NIH Public Access
Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
NIH-PA Author Manuscript
Published in final edited form as:
J Thromb Haemost. 2010 July ; 8(7): 1584–1593. doi:10.1111/j.1538-7836.2010.03883.x.
PECAM-1 is a negative regulator of laminin-induced platelet
activation
Jimmy Crockett1, Debra K. Newman2,3, and Peter J. Newman1,3,4,5
1Blood Research Institute BloodCenter of Wisconsin Milwaukee, WI 53201
2Department
of Microbiology, Medical College of Wisconsin Milwaukee, WI 53226
3Department
of Pharmacology, Medical College of Wisconsin Milwaukee, WI 53226
4Department
of Cellular Biology, Medical College of Wisconsin Milwaukee, WI 53226
5The
Cardiovascular Research Center Medical College of Wisconsin Milwaukee, WI 53226
NIH-PA Author Manuscript
Abstract
Background—Interaction of resting platelets with exposed components of the subendothelial
matrix is an important early activating event that takes place at sites of vascular injury. Platelet
responses to collagen are mediated by the integrin α2β1 and the glycoprotein (GP)VI/Fc receptor
(FcR)γ chain complex, while platelet activation by laminin is mediated by the related integrin,
α6β1, and similarly requires signaling through GPVI/FcRγ.
Objective—Because the cell adhesion and signaling receptor, PECAM-1, has previously been
shown to dampen collagen-induced platelet activation, we sought to determine whether PECAM-1
might similarly regulate platelet activation by laminin.
Methods/Results—We found that PECAM-1 became tyrosine phosphorylated on its
cytoplasmic ITIMs following adhesion of either human or murine platelets to immobilized
laminin. While the presence or absence of PECAM-1 had no effect on either the rate or extent of
platelet adhesion or spreading on laminin, PECAM-1 inhibited laminin-induced phosphorylation
of GPVI/FcRγ chain ITAMs, activation of its downstream effector, Syk kinase, and suppressed
granule secretion.
NIH-PA Author Manuscript
Conclusion—Taken together, these data are consistent with previous findings in platelets and
other blood and vascular cells that PECAM-1 functions by modulating ITAM-mediated signaling
pathways that amplify cellular activation.
Keywords
PECAM-1; laminin; α6β1; GPVI; platelet signaling; ITAM; ITIM
Address correspondence to: Peter J. Newman Blood Research Institute BloodCenter of Wisconsin P.O. Box 2178 638 N. 18th Street
Milwaukee, WI 53201 Phone: (414) 937-6237 Fax: (414) 937-6284 peter.newman@bcw.edu.
AUTHORSHIP
Contribution: JC designed research, performed research, analyzed results and drafted the paper; DKN designed research and analyzed
results; P.J.N. designed research, analyzed results and wrote the paper.
Portions of this work were presented in abstract form at the 49th Annual Meeting of the American Society of Hematology, December
8-11, 2007.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Crockett et al.
Page 2
INTRODUCTION
NIH-PA Author Manuscript
Breach of the vascular endothelium results in rapid adhesion of platelets to the underlying
basement membrane – an extracellular matrix sheet composed of glycosaminoglycans, type
IV collagen, fibronectin, von Willebrand factor, and one or more members of the laminin
family of adhesive glycoproteins [1]. Platelet adhesion to collagen, and to a lesser extent,
laminin, initiates a series of biochemical reactions that result in tyrosine phosphorylation of
cytoplasmic enzymes and adaptor proteins, calcium mobilization from cytosolic stores,
polymerization of cytoskeletal proteins that drive shape change and spreading, rapid
conformational activation of cell-surface integrins that enable high-affinity binding to
adhesive ligands, and secretion of the contents of platelet alpha and dense granules (for a
review, see references [2] and [3]). Together, these events constitute primary hemostasis,
and allow formation of a platelet plug designed to prevent blood loss at sites of vascular
injury.
NIH-PA Author Manuscript
The glycoprotein (GP)VI/FcRγ chain complex is a critical mediator of adhesion-induced
platelet activation, operating via GPVI-associated Src-family kinases that, upon ligand
binding, phosphorylate tyrosine residues located within Immunoreceptor Tyrosine-base
Activation Motifs (ITAMs) located within cytoplasmic domain of the FcRγ chain.
Phosphorylated FcRγ serves as a docking site for the tyrosine kinase Syk which, with the
help of adaptor proteins LAT, SLP76, and Gads, and the tyrosine kinase Btk, activates
phospholipase C (PLC)γ2 and/or PLCγ1, which via their lipase activity generate lipid
products that support a multitude of cellular activation responses, including calcium
mobilization, platelet spreading, integrin activation, and granule secretion [4].
NIH-PA Author Manuscript
While many of the biochemical events associated with adhesion-initiated platelet activation
have been elucidated, the identity and mechanism of action of those receptors that set a
physiological threshold for platelet activation and/or return platelets to a resting state is
much less well-understood. Of these, the Immunoreceptor Tyrosine-based Inhibitory Motif
(ITIM) – bearing inhibitory receptor, PECAM-1 is perhaps the best characterized.
PECAM-1 is a 130 kDa member of the Ig-ITIM family that is expressed on platelets,
leukocytes and endothelial cells. In leukocytes, PECAM-1 has been shown, upon co-ligation
with the TCR, to attenuate calcium release from intracellular stores [5], and can also exert its
inhibitory function by suppressing cytokine production [6-9]. In endothelial cells, PECAM-1
has been shown to inhibit apoptotic signals that activate the intrinsic, Bax-mediated pathway
of programmed cell death [10-13]. In platelets, numerous laboratories have demonstrated
that PECAM-1 inhibits low-dose collagen-induced platelet activation – an effect that has
been observed both in vitro and in vivo [14-18]. Interestingly, it appears that PECAM-1 does
so by regulating granule secretion – a key amplifier of platelet activation and thrombus
formation.
Collagen- and laminin-induced platelet activation have been shown to be mechanisticallylinked via their use of the GPVI/FcRγ-chain complex to send activation signals into the cell
downstream of ligand binding to integrins α2β1and α6β1, respectively [19]. Because
PECAM-1, has previously been shown to dampen collagen-induced platelet activation, we
sought to determine whether PECAM-1 might similarly regulate platelet activation by
laminin. Our findings provide further support for the notion that PECAM-1 functions to
regulate biochemical and cell biological events that amplify platelet activation responses.
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 3
MATERIALS AND METHODS
Reagents and Antibodies
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Laminin from human placenta, bovine serum albumin, tetramethylrhodamine isothiocyanate
(TRITC)-labeled phalloidin, and prostaglandin E1 (PGE1) were purchased from SigmaAldrich (St Louis, MO). Soluble calf skin collagen was obtained from BioData (Horsham,
PA) and collagen-related peptide (CRP) was synthesized by the Protein Chemistry Core
Laboratory of the Blood Research Institute, BloodCenter of Wisconsin. Mouse anti-human
PECAM-1 monoclonal antibody (mAb), PECAM-1.3, and rabbit anti-human PECAM-1
polyclonal antibody (pAb), SEW 32-34 have been previously described [5]. Rat anti-mouse
PECAM-1 mAb, 390, was generously provided by Dr. Steven Albelda (University of
Pennsylvania, Philadelphia, PA) and has also been also previously described [20]. Goat antimouse PECAM-1 (clone M-20) pAb and rabbit anti-SHP-2 (clone C-18) pAb were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Unconjugated and fluorescein
isothiocyanate (FITC)-conjugated rat anti–integrin α6 (clone GoH3) mAbs and
phycoerythrin (PE)-conjugated rat anti-mouse integrin α2 (clone 3H1480) mAbs were
obtained from Abcam (Cambridge, MA). Horseradish peroxidase (HRP)-conjugated mouse
anti–phospho-tyrosine (PY-20) was obtained from Zymed (San Francisco, CA). Rabbit antiFcεRI, anti-FcRγ-chain pAbs and mouse anti–phosphotyrosine (clone 4G10) were obtained
from Upstate (Lake Placid, NY). PE-conjugated rat anti-mouse PECAM-1 (clone MEC
13.3) mAb was obtained from BD Biosciences (Franklin Lakes, NJ). The rat anti-mouse
GPVI (clone JAQ1) mAb and PE-conjugated rat anti-mouse integrin αIIbβ3 (clone JON/A)
mAb were purchased from Emfret Analytics (Würzburg, Germany). JAQ1 antibodies were
further conjugated with Alexa Fluor 647 using a labeling kit (Invitrogen, Eugene, OR).
HRP-conjugated secondary antibodies used for western blot analysis were purchased from
Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). For flow cytometry,
appropriate conjugated and isotype-matched antibodies were purchased either from
Molecular Probes (Invitrogen) or Pharmingen (BD Biosciences). Calcein-AM was
purchased from Molecular Probes and (14C) serotonin was obtained from GE Healthcare
(Piscataway, NJ).
NIH-PA Author Manuscript
Preparation of murine and human platelets—PECAM-1-deficient mice on a C57BL/
6J background were housed and bred in pathogen-free conditions at the Animal Resource
Facility of the Medical College of Wisconsin. PECAM-1-negative mice were genotyped by
polymerase chain reaction (PCR) and either wild-type littermates or age- and sex-matched
wild-type mice used as a controls. Whole blood was obtained from the inferior vena cava
using a syringe containing 0.1 volume of 3.8% sodium citrate. Blood from healthy human
volunteers was obtained by venipuncture into tubes containing 10% acid-citrate-dextrose
(ACD). Blood samples were diluted 1:5 with modified Tyrodes-HEPES buffer (10 mM
HEPES [pH 7.4], 12 mM NaHCO3, 137 mM NaCl, 2.7 mM KCl, 5 mM glucose, 0.25%
bovine serum albumin [BSA]) and prostaglandin E1 (PGE1) added to a final concentration
of 50 ng/mL. Platelet-rich-plasma (PRP) was obtained following centrifugation for 10
minutes at 300g, transferred into a fresh tube, and platelets pelleted at 700g before
resuspension in modified Tyrodes-HEPES buffer at 2 × 107 platelets per ml for platelet
spreading assays, 1 × 108 platelets per ml for adhesion, flow cytometry, and serotonin
release assays, or 6 × 108 platelets per ml for immunoprecipitation/immunoblot analysis.
Quantitative platelet adhesion assays—Adhesion to immobilized substrates was
performed as previously described [14,14], with minor modifications. Briefly, 96 well
Immulon-2HB microtiter plates (Dynex Technologies, Chantilly, VA) were coated overnight
at 4°C with various concentrations of human laminin or calf soluble collagen. After washing
and blocking 3% BSA. Calcein-AM-loaded platelets in modified Tyrodes-HEPES buffer
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 4
NIH-PA Author Manuscript
containing 2 mM MgCl2 were added to each of triplicate wells and incubated at 37°C for 60
minutes under static conditions. Total fluorescence was determined using a fluorescence
plate reader (Cytofluor 4000, PerSeptive Biosystems, Framingham, MA) at 485 nm
excitation and 530 nm emission wavelengths. Wells were rinsed with Tyrodes-HEPES
buffer 3 times and bound fluorescence measured again. Percent adhesion was calculated as
bound fluorescence/total fluorescence × 100.
NIH-PA Author Manuscript
Immunoprecipitation and immunoblot analysis—700 μl of washed human or
murine platelets (6 × 108/mL) suspended in modified Tyrodes-HEPES buffer containing 2
mM Mg2+ were seeded onto laminin or collagen-coated Corning cell culture dishes (Corning
Inc., Corning, NY) and incubated at 37 °C for the indicated time period. Reactions were
terminated by addition of an equal volume of 2x ice-cold lysis buffer (30 mM HEPES, pH
7.4, 300 mM NaCl, 20 mM EGTA, 0.2 mM MgCl2, 2% Triton X-100 containing 2x
protease inhibitor cocktail (CalBiochem, San Diego, CA), 2x phosphatase inhibitor cocktail
set II (CalBiochem), and 4 mM sodium orthovanadate. After 30 minutes on ice, lysates were
precleared with protein G-Sepharose beads before addition of primary antibody. After
overnight incubation at 4°C, immune complexes were captured with protein G-Sepharose
beads, eluted off in reducing SDS loading buffer, and subjected to SDS-PAGE/western blot
analysis. Binding was detected using enhanced chemiluminescence (Amersham, Piscataway,
NJ) and band intensity quantitated using a Kodak 1D imaging system (Scientific Imaging
System, New Haven, CT). The degree of phosphorylation was expressed as a ratio of the
band intensity for the phosphorylated protein relative to protein antigen.
Platelet spreading assays—Washed platelets derived from either wild-type or
PECAM-1-negative mice, or from human blood were resuspended to a concentration of 2 ×
107/mL in modified Hepes/Tyrodes buffer containing 2 mM Mg2+, added to laminin-coated
coverslips, and incubated for the indicated time-points at 37 °C. After a light rinse, adherent
platelets were fixed using 3% paraformaldehyde, permeabilized with 0.1% Triton X-100,
and stained for F-actin using tetramethylrhodamine isothiocyanate (TRITC)-labeled
phalloidin (0.1 μg/ml). Platelets were visualized using a Nikon TE300 inverted microscopy
equipped with a CFI Plan Achromat DL Ph3 100x oil-immersion lens and a Photometrics
SenSys charge-coupled device camera (Photometrics, Tucson, AZ). Eight images,
encompassing a total of 179 – 264 platelets, were randomly chosen for each time-point and
the surface area of each platelet calculated using Metamorph software (Universal Imaging,
Downingtown, PA). All analyses were performed under blinded conditions. Statistical
significance was determined using the Student t-test for unpaired samples and results
expressed at the mean surface area/platelet ± SEM. P values of less than 0.05 were
interpreted as statistically significant.
NIH-PA Author Manuscript
Serotonin release assay—Murine platelets at a concentration of 1 × 108/mL in modified
Tyrodes buffer containing 50 ng/ml PGE1 without calcium or magnesium were loaded with
0.2 μCi/ml of [14C] 5-hydroxytryptamine for 1 hour at 37 °C. Labeled platelets were washed
by centrifugation and resuspended in modified Tyrodes buffer containing 2 mM MgCl2,
added in triplicate to collagen or laminin-coated microtiter wells. Following incubation for
60 minutes at room temperature, non-adherent platelets were removed and the supernatant
containing the released serotonin counted in a scintillation counter.
Statistical analysis—Data analysis was performed using SigmaPlot for Windows,
version 6.0 (SPSS Inc. Chicago, IL) and/or SAS Learning Edition 4.1 (SAS Institute Inc.
Cary, NC). All results were expressed as mean ± SEM. A P-value <0.05 was considered
statistically significant. Quantitative data with normal distributions were compared by
parametric tests, and data with abnormal distributions were analyzed by non-parametric
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 5
NIH-PA Author Manuscript
tests. Multiple groups were analyzed by analysis of variance (ANOVA) or by Kruskall–
Wallis tests. In the case of significant main effects, post hoc analysis was performed using
Student's t-tests for the ANOVA, or the Wilcoxon test for the Kruskall–Wallis tests.
Comparisons between two groups were performed by Student's t-test or Mann–Whitney Utests.
RESULTS
Platelet adhesion to laminin induces tyrosine phosphorylation of PECAM-1
Platelet adhesion to laminin was first described in 1984 by Ill and Ruoslahti [21], and when
performed in the presence of at least 1 mM MgCl2, platelets have consistently been observed
to not only adhere, but to undergo cytoskeletally-mediated, laminin-induced cell spreading
as well [19,22]. Similar to these studies, we found that human platelets bound and spread on
immobilized laminin nearly as well as they did on immobilized collagen (Figure 1B), though
the morphology of platelets bound to the two extracellular matrix substrates differed in the
degree of activation-induced spreading, especially at early time points (Figure 1A).
NIH-PA Author Manuscript
PECAM-1 is thought to function as a negative regulator of collagen-induced platelet
activation [14-17] via ligand binding-induced phosphorylation of its cytoplasmic ITIM
tyrosine, followed by recruitment of the protein-tyrosine phosphatase, SHP-2 [23]. To
determine whether this same negative-feedback pathway might be initiated in response to
platelet adhesion to immobilized laminin, we incubated both human and mouse platelets in
laminin-coated microtiter wells, allowed them to settle, bind, and spread at 37 °C, and
performed immunoblot analysis of cells that were detergent-lysed at various time points up
to one hour. As shown in Figure 2, weak PECAM-1 tyrosine phosphorylation could be
detected as early as 15 minutes, peaking at 45 minutes, and declining by 60 minutes. Similar
to that observed following platelet adhesion to collagen, SHP-2 became associated with the
PECAM-1 cytoplasmic domain following platelet adhesion to immobilized laminin (Figure
3). Laminin, and not contaminants in the laminin preparation, was responsible for activation
of PECAM-1 and its subsequent recruitment of SHP-2, as addition to platelets of the α6β1specficic mAb, GoH3 [24], effectively inhibited both responses. Taken together, these data
support the notion that the inhibitory function of PECAM-1 is enlisted in response to platelet
adhesion to multiple components of the extracellular matrix.
The rate and extent of platelet adhesion and spreading on immobilized laminin are
unaffected by the presence or absence or PECAM-1
NIH-PA Author Manuscript
We employed two different approaches to determine whether PECAM-1 might function to
inhibit platelet adhesion and spreading following exposure to immobilized laminin. In the
first, the rate and extent of spreading of wild-type and PECAM-1-deficient murine platelets
on immobilized laminin was compared. As shown in Figure 4, there was no significant
difference. The second approach took the tact of Cicmil et al. [16] of adding anti-PECAM-1
mAbs to human platelets, either in the presence or absence of secondary anti-mouse
antibodies, to stimulate the inhibitory activity of PECAM-1. As shown in Figure 5, although
mAb PECAM-1.3 was able to induce PECAM-1 ITIM tyrosine phosphorylation, its addition
had no observable effect on either the number of platelets that became adherent to laminin,
or the extent to which they spread following contact.
PECAM-1 negatively regulates laminin-induced amplification pathways leading to granule
secretion
Though PECAM-1 does not appear to control the initial adhesive phenotype of the cell
following exposure to adhesive ligands, signaling downstream of PECAM-1 is thought to
provide negative feedback regulation of amplification events, in particular secretion of
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 6
NIH-PA Author Manuscript
platelet granules. Thus, we [14] and others [15,16] have shown that PECAM-1-deficient
murine platelets exhibit enhanced secretion responses to the collagen→α2β1→GPVI/
FcRγ→Syk→PLCγ2 platelet activation pathway. To determine whether granule secretion
was similarly exaggerated in PECAM-1-deficient platelets exposed to laminin, platelets
from wild-type and PECAM-1 knockout mice were isolated, labeled with 14C serotonin, and
applied to microtiter wells that have been coated with BSA, collagen, or laminin. As shown
in Figure 6, dense granule secretion in response to laminin stimulation was modestly, but
significantly, increased in PECAM-1-deficient, relative to wild-type, platelets. This was not
due to differential expression of the major cell surface receptors known to regulate platelet
responses to collagen, as the expression in PECAM-1-deficient platelets of α6β1 and GPVI
was similar to that found in their wild-type counterparts (Table 1).
Because granule secretion is downstream of the α6β1→GPVI/FcRγ chain→Syk signaling
pathway, we next examined whether the time-course of phosphorylation of these proteins
might be influenced by the presence or absence of PECAM-1. As shown in the top panel of
Figure 7A, phosphorylation the FcRγ-chain occurred earlier in PECAM-1-/- versus
PECAM-1+/+ platelets following exposure to immobilized laminin. The same was found for
Syk (panels of Figure 7). Taken together with the results presented in Figure 6, these data
suggest that the threshold for laminin-induced platelet activation is normally suppressed by
PECAM-1, similar to that previously observed for platelet responses to collagen.
NIH-PA Author Manuscript
DISCUSSION
The extracellular matrix is a complex, interdigitating network comprised of
glycosaminoglycans, structural proteins like collagen and elastin, and multi-adhesive
proteins such as fibronectin and laminin. In addition to serving as a repository for cytokines
and growth factors that initiate intracellular signaling, and as a structural scaffold that
maintains tissue integrity, the matrix also provides a substrate for cell migration and
adhesion. As a major constituent of the basement membrane – a specialized sheet-like
structure that either surrounds individual cells or is interposed underneath epithelial and
endothelial sheets - laminin helps to anchor the matrix to surrounding cells via its
association with cell-surface integrins. Of the 24 known integrin pairs, only α3β1, α6β1,
α7β1, and α6β4 have been shown to bind laminin [25], and of these, the only laminin
receptor expressed on the platelet surface is α6β1 [26], and this integrin has been found to be
one of several that are required for efficient platelet adhesion at sites of vascular injury [27].
NIH-PA Author Manuscript
Laminin was originally isolated in 1979 from a mouse tumor that produced basement
membrane proteins [28], however rather than existing as a single entity, laminin is now
known to be comprised of a family of proteins consisting of α, β, and γ subunits that
assemble into a disulfide-linked, cruciform shaped heterotrimers capable of interacting with
other extracellular matrix components such as sulfated lipids, heparan sulfate proteoglycans,
and collagen [1]. The 5 known α, 3β, and 3γ chains assemble into at least 15 different,
tissue-specific laminin trimers that are named according to their chain composition [29].
Thus, α1β1γ1 is known as laminin 111, α5β1γ1 as 511, and so on. Early studies examining
the ability of platelets to bind to and spread on laminin almost universally employed laminin
111 [21,22,26,30], and while Mg++-dependent adhesion to immobilized laminin has been
commonly observed, the ability to form filopodia and lamellipodia and spread on
immobilized laminin appears to take place only under conditions in which the Mg++
concentration is 1 mM or greater (references[19,22], and this report). In addition, despite the
fact that many studies have examined the interaction of platelets with immobilized laminin
111, the expression of this isoform is highly-restricted, and is in fact not present in the walls
of most blood vessels [1,31]. Laminins 411 and 511, on the other hand, are much more
widely-distributed, produced and secreted by endothelial cells into their basement
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 7
NIH-PA Author Manuscript
membranes, and stored in platelet α-granules [1,30-32]. Placenta is also a rich source of
laminin 511, highly adhesive to platelets [33], and is the source of laminin used in more
recent platelet activation studies [19,33], including this one.
The major findings of the current investigation are that, in addition to the activation events
that have been described in many studies to take place following platelet exposure to
immobilized laminin [19,21,22,26,30,32,33], a negative feedback inhibitory pathway
mediated by PECAM-1 also becomes enlisted, most likely to moderate the effects of
laminin-induced platelet activation in the absence of overwhelming exposure to this
extracellular matrix protein. Thus, following exposure of platelets to immobilized laminin
511 in the presence of 2 mM MgCl2, PECAM-1 was found to become tyrosine
phosphorylated on its cytoplasmic ITIM tyrosines and recruit the protein-tyrosine
phosphatase SHP-2 to the inner face of the plasma membrane (Figures 2 and 3), where it
presumably downregulates the GPVI/FcRγ chain→Syk activation pathway (Figure 7)
leading to dampened granule secretion (Figure 6). These findings, therefore, expand the
scope of PECAM-1's regulatory function to include not only platelet activation by collagen
[14-17] and VWF [34], but now laminin as well.
NIH-PA Author Manuscript
All the experiments were performed under static conditions, and therefore the kinetics of
platelet activation, and of PECAM-1-regulation of this process differ from those found
under in vivo conditions of flow. For example, laminin-induced tyrosine phosphorylation of
PECAM-1, Syk, and the FcRγ chain was observed only after 15-30 minutes of incubation in
microtiter wells (Figures 2 and 7), while we have previously shown that PECAM-1 exerts
its inhibitory effect on thrombus formation in vivo in only 5-10 minutes following vascular
injury. This no doubt reflects the time needed for enough platelets to settle onto the
immobilized matrix under static conditions to measure their cumulative activation, while in
vivo, platelets are continuously forced onto exposed thrombogenic surfaces by the
abundance of red cells that force their margination, resulting in a much faster activation
response. The reductionist approach taken in the present manuscript to examine regulation
of adhesion-initiated signal amplification and control under static conditions, while perhaps
not fully able to mimic kinetics that take place in the vasculature, nonetheless reveal the
contribution of laminin to platelet granule secretion, and the ability of PECAM-1 to regulate
this process.
NIH-PA Author Manuscript
Given that collagen and laminin each employ GPVI to activate platelets, why are all of the
effects of PECAM-1 on collagen-induced platelet activation not also seen with laminin? The
answer is likely due to the multivalent nature of collagen, which is able to activate platelets
in solution, whereas soluble laminin, a much weaker agonist, is without effect unless first
immobilized. Thus, while we and others have shown that PECAM-1 dampens low-dose
collagen- or CRP-induced platelet aggregation as well as dense granule secretion [14,35], in
the present manuscript we were only able to evaluate its effects on granule secretion and a
two key upstream signaling events leading to granule secretion.
A common theme emerging in the field of platelet activation is that large, adhesive ligands
present in the extracellular matrix engage platelet receptors that are able to co-opt one or
more transmembrane adaptor molecules that contain cytoplasmic ITAM tyrosine residues.
These tyrosines become phosphorylated by Src-family kinases shortly after platelets
encounter one or more components of the extracellular matrix, resulting in the assembly of
kinase-containing protein complexes that mediated feed-forward amplification loops. ITIMbearing receptors, due to their ability to recruit tyrosine and lipid phosphatases, have been
increasingly observed to moderate such activation events [36-38], and PECAM-1 regulation
of laminin-induced platelet activation is therefore just the latest example. Future
characterization of activatory/inhibitory receptor pairs involved in the regulation of
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 8
thrombosis, hemostasis and the inflammatory response may yield important new clues
leading to improved intervention and management of these clinically important conditions.
NIH-PA Author Manuscript
Acknowledgments
This work was supported by Program Project Grant HL-44612 (to PJN and DKN) from the National Heart, Lung,
and Blood Institute of the National Institutes of Health.
References
NIH-PA Author Manuscript
NIH-PA Author Manuscript
1. Scheele S, Nystrom A, Durbeej M, Talts JF, Ekblom M, Ekblom P. Laminin isoforms in
development and disease. J Mol Med. 2007; 85:825–36. [PubMed: 17426950]
2. Jackson SP. The growing complexity of platelet aggregation. Blood. 2007; 109:5087–95. [PubMed:
17311994]
3. Newman, PJ.; Newman, DK. Platelets and the Vessel Wall. 7th Edition. 2008. p. 1378-98.
4. Watson SP, Auger JM, McCarty OJ, Pearce AC. GPVI and integrin αIIb β3 signaling in platelets. J
Thromb Haemost. 2005; 3:1752–62. [PubMed: 16102042]
5. Newton-Nash DK, Newman PJ. A new role for PECAM-1 (CD31): Inhibition of TCR-mediated
signal transduction. J Immunol. 1999; 163:682–8. [PubMed: 10395658]
6. Tada Y, Koarada S, Morito F, Ushiyama O, Haruta Y, Kanegae F, Ohta A, Ho A, Mak TW,
Nagasawa K. Acceleration of the onset of collagen-induced arthritis by a deficiency of platelet
endothelial cell adhesion molecule 1. Arthritis Rheum. 2003; 48:3280–90. [PubMed: 14613294]
7. Maas M, Stapleton M, Bergom C, Mattson DL, Newman DK, Newman PJ. Endothelial cell
PECAM-1 confers protection against endotoxic shock. Am J Physiol Heart Circ Physiol. 2005;
288:H159–H164. [PubMed: 15319204]
8. Carrithers M, Tandon S, Canosa S, Michaud M, Graesser D, Madri JA. Enhanced susceptibility to
endotoxic shock and impaired STAT3 signaling in CD31-deficient mice. Am J Pathol. 2005;
166:185–96. [PubMed: 15632011]
9. Goel R, Boylan B, Gruman L, Newman PJ, North PE, Newman DK. The proinflammatory
phenotype of PECAM-1-deficient mice results in atherogenic diet-induced steatohepatitis. Am J
Physiol Gastrointest Liver Physiol. 2007; 293:G1205–G1214. [PubMed: 17932230]
10. Bird IN, Taylor V, Newton JP, Spragg JH, Simmons DL, Salmon M, Buckley CD. Homophilic
PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading
or migration. J Cell Sci. 1999; 112:1989–97. [PubMed: 10343075]
11. Noble KE, Wickremasinghe RG, DeCornet C, Panayiotidis P, Yong KL. Monocytes stimulate
expression of the Bcl-2 family member, A1, in endothelial cells and confer protection against
apoptosis. J Immunol. 1999; 162:1376–83. [PubMed: 9973392]
12. Evans PC, Taylor ER, Kilshaw PJ. Signaling through CD31 protects endothelial cells from
apoptosis. Transplantation. 2001; 71:457–60. [PubMed: 11233910]
13. Gao C, Sun W, Christofidou-Solomidou M, Sawada M, Newman DK, Bergom C, Albelda SM,
Matsuyama S, Newman PJ. PECAM-1 functions as a specific and potent inhibitor of
mitochondrial-dependent apoptosis. Blood. 2003; 102:169–79. [PubMed: 12649141]
14. Patil S, Newman DK, Newman PJ. Platelet endothelial cell adhesion molecule-1 serves as an
inhibitory receptor that modulates platelet responses to collagen. Blood. 2001; 97:1727–32.
[PubMed: 11238114]
15. Jones KL, Hughan SC, Dopheide SM, Farndale RW, Jackson SP, Jackson DE. Platelet endothelial
cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions. Blood. 2001;
98:1456–63. [PubMed: 11520795]
16. Cicmil M, Thomas JM, Leduc M, Bon C, Gibbins JM. Platelet endothelial cell adhesion
molecule-1 signaling inhibits the activation of human platelets. Blood. 2002; 99:137–44.
[PubMed: 11756163]
17. Falati S, Patil S, Gross PL, Stapleton M, Merrill-Skoloff G, Barrett NE, Pixton KL, Weiler H,
Cooley B, Newman DK, Newman PJ, Furie BC, Furie B, Gibbins JM. Platelet PECAM-1 inhibits
thrombus formation in vivo. Blood. 2006; 107:535–41. [PubMed: 16166583]
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 9
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
18. Dhanjal TS, Ross EA, Auger JM, McCarty OJ, Hughes CE, Senis YA, Watson SP. Minimal
regulation of platelet activity by PECAM-1. Platelets. 2007; 18:56–67. [PubMed: 17365855]
19. Inoue O, Suzuki-Inoue K, McCarty OJ, Moroi M, Ruggeri ZM, Kunicki TJ, Ozaki Y, Watson SP.
Laminin stimulates spreading of platelets through integrin α6β1-dependent activation of GPVI.
Blood. 2006; 107:1405–12. [PubMed: 16219796]
20. Sun J, Williams J, Yan H-C, Amin KM, Albelda SM, DeLisser HM. Platelet Endothelial Cell
Adhesion Molecule-1 (PECAM-1) homophilic adhesion is mediated by immunoglobulin-like
domains 1 and 2 and depends on the cytoplasmic domain and the level of surface expression. J
Biol Chem. 1996; 271:18561–70. [PubMed: 8702505]
21. Ill CR, Engvall E, Ruoslahti E. Adhesion of platelets to laminin in the absence of activation. J Cell
Biol. 1984; 99:2140–5. [PubMed: 6501416]
22. Hindriks G, Ijsseldijk MJ, Sonnenberg A, Sixma JJ, de Groot PG. Platelet adhesion to laminin: role
of Ca2+ and Mg2+ ions, shear rate, and platelet membrane glycoproteins. Blood. 1992; 79:928–35.
[PubMed: 1737101]
23. Jackson DE, Ward CM, Wang R, Newman PJ. The protein-tyrosine phosphatase SHP-2 binds
PECAM-1 and forms a distinct signaling complex during platelet aggregation. Evidence for a
mechanistic link between PECAM-1 and integrin-mediated cellular signaling. J Biol Chem. 1997;
272:6986–93. [PubMed: 9054388]
24. Sonnenberg A, Janssen H, Hogervorst F, Calafat J, Hilgers J. A complex of platelet glycoproteins
Ic and IIa identified by a rat monoclonal antibody. J Biol Chem. 1987; 262:10376–83. [PubMed:
3301835]
25. Nishiuchi R, Takagi J, Hayashi M, Ido H, Yagi Y, Sanzen N, Tsuji T, Yamada M, Sekiguchi K.
Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of lamininintegrin interactions using recombinant α3β1, α6β1, α7β1 and α6β4 integrins. Matrix Biol. 2006;
25:189–97. [PubMed: 16413178]
26. Sonnenberg A, Modderman PW, Hogervorst F. Laminin receptor on platelets is the integrin
VLA-6. Nature. 1988; 336:487–9. [PubMed: 2973567]
27. Gruner S, Prostredna M, Schulte V, Krieg T, Eckes B, Brakebusch C, Nieswandt B. Multiple
integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury
in vivo. Blood. 2003
28. Timpl R, Rohde H, Robey PG, Rennard SI, Foidart JM, Martin GR. Laminin--a glycoprotein from
basement membranes. J Biol Chem. 1979; 254:9933–7. [PubMed: 114518]
29. Aumailley M, Bruckner-Tuderman L, Carter WG, Deutzmann R, Edgar D, Ekblom P, Engel J,
Engvall E, Hohenester E, Jones JC, Kleinman HK, Marinkovich MP, Martin GR, Mayer U,
Meneguzzi G, Miner JH, Miyazaki K, Patarroyo M, Paulsson M, Quaranta V, Sanes JR, Sasaki T,
Sekiguchi K, Sorokin LM, Talts JF, Tryggvason K, Uitto J, Virtanen I, von der MK, Wewer UM,
Yamada Y, Yurchenco PD. A simplified laminin nomenclature. Matrix Biol. 2005; 24:326–32.
[PubMed: 15979864]
30. Geberhiwot T, Ingerpuu S, Pedraza C, Neira M, Lehto U, Virtanen I, Kortesmaa J, Tryggvason K,
Engvall E, Patarroyo M. Blood platelets contain and secrete laminin-8 (α4β1γ1) and adhere to
laminin-8 via α6β1 integrin. Exp Cell Res. 1999; 253:723–32. [PubMed: 10585296]
31. Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, Sanes JR. The
laminin α chains: expression, developmental transitions, and chromosomal locations of α1-5,
identification of heterotrimeric laminins 8-11, and cloning of a novel α3 isoform. J Cell Biol.
1997; 137:685–701. [PubMed: 9151674]
32. Chang JC, Chang HH, Lin CT, Lo SJ. The integrin α6β1 modulation of PI3K and Cdc42 activities
induces dynamic filopodium formation in human platelets. J Biomed Sci. 2005; 12:881–98.
[PubMed: 16228294]
33. Nigatu A, Sime W, Gorfu G, Geberhiwot T, Anduren I, Ingerpuu S, Doi M, Tryggvason K,
Hjemdahl P, Patarroyo M. Megakaryocytic cells synthesize and platelets secrete α5-laminins, and
the endothelial laminin isoform laminin 10 (α5β1β1) strongly promotes adhesion but not activation
of platelets. Thromb Haemost. 2006; 95:85–93. [PubMed: 16543966]
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 10
NIH-PA Author Manuscript
34. Rathore V, Stapleton MA, Hillery CA, Montgomery RR, Nichols TC, Merricks EP, Newman DK,
Newman PJ. PECAM-1 negatively regulates GPIb/V/IX signaling in murine platelets. Blood.
2003; 102:3658–64. [PubMed: 12893757]
35. Wilkinson R, Lyons AB, Roberts D, Wong MX, Bartley PA, Jackson DE. Platelet endothelial cell
adhesion molecule-1 (PECAM-1/CD31) acts as a regulator of B-cell development, B-cell antigen
receptor (BCR)-mediated activation, and autoimmune disease. Blood. 2002; 100:184–93.
[PubMed: 12070026]
36. Unkeless JC, Jin J. Inhibitory receptors, ITIM sequences and phosphatases. Curr Opin Immunol.
1997; 9:338–43. [PubMed: 9203414]
37. Bolland S, Ravetch JV. Inhibitory pathways triggered by ITIM-containing receptors. Adv
Immunol. 1999; 72:149–77. [PubMed: 10361574]
38. Gibbins JM. The negative regulation of platelet function: extending the role of the ITIM. Trends
Cardiovasc Med. 2002; 12:213–9. [PubMed: 12161075]
NIH-PA Author Manuscript
NIH-PA Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 11
NIH-PA Author Manuscript
Figure 1. Human platelets adhere to immobilized laminin and collagen, but spread with different
morphology
(A) Visualization of platelets bound to immobilized matrix proteins. Washed human
platelets resuspended at 1 × 108/mL in 2 mM MgCl2 and 0.5 mM CaCl2 were seeded on
surfaces pre-coated with either human placental laminin 511 or soluble calf-skin collagen.
After 30 minutes at 37°C, unbound platelets were removed, adherent platelets fixed with 3%
paraformaldehyde, and visualized using Hoffman modulation contrast. Results shown are
representative of three separate experiments. Note that platelets bind well to immobilized
laminin, but do not spread as well as they do on immobilized collagen. (B) Quantitation of
platelet adhesion to immobilized matrix proteins. Washed human platelets were loaded with
Calcein AM and seeded onto microtiter wells that had been pre-coated with the indicated
concentrations of human laminin or collagen. After a 60 minute incubation at 37°C, the
percentage of adherent platelets was calculated by taking fluorescence measurements both
before and after removal of unbound platelets. Data shown represents mean values ± SEM
from three independent experiments performed using triplicate wells.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 12
NIH-PA Author Manuscript
Figure 2. PECAM-1 becomes tyrosine phosphorylated following adhesion of human and mouse
platelets to immobilized laminin
Washed platelets were resuspended at a concentration of 4 × 108/mL in Tyrodes buffer
containing 2 mM MgCl2 and 0.5 mM CaCl2 and added to culture plates that had been precoated with 50 μg/mL of laminin 511. Platelets were allowed to settle, bind, and spread at
37°C for the indicated times, and then detergent lysed and subjected to immunoprecipitation
analysis using mAbs PECAM-1.3 for human platelets and 390 for murine platelets. Western
blots were developed using the anti-phosphotyrosine mAb PY20 (top panels) and antiPECAM-1 polyclonal antibodies SEW32-34 (human) and M-20 (mouse) to visualize antigen
loading. Maximal phosphorylation of PECAM-1 at the 45 minute time point reflects the
time needed for platelets to settle, make contact with the immobilized laminin, and activate
the platelets. The result shown is representative of three separate experiments.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 13
NIH-PA Author Manuscript
Figure 3. Tyrosine-phosphorylated PECAM-1 recruits SHP-2 following adhesion to immobilized
laminin
NIH-PA Author Manuscript
Top panel: Washed human platelets were added to laminin-coated tissue culture plates as
described in the legend for Figure 2 and incubated for 30-45 minutes at 37°C. Wells coated
with 1 mg/ml BSA or 50 μg/mL collagen served as negative and positive controls,
respectively. Platelets were then lysed and subjected to immunoprecipitation/western blot
analysis as in Figure 2. SHP-2 was detected in the co-immunoprecipitates using polyclonal
antibody C-18. The result shown is representative of three individual experiments. Middle
and bottom panels: Quantitative analysis of PECAM-1 tyrosine phosphorylation and SHP-2
binding following platelet adhesion to immobilized laminin. Band intensity was determined
using a Kodak Molecular Imaging Densitometry System. PECAM-1 tyrosine
phosphorylation levels are expressed as the ratio of PY20:PECAM-1 antigen. Data were
normalized to values obtained from resting platelets adherent to BSA. Data from three
independent experiments were analyzed by the Wilcoxon rank sum test and represented as
mean ± SEM. *P < 0.05. Similar results were obtained for murine platelets (not shown).
NIH-PA Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 14
NIH-PA Author Manuscript
NIH-PA Author Manuscript
Figure 4. Rate and extent of spreading of wild-type versus PECAM-1–deficient platelets on
immobilized laminin
Washed platelets isolated from the anticoagulated whole blood of wild-type or PECAM-1–
deficient mice were seeded on laminin-coated coverslips in the presence of 2 mM Mg++ and
incubated for 15, 30, 45 or 60 minutes at 37°C. Adherent platelets were fixed,
permeabilized, stained with TRITC-labeled phalloidin, and visualized by fluorescence
microscopy. Left panels – representative images taken over a 60 minute period. Upper right
panel - Quantitation of platelet spreading from eight randomly chosen fields (~200 platelets/
field) using Metamorph software. Data represent the mean ± SEM from three independent
experiments. Statistical significance was determined using the Student t-test for unpaired
samples.
NIH-PA Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 15
NIH-PA Author Manuscript
Figure 5. PECAM-1 engagement-induced inhibitory signaling does not affect platelet adhesion or
spreading on immobilized laminin
NIH-PA Author Manuscript
(A) 3 × 108 human platelets/ml were incubated with 5 μg/mL mAb PECAM-1.3 for 10
minutes in the presence or absence of F(ab')2 fragments of anti-mouse IgG Fc and subjected
to immunoprecipitation/western blot analysis. GoH3, a specific mAb for the integrin α6
subunit, was used as a negative control. Note that binding and cross-linking of mAb
PECAM-1.3 on the platelet surface was able to induce tyrosine phosphorylation of
PECAM-1. (B) Effect of antibody-induced inhibitory signaling on platelet spreading on
immobilized laminin. Resting human platelets were treated with GoH3 or mAb PECAM-1.3
and cross-linked (Pecam-1 XL) as described in panel A and the platelets added to culture
dishes that had been the indicated concentration of laminin. After 30 minutes at 37°C,
unbound platelets were removed and the remaining bound platelets were fixed, stained, and
analyzed. Panels C and D - The number and mean surface area of adherent platelets from
three independent experiments were quantitated from 8 randomly chosen fields of 50 – 300
platelets each, analyzed using Metamorph software, and expressed as the mean ± SEM. Note
that although PECAM-1.3 was effective in initiating inhibitory signaling (panel A), this had
no effect on platelet adhesion or spreading.
NIH-PA Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 16
NIH-PA Author Manuscript
Figure 6. PECAM-1 regulates laminin-induced granule secretion
NIH-PA Author Manuscript
Washed platelets obtained from age- and sex-matched wild-type or PECAM-1-deficient
mice were labeled with 14C-serotonin and added in triplicate to wells pre-coated with either
2% BSA, 50 μg/mL of collagen, or 50 μg/mL of laminin. In some cases, platelets were
preincubated with 5 μg/mL of GoH3 10 minutes before seeding onto immobilized laminin.
After 60 minutes at 37°C, the amount of 14C-serotonin that had been released into the
culture media was determined by scintillation counting and expressed as the mean ± SEM.
Data shown is representative of two independent experiments. * P < 0.001.
NIH-PA Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 17
NIH-PA Author Manuscript
Figure 7. Early activation of the GPVI /FcRγ-chain→Syk platelet activation pathway in
PECAM-1-deficient platelets
NIH-PA Author Manuscript
Murine platelets were added to laminin-coated microtiter wells for the indicated times,
lysed, and subjected to western blot analysis using the indicated antibodies. Panels A and B PECAM-1 delays laminin-induced FcRγ-chain phosphorylation. A representative
immunoblot is shown in (A), and cumulative quantitative data derived from three
independent experiments is shown in (B). Panels C and D - PECAM-1 affects the kinetics of
laminin-induced Syk phosphorylation. A representative immunoblot is shown in (C), with
cumulative quantitative data derived from three independent experiments is shown in (D).
Note that PECAM-1 appears to set a higher threshold for platelet activation, as both the
FcRγ chain and Syk become phosphorylated earlier in PECAM-1-deficient platelets.
NIH-PA Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.
Crockett et al.
Page 18
Table 1
Expression of select membrane glycoproteins on the surface of wild-type and PECAM-1-deficient platelets
NIH-PA Author Manuscript
Glycoprotein
PECAM-1+/+ platelets
PECAM-1-/- platelets
Integrin α6β1
32 ± 3
28 ± 1
Integrin α2β1
60 ± 13
61 ± 20
GPVI
46 ± 2
44 ± 2
PECAM-1
21 ± 4
2±2
*Washed platelets at 1 × 108/ml were incubated with 5 μg/ml of the indicated antibody for 15 min at room temperature and then analyzed on a BD
LSR II flow cytometer. Values shown are the median fluorescence intensity of triplicate determinations. With the exception of PECAM-1, none of
the other differences were significant.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
J Thromb Haemost. Author manuscript; available in PMC 2011 July 1.