|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the School of Animal and Microbial Sciences,
University of Reading, United Kingdom; and Institute Pasteur, Paris,
France.
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a
130-kd transmembrane glycoprotein and a member of the growing family of
receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIMs).
PECAM-1 is expressed on platelets, certain T cells, monocytes,
neutrophils, and vascular endothelial cells and is involved in a range
of cellular processes, though the role of PECAM-1 in platelets is
unclear. Cross-linking of PECAM-1 results in phosphorylation of the
ITIM allowing the recruitment of signaling proteins that bind by way of
Src-homology domain 2 interactions. Proteins that have been implicated
in the negative regulation of cellular activation by ITIM-bearing
receptors include the tyrosine phosphatases SHP-1 and SHP-2. Tyrosine
phosphorylation of immunoreceptor tyrosine-based activatory motif
(ITAM)-bearing receptors such as the collagen receptor GPVI-Fc
receptor Platelet endothelial cell adhesion molecule-1
(PECAM-1, CD31) is a 130-kd membrane-spanning glycoprotein whose
expression is restricted to several hematopoietic cell types including
platelets, monocytes, neutrophils, certain T lymphocytes, and vascular
endothelial cells.1-4 The functions of PECAM-1 are diverse
and include angiogenesis,5 vasculogenesis,6
integrin regulation,7,8 transendothelial migration of
leukocytes,9-11 and T- and B-cell antigen receptor function,12,13 though the role of this molecule in
platelets is unclear. When PECAM-1 was cloned, it was assigned to the
family of cell adhesion molecules on the basis of structural
similarities.4 PECAM-1 is involved in adhesion, though
much attention has been directed recently to studying its ability to
participate in signal transduction. The cytoplasmic tail of PECAM-1
contains a conserved motif called an immunoreceptor tyrosine-based
inhibitory motif (ITIM), which underlies its signaling properties and
is shared by a growing family of inhibitory receptors. These include
immunoglobulin G (IgG) receptor Fc The ligand-binding properties of PECAM-1 are complex. It has the
capacity for homophilic interactions3,16 and heterophilic interactions with a number of molecules that include integrin Immunoreceptor tyrosine-based activatory motif (ITAM)-bearing
receptors have a critical place in the regulation of platelet function.28 Indeed, the collagen receptor GPVI-FcR
In this study we examined the effect of PECAM-1 signaling on the
activation of human platelets. We demonstrate that the activation of
PECAM-1 signaling by antibody-mediated cross-linking results in the
inhibition of collagen-mediated activation; similar results were
obtained using a GPVI-selective agonist convulxin (Cvx). Furthermore,
we present evidence to indicate that the inhibitory functions of
PECAM-1 may not be restricted to the inhibition of ITAM-containing
receptor signaling pathways because thrombin-stimulated activation was
also inhibited. Inhibition of platelet activation is accompanied by a
concomitant inhibition of platelet protein tyrosine phosphorylation and
decreased levels of calcium mobilization from intracellular stores. Our
data support the notion that PECAM-1 signaling may be involved in the
regulation of platelet function in vivo.
Materials
Preparation and stimulation of platelets
Immunoprecipitation studies Platelet stimulation was terminated by the addition of an equal volume of ice-cold lysis buffer (2% (vol/vol) Nonidet P40, 20 mM Tris, 300 mM NaCl, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 µg/mL pepstatin A, pH 7.3). Detergent-insoluble debris was removed, and the lysates were precleared by mixing with protein A-Sepharose for 1 hour at 4°C (20 µL of a 50% (wt/vol) suspension of protein A-Sepharose in Tris-buffered saline-Tween (TBS-T; 20 mM Tris, 137 mM NaCl, 0.1% (vol/vol) Tween 20, pH 7.6)). Protein A-Sepharose was removed from the lysates before the addition of anti-PECAM-1 antibody (HC1/6, 1 µg). After rotation at 4°C for 1 hour, 0.5 µL secondary antiserum was added (rabbit anti-mouse IgG) and mixed for a further 30 minutes. Protein A-Sepharose suspension (25 µL) was added to each sample, and mixing continued for 1 hour before the Sepharose pellet was washed in lysis buffer; this was followed by a wash with TBS-T and by the addition of Laemmli sample-treatment buffer. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions using 10% gels and were transferred to polyvinylidene difluoride membranes by semidry Western blotting.Immunoblotting Membranes were blocked by incubation in 10% (wt/vol) bovine serum albumin dissolved in TBS-T. Primary and secondary antibodies were diluted in TBS-T containing 2% (wt/vol) bovine serum albumin and were incubated with membranes for 1 hour at room temperature. Blots were washed for 2 hours in TBS-T after incubation with antibodies for 1 hour at room temperature and were developed using an enhanced chemiluminescence detection system. Primary antibodies were used at a concentration of 1 µg/mL (antiphosphotyrosine, 4G10; anti-PECAM-1, C-20), and horseradish peroxidase-conjugated secondary antibodies were diluted at 1:10 000.5-Hydroxytryptamine secretion assay Platelets were loaded with [3H]5-hydroxytryptamine (5-HT) by incubation with 0.5 µCi/mL (18.5 kBq) platelet-rich plasma for 1 hour at 37°C. Platelets were prepared from the platelet-rich plasma as described above. Stimulation of platelets was terminated by the addition of an equal volume of 6% glutaraldehyde and microcentrifugation, and the level of [3H]5-HT release into the supernatant was determined by scintillation spectrometry. [3H]5-HT release was expressed as a percentage of the total tissue content after subtraction of release under basal conditions.Measurement of [Ca++]i by spectrofluorometry Washed human platelets (prepared as above) were incubated at 2 × 109 cells/mL in calcium-free Tyrode HEPES buffer with 3 µM Fura-2 AM for 45 minutes. Platelets were washed once and resuspended at 2 × 108 cells/mL in modified Tyrode HEPES buffer. Stimulation of platelets (450 µL) in the presence of 2 mM EGTA with Cvx and thrombin (delivered in 50 µL) was performed with constant stirring at 37°C in a luminescence spectrophotometer (LS-50B; Perkin-Elmer) with excitation wavelengths of 340 nm and 380 nm. Fluorescence emission was measured at a wavelength of 510 nm. Where required, PECAM-1 was cross-linked before stimulation with agonist as described above. The ratio of emission values (excitation at 340/380 nm) was calculated and converted to calcium concentration using FLWinLab software (Perkin-Elmer) using the equation [Ca++]i = Kd × (R Rmin)/(Rmax R) × SFB,
where R is emission ratio value (340/380 nm). Rmax, the
maximum 340/380 ratio, was determined by lysing platelets with 25 µM
digitonin in the presence of 1 mM CaCl2. The
Rmin 340:380 nm ratio was obtained by adding 2 mM EGTA.
Kd was the dissociation constant of the
Fura-2/Ca++ complex (224 nM), and SFB was the fluorescence
ratio at 340/380nm Rmin and Rmax).
Statistical analysis Determination of statistical significance was performed using the Student paired t test. Results are expressed as mean ± SEM.
Cross-linking PECAM-1 inhibits collagen-stimulated platelet aggregation An antagonistic relationship has been reported between ITIM- and ITAM-containing receptors when they are expressed in the same cell.12,13,34 Because the platelet collagen receptor GPVI signals through an ITAM on the FcR -chain with which it is associated, we investigated the effect of PECAM-1 signaling on platelet
activation with collagen. PECAM-1 was activated by incubation with
antibodies specific for the ectodomain of PECAM-1 (AB468 (Figure
1) or PECAM1.3, not shown) and was
cross-linked with a secondary antibody, (Fab')2 fragment.
Consistent with findings from other reports,19 this
resulted in increased tyrosine phosphorylation of the protein and did
not result in the stimulation of platelet aggregation (Figure 1A).
Tyrosine phosphorylation of PECAM-1 was maintained on cross-linking in
the presence of EGTA (1 mM), RGDS peptide (0.5 mM), and -chain
peptide of fibrinogen (100 µM), added separately or together (not
shown). This, together with fact that these experiments were performed
on washed platelets, indicates that the tyrosine phosphorylation of
PECAM-1 on cross-linking is not dependent on integrin
 IIb 3 engagement. The effect of PECAM-1
cross-linking for 90 seconds before stimulation with collagen was found
to have a marked inhibitory effect on collagen-stimulated platelet
aggregation. At lower concentrations of collagen (eg, 10 µg/mL),
cross-linking of PECAM-1 before agonist addition completely abolished
aggregation (data not shown). Figure 1Bii shows the marked
inhibitory effect of PECAM-1 cross-linking on a high concentration of
collagen (100 µg/mL). The use of an isotype-matched IgG control and
cross-linker F(ab')2 had no effect on PECAM-1 tyrosine
phosphorylation (not shown) and collagen-stimulated platelet
aggregation (Figure 1Bi). Results are representative of 3 separate
experiments. Similar results were obtained using the alternative
anti-PECAM-1 antibody, PECAM 1.3. In some experiments, the low-affinity
receptor for IgG FcRIIA was blocked before PECAM-1 cross-linking and
agonist stimulation using a saturating concentration of
F(ab')2 fragments of mAb IV.3. The inhibitory effect of
PECAM-1 cross-linking was unaltered under these conditions, indicating
that the inhibitory effect of PECAM-1 using antibodies was not caused
by the activation of FcRIIA. This result was not surprising given
that the activation of FcRIIA by cross-linking with antibodies
results in platelet activation.36
PECAM-1 cross-linking inhibits GPVI- and thrombin receptor-mediated platelet aggregation Given the marked effect of PECAM-1 signaling on collagen-mediated platelet aggregation, we investigated whether this was restricted to GPVI-mediated signaling. GPVI was stimulated using the selective agonist Cvx, a protein purified from the venom of the rattlesnake, C durissus terrificus. Aggregation stimulated with 15 ng/mL Cvx was completely inhibited by prior activation of PECAM-1 (Figure 2Ai), and a partial inhibitory effect was observed at higher concentrations of Cvx (31.25 and 62.5 ng/mL; not shown and Figure 2Aii, respectively). Similar results were observed on the stimulation of platelets with the G protein-coupled receptor agonist thrombin. Complete inhibition of aggregation at 90-second stimulation was observed at a thrombin concentration of 0.05 U/mL (Figure 2Bi) and a partial effect at 0.1 U/mL (Figure 2Bii). The use of an isotype-matched IgG control and cross-linker F(ab')2 had no effect on Cvx- or thrombin-stimulated platelet aggregation (Figure 2Ai-Bi). No inhibitory effect of PECAM-1 activation was observed at higher concentrations of thrombin (eg, 0.5 and 1, U/mL; data not shown). Results are representative of 5 separate experiments. Similar results were obtained using the alternative anti-PECAM-1 antibody PECAM 1.3 and when FcRIIA was blocked before PECAM-1
cross-linking.
Platelet secretion is inhibited by PECAM-1 signaling Platelet activation is accompanied by secretion from dense granules. Dense granule secretion was assessed by measuring the release of [3H]5-HT from preloaded washed platelets. Figure 3 shows the results of experiments to determine the effect of PECAM-1 cross-linking on [3H]5-HT secretion. A significant reduction in secretion was observed in platelets in which PECAM-1 was activated before stimulation with Cvx (81.9% ± 2.9% to 39.8% ± 4.1%; P = .02; n = 3) or thrombin (70.9% ± 4.6% to 37.0% ± 8.0%; P = .01; n = 3). The use of an isotype-matched IgG control and cross-linker F(ab')2 had no effect on Cvx- or thrombin-stimulated dense granule secretion (not shown). Experiments performed in the presence of mAb IV.3 to block the Fc receptor FcRIIA produced similar results.
PECAM-1 inhibits platelet protein tyrosine phosphorylation The effect of PECAM-1 cross-linking on GPVI- and thrombin receptor-stimulated signaling was investigated. Platelets were stimulated with Cvx (15 ng/mL) or thrombin (0.05 U/mL) with or without prior cross-linking of PECAM-1 for 90 seconds. Whole-cell protein tyrosine phosphorylation levels were determined by immunoblotting. Cross-linking PECAM-1 alone had no effect on basal platelet tyrosine phosphorylation levels (Figure 4). Stimulation with Cvx (15 ng/mL) or thrombin (0.05 U/mL) caused an increase in the level of tyrosine phosphorylation of a broad range of proteins, consistent with previous reports.32,37 In samples in which PECAM-1 signaling was stimulated by cross-linking before incubation with Cvx or thrombin, total tyrosine phosphorylation was reduced (Figure 4). The use of an isotype-matched IgG control and cross-linker F(ab')2 had no detectable effect on Cvx- or thrombin-stimulated total tyrosine phosphorylation levels.
PECAM-1 inhibits the mobilization of calcium from intracellular stores Stimulation of the collagen receptor GPVI and thrombin receptors leads to rapid intracellular mobilization of calcium, an effect that is essential for secretion and aggregation.38 Intracellular calcium levels were measured fluorometrically using the calcium-sensitive dye Fura-2 AM. Experiments were performed in the presence of 2 mM EGTA to prevent the entry of extracellular calcium. Stimulation of platelets with Cvx and thrombin resulted in a rapid increase in the levels of intracellular calcium that declined over a period of approximately 5 minutes (Figure 5). Incubation of platelets with control antibody and cross-linker F(ab')2 caused no change in Cvx- and thrombin-stimulated intracellular calcium mobilization (Figure 5A). Cross-linking of PECAM-1 for 90 seconds before incubation with Cvx (62.5 ng/mL) or thrombin (0.1 U/mL) resulted in a markedly reduced level of calcium mobilization (Figure 5Bi-Ci). At the lower concentrations of agonists used (Cvx, 15 ng/mL; thrombin, 0.05 U/mL), calcium mobilization was almost abolished (Figure 5Bii-Cii). The effect of PECAM-1 cross-linking on the reduction of peak intracellular calcium levels for a range of agonist concentrations is shown in Figure 5Biii-Ciii. A reduction of at least 50% in calcium mobilization was observed at all concentrations of Cvx and thrombin tested. Similar results were obtained using the alternative anti-PECAM-1 antibody, PECAM 1.3. Furthermore, experiments performed in the absence of extracellular EGTA indicate that PECAM-1 cross-linking does not inhibit agonist-induced influx of calcium (data not shown).
Rapid and complete activation of platelets at sites of tissue damage is ensured through numerous positive feedback pathways, mainly through the actions of mediators such as thromboxane A2 and ADP that are released from activated platelets. The existence of such a rapid and reactive system emphasizes the need for effective regulation of platelet function to prevent disorders such as thrombosis and hemorrhage. Platelet reactivity is a controlled balance between positive and negative regulatory factors and signaling mechanisms. Much attention has recently been focused on the identification of the receptors and signaling pathways that lead to platelet activation, particularly on exposure to collagen,30,31,33 thrombin,39 and ADP.40 Negative regulatory mechanisms in platelets are less well understood. The effects of endothelium-derived prostacyclin and nitric oxide are recognized for their roles in inhibiting platelet activation in healthy blood vessels. These molecules inhibit activation through cyclic adenosine monophosphate- and cyclic guanosine monophosphate-dependent signaling mechanisms, respectively. We report here on a third negative regulation system that is mediated through a cell-surface ITIM-bearing adhesion receptor, PECAM-1. Platelet collagen receptor GPVI signals through an ITAM present on the
cytoplasmic tail of the FcR PECAM-1 was stimulated through cross-linking using antibodies directed
to the extracellular domain of the receptor. This strategy was chosen
as the most specific manner through which to activate PECAM-1, and
activation was confirmed because cross-linking stimulated its tyrosine
phosphorylation. Confusion surrounds the natural endogenous ligand for
PECAM-1 on platelets in vivo. As described earlier, PECAM-1 has been
shown to participate in homophilic and heterophilic ligand binding. One
would anticipate that results similar to those described here would be
obtained using the recombinant extracellular domain of PECAM-1 as
ligand; however, results of such experiments may be complicated by the
ability of PECAM-1 to bind to other cell surface proteins. As has been
reported previously, PECAM-1 cross-linking stimulates tyrosine
phosphorylation (Figure 1) and association of SHP-2 (not shown) but did
not in itself cause platelet activation. Furthermore, tyrosine
phosphorylation of PECAM-1 on cross-linking appeared not to be
dependent on integrin Experiments were conducted to determine whether the inhibitory effect
of PECAM-1 is restricted to signaling through ITAM-containing receptors. On the contrary, PECAM-1 cross-linking was also found to
inhibit thrombin-stimulated platelet aggregation. It is not possible to
compare directly the potency of 2 different agonists that mediate their
effects through different receptors and signaling pathways. However, no
inhibitory effect of PECAM-1 was observed at moderate concentrations of
thrombin (0.5 and 1 U/mL; results not shown), where dramatic levels of
inhibition were observed at high concentrations of collagen (100 µg/mL). PECAM-1-mediated inhibition of thrombin-stimulated
aggregation was observed only at lower thrombin concentrations
(complete inhibition with 0.05 U/mL and slight inhibition at 0.1 U/mL).
This suggests that PECAM-1 activation inhibits thrombin-stimulated
platelet aggregation less efficiently than it does collagen-stimulated
aggregation, consistent with the general inhibitory effect on platelet
aggregation by an antibody bound to PECAM-1 reported by Wu et
al.42 We also examined the effect of PECAM-1 cross-linking
on platelet aggregation stimulated by other G protein-coupled receptor
agonists. Aggregation in response to low concentrations of the
thromboxane mimetic U46619 was also reduced by PECAM-1 signaling (M.C.
and J.M.G., unpublished results, May 2001). In addition,
preliminary work suggests that ADP-induced platelet aggregation at low
agonist concentrations may also be affected. This is consistent with
PECAM-1 performing a negative regulatory role in the control of
platelet activation stimulated by ITAM- and non-ITAM-containing
receptor agonists. Platelet responses that are not dependent
ITAM-bearing- or G protein-coupled receptors Having established that PECAM-1 cross-linking inhibits platelet
function, we examined the effect of this on some aspects of signal
transduction. Platelet activation by the collagen receptor GPVI is
dependent on tyrosine kinases and consequently is associated with the
rapid tyrosine phosphorylation of a wide variety of platelet proteins.
Stimulation with thrombin results in protein tyrosine phosphorylation,
but to a lesser degree. In this study we have demonstrated that
cross-linking PECAM-1, which in itself does not alter whole-cell
protein tyrosine phosphorylation levels, inhibits substantially the
level of tyrosine phosphorylation induced by subsequent stimulation
with Cvx or thrombin. This is consistent with the reduction observed in
aggregation and secretion. The identities of the phosphoproteins whose
phosphorylation is reduced on PECAM-1 cross-linking is under
investigation. It has been reported that PECAM-1 stimulation by
cross-linking inhibits the mobilization of calcium from intracellular
stores by the T-cell antigen receptor.12 A recent report
suggests that a similar effect is seen with B-cell antigen
receptors.13 Indeed, all the observations presented in
this report have been reproduced in platelets using one of the
anti-PECAM-1 antibodies used in the above 2 studies (PECAM 1.3).
Curiously, cross-linking PECAM-1 on endothelial cells has been reported
to stimulate increases in intracellular calcium concentration.44 In the data presented here, we
demonstrate that underlying the PECAM-1-mediated inhibition of
platelet activation is a significant level of inhibition of calcium
release from intracellular stores. As seen with aggregation assays, at
lower concentrations of Cvx and thrombin (15 ng/mL and 0.05 U/mL,
respectively), PECAM-1 signaling inhibits release almost completely,
whereas a partial effect is observed at higher agonist concentrations.
Calcium mobilization is stimulated through the intracellular generation
of inositol 1,4,5-trisphosphate from phosphatidylinositol
4,5-bisphosphate by phospholipase C (PLC). It is well established that
in platelets, stimulation with collagen leads to phosphorylation and
activation of PLC This report illustrates that PECAM-1, an ITIM-containing receptor, is capable of regulating platelet function. However, its role in vivo remains to be established. It is possible that this molecule provides a mechanism to prevent platelet activation on collision of platelets with healthy endothelium and collisions with other platelets. PECAM-1 signaling may result in a negative feedback on platelet activation pathways and thereby set the threshold stimulation level for platelet activation. PECAM-1 ligation on platelet contact, and between platelets and healthy endothelium, may also be instrumental in limiting the size of a thrombus formation through inactivating platelets on the thrombus periphery. A recent report by Vollmar et al45 using an in vivo thrombosis model in PECAM-1 null mice suggests that PECAM-1 has no role in vascular thrombosis. This is surprising given the in vitro data presented here and ex vivo studies on PECAM-1-deficient platelets.41 This may be explained by the redundancy in activation systems through which platelets may be stimulated given that we show here that thrombin-induced platelet activation is less sensitive to inhibition by PECAM-1. It is also highly likely that additional ITIM-containing receptors are expressed on the platelet, resulting in redundancy in inhibitory regulation systems. The above report does not address the effect of PECAM-1 deficiency on larger arterial vessels in which shear forces are likely to reach higher levels and platelet thrombi may be encountered. This is a complex area, further complicated by the potential roles of PECAM-1 in endothelial cell function, and it requires greater investigation. In conclusion, this report provides evidence for an extension of the known roles of ITIM receptors in the negative regulation of nonimmune cell function. In addition, in this study we have begun to explain at the cell signaling level the inhibitory effects of PECAM-1 on platelet activation. Future work is required to determine precisely how PECAM-1 inhibits the signaling pathways activated by multiple platelet agonists.
We thank Prof Peter Newman for providing the antibody PECAM 1.3 and for valuable discussion regarding this study. We also thank Dr Fiona Barry and Dr Peter Jordan for their advice in the preparation of this report and Tanya Sage for expert technical assistance.
Submitted February 12, 2001; accepted August 17, 2001.
Supported by funding from the Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the University of Reading Research Endowment Trust Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jonathan Gibbins, Cardiovascular Research Group, School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, United Kingdom; e-mail: j.m.gibbins{at}reading.ac.uk.
1.
Ohto H, Maeda H, Shibata Y, et al.
A novel leukocyte differentiation antigen: two monoclonal antibodies, TM2 and TM3, define 120-kDa molecule present on neutrophils, monocytes, platelets, and activated lymphoblasts.
Blood.
1985;66:873-881
2.
Muller WA, Ratti CM, McDonnell SL, Cohn ZA.
A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intracellular junctions.
J Exp Med.
1989;178:449-460
3.
Albelda SM, Oliver PD, Romer LH, Buck CA.
EndoCAM: a novel endothelial cell-cell adhesion molecule.
J Cell Biol.
1990;110:1227-1237
4.
Newman PJ, Berndt MJ, Gorski J, et al.
PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.
Science.
1990;247:1219-1222 5. Matsumura PW, Wolff K, Petzelbauer P. Endothelial cell tube formation depends on cadherin and CD31 interactions with filamentous actin. J Immunol. 1997;158:3408-3416[Abstract].
6.
Brier G, Brevario F, Caveda L, et al.
Molecular cloning and expression of murine vascular endothelial cadherin in early stage development of the cardiovascular system.
Blood.
1996;87:630-641
7.
Tanaka Y, Albelda SM, Horgan KJ, et al.
CD31 expressed on distinctive T cell subsets is a preferential amplifier of
8.
Piali L, Albelda SM, Baldwin HS, et al.
Murine platelet endothelial cell adhesion molecule (PECAM-1/CD31) modulates
9.
Vaporciyan AA, DeLisser HM, Yan H-C, et al.
Involvement of platelet-endothelial cell adhesion molecule-1 in neutrophil recruitment in vivo.
Science.
1993;262:1580-1582 10. Muller WA, Weigl SA, Deng X, Phillips DM. PECAM-1 is required for transendothelial migration of leukocytes. J Exp Med. 1993;178:449-460.
11.
Duncan GS, Andrew DP, Takimoto H, et al.
Genetic evidence for functional redundancy of platelet/endothelial cell adhesion molecule-1 (PECAM-1): CD31- deficient mice reveal PECAM-1-dependent and PECAM-1-independent functions.
J Immunol.
1999;162:3022-3030
12.
Newton-Nash DK, Newman PJ.
A new role for platelet-endothelial cell adhesion molecule-1 (CD31): inhibition of TCR-mediated signal transduction.
J Immunol.
1999;163:682-688
13.
Newman DK, Hamilton CA, Armstrong MJ, Newman PJ.
Inhibition of antigen-receptor signaling by platelet endothelial cell-adhesion molecule-1 (CD31) requires an intact ITIM, SHP-2, and p56Lck.
Blood.
2000;97:2351-2357 14. Newman PJ. Switched at birth: a new family for PECAM-1. J Clin Invest. 1999;103:5-9[Medline] [Order article via Infotrieve]. 15. Vivier E, Daeron M. Immunoreceptor tyrosine-based inhibitory motifs. Immunol Today. 1997;18:286-291[Medline] [Order article via Infotrieve].
16.
Albelda SM, Muller WA, Buck CA, Newman PJ.
Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule.
J Cell Biol.
1991;114:1059-1068
17.
Buckley CD, Doyonnas R, Newton JP, et al.
Identification of
18.
Deaglio S, Morra M, Mallone R, et al.
Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member.
J Immunol.
1998;160:395-402 19. Edmead CE, Crosby DA, Southcott M, Poole AW. Thrombin-induced association of SHP-2 with multiple tyrosine phosphorylated proteins in human platelets. FEBS Lett. 1999;459:27-32[CrossRef][Medline] [Order article via Infotrieve].
20.
Sagawa K, Swaim W, Zhang J, Unsworth E, Siraganian RP.
Aggregation of the high-affinity IgE receptor results in the tyrosine phosphorylation of the surface adhesion protein PECAM-1 (CD31).
J Biol Chem.
1997;272:13412-13418 21. Osawa M, Masuda M, Harada N, Lopes RB, Fugiwara K. Tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) in mechanically stimulated vascular endothelial cell. Eur J Cell Biol. 1997;72:229-237[Medline] [Order article via Infotrieve]. 22. Wang R, Paddock C, Augustine JA, Newman PJ. Oxidative stress triggers PECAM-1-mediated signaling pathways [abstract]. Blood. 1998;92:548.
23.
Cicmil M, Thomas JM, Sage T, et al.
Collagen, convulxin, and thrombin stimulate aggregation-independent tyrosine phosphorylation of CD31 in platelets: evidence for the involvement of Src family kinases.
J Biol Chem.
2000;275:27339-27347
24.
Jackson DE, Ward CM, Wang R, Newman PJ.
The protein-tyrosine phosphatase SHP-2 binds platelet/endothelial cell adhesion molecule-1 (PECAM-1) and forms a distinct signaling complex during platelet aggregation.
J Biol Chem.
1997;272:6986-6993
25.
Jackson DE, Kupcho KR, Newman PJ.
Characterization of phosphotyrosine binding motifs in the cytoplasmic domain of platelet/endothelial cell adhesion molecule-1 (PECAM-1) that are required for the cellular association and activation of the protein-tyrosine phosphatase, SHP-2.
J Biol Chem.
1997;272:24868-24875
26.
Hua CT, Gamble JR, Vadas MA, Jackson DE.
Recruitment and activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial cell adhesion molecule-1 (PECAM-1): identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates.
J Biol Chem.
1998;273:28332-28340
27.
Fukunaga K, Noguchi T, Takeda H, et al.
Requirement for protein-tyrosine phosphatase SHP-2 in insulin-induced activation of c-Jun NH2-terminal kinase.
J Biol Chem.
2000;275:5208-5213 28. Watson SP, Gibbins J. Collagen receptor signalling in platelets: extending the role of the ITAM. Immunol Today. 1998;19:260-264[CrossRef][Medline] [Order article via Infotrieve].
29.
Gibbins J, Asselin J, Farndale R, et al.
Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets.
J Biol Chem.
1996;271:18095-18099
30.
Gibbins JM, Okuma M, Farndale R, Barnes M, Watson SP.
Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor
31.
Poole A, Gibbins JM, Turner M, et al.
The Fc receptor
32.
Gibbins JM, Briddon S, Shutes A, et al.
The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activator of T cells (LAT) in platelets stimulated by collagen and convulxin.
J Biol Chem.
1998;273:34437-34443
33.
Tsuji M, Ezumi Y, Arai M, Takayama H.
A novel association of Fc receptor 34. Daeron M, Latour S, Malbec O, et al. The same tyrosine-based inhibition motif, in the intracytoplasmic domain of Fc gamma RIIB, regulates negatively BCR, TCR, and FcR-dependent cell activation. Immunity. 1995;3:635-646[CrossRef][Medline] [Order article via Infotrieve]. 35. Francischetti IMB, Saliou B, Leduc M, et al. Convulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets. Toxicon. 1997;35:1217-1228[Medline] [Order article via Infotrieve].
36.
Robinson A, Gibbins J, Rodriguez-Linares B, et al.
Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking.
Blood.
1996;88:522-530
37.
Nakamura S, Yamamura H.
Thrombin and collagen induce rapid phosphorylation of a common set of cellular proteins on tyrosine in human platelets.
J Biol Chem.
1989;264:7089-7091 38. Smith JB, Selak MA, Dangelmaier C, Daniel JL. Cytosolic calcium as a second messenger for collagen-induced platelet responses. Biochem J. 1992;288:925-929. 39. Kahn ML, Zheng Y-W, Huang W, et al. A dual thrombin receptor system for platelet activation. Nature. 1998;394:690-694[CrossRef][Medline] [Order article via Infotrieve]. 40. Hollopeter G, Jantzen H-M, Vincent D, et al. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature. 2001;409:202-207[CrossRef][Medline] [Order article via Infotrieve].
41.
Patil S, Newman DK, Newman PJ.
PECAM-1 serves as an inhibitory receptor that modulates platelet responses to collagen.
Blood.
2001;97:1727-1732
42.
Wu X-W, Lian EC-Y.
Binding properties and inhibition of platelet aggregation by a monoclonal antibody to CD31 (PECAM-1).
Arterioscler Thromb Vasc Biol.
1997;17:3154-3158
43.
Nieswandt B, Bergmeier WG, Eckly A, et al.
Evidence for cross-talk between glycoprotein VI and Gi-coupled receptors during collagen-induced platelet aggregation.
Blood.
2001;97:3829-3835 44. Gurubhagavatula I, Amrani Y, Practico D, et al. Engagement of human PECAM-1 (CD31) on human endothelial cells increases intracellular calcium ion concentration and stimulates prostacyclin release. J Clin Invest. 1998;101:212-222[Medline] [Order article via Infotrieve]. 45. Vollmar B, Schmits R, Kunz D, Menger MD. Lack of in vivo function of CD31 in vascular thrombosis. Thromb Haemost. 2001;85:160-164[Medline] [Order article via Infotrieve]. 46. Blake RA, Schieven GL, Watson SP. Collagen stimulates tyrosine phosphorylation of phospholipase C-2 but not phospholipase C-1 in human platelets. FEBS Lett. 1994;353:212-216[CrossRef][Medline] [Order article via Infotrieve]. 47. Daniel JL, Dangelmaier C, Smith JB. Evidence for a role for tyrosine phosphorylation of phospholipase C-2 in collagen-induced platelet cytosolic calcium mobilisation. Biochem J. 1994;302:617-622.
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  J. Rivera, M. L. Lozano, L. Navarro-Nunez, and V. Vicente Platelet receptors and signaling in the dynamics of thrombus formation Haematologica, May 1, 2009; 94(5): 700 - 711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Dasgupta, E. Dufour, Z. Mamdouh, and W. A. Muller A Novel and Critical Role for Tyrosine 663 in Platelet Endothelial Cell Adhesion Molecule-1 Trafficking and Transendothelial Migration J. Immunol., April 15, 2009; 182(8): 5041 - 5051. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J.A. Korporaal, C. A. Koekman, S. Verhoef, D. E. van der Wal, M. Bezemer, M. Van Eck, and J.-W. N. Akkerman Downregulation of Platelet Responsiveness Upon Contact With LDL by the Protein-Tyrosine Phosphatases SHP-1 and SHP-2 Arterioscler Thromb Vasc Biol, March 1, 2009; 29(3): 372 - 379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Wong, Y. Liu, J. Yip, R. Chand, J. L. Wee, L. Oates, B. Nieswandt, A. Reheman, H. Ni, N. Beauchemin, et al. CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo Blood, February 19, 2009; 113(8): 1818 - 1828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Mori, A. C. Pearce, J. C. Spalton, B. Grygielska, J. A. Eble, M. G. Tomlinson, Y. A. Senis, and S. P. Watson G6b-B Inhibits Constitutive and Agonist-induced Signaling by Glycoprotein VI and CLEC-2 J. Biol. Chem., December 19, 2008; 283(51): 35419 - 35427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Tucker, T. Sage, J. M. Stevens, P. A. Jordan, S. Jones, N. E. Barrett, R. St-Arnaud, J. Frampton, S. Dedhar, and J. M. Gibbins A dual role for integrin-linked kinase in platelets: regulating integrin function and {alpha}-granule secretion Blood, December 1, 2008; 112(12): 4523 - 4531. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jones, K. L. Tucker, T. Sage, W. J. Kaiser, N. E. Barrett, P. J. Lowry, A. Zimmer, S. P. Hunt, M. Emerson, and J. M. Gibbins Peripheral tachykinins and the neurokinin receptor NK1 are required for platelet thrombus formation Blood, January 15, 2008; 111(2): 605 - 612. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Randriamboavonjy, F. Pistrosch, B. Bolck, R. H.G. Schwinger, M. Dixit, K. Badenhoop, R. A. Cohen, R. Busse, and I. Fleming Platelet Sarcoplasmic Endoplasmic Reticulum Ca2+-ATPase and {micro}-Calpain Activity Are Altered in Type 2 Diabetes Mellitus and Restored by Rosiglitazone Circulation, January 1, 2008; 117(1): 52 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Rui, X. Liu, N. Li, Y. Jiang, G. Chen, X. Cao, and J. Wang PECAM-1 Ligation Negatively Regulates TLR4 Signaling in Macrophages J. Immunol., December 1, 2007; 179(11): 7344 - 7351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. S. Dhanjal, C. Pendaries, E. A. Ross, M. K. Larson, M. B. Protty, C. D. Buckley, and S. P. Watson A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice Blood, May 15, 2007; 109(10): 4237 - 4244. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Moraes, K. E. Swales, J. A. Wray, A. Damazo, J. M. Gibbins, T. D. Warner, and D. Bishop-Bailey Nongenomic signaling of the retinoid X receptor through binding and inhibiting Gq in human platelets Blood, May 1, 2007; 109(9): 3741 - 3744. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. A. Senis, M. G. Tomlinson, A. Garcia, S. Dumon, V. L. Heath, J. Herbert, S. P. Cobbold, J. C. Spalton, S. Ayman, R. Antrobus, et al. A Comprehensive Proteomics and Genomics Analysis Reveals Novel Transmembrane Proteins in Human Platelets and Mouse Megakaryocytes Including G6b-B, a Novel Immunoreceptor Tyrosine-based Inhibitory Motif Protein Mol. Cell. Proteomics, March 1, 2007; 6(3): 548 - 564. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. A. Lee, M. van Lier, I. A. M. Relou, L. Foley, J.-W. N. Akkerman, H. F. G. Heijnen, and R. W. Farndale Lipid Rafts Facilitate the Interaction of PECAM-1 with the Glycoprotein VI-FcR {gamma}-Chain Complex in Human Platelets J. Biol. Chem., December 22, 2006; 281(51): 39330 - 39338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Falati, S. Patil, P. L. Gross, M. Stapleton, G. Merrill-Skoloff, N. E. Barrett, K. L. Pixton, H. Weiler, B. Cooley, D. K. Newman, et al. Platelet PECAM-1 inhibits thrombus formation in vivo Blood, January 15, 2006; 107(2): 535 - 541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Wee and D. E. Jackson The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin {alpha}IIb{beta}3 in platelets Blood, December 1, 2005; 106(12): 3816 - 3823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J.A. Korporaal, G. Gorter, H. J.M. van Rijn, and J.-W. N. Akkerman Effect of Oxidation on the Platelet-Activating Properties of Low-Density Lipoprotein Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 867 - 872. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Jordan, J. M. Stevens, G. P. Hubbard, N. E. Barrett, T. Sage, K. S. Authi, and J. M. Gibbins A role for the thiol isomerase protein ERP5 in platelet function Blood, February 15, 2005; 105(4): 1500 - 1507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Maas, M. Stapleton, C. Bergom, D. L. Mattson, D. K. Newman, and P. J. Newman Endothelial cell PECAM-1 confers protection against endotoxic shock Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H159 - H164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Graham, J. M. Stevens, N. M. Page, A. D. Grant, S. D. Brain, P. J. Lowry, and J. M. Gibbins Tachykinins regulate the function of platelets Blood, August 15, 2004; 104(4): 1058 - 1065. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Albelda, K. C. Lau, P. Chien, Z.-Y. Huang, E. Arguiris, A. Bohen, J. Sun, J. A. Billet, M. Christofidou-Solomidou, Z. K. Indik, et al. Role for Platelet-Endothelial Cell Adhesion Molecule-1 in Macrophage Fc{gamma} Receptor Function Am. J. Respir. Cell Mol. Biol., August 1, 2004; 31(2): 246 - 255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Maxwell, Y. Yuan, K. E. Anderson, M. L. Hibbs, H. H. Salem, and S. P. Jackson SHIP1 and Lyn Kinase Negatively Regulate Integrin {alpha}IIb{beta}3 Signaling in Platelets J. Biol. Chem., July 30, 2004; 279(31): 32196 - 32204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Gibbins Platelet adhesion signalling and the regulation of thrombus formation J. Cell Sci., July 15, 2004; 117(16): 3415 - 3425. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Coffey, G. E. Jarvis, J. M. Gibbins, B. Coles, N. E. Barrett, O. R.E. Wylie, and V. B. O'Donnell Platelet 12-Lipoxygenase Activation via Glycoprotein VI: Involvement of Multiple Signaling Pathways in Agonist Control of H(P)ETE Synthesis Circ. Res., June 25, 2004; 94(12): 1598 - 1605. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Patrono, F. Bachmann, C. Baigent, C. Bode, R. De Caterina, B. Charbonnier, D. Fitzgerald, J. Hirsh, S. Husted, J. Kvasnicka, et al. Expert Consensus Document on the Use of Antiplatelet Agents: The Task Force on the Use of Antiplatelet Agents in Patients with Atherosclerotic Cardiovascular Disease of the European Society of Cardiology Eur. Heart J., January 2, 2004; 25(2): 166 - 181. [Full Text] [PDF]
|
|
|
|
 |
|
 D. Feng, J. A. Nagy, K. Pyne, H. F. Dvorak, and A. M. Dvorak Ultrastructural Localization of Platelet Endothelial Cell Adhesion Molecule (PECAM-1, CD31) in Vascular Endothelium J. Histochem. Cytochem., January 1, 2004; 52(1): 87 - 102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ragab, S. Bodin, C. Viala, H. Chap, B. Payrastre, and J. Ragab-Thomas The Tyrosine Phosphatase 1B Regulates Linker for Activation of T-cell Phosphorylation and Platelet Aggregation upon Fc{gamma}RIIa Cross-linking J. Biol. Chem., October 17, 2003; 278(42): 40923 - 40932. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. A. M. Relou, G. Gorter, I. A. Ferreira, H. J. M. van Rijn, and J.-W. N. Akkerman Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) Inhibits Low Density Lipoprotein-induced Signaling in Platelets J. Biol. Chem., August 29, 2003; 278(35): 32638 - 32644. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Newman and D. K. Newman Signal Transduction Pathways Mediated by PECAM-1: New Roles for an Old Molecule in Platelet and Vascular Cell Biology Arterioscler Thromb Vasc Biol, June 1, 2003; 23(6): 953 - 964. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. A. Barry and J. M. Gibbins Protein Kinase B Is Regulated in Platelets by the Collagen Receptor Glycoprotein VI J. Biol. Chem., April 5, 2002; 277(15): 12874 - 12878. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
RI),
for example,
ristocetin-induced agglutination