Calpain Regulation of Cytoskeletal Signaling Complexes in Von Willebrand Factor-stimulated Platelets
DISTINCT ROLES FOR GLYCOPROTEIN Ib-V-IX AND GLYCOPROTEIN IIb-IIIa (INTEGRIN alpha IIbbeta 3) IN VON WILLEBRAND FACTOR-INDUCED SIGNAL TRANSDUCTION*

(Received for publication, December 30, 1996, and in revised form, June 3, 1997)

Yuping Yuan , Sacha M. Dopheide Dagger , Chris Ivanidis , Hatem H. Salem and Shaun P. Jackson §

From the Department of Medicine, Monash Medical School, the Australian Centre for Blood Diseases, and the § Department of Pathology, Box Hill Hospital, Victoria 3128, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

The adhesion of platelets to sites of vascular injury is critically dependent on the binding of subendothelial bound von Willebrand factor (vWf) to the platelet surface glycoprotein complexes, GP Ib-V-IX and GP IIb-IIIa (integrin alpha IIbbeta 3). There is growing evidence that the binding of vWf to these receptors is not only essential for stable platelet adhesion but is also important for the transduction of activation signals required for changes in platelet morphology, granule secretion, and platelet aggregation. In this study we have investigated signaling events induced by vWf binding to GP Ib-V-IX in both spreading and aggregated platelets. The adhesion of platelets to vWf resulted in dramatic actin filament reorganization, as assessed by immunofluorescence with fluorescein isothiocyanate-conjugated phalloidin, and the cytoskeletal recruitment of various structural proteins (talin and integrin alpha IIbbeta 3) and signaling enzymes (pp60c-src, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI 3-kinase), and protein-tyrosine phosphatase (PTP)-1B). Time course experiments in both spreading and aggregated platelets revealed that talin, FAK, and PTP-1B were proteolyzed after translocation to the cytoskeleton. The proteolysis of these proteins was dependent on the presence of extracellular calcium and was specifically inhibited by pretreating platelets with the membrane-permeable calpain inhibitors calpeptin, E64d, and MDL 28,170, but not with the membrane-impermeable inhibitors leupeptin, E64, and calpastatin. The cytoskeletal translocation of signaling enzymes in vWf-stimulated platelets was abolished by pretreating platelets with an anti-GP Ib-V-IX antibody but was unaffected by blocking ligand binding to integrin alpha IIbbeta 3. In contrast, calpain activation in vWf-stimulated platelets required ligand binding to both GP Ib-V-IX and integrin alpha IIbbeta 3. The activation of calpain in both spreading and aggregated platelets resulted in a substantial decrease in the level of tyrosine phosphorylation of multiple platelet proteins and was associated with a 50-80% reduction in the amount of cytoskeletal associated talin, integrin alpha IIbbeta 3, PI 3-kinase, FAK, pp60c-src, and PTP-1B. These studies suggest a potentially important role for calpain in regulating the formation and/or stability of cytoskeletal signaling complexes in vWf-stimulated platelets. Furthermore, they demonstrate distinct roles for GP Ib-V-IX and integrin alpha IIbbeta 3 in vWf-induced signal transduction.


INTRODUCTION

Cell adhesion processes play a fundamental role in inflammation, immunity, embryogenesis, and hemostasis. These adhesion processes are regulated by distinct families of cell surface receptors, including the integrins, immunoglobulin gene family, cadherins, selectins, and the leucine-rich glycoprotein gene family, of which the receptor for vWf,1 GP Ib-V-IX, is the best characterized member (1, 2). There is growing evidence that many of these receptors not only mediate cell adhesion but also play an important role in transducing signals that regulate cell morphology, motility, growth, and differentiation (3, 4). The molecular basis by which these receptors transmit signals has not been clearly defined, although there is increasing evidence that the interaction between these receptors and the cytoskeleton is critical for signal transduction (4). For example, studies in human platelets and a range of cultured cells have demonstrated that the ligation and clustering of integrins on the cell surface lead to the cytoskeletal recruitment of various structural proteins (talin, vinculin, alpha -actinin, tensin, paxillin) and signaling molecules (FAK, Src kinases, c-CSK, PI 3-kinase, protein kinase C, phospholipase Cgamma , rasGAP, Grb2, SOS, SHP1, PTP-1B, Rho, Ras, Raf, MEK kinases, ERK1, ERK2 and calpain) (5-15). There is growing evidence that tyrosine phosphorylation events, mediated by the FAK and Src family kinases, play a major role in recruiting these signaling molecules to the cytoskeleton by providing high affinity binding sites for SH2-containing signaling molecules (5, 6, 15-17).

Although considerable progress has been made in identifying several key proteins involved in regulating the formation of cytoskeletal signaling complexes, there is far less information on cellular proteins negatively regulating these signaling events. A potential candidate protein is the ubiquitous thiol protease, calpain. This enzyme has previously been localized to focal adhesions in cultured cells and in the cytoskeletal fraction of thrombin-stimulated platelets (18, 19), where it cleaves a number of focal adhesion structural proteins (talin, beta 3 integrin) (20-22) and several signaling enzymes (FAK, pp60c-src, PTP-1B, phospholipase Cgamma , protein kinase C) (23-27). Recent studies in thrombin-stimulated platelets have revealed that calpain cleavage of FAK and pp60c-src results in a reduction in their autokinase activity (23, 25), whereas PTP-1B cleavage is associated with enhanced phosphatase activity (24), raising the possibility that calpain may indirectly regulate the level of protein-tyrosine phosphorylation within the cell (28). Furthermore, the cleavage of talin, pp60c-src, and FAK is associated with their subcellular relocation (23, 25), suggesting a potentially important role for calpain in regulating the stability of integrin signaling complexes.

We have recently reported that vWf binding to GP Ib-V-IX induces the cytoskeletal association of pp60c-src and PI 3-kinase in aggregated platelets (29). Given the critical role for this receptor in the initial adhesion of platelets to the site of vascular injury, we have investigated the possibility that vWf binding to GP Ib-V-IX can also induce the formation of cytoskeletal signaling complexes in spreading platelets. Our studies indicate that various structural proteins and signaling enzymes associate with the cytoskeletal fraction of platelets spread on a vWf matrix. The formation of these complexes was associated with calpain activation and the cleavage of talin, FAK, pp60c-src, and PTP-1B. Cleavage of these focal adhesion proteins in spreading and aggregated platelets was associated with a substantial reduction in protein-tyrosine phosphorylation and a 50-80% decrease in the cytoskeletal content of integrin alpha IIbbeta 3, talin, FAK, pp60c-src, PTP-1B, and PI 3-kinase. These observations suggest a potentially important role for calpain in modulating the formation and/or stability of cytoskeletal signaling complexes in human platelets.


EXPERIMENTAL PROCEDURES

Materials

Calpeptin was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Arg-Gly-Asp-Ser (RGDS) peptide, bovine serum albumin, and FITC-conjugated phalloidin were purchased from Sigma. Ristocetin was supplied by Paesel and Lorei Inc. (Germany). All other materials were from sources we have described previously (29, 30).

Antibodies

Anti-GP IIb monoclonal antibody (mAb) SZ22 and anti-GP IIIa mAb SZ21 were from Immunotech (France). Anti-GP Ib mAb AK2 was a generous donation from Dr. Michael Berndt (Baker Medical Research Institute, Melbourne, Australia). Anti-talin mAb 8d4 and anti-actin mAb were from Sigma. Anti-p85 (p85 subunit of PI-3 kinase) polyclonal antibody was from Upstate Biotechnology Inc. Anti-pp60c-src mAb 327 was a generous donation of Dr. Joan Brugge (University of Pennsylvania). Anti-phosphotyrosine mAb PY20 was from ICN Biochemicals Inc. Anti-FAK mAb and anti-PTP-1B mAb were from Transduction Laboratories. Anti-calpain (80 kDa) polyclonal antibody was kindly donated by Dr. T. C. Saido (Metropolitan Institute of Medical Science, Tokyo, Japan). Anti-mouse and rabbit peroxidase-conjugated IgG were from Silenus Laboratories (Victoria, Australia).

Preparation of Washed Platelets and Platelet Aggregation Studies

Blood was obtained from healthy donors who had not taken any anti-platelet medication in the preceding 2 weeks. Platelets were isolated and washed as described previously (29). For platelet aggregation studies, washed platelets were resuspended in modified Tyrode's buffer (10-50 mM Hepes, pH 7.5, 12 mM NaHCO3, 137 mM NaCl, 2.7 M KCl, and 5 mM glucose) and then incubated at 37 °C for 30 min before stimulation. Platelets (3 × 108/ml) were activated using either purified vWf (10-50 µg/ml) in the presence of ristocetin (1 mg/ml) or thrombin (1 unit/ml) in the presence of Ca2+ (1 mM). Aggregation was initiated by stirring the platelet reaction mixtures at 950 rpm at 37 °C, in a four-channel automated platelet analyzer (Kyoto Daiichi, Japan). In some experiments platelets were preincubated for 30 min with either vehicle alone (0.5% Me2SO), calpeptin (50 µg/ml), RGDS (1 mM), anti-GP IIIa mAb SZ21 (5 µg/ml) or anti-GP Ib mAb AK2 (5 µg/ml) before stimulation. In other experiments the GP IIb-IIIa complex was disrupted irreversibly by pretreating platelets with 5 mM EDTA for 90 min at 37 °C, as described previously (29).

Immunofluorescence Studies

Glass coverslips were coated with vWf (50 µg/ml) for 2 h at room temperature and then blocked with 0.1% bovine serum albumin for 60 min. Washed platelets (1 × 108/ml) were applied to the coverslips for 60 min in the presence of the platelet inhibitor theophylline (1 mM) to allow platelet adhesion without spreading. The nonadherent platelets were aspirated and the adherent cells washed twice with theophylline-free phosphate-buffered saline (PBS) (20 mM Na2HPO4, 4 mM NaH2PO4, pH 7.4, 150 mM NaCl). Gentle washing was essential to minimize detachment of theophylline-treated platelets from the vWf matrix. After platelets had spread for the indicated times (0, 10, 20, 40, and 60 min), the cells were washed three times with PBS, fixed for 30 min in 3.7% formaldehyde in PBS, then permeabilized with 0.1% Triton X-100 in PBS for 30 min. The permeabilized cells were stained with FITC-conjugated phalloidin (1:1,000 dilution) for 60 min in the dark, then washed three times with PBS. The coverslips were mounted onto glass slides with p-phenylenediamine (1 mg/ml) containing 90% glycerol, 10% PBS (v/v), then visualized with a fluorescence microscope (× 310) (Olympus).

Platelet Adhesion Studies and Subcellular Fractionation

To obtain sufficient platelet protein for immunoblot analysis, platelet adhesion studies were performed on polystyrene tissue culture dishes (100 mm) (Nunc). These dishes were coated with vWf and the washed platelets applied to these dishes in a manner identical to that described above. After platelets had spread for the indicated times on these dishes, the cells were washed three times, lysed with 1% Triton X-100 lysis buffer (10 mM Tris, pH 7.2, 1% Triton X-100, 158 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 µM calpain inhibitor I, 1 mM Na3VO4, and 2 mM benzamidine), then scraped from the dishes with a rubber policeman. Triton X-100-soluble and -insoluble (cytoskeleton) extracts were isolated from the adherent platelets in a manner identical to that used in aggregated platelets (30). The cytoskeletal proteins were extracted from the Triton X-100-insoluble material with radioimmunoprecipitation assay buffer (10 mM Tris, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 158 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 µM calpain inhibitor I, 1 mM Na3VO4 and 2 mM benzamidine) for 60 min at 4 °C. Radioimmunoprecipitation assay buffer-insoluble material was removed by centrifugation at 15,000 × g for 5 min. Whole cell lysates were prepared by lysing adherent platelets with radioimmunoprecipitation assay buffer for 1 h at 4 °C.

Immunoblot Analysis

Equal quantities of platelet extracts were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions according to the method of Laemmli (31), then transferred to polyvinylidene difluoride membranes. Western blots were performed as described by Towbin et al. (32), using specific primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies, and finally developed using Enhanced Chemiluminescence (DuPont). When studies on tyrosine phosphorylation of platelet proteins were performed, cells were lysed in an equal volume of Laemmli reducing buffer (50 mM Tris, pH 6.8, 4 mM EDTA, 2 mM Na3VO4, 2 mM phenylmethylsulfonyl fluoride, 4 mM benzamidine, 10% glycerol, 4% SDS, 10% beta -mercaptoethanol, 0.002% bromphenol blue) and boiled immediately for 5 min before SDS-polyacrylamide gel electrophoresis.

Miscellaneous Methods

vWf was purified from cryoprecipitate as described by Montgomery and Zimmerman (33). Protein concentrations were determined using the Bio-Rad protein assay with bovine serum albumin as a standard.


RESULTS

Formation of Cytoskeletal Signaling Complexes in Spreading Platelets

vWf is essential for effective platelet adhesion at sites of vascular injury, especially under conditions of high shear stress. Stable platelet adhesion under high shear requires coordinated binding of immobilized vWf to both the GP Ib-V-IX and GP IIb-IIIa (integrin alpha IIbbeta 3) complexes on the platelet surface (34). Platelet adhesion is followed by dramatic cytoskeletal reorganization, leading to platelet shape change, spreading, and aggregation. In preliminary studies, we examined the time course for platelet spreading on vWf-coated glass coverslips. Washed platelets (1 × 108/ml) were applied to vWf-coated coverslips in the presence of the platelet activation inhibitor theophylline (1 mM) to allow platelet adhesion without spreading, as described under "Experimental Procedures." Platelet adhesion to vWf under these conditions is potentially reversible, therefore gentle washing conditions were required to minimize the displacement of platelets from the matrix. After removal of theophylline, adherent platelets were allowed to spread for the indicated time points (0, 10, 20, 40, and 60 min), before fixation with formaldehyde, permeabilization with Triton X-100, and staining with FITC-conjugated phalloidin to visualize changes in the actin filament organization. As demonstrated in Fig. 1A, within 10 min of binding to the vWf matrix, multiple filopodial extensions (1-4 µM in length) projected from the platelet surface. By 20 min, broad lamellipodia sheets began extending between these filopodial projections. These lamellipodia continued to radiate out from the cell center until a confluent platelet monolayer had covered the vWf matrix (60 min).


Fig. 1.

Association of integrin alpha IIbbeta 3 (GP IIb-IIIa), talin, pp60c-src, and FAK with the cytoskeletal fraction of spreading platelets. Washed platelets (1 × 108/ml) were applied to a vWf matrix in the presence of theophylline to allow platelet adhesion without spreading. After 60 min, nonadherent platelets were washed from the coverslips and theophylline removed to allow spreading. At the indicated time points (0, 10, 20, 40, and 60 min), spreading platelets were fixed, permeabilized with Triton X-100, and then stained with FITC-conjugated phalloidin to visualize actin filaments (magnification × 310). Panel A, prominent filopodial extensions were evident after 10 min (arrow). Broad lamellipodia sheets were apparent by 20 min (arrow), and by 60 min the lamellipodia had covered the matrix surface. Panel B, immunofluorescent staining with FITC-conjugated phalloidin demonstrating an intact actin filament network after Triton X-100 extraction of spread platelets. These images are representative of the total area. Panel C, immunoblot analysis of cytoskeletal extracts from spreading platelets. Equal amounts of cytoskeletal protein (30 µg) from each of the time points examined were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted with antibodies against GP IIb, talin, pp60c-src, and FAK. Coomassie Brilliant Blue staining of the polyvinylidene difluoride membrane demonstrated the cytoskeletal F-actin content. The cytoskeletal content of GP IIb, talin, pp60c-src, FAK, and actin was quantitated by performing densitometry on the immunoblots or after staining the polyvinylidene difluoride membranes with Coomassie Brilliant Blue (line graph). The proteolytic fragments of talin (190 kDa) and FAK (90 kDa) are highlighted with arrows. These results are from one experiment, representative of four.


[View Larger Version of this Image (39K GIF file)]

We have reported recently that vWf-induced platelet aggregation is associated with the cytoskeletal translocation of pp60c-src and PI 3-kinase (29). We therefore examined whether the changes in the actin filament network induced by vWf binding to GP Ib-V-IX in spreading platelets was associated with the cytoskeletal recruitment of signaling enzymes. To obtain sufficient amounts of cytoskeletal associated protein for immunoblot analysis, platelet adhesion studies were performed on Petri dishes coated with purified vWf. Washed platelets (3 × 108/ml) were applied to this matrix under conditions identical to those used in Fig. 1A. At various time points (0, 10, 20, 40, and 60 min) the adherent platelets were lysed with Triton X-100 lysis buffer and cytoskeletal proteins isolated as described under "Experimental Procedures." We confirmed that the Triton X-100-insoluble residues remaining on the matrix were cytoskeletal structures by performing immunofluorescence with FITC-conjugated phalloidin. As demonstrated in Fig. 1B, these Triton X-100-resistant structures demonstrated an overall similar pattern of immunofluorescence to the permeabilized platelets in Fig. 1A (60 min), except for the loss of actin filament networks lining the surface membrane. SDS-polyacrylamide gel electrophoresis analysis of the proteins extracted from the matrix revealed a protein pattern similar to that reported previously for cytoskeletal extracts from spreading platelets (35), with actin constituting greater than 65% of the total cytoskeletal protein (data not shown). An alternative technique for isolating cytoskeletal proteins in which the Triton X-100-insoluble residue from adherent platelets was extracted with SDS-containing buffers yielded identical results (data not shown).

Immunoblot analysis of the cytoskeletal extracts from spreading platelets revealed a time-dependent increase in the cytoskeletal content of integrin alpha IIbbeta 3 (GP IIb), talin, pp60c-src, FAK (Fig. 1C), the p85 subunit of PI 3-kinase, and PTP-1B (data not shown). Several lines of evidence suggest that this increase represents specific recruitment and enrichment of these proteins within the cytoskeleton rather than simply a nonspecific increase caused by changes in the total amount of cytoskeletal protein. First, at each of the time points examined, equal amounts of cytoskeletal protein (30 µg) were used for immunoblot analysis. Second, the increase in these proteins was disproportionate to changes in the cytoskeletal actin content. In fully spread platelets (60 min) the ratio of integrin alpha IIbbeta 3, talin, pp60c-src, and FAK to actin was substantially greater (at least 10-fold) than at earlier time points of platelet spreading (10 min) (line graph, Fig. 1C). Finally, changes in the level of other cytoskeletal associated proteins, such as GP Ib, did not correlate with the increases in these other proteins (data not shown).

Calpain-mediated Cleavage of Talin, FAK, pp60c-src, and PTP-1B in Spreading Platelets

We noted consistently that the cytoskeletal associated forms of talin, FAK, and PTP-1B underwent proteolytic modification in spreading platelets. Talin was proteolyzed to a 190-kDa fragment, FAK to a 90-kDa fragment, and PTP-1B to a 42-kDa fragment. This pattern of proteolysis is identical to that mediated by calpain in thrombin and ionophore A23187-aggregated platelets (21, 23-26). We did not, however, detect pp60c-src proteolytic fragments in these cytoskeletal extracts even though this kinase has been identified previously as a calpain substrate (25). Previous studies in ionophore A23187-stimulated platelets have demonstrated that the proteolysis of pp60c-src leads to its relocation from the particulate to the soluble fraction of platelets (25). We therefore examined the Triton X-100-soluble extracts from spreading platelets for evidence of pp60c-src proteolysis. As demonstrated in Fig. 2, proteolytic fragments of pp60c-src, FAK, PTP-1B, and talin were evident in these Triton X-100-soluble extracts. The cleavage of each of these proteins was specifically inhibited by pretreating platelets with the calpain inhibitors calpeptin (Fig. 2) or E64d, or by chelating extracellular calcium with EGTA/Mg2+ (data not shown). Inhibiting calpain under each of these experimental conditions had no inhibitory effect on the rate or extent of platelet spreading (data not shown).


Fig. 2. Proteolytic fragments of FAK, pp60c-src, PTP-1B, and talin within the Triton X-100-soluble fraction of spread platelets. Washed platelets (3 × 108/ml) were preincubated with Me2SO or calpeptin (50 µg/ml) for 30 min before their application to the vWf matrix. Platelets were allowed to spread for the indicated time points before lysis. The Triton X-100-soluble extracts were immunoblotted with antibodies against FAK, pp60c-src, PTP-1B, and talin. Note that the PTP-1B immunoblots were overexposed to demonstrate the presence of the 42-kDa fragment. This either reflects poor recognition of the PTP-1B cleavage product by the monoclonal antibody or is caused by rapid cleavage of the 42-kDa fragment to lower molecular forms, not recognized by the primary antibody. We consistently observed an approximately 50% reduction in intact PTP-1B (50 kDa) in spreading and aggregated platelets. These results are from one experiment, representative of five.
[View Larger Version of this Image (39K GIF file)]

All reports to date have indicated that calpain activation by physiological agonists is a postaggregation event critically dependent on fibrinogen binding to integrin alpha IIbbeta 3 (36). Several lines of evidence suggest that the calpain activation observed in spreading platelets is unlikely to be caused by the formation of platelet aggregates in our static adhesion assays. First, phase-contrast microscopy of platelets adherent to the vWf matrix on Petri dishes failed to reveal platelet aggregate formation in 15 independent experiments. Furthermore, we have observed consistently that the formation of small platelet aggregates in vWf-stimulated nonstirred platelets is not associated with pp60c-src cleavage.2 Second, the platelet adhesion studies were performed in the presence of the platelet inhibitor theophylline, as described under "Experimental Procedures." In the presence of theophylline, platelets adhere to the vWf matrix as single cells but do not spread or aggregate. All nonadherent platelets were washed from the matrix before removing the platelet inhibitor to ensure that no platelet aggregates form on the adherent monolayer. Under these assay conditions, cytoskeletal signal complex formation and calpain activation were identical to that observed in platelets not pretreated with theophylline (data not shown).

Previous studies have demonstrated that irreversible platelet adhesion onto a vWf matrix is critically dependent on both GP Ib-V-IX and integrin alpha IIbbeta 3 (37). Using monoclonal antibodies against GP Ib-V-IX we were able to prevent platelet adhesion to the vWf matrix, whereas antibodies against integrin alpha IIbbeta 3 did not prevent platelet adhesion but dramatically reduced platelet spreading. Similar observations were found in Glanzmann's thrombasthenic platelets, which congenitally lack integrin alpha IIbbeta 3 (data not shown). These observations limited our ability to investigate the relative roles of GP Ib-V-IX and integrin alpha IIbbeta 3 in inducing calpain activation and the formation of cytoskeletal signaling complexes in vWf-activated platelets, as these signaling events were critically dependent on both platelet adhesion and spreading (data not shown). We overcame these technical limitations by examining cytoskeletal signal complex formation in suspension-activated platelets. We have demonstrated previously in this system that vWf binding to GP Ib-V-IX and subsequent platelet aggregation are sufficient for pp60c-src translocation to the cytoskeleton, independent of ligand binding to integrin alpha IIbbeta 3 (29). We investigated the cytoskeletal translocation of the two calpain substrates, talin and PTP-1B, in vWf-aggregated platelets. As with spreading platelets, both talin and PTP-1B translocated to the cytoskeleton and underwent proteolytic modification (Fig. 3). Both the translocation and cleavage of PTP-1B and talin required platelet stirring and aggregation and were abolished by anti-GP Ib-V-IX antibodies (data not shown). Proteolysis of both substrates was also abolished by pretreating the platelets with calpeptin (Fig. 3). A potential limitation of these experimental conditions is the requirement for a nonphysiological modulator, such as ristocetin, to induce vWf binding to GP Ib-V-IX. The binding of vWf-ristocetin to GP Ib-V-IX is irreversible and contrasts with the reversible binding of GP Ib-V-IX to immobilized vWf (49). We have reported previously (29) that bovine vWf, which binds human GP Ib-V-IX in the absence of nonphysiological modulators, induces similar signaling events as vWf-ristocetin, suggesting that these kinetic differences do not have a major effect on signaling processes mediated by GP Ib-V-IX.


Fig. 3. Calpain-mediated cleavage of talin and PTP-1B in vWf-aggregated platelets. Washed platelets (3 × 108/ml) were preincubated with Me2SO or calpeptin (50 µg/ml) for 30 min and then aggregated with vWf (10 µg/ml) and ristocetin (1 mg/ml) for 5 min. Cells were lysed and fractionated into Triton X-100-soluble and cytoskeletal extracts as described under "Experimental Procedures." Cytoskeletal proteins were analyzed by immunoblotting with antibodies against talin or PTP-1B. These results are from one experiment, representative of five.
[View Larger Version of this Image (22K GIF file)]

To examine the role of integrin alpha IIbbeta 3 in regulating calpain activation and the formation of cytoskeletal signal complexes in vWf-aggregated platelets, we disrupted ligand binding to this adhesion receptor by preincubating platelets with EDTA for 90 min at 37 °C or by blocking ligand binding with RGDS peptides or an anti-integrin alpha IIbbeta 3 monoclonal antibody. As demonstrated in Fig. 4, abolishing ligand binding to integrin alpha IIbbeta 3 had no inhibitory effect on the ability of talin to translocate to the cytoskeleton, suggesting that cytoskeletal reorganization in response to vWf does not depend on signaling events linked to integrin alpha IIbbeta 3 occupancy. Similar observations were found with pp60c-src, p85, and PTP-1B translocation (data not shown). Unfortunately we could not examine FAK translocation under these assay conditions as ristocetin nonspecifically precipitated this kinase. In contrast to translocation, the cleavage of talin (Fig. 4) and PTP-1B (not shown) was abolished by inhibiting ligand binding to integrin alpha IIbbeta 3, indicating that integrin alpha IIbbeta 3 postligand occupancy events are essential for calpain activation in vWf-stimulated platelets. These findings were observed consistently in both vWf-ristocetin and bovine vWf-activated platelets.


Fig. 4. Role of integrin alpha IIbbeta 3 in regulating the cytoskeletal translocation and cleavage of talin in vWf-aggregated platelets. Washed platelets were pretreated either with Me2SO, calpeptin (50 µg/ml), RGDS peptide (1 mM), or anti-GP IIIa mAb (alpha -GPIIIa) for 30 min at room temperature, or with 5 mM EDTA for 90 min (EDTA-90') at 37 °C as described under "Experimental Procedures." Platelets were aggregated with vWf (50 µg/ml) and ristocetin (1 mg/ml) for the indicated time points while stirring. Platelets were lysed with Triton X-100, fractionated, and equal amounts of cytoskeletal protein subjected to immunoblot analysis with an anti-talin mAb (8d4). These results are from one experiment, representative of four.
[View Larger Version of this Image (18K GIF file)]

Although ligand binding to integrin alpha IIbbeta 3 does not appear to be essential for cytoskeletal signal complex formation in vWf-aggregated platelets, these results do not exclude the possibility that this receptor may participate in the formation of these complexes in the absence of bound ligand. To investigate further the role of integrin alpha IIbbeta 3 in vWf signaling, we examined cytoskeletal signal complex formation and calpain activation in Glanzmann's thrombasthenic platelets. As demonstrated in Fig. 5, A and B, pp60c-src, p85, talin, and PTP-1B all translocated to the cytoskeletal fraction of vWf-aggregated Glanzmann's platelets, strongly suggesting that integrin alpha IIbbeta 3 is not essential for this signaling event. As expected, talin and PTP-1B were not proteolyzed in these platelets. To confirm that the reduction in talin and PTP-1B cleavage in Glanzmann's platelets was the result of the inability of vWf to activate calpain, we immunoblotted whole cell lysates with an antibody that recognizes the full-length (80 kDa) inactive form of calpain (Fig. 5C). Previous studies in thrombin-stimulated platelets have demonstrated that immunoblot analysis with this antibody is a sensitive means of detecting calpain activation within the cell (38, 39). Activation of calpain is associated with the autolytic conversion of its large subunit (80 kDa) to 78- and 76-kDa active forms, which are not recognized by this antibody. As demonstrated in Fig. 5C, thrombin or vWf stimulation of normal platelets leads to a 70-75% reduction in the amount of inactive calpain. In contrast, no reduction in full-length calpain was observed in thrombin or vWf-stimulated Glanzmann's platelets.


Fig. 5. Calpain activation and cytoskeletal association of pp60c-src, PI-3 kinase, talin, and PTP-1B in normal or Glanzmann's thrombasthenic platelets aggregated with vWf. Washed platelets (3 × 108/ml) from a healthy volunteer or from a patient with Glanzmann's thrombasthenia were stirred in the presence of vWf (50 µg/ml) and ristocetin (1 mg/ml), or thrombin (1 unit/ml) for the indicated time points. Aggregated platelets were lysed with Triton X-100 and either analyzed as whole cell lysates or fractionated into Triton X-100-soluble or cytoskeletal extracts. Panel A, immunoblot analysis of the cytoskeletal fraction from normal and Glanzmann's platelets with antibodies against GP IIb, pp60c-src, p85 subunit of PI-3 kinase (panel B) or against talin and PTP-1B. Note that the additional band observed in the p85 immunoblot was a nonspecific band not consistently observed in all experiments. Panel C, whole cell lysates from platelets aggregated with vWf and ristocetin for 20 min were analyzed for calpain activation using an antibody that selectively recognizes the 80-kDa inactive form of calpain. The extent of calpain activation (histogram) was quantitated by performing densitometry on the calpain immunoblots. The results presented are from one experiment, representative of two independent experiments performed in duplicate.
[View Larger Version of this Image (21K GIF file)]

Effects of Calpain Activation on Protein-Tyrosine Phosphorylation

The ability of calpain to regulate the subcellular distribution and enzyme activity of multiple nonreceptor tyrosine kinases and phosphatases in human platelets has suggested a potentially important role for this protease in regulating the level of tyrosine phosphorylation within the cell (28). We investigated the changes in protein-tyrosine phosphorylation in platelets spread on a vWf matrix. As demonstrated in Fig. 6A, only a small number of platelet proteins (60 and 64 kDa) were tyrosine phosphorylated in adherent nonspread platelets, as assessed by anti-phosphotyrosine immunoblot analysis. In contrast, the pattern of protein-tyrosine phosphorylation in spread platelets was dramatically different, with increased phosphorylation of the 60- and 64-kDa proteins and the appearance of 118-, 107-, 100-, 92-, 74-, 70-, 56-, and 49-kDa phosphorylated proteins. Pretreating platelets with calpeptin resulted in a substantial increase in the level of phosphorylation of the 107-, 74-, 70-, and 64-kDa proteins, a modest increase in the phosphorylation of the 92-, 86-, and 60-kDa proteins, and a sharp reduction in the level of phosphorylation of the 49-kDa protein. Similar changes in protein-tyrosine phosphorylation were also observed in vWf-aggregated platelets (Fig. 6B). Time course studies revealed that the tyrosine phosphorylation of platelet proteins in vWf-aggregated platelets was a transient phenomenon with maximal phosphorylation observed within 1 min of stimulation followed by progressive dephosphorylation between 2 and 10 min. As with spreading platelets, pretreating these cells with calpeptin before vWf activation led to a higher and more sustained pattern of tyrosine phosphorylation (Fig. 6B).


Fig. 6. Effect of calpain activation on protein-tyrosine phosphorylation in vWf-stimulated platelets. Washed platelets (1 × 108/ml) were preincubated with Me2SO or calpeptin (50 µg/ml) for 30 min before the performance of (panel A) platelet adhesion or (panel B) platelet aggregation studies. Panel A, adherent platelets were allowed to spread for the indicated time points before lysis in SDS-sample buffer. Whole cell lysates were subjected to immunoblot analysis with an anti-phosphotyrosine mAb (PY20). Panel B, washed platelets (3 × 108/ml) were aggregated with vWf (10 µg/ml) and ristocetin (1 mg/ml) for the indicated time points in the presence or absence of calpeptin (50 µg/ml). Total cell lysates were subjected to anti-phosphotyrosine immunoblot analysis. Panel C, cytoskeletal extracts from vWf-aggregated platelets were immunoblotted with anti-PTP-1B mAb. These results were from one experiment representative of four.
[View Larger Version of this Image (42K GIF file)]

Previous studies have revealed that many of the proteins phosphorylated on tyrosine residues are located within the cytoskeleton (8). We therefore investigated the time course for PTP-1B cytoskeletal translocation and cleavage in vWf-aggregated platelets and correlated this with changes in tyrosine phosphorylation. As demonstrated in Fig. 6C, PTP-1B translocation occurred rapidly within 45 s of platelet activation, whereas cleavage was not observed until after 2 min of stimulation. The time course for PTP-1B cleavage correlated well with the calpain-regulated dephosphorylation of platelet proteins, suggesting a potential role for this phosphatase in this dephosphorylation process.

Effects of Calpain on the Formation of Cytoskeletal Signaling Complexes in Spreading Platelets

Given the potential importance of FAK and pp60c-src in regulating the formation of cytoskeletal signaling complexes, we investigated the possibility that calpain activation may lead to reduced formation and/or stability of cytoskeletal signaling complexes in vWf-stimulated platelets. We compared the cytoskeletal content of integrin alpha IIbbeta 3, talin, pp60c-src, FAK, and PI 3-kinase in spreading platelets pretreated with calpeptin, E64d, or Me2SO (Fig. 7). We consistently observed in these studies a 2-4-fold higher content of each of these proteins in spreading platelets pretreated with calpeptin or E64d compared with Me2SO controls. This increase was specific to these proteins as the cytoskeletal content of F-actin or GP Ib remained essentially unchanged under the same experimental conditions. We performed similar experiments in vWf-aggregated platelets and observed a similar phenomenon (Fig. 3 and data not shown). Collectively, these results suggest a potentially important role for this protease in regulating cytoskeletal signaling complex formation in vWf-stimulated platelets.


Fig. 7. Effect of calpain activation on the cytoskeletal content of GP IIb, talin, pp60c-src, p85, FAK, and actin. Washed platelets (1 × 108/ml) were pretreated with Me2SO or calpeptin (50 µg/ml) for 30 min and then applied to the vWf matrix in the presence of theophylline to allow platelet adhesion without spreading. After 60 min, nonadherent cells were washed from the dish and the theophylline removed to allow platelet spreading. At the indicated time points the adherent platelets were lysed and the cytoskeletal proteins extracted. Immunoblot analysis of equal quantities of these extracts was performed using antibodies against GP IIb, talin, pp60c-src, p85 subunit of PI 3-kinase, FAK, and actin. The cytoskeletal content of each of these proteins (histogram) was quantitated by performing densitometry on each of the immunoblots. Note that unlike FAK and PTP-1B, the proteolysis of talin within the cytoskeletal fraction of platelets was not always completely inhibited by pretreating these cells with calpeptin. These results were derived from one experiment, representative of four.
[View Larger Version of this Image (27K GIF file)]


DISCUSSION

The stable adhesion of platelets to the site of vascular injury, particularly under conditions of high shear stress, is critically dependent on the coordinated binding of GP Ib-V-IX and integrin alpha IIbbeta 3 to subendothelial bound vWf (34). There is growing evidence that the interaction between vWf and these adhesion receptors plays an important role in the initiation of platelet activation, leading to platelet shape change, spreading, granule release, and aggregation (29, 40-42). Despite the fundamental importance of these adhesion events, the signaling processes linked to these receptors, especially GP Ib-V-IX, remain poorly understood. The studies presented in this article reveal the following information regarding signaling processes induced by the binding of vWf to human platelets. First, vWf is capable of inducing the cytoskeletal translocation of multiple structural proteins and signaling enzymes in both spreading and aggregated platelets. The cytoskeletal association of these signaling molecules in aggregated platelets requires vWf binding to GP Ib-V-IX but occurs independently of ligand binding to integrin alpha IIbbeta 3. Second, vWf stimulation of platelets induces calpain activation, leading to the limited proteolysis of talin, PTP-1B, FAK, and pp60c-src. The activation of calpain by vWf is distinct from the formation of cytoskeletal signaling complexes in that it requires ligand binding to both GP Ib-V-IX and integrin alpha IIbbeta 3. Finally, the activation of calpain in vWf-stimulated platelets results in a substantial decrease in the level of tyrosine phosphorylation of multiple platelet proteins and is associated with a 50-80% reduction in the cytoskeletal content of integrin alpha IIbbeta 3, talin, pp60c-src, FAK, PTP-1B, and PI 3-kinase.

Previous studies in suspension-activated platelets have clearly established a central role for integrin alpha IIbbeta 3 in regulating the formation of cytoskeletal signaling complexes. Studies in thrombin-stimulated platelets have suggested that fibrinogen binding to integrin alpha IIbbeta 3 induces cytoskeletal reorganization and the translocation of multiple signaling enzymes to the cytoskeleton (7-13). Ligand binding to integrin alpha IIbbeta 3 per se does not appear to be sufficient for the formation of these signaling complexes, as these events only occur after stirring the platelet suspensions to induce platelet aggregation (8). We have demonstrated a similar phenomenon in vWf-stimulated platelets, whereby the binding of vWf to GP Ib-V-IX induces the translocation of signaling enzymes when these cells are stirred and allowed to aggregate (29). These observations raise the possibility that vWf binding to GP Ib-V-IX induces cytoskeletal changes similar to those observed following fibrinogen binding to integrin alpha IIbbeta 3. Recent studies in Chinese hamster ovary cells transfected with GP Ibalpha , GP Ibbeta , and GP IX support these findings, as they demonstrate that binding of transfected cells to vWf induces cell spreading and the extension of pseudopodia. These changes in cell morphology appear to be regulated in part by the interaction of the cytoplasmic tail of GP Ibalpha with the cytoskeleton, as truncation of this tail resulted in a significant alteration in both the morphology and adhesion of these cells (43).

We have demonstrated under a variety of different experimental conditions that the activation of calpain by vWf requires ligand binding to both GP Ib-V-IX and integrin alpha IIbbeta 3. A similar requirement for integrin alpha IIbbeta 3 in calpain activation has been reported previously in thrombin-stimulated platelets (36), although the mechanism by which this receptor regulates calpain activity has yet to be established. Studies in platelets have suggested an important role for inositol phospholipids and calcium influx in inducing calpain activation (21, 44). The more highly phosphorylated phosphoinositides (phosphoinositide 4-phosphate and 4,5-bisphosphate) appear to be most effective in lowering the calcium requirement for calpain activation (44). Studies in cultured cells and platelets have demonstrated an important role for integrins in regulating the formation of phosphoinositide 4,5-bisphosphate and D-3 phosphoinositides (45, 46). Furthermore, integrin alpha IIbbeta 3 can regulate the influx of calcium in response to a variety of physiological agonists, including vWf (47, 48). Whether these integrin alpha IIbbeta 3-regulated signaling processes are directly responsible for calpain activation in vWf-stimulated platelets will require further investigation.

Studies of calpain activation in human platelets have been confined to suspension-activated platelets. We have demonstrated here that calpain activation occurs in surface-activated platelets, in the absence of added exogenous agonists or platelet aggregation. Several lines of evidence support the hypothesis that the observed proteolysis of talin, FAK, PTP-1B, and pp60c-src occurred in vivo and was not a post-lysis phenomenon. First, proteolysis of these substrates was not only observed when spreading platelets were lysed with Triton X-100, but also when the cells were directly solubilized in SDS reducing buffer and boiled immediately. Second, proteolysis was prevented by pretreating platelets with membrane-permeable calpain inhibitors such as calpeptin, E64d, and MDL and not by membrane-impermeable inhibitors such as leupeptin, E64, or calpastatin. Finally, pretreating platelets with the membrane-permeable inhibitors of calpain resulted in increased cytoskeletal content of integrin alpha IIbbeta 3, talin, PTP-1B, FAK, pp60c-src, and PI 3-kinase and enhanced total cell protein-tyrosine phosphorylation, whereas membrane-impermeable calpain inhibitors had no effect on these signaling events.

The observation that calpain is activated in spreading platelets challenges the view that calpain activation only occurs as a post-platelet aggregation event. The ability of this protease to regulate the cytoskeletal content of a range of focal adhesion structural proteins and signaling enzymes raises the possibility that this protease may regulate cell matrix-adhesive processes. Inhibiting calpain activation with a number of different calpain inhibitors, including calpeptin, calpain inhibitor-1, E64d, and MDL, has had no demonstrable inhibitory effect on the rate of platelet adhesion or spreading on a vWf matrix in our static adhesion assays. These conditions do not, however, mimic the situation in vivo, in which platelets adhere and spread at sites of vascular injury over a wide range of shear stresses. It is possible under these conditions that the calpain-mediated reduction in cytoskeletal-associated adhesion complexes may lead to alterations in the stability of adhesion contacts. Future platelet adhesion studies under a variety of flow conditions will be required to resolve these issues.

Although considerable progress has been made in identifying the key structural elements and signaling molecules involved in the assembly of cytoskeletal signaling complexes (4), there is far less information available regarding potential negative regulators of these processes. The studies presented here, as well as recent observations demonstrating calpain-mediated down-regulation of the kinase activity and cytoskeletal attachment of pp60c-src and FAK (23, 25), suggest a potentially important role for this protease in the assembly and/or stability of cytoskeletal signaling complexes. The close proximity of calpain to focal adhesion contacts (18), the regulation of its protease activity by integrins (36), and its ability to regulate proteolytically several key proteins involved in adhesion receptor signal transduction place this enzyme in a pivotal position to modulate downstream signals induced by cell-cell and cell-matrix interactions.


FOOTNOTES

*   This work was supported in part by a grant from the National Health and Medical Research Council of Australia.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.

The first two authors contributed equally to this work.


Dagger    Recipient of an Australian postgraduate award. To whom correspondence should be addressed: Dept. of Medicine, Monash Medical School, Box Hill Hospital, Arnold St., Box Hill, Victoria 3128, Australia. Tel.: 61-3-9895-0311; Fax: 61-3-9895-0332.
1   The abbreviations used are: vWf, von Willebrand factor; GP, glycoprotein; PI 3-kinase, phosphatidylinositol 3-kinase; PTP, protein-tyrosine phosphatase; RGDS peptide, Arg-Gly-Asp-Ser peptide; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; Me2SO, dimethyl sulfoxide; PBS, phosphate-buffered saline; FAK, focal adhesion kinase.
2   Y. Yuan, S. Dopheide, C. Ivanidis, H. H. Salem, and S. P. Jackson, unpublished observations.

ACKNOWLEDGEMENT

We thank Dr. T. C. Saido for the generous supply of anti-calpain antibodies.


REFERENCES

  1. Ruggeri, Z. M. (1991) Prog. Hemost. Thromb. 10, 35-68 [Medline] [Order article via Infotrieve]
  2. Roth, G. J. (1991) Blood 77, 5-19 [Medline] [Order article via Infotrieve]
  3. Rosales, C., O'Brien, V., Kornberg, L., and Juliano, R. (1995) Biochim. Biophys. Acta 1242, 77-98 [CrossRef][Medline] [Order article via Infotrieve]
  4. Yamada, K. M., and Miyamoto, S. (1995) Curr. Opin. Cell Biol. 7, 681-689 [CrossRef][Medline] [Order article via Infotrieve]
  5. Miyamoto, S., Akiyama, S. K., and Yamada, K. M. (1995) Science 267, 883-885 [Medline] [Order article via Infotrieve]
  6. Miyamoto, S., Teramoto, H., Coso, O. A., Gutkind, J. S., Burbelo, P. D., Akiyama, S. K., and Yamada, K. M. (1995) J. Cell Biol. 131, 791-805 [Abstract]
  7. Grondin, P., Plantavid, M., Sultan, C., Breton, M., Mauco, G., and Chap, H. (1991) J. Biol. Chem. 266, 15705-15709 [Abstract/Free Full Text]
  8. Fox, J. E. B., Lipfert, L., Clark, E. A., Reynolds, C. C., Austin, C. D., and Brugge, J. S. (1993) J. Biol. Chem. 268, 25973-25984 [Abstract/Free Full Text]
  9. Fox, J. E. B. (1993) Thromb. Haemostasis 70, 884-893 [Medline] [Order article via Infotrieve]
  10. Zhang, J., Fry, M. J., Waterfield, M. D., Jaken, S., Liao, L., Fox, J. E. B., and Rittenhouse, S. E. (1992) J. Biol. Chem. 267, 4686-4692 [Abstract/Free Full Text]
  11. Horvath, A. R. E., Muszbek, L., and Kellie, S. (1992) EMBO J. 11, 855-861 [Abstract]
  12. Torti, M., Ramaschi, G., Sinigaglia, F., Lapetine, E. G., and Balduini, C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4239-4243 [Abstract]
  13. Clark, E. A., and Brugge, J. S. (1993) Mol. Cell. Biol. 13, 1863-1871 [Abstract]
  14. Kaplan, B. K., Swedlow, J. R., Morgan, D. O., and Varmus, H. E. (1995) Genes Dev. 9, 1505-1517 [Abstract]
  15. Vuori, K., Hirai, H., Aizawa, S., and Ruoslahti, E. (1996) Mol. Cell. Biol. 16, 2606-2613 [Abstract]
  16. Schaller, M. D., and Parsons, J. T. (1994) Curr. Opin. Cell Biol. 6, 705-710 [Medline] [Order article via Infotrieve]
  17. Schlaepfer, D. D., Hanks, S. K., Hunter, T., and Van der Geer, P. (1994) Nature 372, 786-791 [Medline] [Order article via Infotrieve]
  18. Beckerle, M. C., Burridge, K., DeMartino, G. N., and Croall, D. E. (1987) Cell 51, 569-577 [Medline] [Order article via Infotrieve]
  19. Fox, J. E. B., Santos, G., Zuerbig, S., and Saido, T. C. (1995) Thromb. Haemostasis 72, 987 (abstr.)
  20. White, G. C. (1980) Biochim. Biophys. Acta 631, 130-138 [Medline] [Order article via Infotrieve]
  21. Fox, J. E. B., Reynolds, C. C., and Phillips, D. R. (1983) J. Biol. Chem. 258, 9973-9981 [Abstract/Free Full Text]
  22. Du, X., Saido, T. C., Tsubuki, S., Indig, F. E., Williams, M. J., and Ginsberg, M. H. (1995) J. Biol. Chem. 270, 26146-26151 [Abstract/Free Full Text]
  23. Cooray, P., Yuan, Y., Schoenwaelder, S. M., Mitchell, C. A., Salem, H. H., and Jackson, S. P. (1996) Biochem. J. 318, 41-47 [Medline] [Order article via Infotrieve]
  24. Frangioni, J. V., Oda, A., Smith, M., Salzman, E. W., and Neel, B. G. (1993) EMBO J. 12, 4843-4856 [Abstract]
  25. Oda, A., Druker, B. J., Ariyoshi, H., Smith, M., and Salzman, E. W. (1993) J. Biol. Chem. 268, 12603-12608 [Abstract/Free Full Text]
  26. Banno, Y., Nakashima, S., Hachiya, T., and Nozawa, Y. (1995) J. Biol. Chem. 270, 4318-4324 [Abstract/Free Full Text]
  27. Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, S., Tominaga, M., Kuroda, T., and Nishizuka, Y. (1989) J. Biol. Chem. 264, 4088-4092 [Abstract/Free Full Text]
  28. Ariyoshi, H., Oda, A., and Salzman, E. W. (1995) Thromb. Vasc. Biol. 15, 511-514 [Abstract/Free Full Text]
  29. Jackson, S. P., Schoenwaelder, S. M., Yuan, Y., Rabinowitz, I., Salem, H. H., and Mitchell, C. A. (1994) J. Biol. Chem. 269, 27093-27099 [Abstract/Free Full Text]
  30. Schoenwaelder, S. M., Jackson, S. P., Yuan, Y., Teasdale, M. S., Salem, H. H., and Mitchell, C. A. (1994) J. Biol. Chem. 269, 32479-32487 [Abstract/Free Full Text]
  31. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  32. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  33. Montgomery, R., and Zimmerman, T. (1978) J. Clin. Invest. 61, 1498-1507 [Medline] [Order article via Infotrieve]
  34. Savage, B., Saldivar, E., and Ruggeri, Z. M. (1996) Cell 84, 289-297 [Medline] [Order article via Infotrieve]
  35. Smith, C. M., Burris, S. M., Rao, G. H. R., and White, J. G. (1992) Blood 80, 2774-2780 [Abstract]
  36. Fox, J. E. B., Taylor, R. G., Taffarel, M., Boyles, J. K., and Groll, D. E. (1993) J. Cell Biol. 120, 1501-1507 [Abstract]
  37. Savage, B., Shattil, S. J., and Ruggeri, Z. M. (1992) J. Biol. Chem. 267, 11300-11306 [Abstract/Free Full Text]
  38. Saido, T. C., Suzuki, H., Yamazaki, H., Tanoue, K., and Suzuki, K. (1993) J. Biol. Chem. 268, 7422-7426 [Abstract/Free Full Text]
  39. Saido, T. C., Nagao, S., Shiramine, M., Tsukaguchi, M., Sorimachi, H., Murofushi, H., Tsuchiya, T., Ito, H., and Suzuki, K. (1992) J. Biochem. 111, 81-86 [Abstract]
  40. Weiss, H. J., Rodgers, J., and Brand, H. (1973) J. Clin. Invest. 52, 2697-2707 [Medline] [Order article via Infotrieve]
  41. De Marco, L., Girolami, A., Russel, S., and Ruggeri, Z. M. (1985) J. Clin. Invest. 75, 1198-1203 [Medline] [Order article via Infotrieve]
  42. Kroll, M. H., Harris, T. S., Moake, J. L., Handin, R. I., and Schafer, A. I. (1991) J. Clin. Invest. 88, 1568-1573 [Medline] [Order article via Infotrieve]
  43. Cunningham, J. G., Meyer, S. C., and Fox, J. E. B. (1996) J. Biol. Chem. 271, 11581-11587 [Abstract/Free Full Text]
  44. Saido, T. C., Shibata, M., Takenawa, T., Murofushi, H., and Suzuki, K. (1992) J. Biol. Chem. 267, 24585-24590 [Abstract/Free Full Text]
  45. McNamee, H. M., Ingber, D. E., and Schwartz, M. A. (1992) J. Cell Biol. 121, 673-678 [Abstract]
  46. Sultan, C., Plantavid, M., Bachelot, C., Grondin, P., Breton, M., Mauco, G., Levy-Toledano, S., Caen, J. P., and Chap, H. (1991) J. Biol. Chem. 266, 23554-23557 [Abstract/Free Full Text]
  47. Chow, T. W., Hellums, J. D., Moake, J. L., and Kroll, M. H. (1992) Blood 80, 113-120 [Abstract]
  48. Bertolino, G., Noris, P., Spedini, P., and Balduini, C. L. (1995) Thromb. Haemostasis 73, 689-692 [Medline] [Order article via Infotrieve]
  49. Ruggeri, Z. M. (1997) J. Clin. Invest. 99, 559-564 [Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.