha IIbbeta 3-specific monoclonal antibodies and alpha IIbbeta 3 antagonist peptides (7-9). In addition, a differential engagement of alpha IIbbeta 3 in fibrin clot retraction versus aggregation has been suggested (10, 11). Altogether, these studies have demonstrated that alpha IIbbeta 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 alpha IIbbeta 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 right-arrow Phe747 and Phe759 of the beta 3 cytoplasmic tail exhibited platelets with altered clot retraction ability (17), suggesting a role for beta 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, alpha IIbbeta 3 is central to retraction, based on experiments using alpha IIbbeta 3-specific antagonists or alpha IIbbeta 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 alpha IIbbeta 3-dependent tyrosine dephosphorylation of several polypeptides. Dephosphorylation parallels retraction, is specifically blocked by alpha IIbbeta 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 alpha IIbbeta 3-dependent. Thus, alpha IIbbeta 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 alpha IIbbeta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma -400-411 dodecapeptide, cytochalasin D and E, and the monoclonal anti-actin IgG were from Sigma-Aldrich (Meylan, France). alpha IIbbeta 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 alpha IIbbeta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasma-free Clot Retraction Assay Is Dependent upon alpha IIbbeta 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 alpha IIbbeta 3 engagement. A, kinetics of fibrin clot retraction in buffer and washed platelets and its inhibition by alpha IIbbeta 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-alpha IIbbeta 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-alpha IIbbeta 3, open diamonds, dashed line), Tab (anti-alpha IIb, open circles, dashed line), AP3 (anti-beta 3, open squares, dotted line), 7E3 (anti-beta 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 alpha IIbbeta 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 gamma -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 gamma -400-411 dodecapeptide and nine experiments for Integrilin®. C, defective clot retraction in plasma-free conditions with alpha IIbbeta 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 alpha IIbbeta 3 integrin involvement in retraction of fibrin clot in plasma (7-10). To verify the engagement of alpha IIbbeta 3 in our plasma-free conditions, we used various alpha IIbbeta 3-specific inhibitors (Fig. 1). Among several alpha IIbbeta 3-blocking monoclonal antibodies (Fig. 1A, lower panel), the beta 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 (beta 3-specific) and Tab (alpha IIb-specific), or either one in combination with 10E5 (alpha IIbbeta 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 alpha IIbbeta 3 with fibrin compared with fibrinogen and/or in retraction compared with aggregation.

In Fig. 1B, a comparative analysis was conducted with the alpha IIbbeta 3 antagonists RGDS and gamma -400-411 dodecapeptide and the highly specific peptide Integrilin®. Integrilin® inhibited retraction efficiently (82 ± 11% inhibition at 25 µM), confirming the specific engagement of alpha IIbbeta 3 in fibrin clot retraction. Of note, neither RGDS nor the gamma -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 alpha IIbbeta 3 compared with fibrinogen, possibly due to the polymeric state of the former.

In Fig. 1C, alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 integrin in fibrin clot retraction in plasma-free conditions, and thereby validate our assay and 2) suggest differential engagement of alpha IIbbeta 3 in retraction versus aggregation.

Fibrin Clot Retraction Kinetics Correlates with Protein Tyrosine Dephosphorylation-- We then asked whether differential engagement of alpha IIbbeta 3 corresponded to differential alpha IIbbeta 3 signaling; we thus assessed tyrosine phosphorylation associated with clot retraction, since it is a prominent signaling pathway triggered by alpha IIbbeta 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 gamma -gamma 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.

Tyrosine Dephosphorylation Is Dependent on alpha IIbbeta 3 Integrin Engagement-- Fig. 3 shows that when retraction was inhibited by the retraction-blocking association of anti-alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 engagement in retraction. Interestingly, the initial wave of tyrosine phosphorylation appears alpha IIbbeta 3-independent, since it is preserved in alpha IIbbeta 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 alpha IIbbeta 3-specific monoclonal antibodies or of alpha IIbbeta 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-alpha IIb and anti-beta 3 monoclonal antibodies (20 µg/ml of Tab and AP3, respectively); right panel, platelets from a thrombasthenic patient (no alpha IIbbeta 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-alpha IIbbeta 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.

Cytochalasin D, an Inhibitor of Actin Polymerization, Inhibits Clot Retraction and alpha IIbbeta 3-dependent Protein Tyrosine Dephosphorylation-- Cytochalasins are known to inhibit clot retraction (3). To check if inhibition of actin cytoskeleton affected alpha IIbbeta 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 alpha IIbbeta 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.

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 alpha IIbbeta 3 of a de novo tyrosine phosphatase activity. Alternatively, alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3. It thus follows that alpha IIbbeta 3-dependent dephosphorylation is due to the regulation by alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha IIbbeta 3 integrin engagement. The efficient blocking of clot retraction by the beta 3-specific monoclonal antibody 7E3 and the alpha IIbbeta 3-specific antagonist peptide Integrilin® confirmed the engagement of alpha IIbbeta 3. The inability of several alpha IIbbeta 3-specific aggregation-blocking agents to alter retraction, including RGDS and the gamma -400-411 dodecapeptide, as well as the monoclonals 10E5 and AP2 (not shown), is suggestive of a differential engagement of alpha IIbbeta 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 gamma 408-411 region was deleted did support platelet retraction and not aggregation. The molecular mechanism underlying differential engagement of alpha IIbbeta 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 alpha IIbbeta 3 may be at play.

Protein tyrosine phosphorylation is one of the major "outside-in" signaling events triggered by alpha IIbbeta 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 alpha IIbbeta 3-independent, since neither its intensity nor its pattern were affected when alpha IIbbeta 3 was blocked by Integrilin® or monoclonal antibodies or when alpha IIbbeta 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 alpha IIbbeta 3-independent system, which remains to be determined. In conclusion, as opposed to aggregation, where alpha IIbbeta 3 engagement clearly controls successive waves of tyrosine phosphorylation and dephosphorylation (5, 31, 32), our data are consistent with alpha IIbbeta 3 engagement in the fibrin clot controlling essentially activation of tyrosine dephosphorylation.

Two hypotheses, not necessarily mutually exclusive, could account for alpha IIbbeta 3-dependent tyrosine dephosphorylation: 1) alpha IIbbeta 3 specifically activates one or several tyrosine phosphatase activity(ies), or 2) alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 engagement in clot retraction. Altogether, our results are in favor of the idea that alpha IIbbeta 3-dependent dephosphorylation during clot retraction is the consequence of an alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 engagement were involved in fibrin clot retraction. The signaling and alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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.

Dagger 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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