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Originally published In Press as doi:10.1074/jbc.M212675200 on May 29, 2003

J. Biol. Chem., Vol. 278, Issue 35, 32638-32644, August 29, 2003
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Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Inhibits Low Density Lipoprotein-induced Signaling in Platelets*

Ingrid A. M. Relou {ddagger} §, Gertie Gorter {ddagger}, Irlando Andrade Ferreira {ddagger}, Herman J. M. van Rijn § and Jan-Willem N. Akkerman {ddagger} 

From the {ddagger}Laboratory for Thrombosis and Haemostasis, Department of Haematology and the §Department of Clinical Chemistry, University Medical Center Utrecht and the Institute for Biomembranes, University of Utrecht, 3508 GA Utrecht, The Netherlands

Received for publication, December 12, 2002 , and in revised form, May 9, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
At physiological concentrations, low density lipoprotein (LDL) increases the sensitivity of platelets to aggregation- and secretion-inducing agents without acting as an independent activator of platelet functions. LDL sensitizes platelets by inducing a transient activation of p38MAPK, a Ser/Thr kinase that is activated by the simultaneous phosphorylation of Thr180 and Tyr182 and is an upstream regulator of cytosolic phospholipase A2 (cPLA2). A similar transient phosphorylation of p38MAPK is induced by a peptide mimicking amino acids 3359–3369 in apoB100 called the B-site. Here we report that the transient nature of p38MAPK activation is caused by platelet endothelial cell adhesion molecule 1 (PECAM-1), a receptor with an immunoreceptor tyrosine-based inhibitory motif. PECAM-1 activation by cross-linking induces tyrosine phosphorylation of PECAM-1 and a fall in phosphorylated p38MAPK and cPLA2. Interestingly, LDL and the B-site peptide also induce tyrosine phosphorylation of PECAM-1, and studies with immunoprecipitates indicate the involvement of c-Src. Inhibition of the Ser/Thr phosphatases PP1/PP2A (okadaic acid) makes the transient p38MAPK activation by LDL and the B-site peptide persistent. Inhibition of Tyr-phosphatases (vanadate) increases Tyr-phosphorylated PECAM-1 and blocks the activation of p38MAPK. Together, these findings suggest that, following a first phase in which LDL, through its B-site, phosphorylates and thereby activates p38MAPK, a second phase is initiated in which LDL activates PECAM-1 and induces dephosphorylation of p38MAPK via activation of the Ser/Thr phosphatases PP1/PP2A.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The contact between platelets and low density lipoprotein (LDL)1 particles is known to enhance their responsiveness to aggregation- and secretion-inducing agents (1–5). Part of this sensitization is mediated via activation of p38MAPK and cytosolic phospholipase A2 (cPLA2), which, together with a second stimulus, leads to more liberation of arachidonic acid and the formation of thromboxane A2 (1, 6). A second mechanism involved in sensitization is the activation of p125 focal adhesion kinase (FAK) with its still poorly characterized effects on the formation of focal adhesions and cytoskeletal rearrangements (7, 8). Activation of p38MAPK by LDL is rapid (within 10 s at 1 gram/liter LDL) and occurs at LDL concentrations within the physiological range (0.6–1 gram/liter LDL), suggesting that it may affect platelets in the circulation during diet-induced changes in the lipoprotein profile. p38MAPK is a member of the family of proline-directed serine/threonine kinases that is activated by the simultaneous phosphorylation of Thr180 and Tyr182 (9, 10). LDL-induced p38MAPK activation is insensitive to many inhibitors of signal transduction in platelets including the Ca2+ chelator 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that it is an early step in the activation cascade initiated by LDL (6). An exception is an increase in cAMP, which is a potent inhibitor of p38MAPK activation (6).

The receptor through which LDL initiates p38MAPK and FAK activation in platelets has not yet been characterized but is probably not identical to the classical apoB/E receptor that mediates LDL uptake in fibroblasts and smooth muscle cells, because an antibody directed against the ligand binding domain of the classical LDL-receptor does not change the binding of LDL to platelets (11, 12). Furthermore, LDL binding to platelets is normal in platelets from patients with familial hypercholesterolemia who lack the apoB/E receptor (11).

We have recently identified an activating domain in LDL that induces p38MAPK activation in platelets (13). The major apolipoprotein of LDL is apoB100, a 4563-amino acid-long protein that is wrapped around the lipid particle and possesses a recognition site for the apoB/E receptor in the so called B-site (14). A B-site peptide mimetic consisting of 11 amino-acids with a strong positive charge induced a rapid phosphorylation of p38MAPK, reaching a peak value after 30 s and returning to pre-stimulation values 5–10 min later (13). The extent of p38MAPK activation was in the range found with 1 gram/liter LDL, and the sensitivity to inhibitors of the signaling steps also resembled that of LDL. This is an important observation, because variations between donors, a long isolation procedure, and, specifically, its susceptibility to oxidative modification have led to conflicting interpretations with respect to the platelet-activating properties of LDL (15).

Because platelet sensitization by LDL might be one of the factors that contribute to the development of thrombo-atherosclerotic disease, its down-regulation is equally important as it prevents persistent platelet sensitization. At present, there is little insight into this mechanism. The presence of platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is of specific interest because its cytoplasmic tail contains a so-called immunoreceptor tyrosine-based inhibitory motif (ITIM), which is a characteristic of receptors that mediate inhibitory signals (16–18). The ITIM family comprises several members such as PECAM-1, Fc{gamma}RIIb, signal-regulating protein, CD22, and killer inhibitory receptor; of these, only PECAM-1 is present on human platelets (16, 19). PECAM-1 is a 130-kDa transmembrane glycoprotein of the immunoglobulin superfamily of cell adhesion molecules (20). Its 574-amino acid extracellular domain is organized into six Ig-like homology domains (21). There is a single transmembrane domain and a 118-amino acid cytoplasmic tail. The ITIM motif is characterized by the consensus sequence (L/I/V/S)XYXX(L/V) (16). The expression of PECAM-1 is restricted to hematopoietic and vascular cell types such as platelets, monocytes, neutrophils, and endothelial cells (20). The functions of PECAM-1 are diverse and include roles in angiogenesis, vasculogenesis, integrin regulation, and the transendothelial migration of leukocytes (22). The activation of PECAM-1 is accompanied by phosphorylation of the cytoplasmic part of the molecule. The cytoplasmic tail of PECAM-1 contains twelve serine, four threonine, and five tyrosine residues (20). Which of these residues are phosphorylated depends on the type of agonist. Both resting and thrombin receptor activating peptide (TRAP)-stimulated platelets show predominantly serine phosphorylation of PECAM-1, provided that the cells do not aggregate. When suspensions are stirred and aggregates are formed, PECAM-1 becomes tyrosine-phosphorylated (23). The contact of platelets with collagen leads to strong PECAM-1 phosphorylation on tyrosine residues (24). A second means to activate PECAM-1 in platelets is by cross-linking with the specific antibody PECAM-1.3, which results in tyrosine phosphorylation of the cytoplasmic tail.

Upon receptor activation, the cytoplasmic ITIM-motif recruits and activates a Src-homology 2 (SH2) domain containing protein-tyrosine phosphatases such as SHP-1 and SHP-2 (25). Both SHP-1 and SHP-2 are essential components in PECAM-1-mediated generation of inhibitory signals, and their recruitment and activation depend on the phosphorylation of tyrosine residues on the PECAM-1 cytoplasmic tail (26).

Earlier studies have shown that p38MAPK activation by the B-site in LDL is maximal after 1–2 min and is followed by a gradual decline to pre-activation values. In the present study we investigated whether PECAM-1 takes part in the down-regulation of LDL-induced p38MAPK activation. Furthermore, we determined whether the down-regulation of p38MAPK activation was mediated by the action of phosphatases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—The monoclonal antibody PECAM-1.3 and the polyclonal antibody SEW, both directed against PECAM-1, were kindly provided by Prof. P. J. Newman (The Blood Center of Southeastern Wisconsin, Milwaukee, WI). Anti-mouse F(ab')2 fragments were from Southern Biotechnology Associates, Birmingham, AL. The goat polyclonal anti-PECAM-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody IV.3 was purified from the hybridoma cell culture medium. Anti-phosphotyrosine monoclonal antibody 4G10 and the anti-PP2A A-subunit (clone 4G7) were from Upstate Biotechnology. The antibody directed against Ser(P) was kindly provided by Dr. B.M.T. Burgering (Laboratory for Physiological Chemistry, UMC-Utrecht, The Netherlands). A monoclonal antibody directed against cPLA2 (4–4B-3C) and a polyclonal antibody directed against c-Src were from Santa Cruz Biotechnology. Polyclonal antibodies against p38MAPK and dual phosphorylated p38MAPK (PhosphoPlus p38MAPK) and horseradish peroxidase-labeled anti-rabbit IgG were from New England Biolabs. The antibody against total focal adhesion kinase was from BD Transduction Laboratories. Renaissance chemiluminescence Western blot reagent was from PerkinElmer Life Sciences. Nonfat dry milk was obtained from Nutricia (Zoetermeer, The Netherlands). PP1 was obtained from Alexis Biochemicals (San Diego, CA). Okadaic acid was obtained from Calbiochem, and vanadate was from Sigma. Human {alpha}-thrombin was purchased from Kordia Life Science (Leiden, The Netherlands)

The peptide RLTRKRGLKLA (molecular weight, 1311), designated the B-site peptide, represents the apoB receptor-binding domain (Arg3359 to Ala3369) of apoB100. The peptide was synthesized by standard solid-phase peptide synthesis and purified by C18 reverse-phase chromatography (HPLC, Genosphere Biotechnologies, Paris, France). The purity of the peptide was >99% as determined by high pressure liquid chromatography, and the molecular weight was verified by matrix-assisted laser desorption mass spectrometry by the manufacturer.

Lipoprotein Isolation—Lipoproteins were isolated as described previously (27). In short, fresh, non-frozen plasma from four healthy subjects each containing less than 100 mg of lipoprotein(a) per liter was pooled, and LDL (density range, 1.019–1.063 kg/liter) was isolated by sequential flotation in a Beckman L-70 ultracentrifuge. Centrifugations (20 h; 17,5000 x g; 10 °C) were carried out in the presence of NaN3 and EDTA. The LDL preparations contained only minimal amounts of thiobarbituric acid reactive substances (TBARS) (0.20 ± 0.07 nmol/mg), lipid peroxides (6.7 ± 1.9 nmol/mg), and contaminating plasma proteins (below or within reported values for native LDL). Lp(a) concentrations, determined with the use of a specific antibody (Apotech, Organon Technika), were below 14 mg/liter. Lipoproteins were stored at 4 °C under nitrogen for no longer than 14 days, and before each experiment they were dialyzed overnight against 104 volumes of 150 mmol/liter NaCl. ApoB100 and lipoprotein(a) concentrations were measured using the Behring Nephelometer 100. The concentration of LDL was expressed as grams of apoB100 protein per liter.

Platelet Isolation—Freshly drawn venous blood from healthy volunteers was collected with informed consent into 130 mmol/liter trisodium citrate (1:10, v/v). The donors claimed not to have taken any medication during the 2 weeks prior to blood collection. Platelet-rich plasma was prepared by centrifugation (200 x g; 15 min; 20 °C). Gel-filtered platelets were isolated by gel filtration through Sepharose 2B equilibrated in Ca2+-free Tyrode's solution (137 mmol/liter NaCl, 2.68 mmol/liter KCl, 0.42 mmol/liter NaH2PO4, 1.7 mmol/liter MgCl2, and 11.9 mmol/liter NaHCO3, pH 7.25) containing 0.2% bovine serum albumin and 5 mmol/liter glucose. Gel-filtered platelets were adjusted to a final count of 2 x 1011 platelets/liter and incubated with LDL and other agonists with and without stirring (900 rev·min–1) at 37 °C as indicated under "Results."

Measurement of p38MAPK and cPLA2—Gel-filtered platelets were incubated at 37 °C with LDL, B-site peptide, or thrombin as indicated. After incubation, 100-µl aliquots were mixed (1:10, v/v) with cold lysis buffer (radioimmune precipitation assay buffer containing 10% protease inhibitor mixture and 5 mM NaVO3) and subsequently taken up in Laemmli sample buffer. Samples were heated prior to SDS-PAGE (12%). Proteins were electrophoretically transferred (1 h; 100 volts) to a nitrocellulose membrane using a mini-protean system (Bio-Rad). The blots were blocked in 5% nonfat dry milk and 0.1% Tween 20 in phosphate-buffered saline (1 h; 4 °C) and incubated with the PhosphoPlus p38MAPK (Thr180/Tyr182) or p38MAPK antibody (1:2000 in 1% nonfat dry milk and 0.1% Tween in PBS; 16 h; 4 °C). Both antibodies are raised against residues 171–186 of human p38MAPK. After washing, the membranes were incubated with horseradish peroxidase-labeled anti-rabbit (1:2000; 1 h; 4 °C), and p38MAPK was visualized using the enhanced chemiluminescence reaction. For semi-quantitative determination of the amount of dual phosphorylated or total p38MAPK, the density of the bands was analyzed using ImageQuant software (Amersham Biosciences).

For the measurement of cPLA2 phosphorylation, samples were withdrawn and collected in Laemmli sample buffer. Measurement of cPLA2 was based on the mobility shift on SDS-PAGE that accompanies phosphorylation of the protein (28). The running buffer for electrophoresis of cPLA2 was pH 8.3. cPLA2 was detected using the monoclonal antibody 4-4B-3C. Immune complexes were detected by enhanced chemiluminescence.

PECAM-1 Tyrosine Phosphorylation and Coprecipitation with PP2A—Platelets were incubated with LDL, the B-site peptide, or thrombin as indicated (37 °C). After incubation, 500-µl aliquots were mixed with cold lysis buffer (1:10, v/v) as described above and precipitated with goat polyclonal anti-PECAM-1 (1 µg) and protein G-Sepharose for 3 h (4 °C). After washing with lysis buffer, samples were taken up in Laemmli sample buffer. Samples were heated (5 min; 100 °C) prior to SDS-polyacrylamide gel electrophoresis (7.5%) and the Western blotting procedure. The blots were blocked in 5% BSA and 0.05% Tween 20 in Tris-buffered saline (TBS) (1 h; 4 °C) and incubated with monoclonal 4G10 antibody (1:2000 in 1% BSA and 0.05% Tween 20 in TBS; 16 h; 4 °C) or polyclonal SEW antibody (1:2000 in 1% BSA and 0.05% Tween 20 in TBS; 16 h; 4 °C). Co-immunoprecipitation of PP2 was determined by incubating the blots with the anti-PP2A antibody (1: 1000 in 1% BSA and 0.05% Tween in TBS; 16 h; 4 °C). After washing, the membranes were incubated with peroxidase-linked anti-mouse (1: 5000; 1 h; 4 °C) or anti-rabbit IgG (1:10000; 1 h; 4 °C), and phosphorylation was visualized using the enhanced chemiluminescence reaction.

Statistics—Data are expressed as means ± S.D. with the number of observations (n).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PECAM-1 Inhibits LDL-induced Phosphorylation of p38MAPK—LDL (1 gram/liter) induced a rapid phosphorylation of p38MAPK, reaching a peak value after 30 s and returning to pre-stimulation values after 10–20 min (Fig. 1A, left panel). Thus, following a first phosphorylation step, phosphatases were activated that brought p38MAPK back to the range found in resting platelets. This dephosphorylation of p38MAPK is distinctly faster than that observed with LDL preparations isolated in the presence of the anti-bacterial agent thimerosal, which was commonly used in earlier studies (6, 29). Thus, following an initial phase in which p38MAPK was phosphorylated, both LDL and a peptide mimetic of the B-site of apoB100 (13) induced dephosphorylation of p38MAPK, bringing the enzyme back to the state found in resting platelets.



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  		FIG. 1.
Activation of PECAM results in down-regulation of LDL-induced signaling to p38MAPK. A, left panels, platelets were incubated with LDL (1gram/liter; 37 °C), and dual phosphorylated p38MAPK was measured by SDS-PAGE and Western blotting using a phospho-specific anti-p38MAPK polyclonal antibody (p38MAPK-P) and an anti-total p38MAPK antibody as a control for equal lane loading. Right panels, platelets were incubated with anti-PECAM-1.3 antibody (10 µg/ml; 10 min) and PECAM-1.3 antibody and, in addition, Fab fragments (30 µg/ml; 90 s; 37 °C) to cross-link the antibody prior to stimulation with LDL (1 gram/liter; 5 min; 37 °C). PECAM-1 was immunoprecipitated (IP) from platelet lysates and applied to gel. Tyr phosphorylation (P-Y) of PECAM-1 was analyzed with antibody 4G10. The bottom right panel shows PECAM-1 detected with a polyclonal antibody against this receptor as a control for equal lane loading. B, platelets were incubated with LDL with mild (PECAM-1.3, 10 µg/ml; 10 min; 37 °C) and firm (PECAM-1.3, 10 µg/ml; 10 min; 37 °C and anti-mouse IgG F(ab')2, 30 µg/ml; 1,5 min; 37 °C) cross-linking (XL) of PECAM-1. Subsequently, p38MAPK- and cPLA2-phosphorylation were detected by Western blotting. The bottom panel shows samples analyzed with an antibody directed against the total amount of p38MAPK as a control for equal lane loading. The blots were semi-quantified, and the data were expressed as percentage of the p38MAPK or cPLA2 phosphorylation without PECAM-1 antibodies. Data are expressed as means ± S.D., n = 3. C, control; WB, Western blot.

 

To investigate whether PECAM-1 played a role in the dephosphorylation of p38MAPK, the receptor was activated with the antibody PECAM-1.3, which is specific for the ectodomain of PECAM-1. Incubation of platelets with this antibody (1 µg/ml) for 10 min resulted in tyrosine phosphorylation of PECAM-1, illustrating that the receptor was activated (Fig. 1A, right panel). No further activation of PECAM-1 was found by cross-linking the PECAM-1.3 antibody with anti-mouse Fab fragments.

When platelets were first treated with the PECAM-1.3 antibody for 10 min and thereafter stimulated with LDL (1 gram/liter) for 1 min, the phosphorylation of p38MAPK was reduced to 70% compared with platelets that were not treated with the PECAM-1.3 antibody (Fig. 1B). A further cross-linking of PECAM-1 resulted in a reduction in phosphorylation of p38MAPK to 40%. A similar inhibition by PECAM was observed at the level of cPLA2, which is a downstream target of p38MAPK in LDL-induced signaling (6).

To assess a possible involvement of the Fc{gamma}RIIa receptor-mediated pathway in the anti-PECAM-1.3-induced activation of PECAM-1 and the concomitant dephosphorylation of p38MAPK, studies were repeated in the presence of antibody IV.3, an inhibitor of Fc{gamma}RIIa (data not shown). The inhibition by PECAM-1 activation was unchanged. This observation, together with literature data that show that p38MAPK is activated by Fc{gamma}RIIa activation rather than inhibited by it illustrate that the inhibition by PECAM of LDL-induced p38MAPK phosphorylation is independent of Fc{gamma}RIIa.

LDL Activates PECAM-1—The observation that PECAM inhibited the LDL-induced phosphorylation of p38MAPK raised the question of whether LDL itself was capable of activating PECAM-1, thereby down-regulating the initial activation of p38MAPK-activating pathways. Platelets were incubated with LDL, and the phosphorylation of PECAM-1 was visualized on Western blots after immunoprecipitation of the receptor (Fig. 2A). As a control, platelets were stimulated with thrombin (0.5 units/ml; 2 min; 37 °C) with and without stirring. Stirring resulted in an enhanced tyrosine phosphorylation of PECAM-1, whereas serine phosphorylation was equal under both conditions, which was in agreement with earlier observations (23). Western blots with an appropriate antibody revealed that LDL induced tyrosine phosphorylation of PECAM-1 but was incapable of inducing phosphorylation of serine residues. The LDL (1 gram/liter)-induced tyrosine phosphorylation of PECAM-1 was transient, showing a maximum after 1 min of stimulation and down-regulation after prolonged incubation (Fig. 2B). Also, the B-site peptide induced tyrosine phosphorylation of PECAM-1, which is in line with the concept that the B-site mediates the activation of PECAM-1 by LDL. After immunoprecipitation of PECAM-1, an additional band at ~125 kDa was co-precipitated. Reprobing of the blots with the polyclonal anti-PECAM-1 (SEW) and an antibody against focal adhesion kinase revealed that the upper band represents PECAM-1 and the lower band focal adhesion kinase (data not shown).



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  		FIG. 2.
LDL induces tyrosine phosphorylation of PECAM-1. A, platelets were incubated with LDL (1 gram/liter; 37 °C) or thrombin without stirring (Thrombin –, 0.5 units/ml; 2 min; 37 °C) or thrombin with stirring (Thrombin +, 0.5 units/ml; 900 rpm; 2 min; 37 °C). PECAM-1 was immunoprecipitated (IP) from platelet lysates and applied to gel. With antibody 4G10 the tyrosine phosphorylation (P-Y; top left panel) and the anti-Ser antibody serine phosphorylation (P-S; top right panel) of PECAM-1 were detected. The bottom panels show similar samples in which PECAM-1 was detected with a polyclonal antibody against this receptor as a control for equal lane loading. B, left panel, platelets were incubated with LDL (1 gram/liter; 37 °C), the B-site peptide (100 µmol/liter, 37 °C) for the indicated time-periods or with thrombin under stirred conditions (Thrombin +, 900 rpm; 2 min; 37 °C), and the tyrosine phosphorylation (P-Y) of PECAM-1 was analyzed. Right panel, time courses of the phosphorylation of p38MAPK and PECAM-1 based on the conditions indicated for the left panel here and the left panel in Fig. 1A. Platelets were incubated with LDL (1 gram/liter; 37 °C; • or {triangledown}) or the B-site peptide (100 µmol/liter; 37 °C; {circ}) and the phosphorylation of p38MAPK (• and {circ}) and PECAM-1 ({triangledown} – {triangledown}) were measured. The blots were semi-quantified, and the data were expressed as percentage of the phosphorylation at 1 min (100%, open symbols). Data are expressed as means ± S.D., n >= 4. C, left panel, platelets were incubated with the Src family tyrosine kinases inhibitor PP1 (10 µmol/liter; 15 min) prior to incubation with LDL (1 gram/liter; 5 min; 37 °C), and PECAM-1 tyrosine phosphorylation (P-Y) was detected. Right panel, complex formation between PECAM-1 and Src family tyrosine kinases was measured by incubating platelets with LDL (1 gram/l, 37 °C) for the indicated time periods or with thrombin (1 unit/ml; 2 min; 37 °C; 900 rpm) followed by immunoprecipitation with polyclonal antibody against PECAM-1 and Western blotting with an antibody against c-Src. The 60-kDa band was identified as c-Src. As a control for equal lane loading, blots were reprobed with a monoclonal antibody against total PECAM (bottom right panel). C, control; WB, Western blot.

 

The inhibitor of Src kinases, PP1 (10 µM), abrogated tyrosine phosphorylation of PECAM-1, suggesting a role for Src kinases (Fig. 2C). Indeed, upon LDL stimulation, a 60-kDa band co-precipitated with PECAM-1 upon LDL stimulation. After reprobing with a specific antibody, this band could be identified as c-Src, indicating that a complex was formed between c-Src and PECAM-1 upon stimulation by LDL.

Down-regulation of LDL Signaling by PECAM-1 Depends on Serine/Threonine Phosphatases—PECAM-1 is known to initiate inhibitory signaling pathways by recruitment of the tyrosine phosphatases SHP-1 and SHP-2. As p38MAPK is activated upon phosphorylation of both threonine and tyrosine residues, the involvement of serine/threonine phosphatases as well as tyrosine phosphatases was investigated.

To investigate the contribution of serine/threonine phosphatases, platelets were incubated for 0.5 min with 1 µM okadaic acid, a concentration known to inhibit the serine/threonine phosphatases PP1 and PP2A (30, 31). This treatment resulted in a strong potentiation of p38MAPK activation by LDL (1 gram/liter) and the B-site peptide (100 µM) and changed the transient activation into a more sustained activation compared with LDL-stimulated p38MAPK in the absence of the inhibitor (Fig. 3A).



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  		FIG. 3.
Role of Ser/Thr and Tyr-phosphatase in LDL-signaling to p38MAPK and PECAM-1. Platelets were incubated with LDL (1 gram/liter; 37 °C) or the B-site peptide (100 µmol/liter; 37 °C) for the indicated time periods with and without okadaic acid (1 µM; 30 s) (A) or sodium vanadate (NaVO3; 100 µM; 30 min) (B), and dual phosphorylated p38MAPK (p38 MAPK -P) was identified by Western blotting. C, platelets were incubated with and without sodium vanadate (100 µM; 30 min) prior to incubation with LDL (1 gram/liter; 37 °C) or B-site peptide (100 µmol/liter; 37 °C), and the tyrosine phosphorylation (P-Y) of PECAM-1 was analyzed. IP, immunoprecipitation; WB, Western blot.

 

We next investigated whether inhibition of tyrosine phosphatases with vanadate interfered with the phosphorylation state of p38MAPK. Platelets were preincubated for 30 min with 100 µM sodium vanadate. This treatment completely abolished the phosphorylation of p38MAPK by LDL and the B-site peptide (Fig. 3B). Furthermore, the tyrosine phosphorylation of PECAM-1 induced by LDL and the B-site peptide was preserved by vanadate treatment and even increased to levels above the range found in the absence of the inhibitor (Fig. 3C). Thus, inhibition of tyrosine phosphatases augmented the Tyr phosphorylation of PECAM-1 induced by LDL and the B-site peptide, thereby potentiating the dephosphorylation of p38MAPK to such an extent that any rise in phosphorylated p38MAPK was prevented.

A combination of the two inhibitors induced a dual response. During the first 5 min there was complete inhibition of p38MAPK phosphorylation as observed with vanadate alone. Thereafter, a slight recovery became detectable, but the recovery was later and considerably lower than that seen with okadaic acid alone (Fig. 4A). Thus, following a period in which inhibition by vanadate was predominant, a second phase became apparent in which dephosphorylation of p38MAPK by PECAM-1 was opposed by okadaic acid. Concurrent analysis of PECAM-1 phosphorylation showed that, in the period of p38MAPK inhibition, PECAM-1 was maximally phosphorylated. The later recovery of p38MAPK phosphorylation was accompanied by a fall of phosphorylated PECAM-1, which was slightly faster than in the presence of vanadate alone (Fig. 4B).



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  		FIG. 4.
Combined inhibition of Ser/Thr and Tyr phosphatases during LDL signaling to p38MAPKand PECAM-1. Platelets were incubated (incub) with LDL (1 gram/liter, 37 °C) for the the indicated time periods in the presence of okadaic acid (O) (1 µM; 30 s) and sodium vanadate (V) (100 µM; 30 min), and dual phosphorylated p38MAPK (p38MAPK-P) (A) and tyrosine phosphorylation of PECAM-1 (PECAM-P) (B) were analyzed. Data are means ± S.D., n = 4. Further details as described in the legends to Figs. 1 and 2.

 

Because the effect of okadaic acid on the dephosphorylation of p38MAPK suggested the involvement of a phosphatase of the PP1/PP2a type, a possible co-association between PECAM-1 and PP2A was investigated. Stimulation by LDL induced a transient co-association between Tyr-phosphorylated PECAM-1 and PP2A, supporting the conclusion that PECAM-1 regulates dephosphorylation of p38MAPK through PP2A (Fig. 5).



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  		FIG. 5.
Co-association between PECAM-1 and PP2A. Platelets were incubated without and with LDL (1 gram/liter; 37 °C), and PECAM-1 was immunoprecipitated (IP) from platelet lysates and applied to gel. Following PAGE, blots (WB) were incubated with an antibody against PP2A, an anti-total PECAM antibody as a control for equal lane loading, and 4G10, which detects tyrosine phosphorylated proteins.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
ApoB100 is the main protein constituent of LDL and consists of 4563 amino acids wrapped around the lipid particle. The B-site is the domain in apoB100 that binds to the apoB/E receptors on cells that remove plasma cholesterol from the circulation. A peptide that mimics this region between Arg3359 and Ala3369 of apoB100 induced a transient activation of platelet p38MAPK, showing a maximal phosphorylation after 1 min of stimulation (13). Also, LDL induced a rapid and transient phosphorylation of p38MAPK with approximately similar kinetics as observed with the B-site peptide. It is possible that this down-regulation at prolonged incubation times might reflect a protection mechanism that prevents extensive platelet activation in the circulation by LDL.

Here we report that the down-regulation of phosphorylated p38MAPK is mediated by PECAM-1. PECAM-1 activation by a specific receptor cross-linking antibody led to Tyr-phosphorylation of PECAM-1 and p38MAPK dephosphorylation. A second cross-linking Fab fragment in addition to the PECAM-1 antibody did not induce much more PECAM-1 tyrosine phosphorylation, although literature data suggest that Fab fragments are required for optimal PECAM-1 activation (32). Activation of PECAM-1 with the PECAM-1.3 antibody resulted in a decrease in LDL-induced phosphorylation of p38MAPK, and a further activation of PECAM with additional Fab fragments led to a further reduction in the phosphorylation of the enzyme. These observations make PECAM-1 an important inhibitor of LDL-induced p38MAPK phosphorylation. P38MAPK is activated by dual phosphorylation of the p38MAPK of Thr180 and Tyr182, and the inhibition by PECAM-1 is therefore likely to impair further signaling to downstream effectors. Indeed, activation of PECAM-1 also inhibits the phosphorylation of cPLA2, which is a key step in the mobilization of arachidonic acid and the further formation of thromboxane A2. This makes PECAM-1 a key factor in the control of LDL-induced platelet sensitization.

There is little insight into the mechanisms by which PECAM-1 is activated under physiological conditions. The activation of PECAM-1 could be the result of agonists present in the circulation that induce a protection mechanism against cardiovascular complications. Platelet activation through the collagen receptor glycoprotein VI results in PECAM-1 tyrosine phosphorylation, which may function to negatively limit growth of platelet thrombi on collagen surfaces (18, 32–34). Also, platelet activation through thrombin receptors leads to PECAM-1 activation. Activated non-aggregated platelets exhibit serine phosphorylation of PECAM-1 in contrast to aggregated platelets, which show tyrosine phosphorylated PECAM-1 (23). The present experiments suggest a role for LDL as an agonist for PECAM-1-mediated platelet inhibition. Incubation of platelets with LDL or the B-site peptide induced tyrosine phosphorylation of PECAM-1 with an optimum after 2–5 min. Neither LDL nor the B-site peptide induced phosphorylation of serines on PECAM-1, which is in contrast to PECAM-1 stimulation by thrombin. Our data also reveal that in non-aggregated platelets PECAM-1 can be tyrosine phosphorylated, because LDL is not an aggregation-inducing agent.

Upon incubation of platelets with LDL, a band with a slightly lower molecular weight compared with PECAM-1 co-precipitates with this receptor. This band interacted strongly with an antibody against focal adhesion kinase. LDL is a potent activator of focal adhesion kinase via the Src kinase Fgr and triggers the phosphorylation of the six tyrosine residues with kinetics that differ greatly form those induced by thrombin (35).

Okadaic acid is an inhibitor of the serine/threonine phosphatases PP1 and PP2A (36). When platelets were preincubated with this inhibitor, the transient phosphorylation of p38MAPK by LDL and the B-site peptide changed into a persistent activation and reached levels far above the maximal phosphorylation observed in untreated platelets. A recent study on p38MAPK phosphorylation by collagen revealed that PP2A mediates the dephosphorylation, and a similar role might be present in platelets stimulated by LDL (37). p38MAPK is activated by dual phosphorylation on tyrosine and threonine residues by dual specific MAPK kinases 3 and 6 (MKK3/6) (38, 39). MKK6 is activated by phosphorylation on Ser151 and Thr155 by an upstream enzyme termed MKK kinase. Thus, the dephosphorylation of p38MAPK by PP1/PP2A might be the result of a direct effect of these phosphatases on p38MAPK, but an indirect effect caused at the level of upstream kinases is equally feasible.

Vanadate is an inhibitor of tyrosine phosphatases. When platelets were preincubated with this inhibitor, the transient tyrosine phosphorylation of PECAM-1 was increased and more sustained, enabling the receptor to transmit a stronger inhibitory signal into the cell. Apparently, vanadate did not increase the phosphorylated state of p38MAPK, although the enzyme is phosphorylated on threonine and tyrosine residues. Instead, vanadate treatment completely abolished the increase of phosphorylated p38MAPK by LDL and the B-site peptide. This illustrates the potent inhibitory action of PECAM-1, which, through its strong activation of PP1/PP2A, abolished the accumulation of phosphorylated p38MAPK. Also, when both inhibitors are present the inhibition by vanadate is predominant, and only after prolonged incubation with LDL does the effect of okadaic acid become detectable. Together, these results support the concept that PECAM-1 is a negative regulator of LDL-induced signaling in platelets by activating a phosphatase sensitive to okadaic acid. Indeed, immunoprecipitates reveal co-association with PP2A. Together with the coprecipitation of focal adhesion kinase (Ref. 35 and this study), the Src-kinases Fgr (35), and c-Src (this study), LDL-induced tyrosine phosphorylation of PECAM appears central in the formation of a complex containing multiple signaling elements.

Signal transduction by PECAM-1 depends on the ITIM motif, which becomes tyrosine phosphorylated. Because PECAM-1 does not appear to be autophosphorylated, a kinase is thought to phosphorylate the cytoplasmic tyrosine residue (40, 41). A candidate responsible for the tyrosine phosphorylation is a member of the Src family tyrosine kinase (34, 41). Immunoprecipitation studies after the incubation of platelets with LDL showed co-precipitation of c-Src with PECAM-1. P38MAPK activation was not affected by the okadaic acid treatment until the platelets had been stimulated with LDL for 1 min or more. This is in accordance with the time-dependent activation of PECAM-1 by LDL and the recruitment of c-Src. This suggests that c-Src recruitment to the cytoplasmic tail of PECAM-1 induces ITIM-tyrosine phosphorylation and the subsequent recruitment of PP2A.

In conclusion, the results reported in this study are best explained by assuming a model in which LDL affects platelet signaling mechanisms in two phases. During the initial phase, LDL binds via its B-site in apoB100 to the putative LDL receptor on platelets, thereby inducing the dual phosphorylation of p38MAPK and activating the enzyme. This step leads to activation of cPLA2 and the formation of thromboxane A2. This mechanism explains why aspirin or related drugs abolish at least in part the sensitization of platelets by LDL. This activation phase is followed by a second phase in which LDL, either directly or bound in a complex with the LDL receptor, activates PECAM-1. This results in recruitment of c-Src and the phosphatases PP1/PP2A, thereby down-regulating the phosphorylation of p38MAPK.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by the Netherlands Thrombosis Foundation and to whom correspondence should be addressed: Dept. of Haematology, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Tel.: 31-30-2506512; Fax: 31-30-2511893; E-mail: j.w.n.akkerman{at}azu.nl.

1 The abbreviations used are: LDL, low density lipoprotein; PECAM, platelet endothelial cell adhesion molecule; ITIM, immunoreceptor tyrosine-based inhibitory motif; cPLA2, cytosolic phospholipase A2; PP1, protein Ser/Thr phosphatase 1; PP2A, protein Ser/Thr phosphatase 2A; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase; BSA, bovine serum albumin; TBS, Tris-buffered saline. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Surya, I. I., Gorter, G., Mommersteeg, M., and Akkerman, J. W. N. (1992) Biochim. Biophys. Acta 1165, 19–26[Medline] [Order article via Infotrieve]
  2. Surya, I. I., and Akkerman, J. W. N. (1993) Am. Heart J. 125, 272–275[CrossRef][Medline] [Order article via Infotrieve]
  3. van Willigen, G., Gorter, G., and Akkerman, J. W. N. (1994) Arterioscler. Thromb. 14, 41–46[Abstract/Free Full Text]
  4. Andrews, H. E., Aitken, J. W., Hassall, D. G., Skinner, V. O., and Bruckdorfer, K. R. (1987) Biochem. J. 242, 559–564[Medline] [Order article via Infotrieve]
  5. Curtiss, L. K., and Plow, E. F. (1984) Blood 64, 365–374[Abstract/Free Full Text]
  6. Hackeng, C. M., Relou, I. A., Pladet, M. W., Gorter, G., van Rijn, H. J., and Akkerman, J. W. (1999) Thromb. Haemost. 82, 1749–1756[Medline] [Order article via Infotrieve]
  7. Hackeng, C. M., Pladet, M. W., Akkerman, J. W., and van Rijn, H. J. (1999) J. Biol. Chem. 274, 384–388[Abstract/Free Full Text]
  8. Schaller, M. D. (2001) Biochim. Biophys. Acta 1540, 1–21[Medline] [Order article via Infotrieve]
  9. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) J. Biol. Chem. 270, 7420–7426[Abstract/Free Full Text]
  10. Ono, K., and Han, J. (2000) Cell Signal. 12, 1–13[CrossRef][Medline] [Order article via Infotrieve]
  11. Pedreno, J., de Castellarnau, C., Cullare, C., Sanchez, J., Gomez Gerique, J., Ordonez Llanos, J., and Gonzalez Sastre, F. (1992) Arterioscler. Thromb. 12, 1353–1362[Abstract/Free Full Text]
  12. Brown, M. S., and Goldstein, J. L. (1986) Science 232, 34–47[Free Full Text]
  13. Relou, I. A. M., Gorter, G., van Rijn, H. J., and Akkerman, J. W. N. (2002) Thromb. Haemost. 87, 880–887[Medline] [Order article via Infotrieve]
  14. Boren, J., Lee, I., Zhu, W., Arnold, K., Taylor, S., and Innerarity, T. L. (1998) J. Clin. Invest. 101, 1084–1093[Medline] [Order article via Infotrieve]
  15. Malle, E., and Sattler, W. (1994) Platelets 5, 70–83
  16. Newman, P. J. (1999) J. Clin. Invest. 103, 5–9[Medline] [Order article via Infotrieve]
  17. Newton-Nash, D. K., and Newman, P. J. (1999) J. Immunol. 163, 682–688[Abstract/Free Full Text]
  18. Patil, S., Newman, D. K., and Newman, P. J. (2001) Blood 97, 1727–1732[Abstract/Free Full Text]
  19. DeLisser, H. M., Newman, P. J., and Albelda, S. M. (1993) Curr. Top. Microbiol. Immunol. 184, 37–45[Medline] [Order article via Infotrieve]
  20. Newman, P. J., Berndt, M. C., Gorski, J., White, G. C., Lyman, S., Paddock, C., and Muller, W. A. (1990) Science 247, 1219–1222[Abstract/Free Full Text]
  21. Guselnikov, S. V., Ershova, S. A., Mechetina, L. V., Najakshin, A. M., Volkova, O. Y., Alabyev, B. Y., and Taranin, A. V. (2002) Immunogenetics 54, 87–95[CrossRef][Medline] [Order article via Infotrieve]
  22. DeLisser, H. M., Newman, P. J., and Albelda, S. M. (1994) Immunol. Today 15, 490–495[CrossRef][Medline] [Order article via Infotrieve]
  23. Modderman, P. W., von dem Borne, A. E., and Sonnenberg, A. (1994) Biochem. J. 299, 613–621[Medline] [Order article via Infotrieve]
  24. Jackson, D. E., Ward, C. M., Wang, R., and Newman, P. J. (1997) J. Biol. Chem. 272, 6986–6993[Abstract/Free Full Text]
  25. Hua, C. T., Gamble, J. R., Vadas, M. A., and Jackson, D. E. (1998) J. Biol. Chem. 273, 28332–28340[Abstract/Free Full Text]
  26. Henshall, T. L., Jones, K. L., Wilkinson, R., and Jackson, D. E. (2001) J. Immunol. 166, 3098–3106[Abstract/Free Full Text]
  27. Hackeng, C. M., Huigsloot, M., Pladet, M. W., Nieuwenhuis, H. K., van Rijn, H. J., and Akkerman, J. W. N. (1999) Arterioscler. Thromb. Vasc. Biol. 19, 239–247[Abstract/Free Full Text]
  28. Hackeng, C. M., Franke, B., Relou, I. A., Gorter, G., Bos, J. L., van Rijn, H. J., and Akkerman, J. W. (2000) Biochem. J. 349, 231–238[CrossRef][Medline] [Order article via Infotrieve]
  29. Weidtmann, A., Scheithe, R., Hrboticky, N., Pietsch, A., Lorenz, R., and Siess, W. (1995) Arterioscler. Thromb. Vasc. Biol. 15, 1131–1138[Abstract/Free Full Text]
  30. Cohen, P. (1989) Annu. Rev. Biochem. 58, 453–508[CrossRef][Medline] [Order article via Infotrieve]
  31. Haystead, T. A., Sim, A. T., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989) Nature 337, 78–81[CrossRef][Medline] [Order article via Infotrieve]
  32. Cicmil, M., Thomas, J. M., Leduc, M., Bon, C., and Gibbins, J. M. (2002) Blood 99, 137–144[Abstract/Free Full Text]
  33. Jones, K. L., Hughan, S. C., Dopheide, S. M., Farndale, R. W., Jackson, S. P., and Jackson, D. E. (2001) Blood 98, 1456–1463[Abstract/Free Full Text]
  34. Cicmil, M., Thomas, J. M., Sage, T., Barry, F. A., Leduc, M., Bon, C., and Gibbins, J. M. (2000) J. Biol. Chem. 275, 27339–27347[Abstract/Free Full Text]
  35. Relou, I. A. M., Bax, L. A. B., van Rijn, H. J. M., and Akkerman, J. W. N. (2003) Biochem. J. 369, 407–416[CrossRef][Medline] [Order article via Infotrieve]
  36. Fernandez, J. J., Candenas, M. L., Souto, M. L., Trujillo, M. M., and Norte, M. (2002) Curr. Med. Chem. 9, 229–262[Medline] [Order article via Infotrieve]
  37. Sundaresan, P., and Farndale, R. (2002) FEBS Lett. 528, 139–144[CrossRef][Medline] [Order article via Infotrieve]
  38. Cuenda, A., Alonso, G., Morrice, N., Jones, M., Meier, R., Cohen, P., and Nebreda, A. R. (1996) EMBO J. 15, 4156–4164[Medline] [Order article via Infotrieve]
  39. Han, J., Lee, J. D., Jiang, Y., Li, Z., Feng, L., and Ulevitch, R. J. (1996) J. Biol. Chem. 271, 2886–2891[Abstract/Free Full Text]
  40. Cao, M. Y., Huber, M., Beauchemin, N., Famiglietti, J., Albelda, S. M., and Veillette, A. (1998) J. Biol. Chem. 273, 15765–15772[Abstract/Free Full Text]
  41. Newman, D. K., Hamilton, C., and Newman, P. J. (2001) Blood 97, 2351–2357[Abstract/Free Full Text]

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