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J. Biol. Chem., Vol. 278, Issue 35, 32638-32644, August 29, 2003
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¶
From the
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.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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
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.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 FcRIIa 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 FcRIIa (data not shown).
The inhibition by PECAM-1 activation was unchanged. This observation, together
with literature data that show that p38MAPK is activated by
FcRIIa activation rather than inhibited by it illustrate that the
inhibition by PECAM of LDL-induced p38MAPK phosphorylation is
independent of FcRIIa.
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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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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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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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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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.
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FOOTNOTES |
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¶ 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.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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)
or the B-site peptide (100 µmol/liter; 37 °C; 
) and the
phosphorylation of p38MAPK (• and 
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.
