ADAP is required for normal alphaIIbbeta3 activation by VWF/GP Ib-IX-V and other agonists.

Abstract

Interaction between von Willebrand factor (VWF) and platelet GP Ib-IX-V is required for hemostasis, in part because intracellular signals from VWF/GP Ib-IX-V activate the ligand-binding function of integrin alphaIIbbeta3. Because they also induce tyrosine phosphorylation of the ADAP adapter, we investigated ADAP's role in GP Ib-IX-V signal transduction. Fibrinogen or ligand-mimetic POW-2 Fab binding to alphaIIbbeta3 was stimulated by adhesion of ADAP+/+ murine platelets to dimeric VWF A1A2 but was significantly reduced in ADAP-/- platelets (P<.01). alphaIIbbeta3 activation by ADP or a Par4 thrombin receptor agonist was also decreased in ADAP-/- platelets. ADAP stabilized the expression of another adapter, SKAP-HOM, via interaction with the latter's SH3 domain. However, no abnormalities in alphaIIbbeta3 activation were observed in SKAP-HOM-/- platelets, which express normal ADAP levels, further implicating ADAP as a modulator of alphaIIbbeta3 function. Under shear flow conditions over a combined surface of VWF A1A2 and fibronectin to test interactions involving GP Ib-IX-V and alphaIIbbeta3, respectively, ADAP-/- platelets displayed reduced alphaIIbbeta3-dependent stable adhesion. Furthermore, ADAP-/- mice demonstrated increased rebleeding from tail wounds. These studies establish ADAP as a component of inside-out signaling pathways that couple GP Ib-IX-V and other platelet agonist receptors to alphaIIbbeta3 activation.

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HEMOSTASIS, THROMBOSIS,AND VASCULAR BIOLOGY
ADAPisrequiredfornormal?IIb?3activationbyVWF/GPIb-IX-V
andotheragonists
Ana Kasirer-Friede,1Barry Moran,1Jennifer Nagrampa-Orje,2Ken Swanson,3Zaverio M. Ruggeri,2Burkhart Schraven,4
Benjamin G. Neel,3Gary Koretzky,5and Sanford J. Shattil1
1Department of Medicine, University of California San Diego, La Jolla;2Department of Molecular and Experimental Medicine, The Scripps Research Institute, La
Jolla, CA;3Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA;4Institute of Immunology, University of
Magdeburg, Germany;5Abramson Family Cancer Research Institute and Division of Rheumatology, University of Pennsylvania, Philadelphia
Interaction between von Willebrand fac-
tor (VWF) and platelet GP Ib-IX-V is re-
quired for hemostasis, in part because
intracellular signals from VWF/GP Ib-IX-V
activate the ligand-binding function of
integrin ?IIb?3. Because they also induce
tyrosine phosphorylation of the ADAP
adapter, we investigated ADAP’s role in
GP Ib-IX-V signal transduction. Fibrino-
gen or ligand-mimetic POW-2 Fab binding
to ?IIb?3 was stimulated by adhesion of
ADAP?/?murine platelets to dimeric VWF
A1A2 but was significantly reduced in
ADAP?/?platelets(P < .01).?IIb?3activa-
tion by ADP or a Par4 thrombin receptor
agonist was also decreased in ADAP?/?
platelets. ADAP stabilized the expression
of another adapter, SKAP-HOM, via inter-
action with the latter’s SH3 domain. How-
ever, no abnormalities in ?IIb?3 activa-
tion were observed in SKAP-HOM?/?
platelets, which express normal ADAP
levels, further implicating ADAP as a
modulatorof?IIb?3function.Undershear
flow conditions over a combined surface
of VWFA1A2 and fibronectin to test inter-
actions involving GP Ib-IX-V and ?IIb?3,
respectively, ADAP?/?platelets displayed
reduced ?IIb?3-dependent stable adhe-
sion. Furthermore,ADAP?/?mice demon-
strated increased rebleeding from tail
wounds. These studies establish ADAP
as a component of inside-out signaling
pathwaysthatcoupleGPIb-IX-Vandother
plateletagonistreceptorsto?IIb?3activa-
tion. (Blood. 2007;109:1018-1025)
© 2007 by TheAmerican Society of Hematology
Introduction
Binding of von Willebrand factor (VWF) to GP Ib-IX-V is
important for the initial capture of platelets to the extracellular
matrix of injured blood vessels at high shear rates.1-3GPIb-IX-V is
a complex of 4 transmembrane polypeptides.4-6While the cytoplas-
mic tail of each subunit lacks catalytic function, each may interact
directly or indirectly with proteins that can transmit intracellular
signals. For example, the cytoplasmic tail of GP Ib? can interact
directly with filamin; GP Ib? and Ib? tails can interact with
14-3-3-?; and GP Ib? and GP V tails can interact with calmodu-
lin.7-10GP Ib-IX-V can be coimmunoprecipitated from platelets
withsignalingmolecules,includingSrcfamilykinases,11phosphati-
dyl inositol (PI) 3-kinase,12and SHIP-2.13Furthermore, VWF-
dependent platelet activation may require localization of GP
Ib-IX-V to lipid rafts, membrane structures implicated in cellular
signaling.14In a recent study, we demonstrated that the interaction
of GP Ib-IX-V with the dimericA1 domain of VWF is sufficient to
trigger a sequence of reactions in platelets that leads to activation of
?IIb?3 (inside-out signaling), even when costimulatory inputs
from other agonist receptors are blocked.15These reactions include
activation of Src family kinases, intracellular Ca2?oscillations,
and the actions of PI 3-kinase and protein kinase C. An unantici-
pated finding was the tyrosine phosphorylation of ADAP, a Src
substrate not previously recognized as a potential effector in GP
Ib-IX-V signaling.
ADAP (adhesion and degranulation promoting adapter protein;
previously called Fyb or SLAP-130) is a variably spliced 120 or
130 kDa protein expressed in T cells and other hematopoietic
cells.16-18Notable domains or motifs in ADAP include 2 potential
nuclear localization sequences16; polyproline-rich regions in the
amino terminal half of the molecule that can interact with the SH3
domains of SKAP5519and SKAP-HOM20,21; and several tyrosines,
including Y595 and Y651, which when phosphorylated enable
binding to the SH2 domains of SLP-76 or Fyn in T cells.17,18,22
ADAP can potentially modulate cytoskeletal interactions through
an FP4 motif that recognizes the EVH1 domain of VASP23and a
polyproline-richregionthatrecognizestheSH3domainofmamma-
lian actin binding protein 1 (mAbp1).24Furthermore, ADAP can
coimmunoprecipitate with WASPandArp 2/3, known regulators of
actin polymerization.23,25Thus, ADAP is a candidate linker be-
tweencellactivationeventsattheplasmamembraneandintracellu-
lar events that effect changes in actin polymerization, dynamics,
and organization.
Studies in ADAP knockout mice or ADAP-deficient T cells
have indicated a necessary role forADAPin the regulation ofT-cell
proliferation and IL-2 production26and the promotion of cell
adhesion through clustering and valency modulation of ?1 and ?2
integrins.27-32However, no information exists as to whetherADAP
is necessary for integrin affinity modulation in general or for
?IIb?3 function in platelets. Given the aforementioned tyrosine
phosphorylation of ADAP in response to engagement of GP
Ib-IX-V,15we investigated here whether this adapter is required for
GP Ib-IX-V–triggered activation of ?IIb?3. The results using
Submitted May 9, 2006; accepted September 9, 2006. Prepublished online as
Blood First Edition Paper, September 26, 2006; DOI 10.1182/blood-2006-05-
022301.
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 USC section 1734.
© 2007 by TheAmerican Society of Hematology
1018BLOOD, 1 FEBRUARY 2007?VOLUME 109, NUMBER 3

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ADAP?/?mice establish that ADAP is involved in receptor-
mediated affinity modulation of ?IIb?3, with consequences for
platelet adhesive function ex vivo and in vivo.
Materials and methods
Reagents and antibodies
Preparation of dimeric, murineA1A2VWF (dmA1A2VWF) was similar to
that of human dA1 VWF33except that it encompassed residues 445 to 909
of murine VWF. POW-2, an antibody Fab fragment to murine activated
?IIb?3, has been described.34-36Monoclonal antibody 1B5 against murine
?IIb?3 was from Dr Barry Coller (Rockefeller University, New York,
NY)37; polyclonal antibodies developed against the amino-terminus of
murine ADAP and SKAP-HOM have been described17,20,38,39; monoclonal
antibodies against murine CD62P (P selectin) and anti-CD41(?IIb) were
from BD Biosciences (San Diego, CA); and a monoclonal antibody against
murineGPIb?wasfromEmfretAnalytics(Eibelstadt,Germany).Fibronec-
tin was from EMD Biosciences (San Diego, CA) and fibrinogen from
Enzyme Research Laboratories (South Bend, IN). QiaAMP DNAMini Kit,
plasmid vectors pcDNA 3.1 and pIRESeEGFP, and Lipofectamine were
from Invitrogen (Carlsbad, CA). Convulxin was from Alexis Biochemicals
(Lausen, Switzerland). PPACK was from Haematologic Technologies
(Essex Junction, VT). SuperSignal WestPico reagent was from Pierce
Chemicals (Rockford, IL). All other reagents were from Sigma Chemical
(St Louis, MO).
Mouse strains and bleeding times
ADAP?/?and SKAP-HOM?/?mice have been described.28,40ADAP?/?
and SKAP-HOM?/?mice represent littermate controls. Bleeding time
assays were performed by placing the tails of mice in a Plexiglas restraining
device, prewarming the tails to 37°C in normal saline, and cutting
transversely 0.5 cm from the tip.After immediate reimmersion in the saline
solution, the time to arrest of bleeding was noted. Tail bleeding was further
monitored for at least an additional 60 seconds to determine if rebleeding
occurred. Statistical analyses for bleeding times and rebleeding times were
performed using the Student t test and the ?2test, respectively.
Platelet preparation
Mouse blood was obtained by cardiac puncture and diluted with an equal
volume of modified Tyrode buffer (140 mM NaCl, 2.7 mM KCl, 0.4 mM
NaH2PO4.H2O, 10 mM NaHCO3, 5 mM dextrose, pH 6.5) containing
acid-citrate dextrose anticoagulant and 1.5 U/mL apyrase.After centrifuga-
tion for 4 minutes at 450g, the platelet-rich plasma was removed,
centrifuged for 15 minutes at 800g in the presence of 0.5 U/mLapyrase and
1 ?M PGE1, and platelet pellets were resuspended inWalsh buffer (137 mM
NaCl, 2.7 mM KCl, 1 mM MgCl2.6H2O, 3.3 mM NaH2PO4.H2O, 3.8 mM
HEPES, 0.1% bovine serum albumin [BSA], 0.1% dextrose, pH 7.35).41
Characterization of ADAP?/?platelets
To determine the surface expression of ?IIb?3 and GP Ib?, platelets were
incubated with an antibody against murine anti-CD41 (?IIb) or against
murine GP Ib? in Tyrode buffer (137 mM NaCl, 12 mM NaHCO3, 26 mM
KCl, 5.5 mM glucose, 0.1% BSA, 5.0 mM Hepes, 0.5 mM CaCl2,1 mM
MgCl2, pH 7.4) for 30 minutes. Platelet suspensions were diluted with
buffer, and the amount of antibody bound to platelets was determined by
flow cytometry. CD62P (P selectin) surface expression, reporting on
?-granule secretion, was determined by flow cytometric analysis of binding
of an antibody against murine CD62P in platelets that were resting or
stimulated with 250 ?M Par4 receptor-activating peptide (AYPGKF).
Statistical analyses were performed using the Student t test.
Measurements of ?IIb?3 affinity state
Binding of FITC-fibrinogen or POW-2 Fab41to mouse platelets adherent
to dmA1A2 VWF was evaluated by deconvolution microscopy. Glass
coverslips were coated with 15 ?g/mL dmA1A2 VWF for 1 hour at
room temperature and blocked with 1% denatured BSA. Platelets were
added to the coverslips for 40 minutes at 37°C, routinely in the presence
of FITC-fibrinogen or POW-2 Fab, 2 U/mL apyrase, 10 ?M indometha-
cin, and a phycoerythrin-conjugated, noninhibitory antibody to ?3, the
latter to facilitate discrimination of the cell outline. Nonspecific
fibrinogen or POW-2 Fab binding was assessed in the presence of 5 mM
EDTA. Adherent platelets were washed gently with phosphate-buffered
saline, fixed with 3.7% formaldehyde, and stained with Alexa-488–
conjugated anti–murine H ? L chain–specific IgG (for POW-2 Fab).
Images were captured with a Deltavision deconvolution microscope
(Applied Precision, Issaquah, WA) using a Photometrics (Tucson, AZ)
Sony Coolsnap HQ charged-coupled device camera system (Sony, Park
Ridge, NJ) attached to an inverted, wide-field fluorescence microscope
(Nikon [Melville, NY] TE-200) equipped with a 100? Nikon (NA 1.4)
oil-immersion objective. Identical settings were used for slides from all
experimental conditions. Platelet fluorescence intensity range was
standardized in deconvolution micrographs using the Softworks Analy-
sis Program (Applied Precision). Single-cell analysis was performed by
calculating fluorescence intensities for FITC-fibrinogen bound to each
cell using Image-Pro Plus Software (Media Cybernetics, Silver Spring,
MD). The mean for each group was determined from data derived from
50 to 300 cells per experimental condition for both ADAP?/?and
ADAP?/?platelets. Results are expressed as the percentage of average
ADAP?/?fluorescence ? standard error of the mean (SEM). To
determine the role of ADAP in ?IIb?3 activation through agonists other
than GP Ib-IX-V, platelets from ADAP?/?and ADAP?/?mice were
stimulated with varying concentrations of ADP, Par4-activating pep-
tide,42or convulxin in the presence of FITC-fibrinogen, and bound
FITC-fibrinogen was assessed by flow cytometry at equilibrium binding
conditions. Platelet aggregation was measured as previously described.43
Statistical analyses were performed using the Student t test.
Platelet adhesion under flow conditions
Whole blood was drawn from the retro-orbital plexus and anticoagulated
with 40 ?g/mL PPACK. Platelet number was adjusted in blood from
ADAP?/?mice to correspond to the reduced counts in ADAP?/?mice
by centrifuging for 8 minutes at 750g and carefully depleting platelets
from the buffy coat, thus maintaining hematocrit. Remaining cells and
plasma were reblended very gently. Glass coverslips were coated with
either 20 ?g/mL dmA1A2 VWF, 25 ?g/mL fibronectin, or a combina-
tion of the two for 45 minutes at room temperature, rinsed with buffer
containing 20 mM HEPES and 150 mM NaCl, and mounted in a parallel
plate flow chamber maintained at 37°C. Platelet dense granules were
labeled by addition of 20 ?g/mL mepacrine to whole blood to allow
platelet visualization with an inverted epifluorescence microscope.1
Blood was aspirated through the chamber at a shear rate of 1500 s?1. No
platelets were in contact with the surface prior to initiation of shear flow.
Analog videotaped images of platelets directly captured to the surface
from shear flow were subsequently digitized using Adobe Premiere
(Adobe Systems, San Jose, CA) and analyzed using Metamorph
software (Universal Imaging, West Chester, PA). The number of
platelets that remain stationary (displaced by less than 1 cell diameter)
for the specified time period was calculated and expressed as a
percentage of the total number of platelets contacting the surface over
the period of observation. Data are shown for platelets adherent at 20
seconds, 1 minute, or 2 minutes following initiation of flow. From 800 to
1500 platelets were analyzed for each data point shown. Following flow,
platelets were fixed in situ by flowing 3.7% formaldehyde, rinsed,
permeabilized, and stained with a noninhibitory, FITC-conjugated
antibody against ?IIb.
Studies of ADAP and SKAP-HOM in a CHO cell model system
A5 CHO cells that stably express ?IIb?3 were a gift from Dr M.
Ginsberg (University of California San Diego) and cultured as de-
scribed.44The following mammalian expression plasmids were used:
pEF-BOS/Flag-tagged SKAP-HOM20and pEF/Flag-tagged ADAP.17
ADAP IN ?IIb?3ACTIVATION1019BLOOD, 1 FEBRUARY 2007?VOLUME 109, NUMBER 3

Page 3

The SKAP-HOM plasmid was used as a polymerase chain reaction
(PCR) template to obtain full-length SKAP-HOM and SKAP-HOM
lacking the amino terminal SH3 domain, and PCR products were
subcloned into pIRES2-EGFP (Invitrogen) at 5? XhoI and 3? SacI
restriction sites. Coding sequences were confirmed by automated DNA
sequencing. For transfection, subconfluent cells in 100 mm plates were
transfected with a maximum of 6 ?g plasmid DNA using Lipofectamine
according to the manufacturer’s instructions (Invitrogen). When re-
quired, pcDNA3.1 (Invitrogen) was used to maintain equal quantities of
cDNA per transfection. Cells were examined for ADAP and SKAP-
HOM expression 48 hours after transfection, a time point verified to
produce maximal recombinant protein levels.
Results and discussion
ADAP-deficient mice rebleed from tail wounds
To address the role of ADAP in platelet function, we studied
mice deficient in this protein. Tail bleeding times in the mouse
can be prolonged if there are coagulation abnormalities or
platelet dysfunction, but not mild thrombocytopenia,45although
subtle defects in platelet function may yield only an increase in
tail rebleeding frequency due to thrombus fragility.43ADAP?/?
mice exhibited bleeding times comparable to their ADAP?/?
littermates (Figure 1A), confirming that the mild thrombocytope-
nia in these mice28was not itself sufficient to prolong bleeding.
However, whereas only 21.9% of ADAP?/?mice showed
rebleeding from tail wounds within 1 minute of initial arrest,
61.8% of ADAP?/?mice rebled (P ? .01 in a ?2test) (Figure
1B). Plasma from ADAP?/?mice showed normal thrombin and
activated partial thromoboplastin times (not shown), suggesting
that the higher rebleeding rate in ADAP?/?mice was likely due
to platelet dysfunction.
Characterization of ADAP?/?platelets
ExpressionofGPIb-IX-Vand?IIb?3wascomparableinADAP?/?
and ADAP?/?platelets as assessed by flow cytometry (Figure
2A-B). The botrocetin-mediated binding of FITC-VWF was also
comparable, indicating that GP Ib-IX-V can interact normally with
VWF (not shown). ADAP?/?platelets also exhibited normal
?-granule secretion in response to Par4-activating peptide, as
measured by surface expression of P selectin. Thus, to the extent
that ?-granule secretion may contribute to thrombus growth or
stability, a defect in this process is not likely to be the primary cause
of the increased frequency of tail rebleeding observed in the
ADAP?/?mice.
ADAP is required for normal ?IIb?3 activation
Primary hemostasis is reliant on initial capture of platelets to the
vessel wall, platelet activation, and recruitment of additional
platelets into growing thrombi. GP Ib-IX-V is important from this
standpoint, and Bernard-Soulier patients and mice deficient in GP
Ib? exhibit severe bleeding defects.46GP Ib-IX-V is the only
known receptor that can successfully mediate platelet capture
across a wide range of physiologically relevant shear stresses.47,48
Furthermore, its signaling functions are important for ?IIb?3
activation and for the resulting stable platelet adhesion and
aggregation. To investigate the contribution of ADAP to GP
Ib-IX-V–induced ?IIb?3 activation, platelets from ADAP?/?or
ADAP?/?mice were allowed to adhere to a GP Ib-IX-V–specific
ligand, dmA1A2 VWF, in the presence of soluble FITC-fibrinogen,
a ligand for activated ?IIb?3.
Apyrase and indomethacin were present to prevent costimu-
latory effects of ADP and thromboxane A2, respectively.15
Adhesion of wild-type ADAP?/?platelets to dmA1A2 VWF
induced the binding of FITC-fibrinogen, as evidenced by
patches of green fluorescence associated with the platelets
(Figure 3A). Fibrinogen binding was ?IIb?3 specific because it
was prevented by EDTA (Figure 3B) and by 1B5, a function-
blocking antibody to ?IIb?3 (data not shown). In contrast, there
was much less specific FITC-fibrinogen binding to ADAP?/?
platelets under the same conditions (Figure 3D-E). A quantita-
tive analysis indicated a statistically significant 64% ? 10%
decrease in FITC-fibrinogen binding to ADAP?/?platelets
compared with ADAP?/?platelets (P ? .01) (Figure 3G).
Addition of phorbol myristate acetate (PMA), which circum-
vents platelet agonist receptors by directly stimulating intracel-
lular mediators, such as protein kinase C49and CalDAG-GEFI,50
Figure 1. Bleeding times in wild-type or ADAP knockout mice. Tails of ADAP?/?
or ADAP?/?mice were warmed and then transected 0.5 mm from the end and
immersed in 0.9% isotonic saline solution at 37°C. Blood flow was monitored, and the
time when bleeding arrested initially was noted as the bleeding time. Tails were
monitored further for a minimum of 60 seconds to determine if bleeding recurred. (A)
Bleeding times ? standard error of the mean (SEM). (B) Percentage of mice that
showed rebleeding. Results are calculated from measurements on a total of 33
ADAP?/?and 37ADAP?/?mice.
Figure 2. Characterization of ADAP?/?platelets. Washed platelets from ADAP?/?
and ADAP?/?mice were incubated with the appropriate antibodies, and binding was
determined by flow cytometry. (A) ?IIb?3 surface expression. (B) GP Ib? surface
expression. (C) ?-granule secretion. Platelets were resting, or activated with 250 ?M
Par4-activating peptide, and the binding of an anti–murine P selectin antibody was
determined. There was no statistically significant difference in P selectin expression
between ADAP?/?and ADAP?/?platelets in response to Par4-activating peptide
despite statistically significant increases in P selectin expression compared with
resting platelets. Results shown represent a summary of at least 4 experiments and
show the average ligand binding ? standard error of the mean (SEM).
1020KASIRER-FRIEDE et al BLOOD, 1 FEBRUARY 2007?VOLUME 109, NUMBER 3

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caused increased FITC-fibrinogen binding to bothADAP?/?and
ADAP?/?platelets adherent to dmA1A2 VWF. This indicates
that ?IIb?3 is inherently functional in ADAP?/?mouse plate-
lets. PMA also induced increased platelet spreading (Figure
3C,F). To confirm that adhesion to dmA1A2 VWF stimulated an
increase in ?IIb?3 affinity, the binding of POW-2, a ligand-
mimetic, monovalent anti-?IIb?3 antibody Fab, was assessed.
Compared with ADAP?/?platelets, there was a 94% ? 5%
decrease in POW-2 binding to ADAP?/?platelets (Figure 3H;
P ? .01). Taken together, these results indicate that ADAP is
involved in the modulation of ?IIb?3 affinity in response to
ligation of GP Ib-IX-V by dmA1A2 VWF.
Because GP Ib-IX-V is important for mediating initial
platelet surface interactions under shear flow, we tested whether
ADAPwas required for activation of ?IIb?3 and stable adhesion
in platelets captured from shear flow through GP Ib-IX-V. In
preliminary experiments, we verified that adhesion and translo-
cation on dmA1A2 VWF were normal in ADAP?/?platelets,
and therefore any differences observed in stable adhesion would
not be attributable to defective VWF/GP Ib-IX-V interactions.
Blood was perfused at a wall shear rate of G ? 1500 s?1over a
surface of either dmA1A2 VWF, fibronectin, or a combination
of the two. Unlike for adhesion to immobilized fibrinogen,1
?IIb?3 must be in a high-affinity state to stably adhere to
fibronectin in shear flow.51Thus, with a mixed dmA1A2
VWF/fibronectin surface, we expected that initial platelet
contacts through the GP Ib-IX-V/VWF A1 domain interaction
would be reinforced by contacts through the ?IIb?3/fibronectin
interaction only if ?IIb?3 had become activated. Indeed, this
was the case forADAP?/?platelets, which displayed an increase
in the average time that they remained stably adherent to the
combined dmA1A2 VWF/fibronectin surface when compared
with the single dmA1A2 VWF surface (Figure 4) (P ? .05). The
function-blocking anti-?IIb?3 antibody 1B5 reduced stable
adhesion to the combined surface to levels observed with
Figure 3. Integrin activation induced by GP Ib-IX-V. Washed platelets from ADAP?/?(A-C) or ADAP?/?(D-F) mice were allowed to settle on dmA1A2 VWF. The
activation state of ?IIb?3 was reported by the binding of soluble FITC-fibrinogen (green).Anoninhibitory antibody to ?IIb?3 was used to view the cell outline (red). (Aand
D) Platelets were on dmA1A2 VWF in the presence of a cocktail of inhibitors against ADP and thromboxane A2. (B and E) As in panels A and D, with the addition of
EDTA. (C and F) Platelets were on dmA1A2 VWF and activated with PMA without inhibitors. Results shown are representative of at least 3 experiments with similar
results. (G-H) Quantification of specific, activation-dependent FITC-fibrinogen (G) or POW-2 Fab (H) binding to ?IIb?3 in response to GP Ib-IX-V ligation, as shown in
panels A to F for FITC-fibrinogen. Results for ADAP?/?(white bars) are depicted as a percentage of the soluble ligand that is specifically bound by ADAP?/?platelets
(black bars) ? SEM. Data represent a summary of 4 experiments.
Figure 4. GP Ib-IX-V–induced ?IIb?3 activation under shear flow. Whole blood
from ADAP?/?or ADAP?/?mice was adjusted to correct for thrombocytopenia of
ADAP?/?mouse blood and aspirated over a coverslip coated with dmA1A2 VWF, with
or without fibronectin (FN), and placed in a parallel plate flow chamber. The total
number of platelets tethering to the surface was measured, and the percentage of
these platelets that remained stationary for 3.5 seconds or 10 seconds was
determined at 20 seconds, 1 minute, or 2 minutes after the initiation of flow. Results
represent stationary platelets, as a percentage of the total number of platelets
contacting the surface within the sampling time, ? SEM. (A) Platelets stationary for
3.5 seconds. (B) Platelets stationary for 10 seconds. Stationary adhesion (dependent
on ?IIb?3 activation) is much higher for platelets on the combined dmA1A2 VWF/FN
surface than on dmA1A2 VWF alone. Results shown are the summary of at least 3
experiments. *P ? .05 in a Student t test for ADAP?/?platelets compared with
ADAP?/?platelets.
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dmA1A2 VWF alone (not shown) (P ? .05), confirming that
this response was ?IIb?3 dependent. Almost no platelet adhe-
sion to a surface of fibronectin alone or soluble thrombi was
observed at this shear rate, indicating that platelets that were
stably adherent had not simply become preactivated during
preparative procedures. In contrast to ADAP?/?platelets, stable
adhesion of ADAP?/?platelets to the combined surface was
impaired (Figure 4). While on the dmA1A2 VWF surface,
ADAP?/?and ADAP?/?platelets showed similar levels of
adhesion, on the combined surface with the ?IIb?3 ligand,
fibronectin, ADAP?/?platelets were less able to adhere stably.
Whereas there was a 27% to 43% decrease in the percentage of
ADAP?/?platelets stably adherent for at least 3.5 seconds
(Figure 4A), the decrease in the percentage of platelets stably
adherent for at least 10 seconds was 50% to 85% (P ? .05) at
the time points after initiation of flow shown (Figure 4B). It is
possible that multimeric VWF may have provided a better
substrate than a combined matrix of dmA1A2 VWF and
fibronectin, because GP Ib-IX-V and ?IIb?3 binding sites are
optimally present within the same molecule. However, VWF
was not used as a surface because murine GP Ib? does not
recognize human VWF directly, and murine VWF was not
available. Nevertheless, under conditions of shear flow we were
able to distinguish small but significant increases in stable
adhesion when the fibronectin ?IIb?3 ligand was presented on
the surface along with dmA1A2 VWF. Thus, the results are
consistent with a role for ADAP in inside-out signaling and
activation of ?IIb?3 initiated by platelet adhesion through GP
Ib-IX-V. The differences observed in stable adhesion between
ADAP?/?and ADAP?/?platelets may also be due in part to
differences in response to generated secondary agonists, stabili-
zation of ligand-receptor
reinforcements.52-55
One consequence of ?IIb?3 interaction with ligands such as
fibrinogen is outside-in signaling, leading to cytoskeletal rear-
rangements and the development of filopodial and lamellipodial
extensions. In the absence of soluble agonists such as ADP,
mouse platelets adherent to fibrinogen exhibit more filopodia
and fewer lamellipodia compared with human platelets. Spread-
ing of ADAP?/?platelets on the combined dmA1A2 VWF/
fibronectin surface under shear flow was limited mostly to
filopodial extension (Figure 5A), a response that was reduced in
ADAP?/?platelets (Figure 5B). Thus, while 51% ? 7% of
contacts,and cytoskeletal
ADAP?/?cells showed one or more filopodia on dmA1A2
VWF/fibronectin, only 27% ? 3% ofADAP?/?platelets showed
filopodia (Figure 5C; P ? .05). No such difference between
wild-type and knockout platelets was noted on dmA1A2 VWF
alone (25% and 24%, respectively) (Figure 5B-C). Additionally,
the filopodia of ADAP?/?platelets on the combined surface
tended to be longer than the few produced on the single surface
or the ones exhibited by ADAP?/?platelets on either surface
(Figure 5A-B). Altogether, these results indicate that ADAP?/?
platelets undergo GP Ib-IX-V–dependent activation of ?IIb?3
leading to increased filopodial extension, whereas such re-
sponses are considerably diminished in ADAP?/?platelets. The
reduced length of filopodia in ADAP?/?platelets under shear
flow adherent to the combined dmA1A2 VWF/fibronectin
surface suggests that ADAP may additionally mediate ?IIb?3-
dependent outside-in signals for cytoskeletal rearrangements.
To determine if ADAP is involved in ?IIb?3 activation
downstream of excitatory receptors other than GP Ib-IX-V,
platelets were stimulated with ADP, convulxin, or the Par4
receptor-activating peptide, AYPGKF. Platelets from ADAP?/?
mice bound FITC-fibrinogen in a dose-dependent manner in
response to each agonist. By comparison, ADAP?/?platelets
exhibited a 45% to 75% reduction in FITC-fibrinogen binding to
ADP-activated platelets (P ? .05) (Figure 6A), an approxi-
mately 75% reduction in binding to Par4 peptide-activated
platelets (P ? .01) (Figure 6B), and an approximately 15% to
29% reduction to convulxin-activated platelets (P ? .05) (Fig-
ure 6C) despite normal ?IIb?3 expression (Figure 2C).Aggrega-
tion of stirred platelets stimulated with ADP or Par4 resulted in
normal shape change in ADAP?/?and ADAP?/?platelets. At
lower agonist concentrations, ADAP?/?platelets showed an
18% to 50% decrease in the maximum aggregation at 2 minutes,
and after 2 to 5 ?M ADP the ADAP?/?platelets disaggregated
while the wild-type platelets did not (not shown). Because
ADAP deficiency impairs ?IIb?3 activation by several agonists,
ADAP may transduce signals through modulators, such as talin,
or stabilize signaling complexes that converge on the integrin
cytoplasmic tails to modulate affinity.36,56It has been suggested
that talin interaction with integrins may be regulated by
phosphorylation, binding of PIP2, or calpain-mediated proteoly-
sis.57-60ADAP could participate in talin regulation by increasing
intracellular calcium levels and activating protein kinase C61or
calpain.59Alternatively, ADAP might regulate access of PIP2 to
Figure 5. Cytoskeletal rearrangements in response to adhesion to dmA1A2 VWF and fibronectin under shear flow. Platelets were allowed to interact with dmA1A2
VWF alone or together with FN as described in Figure 4. Cells were fixed after 2 minutes of flow, stained for ?IIb, imaged by deconvolution microscopy, and scored for filopodial
extensions. (A)ADAP?/?or (B)ADAP?/?platelets adherent on the combinedA1A2 VWF/FN surface.Arrowheads illustrate filopodia, which were shorter and less numerous in
panel B. (C) Quantitative representation of the percentage of cells with filopodial protrusions on dmA1A2 VWF alone or together with FN ? SEM (*P ? .05). Results shown are
the summary of 3 separate experiments.
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Page 6

talin by interacting with PIP2 through the ADAP extended
helical SH3 domain.62Finally, the small GTPase, Rap1, has
been implicated in promoting ligand binding to integrins.63
Perhaps ADAP influences the localization of Rap1b effectors to
talin. Further studies are required to assess these possibilities.
The ADAP-binding partner, SKAP-HOM, is not required
for ?IIb?3 activation
ADAP and SKAP-HOM, its binding partner, have been detected in
macromolecular complexes upon platelet activation through GP
VI.64In the process of evaluating ADAP-binding proteins, we
discovered that SKAP-HOM protein levels were undetectable in
ADAP?/?mouse platelets (Figure 7A). This is similar to observa-
tions that ADAP may be required for regulating levels of SKAP55
in a Jurkat T-cell line65and SKAP-HOM levels in lymphocytes.66
To investigate whether ADAP can regulate SKAP-HOM indepen-
dently of other proteins with which it may be associated in
hematopoietic cells, we transfected CHO cells with expression
vectors for each of these proteins. Very little SKAP-HOM protein
was detected when it was transfected without ADAP (Figure 7B).
However, SKAP-HOM was readily detected when cotransfected
with ADAP. This was not observed with a SKAP-HOM mutant
lacking the SH3 domain (Figure 7B). Thus, ADAP stabilizes
SKAP-HOM protein levels in a manner dependent on the SKAP-
HOM SH3 domain.
SKAP55, a protein homologous to SKAP-HOM, is not
expressed in platelets.64However, it associates with ADAP in T
cells20and has been implicated in integrin-mediated T-cell
adhesion.27,28Therefore, the absence of SKAP-HOM inADAP?/?
platelets raised the possibility that it and not ADAP is required
for ?IIb?3 activation. To address this possibility, SKAP-
HOM?/?mice were studied.40These mice exhibited platelet
numbers and levels of platelet ADAP similar to wild-type
littermates. However, unlike the results withADAP?/?platelets,
SKAP-HOM?/?platelets bound fibrinogen normally after adhe-
sion to dmA1A2 VWF or stimulation with PMA (Figure 7C). In
addition, SKAP-HOM?/?platelets bound FITC-fibrinogen nor-
mally in response to 10 ?M ADP or 250 ?M Par4 receptor-
activating peptide (not shown). Thus, ADAP and not SKAP-
HOM is required for signaling through GP Ib-IX-V and other
agonists to ?IIb?3.
Taken together, these experiments establish that ADAP is
involved in (1) normal GP Ib-IX-V–induced signaling to
?IIb?3, both under static and shear flow conditions; (2) ?IIb?3
activation downstream of agonists acting through G-protein–
coupled receptors; (3) regulation of platelet expression of
SKAP-HOM; and (4) normal platelet function in vivo as
assessed by rebleeding from tail wounds. Thus, ADAP defi-
ciency has several consequences for platelet function through
the promotion of integrin activation and stable adhesion under
shear flow. This discovery raises new questions. Does the
increased frequency of rebleeding from tail wounds inADAP?/?
mice mean that defects in GP Ib-IX-V signaling can lead to
unstable thrombi in vivo? If so, would it be possible to
selectively block this signaling pathway to achieve an antithrom-
botic effect with minimal disruption of hemostasis? DoesADAP
influence platelet function through interactions with known
modulators of ?IIb?3 affinity, such as talin or Rap1b, or does it
regulate ?IIb?3 affinity by a novel mechanism? The answers to
Figure 6. Soluble FITC-fibrinogen binding to platelets activated by ADP, Par4 receptor-activating peptide, or convulxin. Platelets were stimulated with varying
concentrations of agonist, and the binding of soluble FITC-fibrinogen to platelets was determined by flow cytometry.ADAP?/?orADAP?/?platelets stimulated with (A)ADP, (B)
Par4, or (C) convulxin. Results are shown as the percentage of ADAP?/?FITC-fibrinogen binding to ADAP?/?cells for a given agonist concentration ? SEM (*P ? .05;
**P ? .01). Results are a summary of at least 4 separate experiments.
Figure 7. SKAP-HOM protein expression and function in ADAP?/?or ADAP?/?
mice. (A) Washed platelets fromADAP?/?orADAP?/?mice were lysed, subjected to
SDS gel electrophoresis, and Western blotted for SKAP-HOM. Blots were then
reprobed for ADAP and integrin ?3, the latter to monitor gel loading. Two separate
anti–SKAP-HOM antibodies were used to confirm results. (B) SKAP-HOM regulation
by ADAP in CHO cells. CHO cells were transfected with 1 ?g ADAP alone, 3 ?g
EGFP-SKAP-HOM or EGFP-SKAP-HOM lacking the SH3 domain (dSH3) alone, or a
combination ofADAP and EGFP-SKAP-HOM.After 48 hours, cells were subjected to
SDS polyacrylamide gel electrophoresis and probed for ADAP and SKAP-HOM.
Platelet lysate from wild-type mice, prepared as in Figure 6A, was used as a positive
control, and blots were reprobed with a ?-actin antibody to monitor gel loading. (C)
Platelets from wild-type or SKAP-HOM knockout mice were allowed to adhere to
dmA1A2 VWF as described in Figure 2, and integrin activation was assessed by
FITC-fibrinogen binding in the presence or absence of a cocktail of inhibitors or PMA.
Results in panels A and B are representative of at least 3 experiments, with similar
results. Results in panel C are for ADAP?/?platelets (white bars), depicted as a
percentage of soluble ligand that is specifically bound by ADAP?/?platelets (black
bars) ? SEM, and are a summary of 3 separate experiments.
ADAP IN ?IIb?3ACTIVATION1023 BLOOD, 1 FEBRUARY 2007?VOLUME 109, NUMBER 3

Page 7

these questions will illuminate how one major class of platelet
adhesion receptor communicates with another.
Acknowledgments
This work was supported by grants from the National Institutes of
Health (HL31950, HL42846, HL7874, HL78784, DK50654-09),
the German Research Foundation, German Israelian Foundation,
and the state of Saxony-Anhalt and anAmerican HeartAssociation
fellowship (0425107Y).
The authors are grateful to Drs Barry Coller and Mark Ginsberg
for supplying reagents.
Authorship
Contribution: A.K.-F. designed research, performed research, ana-
lyzed data, and wrote the paper; B.M. and J.N.-O. collected data;
K.S. and B.S. supplied vital reagents; Z.M.R. supplied vital
reagents, designed research, and wrote the paper; B.G.N. and G.K.
supplied vital reagents and wrote the paper; and S.J.S. designed
research and wrote the paper.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Ana Kasirer-Friede, Department of Medicine,
University of California San Diego, 9500 Gilman Dr, Mail Code
0726, La Jolla, CA92093-0726; e-mail: akasirer@ucsd.edu.
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