JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M106129200 on August 24, 2001

J. Biol. Chem., Vol. 276, Issue 45, 42226-42232, November 9, 2001
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/45/42226    most recent
M106129200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Z.
Right arrow Articles by Du, X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Z.
Right arrow Articles by Du, X.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

A Mitogen-activated Protein Kinase-dependent Signaling Pathway in the Activation of Platelet Integrin alpha IIbbeta 3*

Zhenyu LiDagger, Xiaodong XiDagger, and Xiaoping Du§

From the Department of Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60612

Received for publication, July 2, 2001, and in revised form, August 15, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have recently shown that the platelet integrin alpha IIbbeta 3 is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin alpha IIbbeta 3 is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin alpha IIbbeta 3. Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The integrin alpha IIbbeta 3 mediates platelet adhesion, spreading, and aggregation and thus plays a critical role in thrombosis and hemostasis (1). In normal circulating platelets, the integrin alpha IIbbeta 3 is in a resting state with a low affinity for its ligands such as fibrinogen and von Willebrand factor (vWF).1 At sites of vascular injury, exposure of platelets to soluble agonists (such as thrombin and ADP) or to matrix-bound adhesive proteins (such as collagen and vWF) induces platelet activation. A common consequence of platelet activation is the activation of the ligand binding function of the integrin alpha IIbbeta 3 (2, 3). Under high shear rate flow conditions, such as in stenotic atherosclerotic arteries, initial platelet adhesion and activation are dependent on the interaction between subendothelium-bound vWF and its receptor, the glycoprotein Ib-IX (GPIb-IX) complex (4-10). GPIb-IX not only mediates the physical adherence of platelets to the site of vascular injury but also initiates signal transduction, leading to activation of ligand binding function of the platelet integrin alpha IIbbeta 3 (11-15). In addition, GPIbalpha binds thrombin and is required for the low dose thrombin-induced integrin activation and platelet aggregation (16-22). The importance of GPIb-IX pathway in platelet function is manifested in Bernard-Soulier syndrome, in which genetic deficiency in GPIb-IX resulted in defects in platelet adhesion and activation (10). The mechanism of GPIb-IX-mediated integrin activation is not fully understood. However, we have recently shown that the cGMP-dependent protein kinase (protein kinase G (PKG)) is an important stimulatory mediator in the GPIb-IX-induced activation of integrin alpha IIbbeta 3.2

Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine kinase activated by many extracellular stimuli including growth factors and hormones. Four distinct subgroups within the MAPK family have been described, including the extracellular signal-regulated kinases (ERKs), the c-Jun NH2-terminal kinases, ERK5/big MAP kinase (BMK1), and p38 group of protein kinases. At least three subgroups of these MAP kinases, ERK1/ERK2 (23), p38 (24), and c-Jun NH2-terminal kinase (25), have been identified in platelets and shown to be activated when platelets are stimulated by different agonists such as thrombin and collagen. The prototype MAPK pathway, ERK pathway, consists of a cascade of protein kinases, Raf1, MEK1, and ERK1/ERK2, which sequentially activate a downstream kinase. Raf-1 activation involves Ras and 14-3-3 protein (26). The functions of ERK MAPK pathway in platelets have not been fully understood. ERK has been shown to phosphorylate and activate cytoplasmic phospholipase A2, which is a rate-limiting enzyme in synthesis of thromboxane A2 (TXA2) (27). However, it was reported that ERK pathway is not required in the cytoplasmic phospholipase A2 activation in platelets stimulated by thrombin, as MEK1 inhibitor PD98059 did not abolish thrombin-induced arachidonic acid release (28). It has been reported that ERK pathway is not required for primary platelet response to high doses of collagen and thrombin (28), although MEK1 inhibitor PD98059 has been shown in other studies to inhibit platelet aggregation induced by low doses of collagen and ADP (29). The interpretation of these data has been complicated by the report suggesting that PD98059 may directly inhibit cyclooxygenases in TXA2 synthesis pathway, which is important in platelet aggregation induced by low dose collagen and ADP (29).

In this study, we have examined the roles of ERK pathway in GPIb-IX-dependent platelet activation using a combination of molecular biology and pharmacological approaches. We show that ERK pathway is important in GPIb-IX-dependent integrin activation signaling. We further show that activation of PKG is sufficient to activate ERK pathway and is necessary for GPIb-IX-mediated activation of ERK. These data, combined with our recent finding that PKG mediates GPIb-IX-dependent platelet activation,2 delineate a novel signaling pathway of platelet activation in which ligand binding to GPIb-IX sequentially activates PKG pathway and ERK MAP kinase pathway leading to integrin activation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Recombinant cDNA Constructs in Chinese Hamster Ovary (CHO) Cells-- CHO cells coexpressing integrin alpha IIbbeta 3 and GPIb-IX complex (123 cells), pcDNA3.1 vector-transfected 123 cells, or 123 cells expressing recombinant PKG Ialpha were described previously (15).2 A mutant of MEK1 (K97M) (kinase activity-deficient) was a generous gift from Dr. Gary Johnson, University of Colorado (30). A kinase activity-deficient mutant of Raf-1 (Raf301) was constructed as described previously (31) and was kindly provided by Dr. Mark Renshaw, The Scripps Research Institute. These constructs were subcloned in pcDNA3.1/Zeo+ vector and transfected into 123 cells using LipofectAMINE plus (Life Technologies, Inc.). Stable cell lines were established by selection with 0.4 mg/ml zeocin added to culture media. Expression of these proteins was assessed by Western blotting with anti-MEK1/2 or anti-Raf-1 antibodies (Santa Cruz Biotechnology).

GPIb-IX-mediated Integrin Activation in Reconstituted CHO Cell Model-- GPIb-IX-mediated activation of integrin alpha IIbbeta 3 was examined by flow cytometry analysis of Oregon Green 488-labeled fibrinogen (Molecular Probes) binding to integrin alpha IIbbeta 3 as described previously (15). Briefly, transfected CHO cells were detached from the tissue culture plates by 0.5 mM EDTA in phosphate-buffered saline. After washing, the cells were resuspended in modified Tyrode's solution (2.5 mM Hepes, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 5.5 mM D-glucose, 1 mM CaCl2, 1 mM MgCl2, 1 mg/ml bovine serum albumin, pH 7.4) as described previously (15). The cells were incubated with Oregon Green-labeled fibrinogen and ristocetin (a cofactor that activates vWF binding to GPIb-IX) in the presence or absence of purified human vWF (20 µg/ml) at 22 °C for 30 min and analyzed by flow cytometry. Nonspecific binding of fibrinogen was estimated by measuring fibrinogen binding in the presence of a specific integrin inhibitor RGDS peptide (1 mM). We have previously shown that RGDS but not RGES peptides specifically inhibit fibrinogen binding to integrin alpha IIbbeta 3 (32).

Preparation of Platelets-- Fresh venous blood was anti-coagulated with <FR><NU>1</NU><DE>10</DE></FR> volume of 3.8% trisodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation at 300 × g for 20 min at 22 °C. To prepare washed platelets, blood was anti-coagulated with ACD (2.5% trisodium citrate, 2% D-glucose, and 1.5% citric acid) (1/7 volume). PRP was further centrifuged at 1500 × g for 20 min. Platelets were then washed twice with CGS (0.12 M NaCl, 0.0129 M trisodium citrate, and 0.03 M D-glucose, pH 6.5) containing 0.1% bovine serum albumin, resuspended in modified Tyrode's solution, and allowed to recover to resting state at 37 °C for 1-2 h as previously described (32). In experiments to detect ERK phosphorylation, platelets resuspended in Hepes buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM Hepes, and 10 mM D-glucose, pH 7.4) (5 × 108/ml) were incubated with vWF (20 µg/ml) and botrocetin (5 µg/ml, a cofactor that activates vWF binding to GPIb-IX) or incubated with 8-bromo-cGMP (0.1 mM), 8-pCPT-cGMP (0.1 mM), or glyco-SNAP1 (N-(beta -D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide, Calbiochem) (0.1 mM) at 37 °C for various lengths of time and then analyzed for ERK phosphorylation as described below.

Detection of ERK Phosphorylation-- Transfected CHO cells were detached from tissue culture plates as described above and resuspended in modified Tyrode's buffer (5 × 106 cells/ml). Resuspended cells were stimulated with vWF (20 µg/ml) and botrocetin (5 µg/ml) (or 1 mg/ml ristocetin) at 22 °C for 5 min. In some experiments, the cells were also incubated for 5 min after adding 8-bromo-cGMP (0.1 mM), 8-pCPT-cGMP (0.1 mM), or SNAP1 (0.1 mM) to stimulate PKG activity. When examining the effect of PKG inhibitors, Rp-8-pCPT-cGMP (200 µM) was preincubated with cells at 22 °C for 5 min before the addition of vWF and botrocetin. Cells were solubilized by adding an equal volume of SDS-polyacrylamide gel electrophoresis sample buffer containing 0.2 mM E64, 20 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride, and nuclear DNA was removed by centrifugation. Platelet lysates were prepared as described above. Proteins in lysates were separated by SDS-polyacrylamide gel electrophoresis on a 4-15% gradient polyacrylamide gel and then electrotransferred to polyvinylidene difluoride membranes. The membranes were immunoblotted with an anti-ERK2 antibody (Santa Cruz) or an anti-ERK antibody recognizing the phosphorylation-dependent epitope in the Thr-202/Tyr-204 site of ERKs (New England Biolabs), and reactions were visualized using the enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Platelet Aggregation-- Platelet aggregation was measured using a turbidometric platelet aggregometer at 37 °C with a stirring speed at 1000 rpm. vWF-dependent platelet aggregation was induced by the addition of vWF modulators, ristocetin, or botrocetin to PRP. For alpha -thrombin-induced platelet aggregation, washed platelets were used. In some experiments, aspirin (100 µg/ml), RGDS (1 mM), or MEK inhibitors PD98059 or U0126 were preincubated with platelets at 37 °C for 5 min before the addition of agonists.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inhibition of GPIb-IX-mediated Activation of Integrin by Dominant Negative Mutants of MEK1 and Raf-1-- We have recently shown that activation of the platelet integrin alpha IIbbeta 3 can be reconstituted in CHO cells expressing both recombinant human GPIb-IX and integrin alpha IIbbeta 3 (123 cells) (15). Similar results has also been shown by Zaffran et al. (33). In 123 cells, binding of vWF to GPIb-IX triggers activation of integrin alpha IIbbeta 3, allowing specific binding of fibrinogen, which is a physiological ligand of integrin alpha IIbbeta 3. Reconstitution of integrin activation in this cell line thus allows analysis of integrin activation pathways using molecular biology techniques. To identify the roles of MAPK pathway in GPIb-IX-mediated integrin activation, 123 cells were transfected with cDNA constructs encoding dominant negative mutants of Raf-1 (Raf301) (31) or MEK1 (MEK1 K97M) (30). Immunoblotting analysis confirmed that Raf301 or MEK1 K97M mutants were expressed in the respective cell lines (Fig. 1, A and B). Three different clones each of Raf301- or MEK1 K97M-expressing cells were examined for vWF-induced integrin activation, as indicated by specific binding of soluble fibrinogen. In comparison with the vector-transfected cells, vWF-induced fibrinogen binding to integrin alpha IIbbeta 3 in either Raf301- or MEK1 K97M-expressing cells was significantly inhibited (Fig. 1C). The inhibitory effects of the dominant negative mutants did not result from their effects on the expression of integrin alpha IIbbeta 3 or GPIb-IX, as expression levels of these two receptors in vector- or mutant-transfected cell lines were comparable (Fig. 1D). Also, brief preincubation (5 min) with a MEK inhibitor, PD98059 (20 µM), inhibited vWF-induced fibrinogen binding to 123 cells (not shown). These results indicate that the ERK pathway is important in the signaling pathway of GPIb-IX-mediated integrin activation.


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 1.   Expression of dominant negative mutant of MEK1 or Raf-1 inhibited GPIb-IX-mediated integrin activation. A and B, CHO cells expressing GPIb-IX and integrin alpha IIbbeta 3 (123 cells) were transfected with dominant negative mutants of Raf-1 or MEK1, Raf301, or MEK1 K97M, respectively. As a control, the same cells were transfected with pCDNA 3.1 vector (Vector). Expression of Raf301 (A) or MEK1K97M (B) were shown by Western blotting using an anti-human Raf-1 or MEK1 antibodies. C, 123 cells expressing vector, Raf301, or MEK1 K97M were incubated with Oregon Green-labeled fibrinogen in the presence of ristocetin and vWF. As a negative control, these cells were also incubated with Oregon Green-labeled fibrinogen in the presence of ristocetin but not vWF. Cells were analyzed by flow cytometry. Nonspecific binding of fibrinogen was determined by measuring fibrinogen binding in the presence of 1 mM RGDS peptides. Quantitative results from eight experiments are expressed as fibrinogen binding indices (total bound fluorescence (geo mean)/nonspecifically bound fluorescence (geo mean)). D, cells expressing vector, Raf301, or MEK1 K97M were also analyzed by flow cytometry for the expression levels of GPIb-IX using an anti-GPIbalpha monoclonal antibody, SZ2, and for expression levels of integrin alpha IIbbeta 3 using a monoclonal antibody against alpha IIbbeta 3 complex, D57. Note that the expression levels of GPIb-IX and alpha IIbbeta 3 between these cell lines are comparable.

GPIb-IX-mediated Activation of the ERK Pathway in the Reconstituted CHO Cell Model-- If the ERK pathway mediates GPIb-IX signaling, ligand binding to GPIb-IX is expected to stimulate the MAPK cascade, leading to ERK activation. It is known that activation of ERK is a consequence of ERK phosphorylation at Thr-202 and Tyr-204 by its upstream kinase MEK (34). Thus, to determine whether the ERK pathway is activated after ligand binding to GPIb-IX, we examined phosphorylation of ERK using an anti-ERK antibody recognizing the phosphorylated Thr-202/Tyr-204 site of ERK. Phosphorylation of ERK1 or ERK2 was minimal in suspended 123 cells or in 123 cells treated with ristocetin alone (Fig. 2A). Stimulation of 123 cells with vWF in the presence of ristocetin significantly enhanced phosphorylation of ERK2 (Fig. 2A). GPIb-IX-induced ERK2 phosphorylation was inhibited in cells expressing either Raf301 or MEK1 M97K mutants (Fig. 2A). Thus, vWF binding to GPIb-IX stimulates MAPK pathway leading to ERK2 phosphorylation, which is inhibited by dominant negative mutants of the upstream kinases.


View larger version (59K):
[in this window]
[in a new window]
  		Fig. 2.   GPIb-IX-mediated ERK2 phosphorylation in the CHO cell model. A, vector-, Raf301-, or MEK1 M97K-transfected 123 cells were incubated with buffer (Control), ristocetin (1 mg/ml) (Risto), ristocetin and vWF (20 µg/ml) (vWF) at 22 °C for 5 min. Cells were solubilized by adding equal volume of 2× SDS-sample buffer. Phosphorylation of ERK (P-ERK2) was analyzed by Western blotting using an antibody specific for phosphorylated Thr-202/Tyr-204 of ERKs (New England Biolabs). Equal sample loading was assessed by Western blotting using an anti-ERK2 antibody (Santa Cruz Biotechnology). Note that vWF stimulates ERK2 phosphorylation in vector-transfected but not in Raf301- or MEK1 M97K-transfected 123 cells. B, vector- or PKG Ialpha -transfected 123 cells were incubated with buffer (Control), ristocetin (1 mg/ml) (Risto), ristocetin and vWF (vWF) at 22 °C for 5 min and then immunoblotted to detect ERK2 phosphorylation as described in A. Note that ERK2 phosphorylation is significantly enhanced in PKG-expressing cells. C and D, PKG-transfected 123 cells were preincubated with mouse IgG (40 µg/ml) or a monoclonal antibody against GPIbalpha , SZ2 (40 µg/ml), or RGDS peptide (1 mM) at 22 °C for 5 min and then analyzed for ERK2 phosphorylation as described in A. A representative result is shown in C. Shown in D are the results from four experiments that were scanned and quantitated using NIH Image software. The difference between control (mouse IgG) and SZ2-treated samples is statistically significant (p < 0.005).

The Roles of PKG in GPIb-IX-mediated Activation of ERK Pathway-- We have shown that PKG I is an important stimulatory mediator in GPIb-IX-mediated integrin activation.2 In 123 cells, expression of recombinant human PKG I significantly enhances GPIb-IX-mediated integrin activation. To determine if expression of PKG I also enhances GPIb-IX-induced activation of MAPK pathway, PKG Ialpha -expressing 123 cells (123PKGIalpha cells) were treated with or without vWF and ristocetin and then analyzed for ERK2 phosphorylation. Expression of PKG Ialpha dramatically enhanced vWF-stimulated ERK2 phosphorylation in comparison with vector-transfected 123 cells (Fig. 2B), indicating that PKG promotes GPIb-IX-induced activation of ERK pathway. To determine whether phosphorylation of ERK2 in 123PKGIalpha cells is GPIb-IX-dependent, cells were preincubated with an anti-GPIbalpha monoclonal antibody, SZ2, before the addition of vWF and ristocetin. vWF-induced ERK2 phosphorylation was inhibited by SZ2 (Fig. 2C). In contrast, mouse IgG had no significant effect (Fig. 2C). Furthermore, an integrin inhibitor, RGDS peptide, had no inhibitory effect on vWF-induced activation of ERK2, excluding a possible role for integrin outside-in signaling in ERK2 phosphorylation (Fig. 2C). Thus, PKG promotes GPIb-IX-dependent ERK2 phosphorylation.

To determine whether PKG is required for vWF-induced ERK2 activation, we attempted to determine if a specific inhibitor of PKG, Rp-8-pCPT-cGMP, could inhibit GPIb-IX-mediated phosphorylation of ERK2. Rp-8-pCPT-cGMP inhibited phosphorylation of ERK2 induced by vWF. Thus PKG activity is required for GPIb-IX-induced activation of ERK2 (Fig. 3A). To further examine whether activation of PKG is sufficient to stimulate ERK2 phosphorylation, 123 cells or 123PKGIalpha cells were treated with PKG activators (8-bromo-cGMP, 8-pCPT-cGMP, or glyco-SNAP1) and then examined for ERK2 phosphorylation. All these PKG activators induced phosphorylation of ERK2 in 123 cells (Fig. 3B), and the phosphorylation of ERK2 induced by PKG activators was dramatically increased in 123PKGIalpha cells (Fig. 3B). Thus, activation of ERK2 is downstream from PKG in the GPIb-IX-mediated integrin activation pathway.


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 3.   Effects of PKG inhibitor and activators on ERK2 phosphorylation. A, PKG-transfected 123 cells were incubated with ristocetin alone (Control) or stimulated with vWF in the presence of ristocetin (vWF) for 5 min. The cells were also preincubated with a PKG inhibitor, Rp-8pCPT-cGMP (200 µM) at 22 °C for 5 min, and then stimulated with vWF and ristocetin (Rp+vWF). Cells were then solubilized, analyzed by SDS-polyacrylamide gel electrophoresis, and immunoblotted with the phosphorylation-specific anti-ERK antibody (P-ERK2) to detect ERK phosphorylation or an anti-ERK2 antibody (ERK2) to show equal loading. B, vector- or PKG-transfected cells were incubated with PKG activators, 100 µM 8-bromo-cGMP (Bromo), 100 µM 8-pCPT-cGMP (pCPT), or 100 µM glyco-SNAP1 (SNAP1) at 22 °C for 5 min and then analyzed for ERK2 phosphorylation as described in A.

The Role of ERK Pathway in GPIb-IX-dependent Integrin Activation in Human Platelets-- The above-described data indicate that ERK pathway is downstream from PKG in the GPIb-IX-mediated integrin activation in a reconstituted CHO cell model. To determine if this finding appropriately reflects the role of MAPK pathway in vWF-induced integrin activation in human platelets, we examined the effect of MEK inhibitors PD98059 or U0126 on vWF-induced, integrin-dependent platelet aggregation. As shown previously, ristocetin-induced vWF binding to GPIb-IX induces activation of integrin alpha IIbbeta 3 and a reversible aggregation/agglutination of platelets (partially inhibited by RGDS (Fig. 4)), leading to a totally integrin-dependent second wave of platelet aggregation. The addition of either PD98059 or U0126 dose-dependently inhibited integrin-dependent platelet aggregation induced by ristocetin (Fig. 4, A and B) or botrocetin (not shown). To examine if the inhibitory effect of PD98059 is dependent on its possible effect on cyclooxygenases, ristocetin-induced platelet aggregation/agglutination was examined in the presence of cyclooxygenase inhibitor aspirin. As expected, a TXA2-dependent second wave of platelet aggregation was diminished in the presence of aspirin. However, the ristocetin-induced first wave of platelet aggregation/agglutination was not significantly affected by aspirin but was significantly (but partially) inhibited by RGDS peptide even in the presence of aspirin, suggesting that vWF-induced integrin activation precedes the second wave of platelet aggregation and is independent of TXA2 pathway (Fig. 4C). The addition of PD98059 to the aspirin-treated platelets inhibited ristocetin-induced platelet aggregation in a manner similar to RGDS (Fig. 4C). These results exclude the possibility that the inhibitory effects of PD98059 on GPIb-IX-mediated integrin activation are caused by its effect on cyclooxygenases. Thus, ERK pathway is important in GPIb-IX-mediated integrin activation in platelets.


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 4.   Effects of MEK inhibitors on integrin-dependent platelet aggregation induced by vWF-GPIb-IX interaction. A and B, PRP was preincubated at 37 °C for 5 min with various concentrations of MEK inhibitors, PD98059 (A) or U0126 (B). PRP was also incubated with Me2SO (DMSO) (vehicle for PD98059 and U0126). The vWF modulator, ristocetin, was then added to induce vWF-GPIb-IX interaction. Ristocetin-induced platelet aggregation/agglutination was recorded using a platelet turbidometric aggregometer. C, PRP was incubated without (Control) or with aspirin (100 µg/ml) and Me2SO at 37 °C for 5 min. Aspirin-treated platelets were also incubated in the presence of PD98059 (20 µM) or RGDS (1 mM). Ristocetin (1.25 mg/ml) was then added to PRP to induce platelet aggregation/agglutination.

It is known that GPIb-IX pathway is not only required for vWF-induced platelet activation but also for platelet activation induced by low dose thrombin. To examine whether the MAP kinase pathway was required for low dose-thrombin-induced platelet activation, washed platelets were incubated with PD98059 or U0126 and then stimulated with a low dose of thrombin (0.05 units/ml). Fig. 5 shows that thrombin-induced platelet activation was inhibited by these MEK1 inhibitors. To exclude the possibility that the inhibitory effects of PD98059 resulted from a possible effect on cyclooxygenases (29), platelets were preincubated with aspirin and then stimulated with the same concentration of thrombin. We found that aspirin had no inhibitory effects on low dose thrombin-induced platelet aggregation (Fig. 5). Thus, the inhibitory effect of PD98059 is unlikely to result from its effect on cyclooxygenases. Taken together, these data indicate that MAP kinase pathway is indeed important in GPIb-IX-dependent activation of integrin alpha IIbbeta 3 in platelets.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of MEK inhibitors and aspirin on low dose thrombin-induced platelet aggregation. Washed platelets were preincubated with aspirin (100 µg/ml), PD98059 (20 µM), or U0126 (2 µM) for 5 min. Platelets were also preincubated with Me2SO (DMSO) as a control for MEK inhibitors. A low dose of alpha -thrombin (0.05 units/ml) was then added to induce platelet aggregation.

GPIb-IX-dependent Activation of the ERK Pathway in Platelets-- To determine if the ERK pathway is activated after ligand binding to GPIb-IX, we examined vWF-induced ERK phosphorylation in platelets. ERK phosphorylation was minimal in resting platelets or control platelets treated with botrocetin (or ristocetin) alone (Fig. 6A). When vWF was added in the presence of botrocetin (or ristocetin), however, ERK2 phosphorylation was significantly enhanced (Fig. 6A). Phosphorylation of ERK2 reached maximum only 30 s after the addition of vWF and was significantly reduced after 5 min, suggesting that ERK2 phosphorylation is an early signaling event (Fig. 6A). These data indicate that vWF binding to GPIb-IX activates ERK pathway in human platelets.


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 6.   Phosphorylation of ERK2 in platelets. A, washed platelets (200 µl) were stimulated under stirring for 0.5 or 5 min in a platelet aggregometer with buffer (Ctrl), 5 µg/ml botrocetin only (Botro), or botrocetin and vWF (20 µg/ml) (vWF). B, platelets were incubated with botrocetin alone (Control) or stimulated with vWF in the presence of botrocetin (vWF) for 0.5 min. The platelets were also preincubated with a PKG inhibitor, Rp-8pCPT-cGMP (200 µM) at 22 °C for 5 min, and then stimulated with vWF and botrocetin (Rp+vWF). C, platelets were incubated with 2 µl of water (Control) or PKG activators, 8-bromo-cGMP (100 µM), 8-pCPT-cGMP (100 µM), or SNAP1 (100 µM) at 22 °C for 0.5 min. The platelets were then solubilized and analyzed by immunoblotting with a rabbit antibody recognizing the phosphorylated form of ERK to detect ERK phosphorylation (P-ERK2) and with a rabbit anti-ERK2 antibody to indicate comparable loading levels (ERK2).

Activation of ERK Pathway Is Downstream from PKG in the GPIb-IX-mediated Integrin Activation in Platelets-- To further investigate whether activation of MAPK is downstream from PKG in the GPIb-IX-signaling pathway in platelets, we examined the effects of PKG activators and inhibitors on the phosphorylation of ERK2 in platelets. Again, Rp-8-pCPT-cGMP, a specific inhibitor of PKG, abolished vWF-induced phosphorylation of ERK2 (Fig. 6B), indicating that PKG is important in GPIb-IX-dependent activation of the ERK pathway. PKG activators (8-bromo-cGMP, 8-pCPT-cGMP, or SNAP1) induced phosphorylation of ERK2 in platelets without requiring other stimuli (Fig. 6C), indicating that PKG activation is sufficient to activate ERK pathway in platelets. Taken together, we have identified a novel signaling pathway of platelet activation; ligand binding to GPIb-IX induces PKG activation. PKG stimulates ERK pathway leading to activation of integrin alpha IIbbeta 3.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we found that 1) ligand binding to GPIb-IX activates ERK pathway, which is important in mediating GPIb-IX-induced activation of integrin alpha IIbbeta 3, and 2) the ERK pathway is downstream from PKG in the GPIb-IX-induced integrin activation. Together with our recent finding that GPIb-IX-induced platelet activation requires the cGMP-PKG pathway, these data delineate a novel integrin activation signaling pathway in which ligand binding to GPIb-IX induces activation of PKG, which stimulates ERK pathway, leading to activation of integrin alpha IIbbeta 3 (Fig. 7).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 7.   Signaling pathways of GPIb-IX-induced integrin activation. GPIb-IX-induced integrin activation requires at least two different signaling pathways, 1) a novel cGMP-PKG-ERK pathway as shown in this study and 2) a signaling pathway involving Fcgamma receptor IIA (Fcgamma RII) or Fc receptor gamma  chain (FcRgamma ) coupled to tyrosine kinase syk, phospholipase Cgamma 2, diacylglycerol (DAG), inositol triphosphate (IP3), protein kinase C (PKC), and Ca2+ influx. GPIb-IX has also been shown to be associated with several signaling and cytoskeletal molecules potentially involved in GPIb-IX signaling: 14-3-3, calmodulin (CaM), phosphoinositol 3-kinase (PI3K), and filamin.

The elements of ERK MAPK pathway are abundant in platelets. Previous studies indicate that ERK pathway is stimulated during platelet activation induced by platelet agonists such as ADP, collagen, and thrombin (23, 28). However, understanding the roles of MAPK in platelet activation has been complicated by the report that the commonly used inhibitor of ERK pathway, PD98059, also inhibits cyclooxygenases and, thus, TXA2 synthesis (29). In this study, we took advantage of our newly reconstituted integrin activation model in CHO cells (15) and examined the effects of dominant negative mutants of Raf-1 and MEK1 on GPIb-IX-mediated integrin activation in this system. We conclude that ERK pathway is important in integrin activation via the GPIb-IX signaling pathway. This conclusion is supported by the following findings. 1) Dominant negative mutants of MAP kinase pathway, Raf301 and MEK1 M97K, inhibited GPIb-IX-mediated activation of integrin alpha IIbbeta 3 in a reconstituted CHO cell expression model; 2) MEK inhibitors PD98059 and U0126 inhibited vWF and low dose thrombin-induced, integrin-dependent platelet aggregation in platelets in a cyclooxygenase-independent manner; and 3) vWF binding to GPIb-IX induced phosphorylation of ERK2 at the Thr-Glu-Tyr sequence, indicating that the ERK MAPK pathway is activated. The results obtained in the CHO cell model using recombinant DNA technology and results obtained in platelets using various MEK1 inhibitors and biochemical assays are highly consistent. Inhibition of integrin activation by the dominant negative mutants of MAPK pathway excludes the possible nonspecific effects of pharmacological agents, and results obtained in human platelets indicate that the reconstituted CHO cell model appropriately reflects the role of MAPK pathway in platelets. Furthermore, our data indicate that the inhibitory effect of MEK inhibitor PD98059 is not likely to result from its effects on cyclooxygenases because the inhibitory effect of PD98059 was seen when cyclooxygenases were already inhibited by high concentrations of aspirin (Fig. 4). Furthermore, aspirin had no inhibitory effect on vWF-induced integrin activation in CHO cells (not shown), vWF-induced the first wave of platelet aggregation (Fig. 4), or low-dose thrombin-induced platelet aggregation (Fig. 5), all of which were inhibited by PD98059. Consistent with our results, it was previously shown that ristocetin-induced production of TXA2 was inhibited by anti-integrin monoclonal antibodies, suggesting that integrin activation precedes vWF-induced TXA2 production (35). Thus, MAPK pathway stimulates GPIb-IX-dependent integrin activation and platelet aggregation in a TXA2-independent manner.

We have recently shown that the PKG pathway plays an important role in GPIb-IX-mediated activation of the platelet integrin alpha IIbbeta 3.2 Here we show that expression of recombinant PKG in the CHO cell model significantly enhances GPIb-IX-induced ERK pathway activation, and inhibition of PKG abolished GPIb-IX-induced ERK pathway activation. Furthermore, activation of PKG by cGMP analogs is sufficient to activate ERK pathway. Thus we conclude that the ERK pathway is downstream from PKG in the GPIb-IX-signaling pathway. Although the classic MAPK activation pathway is mediated by the receptor tyrosine kinases via Ras, it has been shown that the ERK pathway is also regulated by multiple cellular signals, including PKG (36-38). However, there have been controversies about whether PKG activates or inhibits MAPK pathway. In one study, ERK activity was reportedly reduced after prolonged incubation (30 min) with cGMP analogs in baby hamster kidney cells (36), However, others found that PKG activated MAPK pathway in endothelial cells and smooth muscle cells (37, 38). PKG directly phosphorylates and activates Raf-1, the upstream kinase in the ERK pathway (37). Our results are consistent with the latter studies. One possible reason for the apparent contradicting results is the difference in the incubation time after the addition of cGMP analogs. Our data indicate that vWF or cGMP analogs induce a rapid increase in ERK2 phosphorylation in platelets that peaks at 30 s (1 min in some donors) and decreases after prolonged incubation (Fig. 6), suggesting that PKG-mediated ERK phosphorylation is a transient event. This is consistent with our observations that cGMP analogs promoted platelet activation only when added simultaneously or immediately after the addition of an agonist such as vWF or thrombin.2 Prolonged preincubation of cGMP analogs with platelets inhibits subsequent platelet response to platelet agonists in a protein kinase A-dependent manner and induces protein kinase A-mediated phosphorylation of vasodilator-stimulated phosphoprotein.2 Thus, it is possible that after a prolonged incubation, cGMP-induced activation of a cAMP-protein kinase A pathway may diminish the function of PKG to induce ERK pathway activation. It is also possible that after the initial activation of MAPK pathway and the integrin, the outside-in signaling of integrin may cause MAPK dephosphorylation, since ligand binding to integrin has been shown to negatively regulate agonist-stimulated ERK activity in platelets (39).

There have been several interesting observations on the relationship between MAPK pathway and integrin signaling in recent years. Although Zhang et al. (40) reports that Ra-Ras enhanced integrin activity, Hughes et al. (41) find that the activity of a constitutively active alpha IIb/alpha 5beta 3/beta 1 chimera integrin expressed in CHO cells was enhanced by the expression of a dominant negative mutant of Ha-Ras but was inhibited by the constitutively active Raf mutant or Ha-Ras. The results by Hughes et al. (41) apparently contradict our data that dominant negative mutants of Raf and MEK inhibited alpha IIbbeta 3 activation induced by GPIb-IX. However, there is a major difference between the experiments of Hughes et al. (41) and ours. The integrin mutant used by Hughes et al. (41) was a chimera integrin with the cytoplasmic domain of the alpha IIb replaced by the cytoplasmic domain of alpha 5 (or alpha  subunits other than alpha IIb) and the cytoplasmic domain of the beta 3 replaced by the cytoplasmic domain of beta 1. Thus, in effect, the cytoplasmic regulatory domain of this integrin mutant was not an alpha IIbbeta 3 but a beta 1 integrin or a hybrid between beta 3 and one of the alpha  subunits that are constitutively active in cells. This may explain why the chimera mutants, unlike wild type integrin alpha IIbbeta 3, are constitutively active. In contrast, we used wild type integrin alpha IIbbeta 3 coexpressed with platelet GPIb-IX. The wild type alpha IIbbeta 3 is normally in resting state but becomes activated only after agonist stimulation. Thus, the results from Hughes et al. (41) may reflect a negative regulation of constitutively active integrins (such as beta 1 integrins), but our results reflect stimulatory roles of MAPK pathway in the inside-out signaling of the integrin alpha IIbbeta 3. In this respect, it is interesting to note that the effects of integrin outside-in signaling on ERK pathway is also different between different integrins. Although beta 1 integrin stimulates activation of ERK pathway in several cell types (42), ligand binding to alpha IIbbeta 3 has been shown to inhibit ERK pathway in platelets (39). It would be interesting to further investigate the mechanisms of the different interrelationships between MAPK pathway and different members of the integrin family.

Although we have identified that the cGMP-PKG-MAPK pathway mediates GPIb-IX-dependent integrin activation signaling, it is important to note that activation of PKG-MAPK pathway by adding cGMP analogs alone in the absence of vWF or thrombin stimulation is not sufficient to activate integrin alpha IIbbeta 3.2 Thus, it appears that additional parallel signaling pathways induced by vWF or thrombin is required for integrin activation in addition to the cGMP-PKG-MAPK pathway. In this respect, it has been reported that vWF-induced platelet activation requires activation of the Fc receptor gamma II (or Fc receptor gamma -chain)-protein-tyrosine kinase Syk-signaling pathway (Fig. 7) (43-46). In addition, GPIb-IX is associated with several intracellular signaling proteins including 14-3-3zeta (47, 48), phosphatidylinositol 3-kinase (via 14-3-3zeta ) (49), and calmodulin (50). Although the roles of these proteins in GPIb-IX signaling are still unclear, it is possible that these proteins are also involved in GPIb-IX-mediated signaling. Thus, GPIb-IX-mediated integrin activation requires coordination of two or more signaling pathways (Fig. 7), one of which is the cGMP-PKG-MAPK-signaling pathway.

The downstream signaling pathway of MAPK-mediated integrin activation is still unknown. Because ERK has been shown to phosphorylate and activate cytoplasmic phospholipase A2 (27), it is a possibility that activation of cytoplasmic phospholipase A2 may be a downstream link between ERK and integrin activation. However, previous studies suggest that the release of arachidonic acid during platelet activation is not affected by MEK inhibitor PD98059 (29), and vWF-induced TXA2 production is preceded by integrin activation (35). We show that vWF-induced integrin activation and low dose-thrombin-induced platelet aggregation are not inhibited by aspirin. These data suggest that ERK-dependent activation of integrin does not require the TXA2 pathway. Although TXA2 is important in the irreversible second wave of platelet aggregation induced by certain agonists, the second wave of platelet aggregation not only requires integrin activation but also requires integrin-dependent outside-in signaling and release of granule contents. It would be interesting to further investigate the downstream mechanism of the MAP kinase-dependent integrin activation pathway.

    ACKNOWLEDGEMENTS

We thank Drs. Mark Ginsberg, Chenggeng Ruan, and Michael C. Berndt for providing reagents.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL52547 and HL62350 and by a grant-in-aid from the American Heart Association.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 These authors contributed equally to this manuscript.

§ An Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612. Tel.: 312-355 0237; Fax: 312-996-1225; E-mail: xdu@uic.edu.

Published, JBC Papers in Press, August 24, 2001, DOI 10.1074/jbc.M106129200

2 Z. Li, X. Xi, M. Gu, R. Ye, and X. Du, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: vWF, von Willebrand factor; GPIb-IX, glycoprotein Ib-IX; PKG protein kinase G, MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; TXA2, thromboxane A2; CHO, Chinese hamster ovary; PRP, platelet-rich plasma; pCPT, 8-(4-chlorophenylthio)-cGMP; SNAP1, N-(beta -D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Ginsberg, M. H., Du, X., O'Toole, T. E., and Loftus, J. C. (1995) Thromb. Haemostasis 74, 352-359[Medline] [Order article via Infotrieve]
2. Phillips, D. R., Charo, I. F., and Scarborough, R. M. (1991) Cell 65, 359-362[CrossRef][Medline] [Order article via Infotrieve]
3. Shattil, S. J., Kashiwagi, H., and Pampori, N. (1998) Blood 91, 2645-2657[Free Full Text]
4. Weiss, H. J., Turitto, V. T., and Baumgartner, H. R. (1978) J. Lab. Clin. Med. 92, 750-764[Medline] [Order article via Infotrieve]
5. Sakariassen, K. S., Bolhuis, P. A., and Sixma, J. J. (1979) Nature 279, 636-638[CrossRef][Medline] [Order article via Infotrieve]
6. Sakariassen, K. S., Nievelstein, P. F., Coller, B. S., and Sixma, J. J. (1986) Br. J. Haematol. 63, 681-691[Medline] [Order article via Infotrieve]
7. Savage, B., Saldivar, E., and Ruggeri, Z. M. (1996) Cell 84, 289-297[CrossRef][Medline] [Order article via Infotrieve]
8. Savage, B., Almus-Jacobs, F., and Ruggeri, Z. M. (1998) Cell 94, 657-666[CrossRef][Medline] [Order article via Infotrieve]
9. Ruggeri, Z. M. (1991) Prog. Hemostasis Thromb. 10, 35-68[Medline] [Order article via Infotrieve]
10. Lopez, J. A., Andrews, R. K., Afshar-Kharghan, V., and Berndt, M. C. (1998) Blood 91, 4397-4418[Free Full Text]
11. De Marco, L., Girolami, A., Russell, S., and Ruggeri, Z. M. (1985) J. Clin. Invest. 75, 1198-1203
12. De Marco, L., Girolami, A., Zimmerman, T. S., and Ruggeri, Z. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7424-7428[Abstract/Free Full Text]
13. Kroll, M. H., Harris, T. S., Moake, J. L., Handin, R. I., and Schafer, A. I. (1991) J. Clin. Invest. 88, 1568-1573
14. Savage, B., Shattil, S. J., and Ruggeri, Z. M. (1992) J. Biol. Chem. 267, 11300-11306[Abstract/Free Full Text]
15. Gu, M., Xi, X., Englund, G. D., Berndt, M. C., and Du, X. (1999) J. Cell Biol. 147, 1085-1096[Abstract/Free Full Text]
16. Okumura, T., and Jamieson, G. A. (1976) Thromb. Res. 8, 701-706[CrossRef][Medline] [Order article via Infotrieve]
17. Jamieson, G. A., and Okumura, T. (1978) J. Clin. Invest. 61, 861-864
18. Yamamoto, N., Kitagawa, H., Tanoue, K., and Yamazaki, H. (1985) Thromb. Res. 39, 751-759[CrossRef][Medline] [Order article via Infotrieve]
19. Katagiri, Y., Hayashi, Y., Yamamoto, K., Tanoue, K., Kosaki, G., and Yamazaki, H. (1990) Thromb. Haemostasis 63, 122-126[Medline] [Order article via Infotrieve]
20. De Marco, L., Mazzucato, M., Masotti, A., Fenton, J. d., and Ruggeri, Z. M. (1991) J. Biol. Chem. 266, 23776-23783[Abstract/Free Full Text]
21. Mazzucato, M., Marco, L. D., Masotti, A., Pradella, P., Bahou, W. F., and Ruggeri, Z. M. (1998) J. Biol. Chem. 273, 1880-1887[Abstract/Free Full Text]
22. Ramakrishnan, V., DeGuzman, F., Bao, M., Hall, S. W., Leung, L. L., and Phillips, D. R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 1823-1828[Abstract/Free Full Text]
23. Papkoff, J., Chen, R. H., Blenis, J., and Forsman, J. (1994) Mol. Cell. Biol. 14, 463-472[Abstract/Free Full Text]
24. Saklatvala, J., Rawlinson, L., Waller, R. J., Sarsfield, S., Lee, J. C., Morton, L. F., Barnes, M. J., and Farndale, R. W. (1996) J. Biol. Chem. 271, 6586-6589[Abstract/Free Full Text]
25. Bugaud, F., Nadal-Wollbold, F., Levy-Toledano, S., Rosa, J. P., and Bryckaert, M. (1999) Blood 94, 3800-3805[Abstract/Free Full Text]
26. Morrison, D. K., and Cutler, R. E. (1997) Curr. Opin. Cell Biol. 9, 174-179[CrossRef][Medline] [Order article via Infotrieve]
27. Lin, L. L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. (1993) Cell 72, 269-278[CrossRef][Medline] [Order article via Infotrieve]
28. Borsch-Haubold, A. G., Kramer, R. M., and Watson, S. P. (1996) Biochem. J. 318, 207-212
29. Borsch-Haubold, A. G., Pasquet, S., and Watson, S. P. (1998) J. Biol. Chem. 273, 28766-28772[Abstract/Free Full Text]
30. Gardner, A. M., Vaillancourt, R. R., Lange-Carter, C. A., and Johnson, G. L. (1994) Mol. Biol. Cell 5, 193-201[Abstract]
31. Qureshi, S. A., Rim, M., Bruder, J., Kolch, W., Rapp, U., Sukhatme, V. P., and Foster, D. A. (1991) J. Biol. Chem. 266, 20594-20597[Abstract/Free Full Text]
32. Du, X. P., Plow, E. F., Frelinger, A. L. I., O'Toole, T. E., Loftus, J. C., and Ginsberg, M. H. (1991) Cell 65, 409-416[CrossRef][Medline] [Order article via Infotrieve]
33. Zaffran, Y., Meyer, S. C., Negrescu, E., Reddy, K. B., and Fox, J. E. (2000) J. Biol. Chem. 275, 16779-16787[Abstract/Free Full Text]
34. Payne, D. M., Rossomando, A. J., Martino, P., Erickson, A. K., Her, J. H., Shabanowitz, J., Hunt, D. F., Weber, M. J., and Sturgill, T. W. (1991) EMBO J. 10, 885-892[Medline] [Order article via Infotrieve]
35. Ruan, C. G., Du, X. P., Xi, X. D., Castaldi, P. A., and Berndt, M. C. (1987) Blood 69, 570-577[Abstract/Free Full Text]
36. Suhasini, M., Li, H., Lohmann, S. M., Boss, G. R., and Pilz, R. B. (1998) Mol. Cell. Biol. 18, 6983-6994[Abstract/Free Full Text]
37. Hood, J., and Granger, H. J. (1998) J. Biol. Chem. 273, 23504-23508[Abstract/Free Full Text]
38. Komalavilas, P., Shah, P. K., Jo, H., and Lincoln, T. M. (1999) J. Biol. Chem. 274, 34301-34309[Abstract/Free Full Text]
39. Nadal, F., Levy-Toledano, S., Grelac, F., Caen, J. P., Rosa, J. P., and Bryckaert, M. (1997) J. Biol. Chem. 272, 22381-22384[Abstract/Free Full Text]
40. Zhang, Z., Vuori, K., Wang, H., Reed, J. C., and Ruoslahti, E. (1996) Cell 85, 61-69[CrossRef][Medline] [Order article via Infotrieve]
41. Hughes, P. E., Renshaw, M. W., Pfaff, M., Forsyth, J., Keivens, V. M., Schwartz, M. A., and Ginsberg, M. H. (1997) Cell 88, 521-530[CrossRef][Medline] [Order article via Infotrieve]
42. Chen, Q., Kinch, M. S., Lin, T. H., Burridge, K., and Juliano, R. L. (1994) J. Biol. Chem. 269, 26602-26605[Abstract/Free Full Text]
43. Sullam, P. M., Hyun, W. C., Szollosi, J., Dong, J., Foss, W. M., and Lopez, J. A. (1998) J. Biol. Chem. 273, 5331-5336[Abstract/Free Full Text]
44. Falati, S., Edmead, C. E., and Poole, A. W. (1999) Blood 94, 1648-1656[Abstract/Free Full Text]
45. Wu, Y., Suzuki-Inoue, K., Satoh, K., Asazuma, N., Yatomi, Y., Berndt, M. C., and Ozaki, Y. (2001) Blood 97, 3836-3845[Abstract/Free Full Text]
46. Canobbio, I., Bertoni, A., Lova, P., Paganini, S., Hirsch, E., Sinigaglia, F., Balduini, C., and Torti, M. (2001) J. Biol. Chem. 276, 26022-26029[Abstract/Free Full Text]
47. Du, X., Harris, S. J., Tetaz, T. J., Ginsberg, M. H., and Berndt, M. C. (1994) J. Biol. Chem. 269, 18287-18290[Abstract/Free Full Text]
48. Du, X., Fox, J. E., and Pei, S. (1996) J. Biol. Chem. 271, 7362-7367[Abstract/Free Full Text]
49. Munday, A. D., Berndt, M. C., and Mitchell, C. A. (2000) Blood 96, 577-584[Abstract/Free Full Text]
50. Andrews, R. K., Munday, A. D., Mitchell, C. A., and Berndt, M. C. (2001) Blood 98, 681-687[Abstract/Free Full Text]

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 BloodHome page
H. Yin, J. Liu, Z. Li, M. C. Berndt, C. A. Lowell, and X. Du
Src family tyrosine kinase Lyn mediates VWF/GPIb-IX-induced platelet activation via the cGMP signaling pathway
Blood, August 15, 2008; 112(4): 1139 - 1146.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
X. Su, J. Mi, J. Yan, P. Flevaris, Y. Lu, H. Liu, Z. Ruan, X. Wang, N. Kieffer, S. Chen, et al.
RGT, a synthetic peptide corresponding to the integrin {beta}3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin {alpha}IIb{beta}3 with Src kinase
Blood, August 1, 2008; 112(3): 592 - 602.
[Abstract] [Full Text] [PDF]