αIIbβ3-expressing CHO Chinese hamster ovary cells on immobilized fibrinogen show activation of the MAP ki-nase family member ERK2, with an enhanced ERK2 activity in Pro33 cells compared
Trang 1Original Paper
Acta Haematol 2017;137:44–50 DOI: 10.1159/000450783
Integrin αIIbβ3-Dependent ERK Signaling Is
Regulated by Src and Rho Kinases in Both Leu33
and Pro33 Polymorphic Isoforms
Khon C Huynh a, c Thi-Hiep Nguyen a Dinh Chuong Pham b
Huong T.T Nguyen c Toi Van Vo a Marianna Gyenes c Volker R Stoldt c
a Biomedical Engineering Department, International University, Vietnam National University, Ho Chi Minh City,
Vietnam; b Faculty of Applied Sciences, Ton Duc Thang University, Ho Chi Minh City , Vietnam; c Department of
Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine University Medical Center, Düsseldorf , Germany
adhesion Our data showed that Src family and rho kinases play a crucial role in the integrin αIIbβ3-dependent
outside-in signaloutside-ing to ERK2 © 2016 S Karger AG, Basel
Introduction
The major platelet integrin, the fibrinogen receptor αIIbβ3, interacts with numerous plasma and extracellular matrix proteins and thus plays an important role in plate-let adhesion and aggregation during hemostasis and thrombosis Upon ADP or thrombin activation of the platelets, integrin αIIbβ3 becomes activated (inside-out signaling), and it can bind soluble fibrinogen, which in turn induces the activation of various cellular responses such as spreading and aggregation (outside-in signaling) [1–3] The β3 subunit of αIIbβ3 is polymorphic at residue
33, and these alleles encode either Leu (HPA-1a) or Pro (HPA-1b) Platelets expressing the Pro33 phenotype show
an increased αIIbβ3 function, e.g., enhanced aggregation, shorter bleeding times, and a greater affinity on immobi-lized fibrinogen [4–7] The possible clinical aspects of this polymorphism have been published in several studies
Keywords
αIIbβ3 · ERK signaling · Leu33 · Pro33 · Polymorphisms ·
Rho kinase · Src signaling
Abstract
Platelet integrin αIIbβ3 possesses a Leu/Pro polymorphism
at residue 33 (Leu33/HPA-1a or Pro33/HPA-1b) The Pro33
isoform has been suggested to exhibit prothrombotic
fea-tures αIIbβ3-expressing CHO (Chinese hamster ovary) cells
on immobilized fibrinogen show activation of the MAP
ki-nase family member ERK2, with an enhanced ERK2 activity in
Pro33 cells compared to Leu33 cells In our present work, we
examined how the Leu/Pro polymorphism modulates the
ERK2 activation stimulated by 2 differently triggered
out-side-in signalings We either treated the CHO cells with Mn 2+
or allowed them to adhere to fibrinogen Moreover, we
stud-ied which signaling cascades are involved in ERK2 activation
In contrast to immobilized fibrinogen, Mn 2+ did not
signifi-cantly increase ERK2 activation However, Mn 2+ had a
syner-gistic effect on ERK2 phosphorylation when combined with
immobilized fibrinogen Pro33 cells adherent to fibrinogen
exhibited a significantly greater ERK2 activity than Leu33
cells in the presence of Mn 2+ , which peaked after 10 min of
Received: June 27, 2016 Accepted after revision: September 8, 2016 Published online: December 7, 2016
Trang 2demonstrating a potential association between these
symptoms, acute coronary syndromes [8] , and a
prema-ture myocardial infarction by patients with coronary
ar-tery disease who are carriers of HPA-1b/1b [9]
Although the activation of integrin αIIbβ3 mostly
oc-curs via inside-out signaling, adhesive ligand occupation
(i.e., immobilized fibrinogen/fibronectin) to the integrin
can also generate the active conformation of the integrin
leading to outside-in signaling [2] Divalent Mn 2+ cations
have also been reported to induce an active
conforma-tional state of αIIbβ3 [10] and to generate a subsequent
outside-in signaling [11, 12] Src tyrosine kinase is
associ-ated with the cytoplasmic tail of the β3 subunit and has
been reported to play a crucial role in the
integrin-medi-ated outside-in signaling [13, 14] A number of other
sig-nal molecules and pathways have also been identified to
participate in the integrin-mediated outside-in signaling,
among others the mitogen-activated protein kinase
(MAPK) family member ERK2, whose Tyr/Thr
phos-phorylation regulates various cellular processes,
includ-ing the release of stored Ca 2+ in platelets [15] , cell
adhe-sion, and spreading [16] Via its substrate, the myosin
light chain kinase (MLCK), ERK2 modulates the myosin
function and thereby the cytoskeletal clustering of
integ-rins, shape changes, and secretion in platelets [17–19]
Previously, it has been reported that the substitution of
Leu to Pro at residue 33 enhances signaling to ERK2,
MLCK, and the extent of the phosphorylated state of the
regulatory subunit in the myosin phosphatase [3, 20, 21]
As these signal proteins are essential for cytoskeletal
rear-rangement, adhesion, and spreading, these results
corre-late well with the increased αIIbβ3 activity observed in the
HPA-1b/1b isoform In our work, we examined how the
Leu33Pro polymorphism modulates ERK2 activation in
outside-in signaling In addition, we studied which
sig-naling pathways are involved in αIIbβ3-mediated ERK
activation
Materials and Methods
Antibodies and Reagents
Anti-Src pY418 was from Invitrogen (Darmstadt, Germany),
anti-v-Src from Calbiochem (Darmstadt, Germany),
phosphory-lated ERK1/2 from Cell Signaling Technology (Danvers, MA,
USA), and anti-ERK1/2 from Promega (Mannheim, Germany)
Nonconjugated IgG mouse was from Sigma (Taufkirchen,
Ger-many), secondary antibody rabbit HRG and mouse HRG from GE
Healthcare (Munich, Germany), FITC-conjugated clone P2
anti-body and clone SZ21 antianti-body from Immunotech (Krefeld,
Ger-many), and FITC-conjugated nonspecific mouse IgG from
Bec-ton-Dickinson (Heidelberg, Germany) Alfazyme was from PAA
Laboratories GmbH (Pasching, Germany), PP1 from Biomol (Hamburg, Germany), PP3 from Merck (Darmstadt, Germany), and the staining kit from Bio-Rad (Munich, Germany) Protease and phosphatase inhibitors, apyrase, PGE1, human fibrinogen, and all other reagents were from Sigma.
Flow Cytometry
Two CHO cell clones stably expressing αIIbβ3 isoforms Leu33 and Pro33 were obtained from the Department of Hemostasis, Hemotherapy, and Transfusion Medicine, Heinrich Heine Uni-versity Medical Center, Düsseldorf, Germany [22] To check the expression of αIIbβ3 isoforms, cells were resuspended in PBS, in-cubated with either FITC-conjugated CD-41 clone P2 antibody (1: 10) or FITC-conjugated HPA-1a-specific antibody (CD61 clone
As a control, nonspecific mouse IgG-FITC was used The labeled cells were analyzed on a FACScalibur flow cytometer (Becton Dickinson).
Cell Adhesion to Immobilized Fibrinogen
CHO cells were grown to 70–80% confluence, detached by
7 min at room temperature, and resuspended in Tyrode’s buffer
tissue plates were coated with 500 μL (100 μg/mL) fibrinogen or
were added to each well and incubated for the indicated time
sodium deoxycholate; pH 7.4) supplied with 250 μg/mL AEBSF, 15 μg/mL pepstatin, chymostatin, antipain, 55 μg/mL leupeptin, and a phosphatase inhibitor mixture For immunoprecipitation, we used
(pH 7.4) supplied with protease and phosphatase inhibitors A non-adherent cell suspension was added to ice-cold 2× lysis buffer The lysates were chilled for 30 min on ice and clarified by centrifugation
con-centration was determined by the Bradford method.
Gel Electrophoresis and Western Blotting
Equal amounts of protein were subjected to electrophoresis, and all samples were electrophoresed either in 8% (for Src) or 10% (for ERK) acrylamide gel for SDS-PAGE, transferred onto PVDF membranes, and subjected to immunoreaction The signals were densitometrically visualized with a chemiluminescence ECL (Am-ersham Biosciences) system and quantified using an Azure c300 Imaging System (Azure Biosystems).
Results
Expression of αIIbβ3 Isoforms in CHO Cells
We obtained αIIbβ3-transfected CHO cells with the appropriate αIIbβ3 isoforms (Leu33 or Pro33) [22] The
Trang 32 clones were confirmed for an equivalent expression
lev-el of Leu33 and Pro33 isoforms prior to adhesion
experi-ments Flow-cytometric analysis with FITC-conjugated
anti-αIIbβ3 antibody P2 demonstrated equivalent
recep-tor expression in the cell lines generated with the Leu33
(HPA-1a)- and Pro33 (HPA-1b)-containing αIIbβ3
iso-forms, respectively SZ21, a specific monoclonal antibody
to the HPA-1a isoform, presented a substantially lower
affinity for the Pro33 cells than for the Leu33 cells ( Fig. 1 )
pERK2 Activity in αIIbβ3-Transfected CHO Cells
To study the modulation of the polymorphism onto
the integrin-mediated outside-in signaling, we
investigat-ed the activation of ERK in αIIbβ3-expressing CHO cells
on immobilized fibrinogen After placing cells onto 100
μg/mL immobilized fibrinogen, we allowed them to
ad-here for 10 min followed by ERK activation analysis
Pro33 cells exhibit higher ERK activation than Leu33 cells
( Fig. 2 )
Mn 2+ is also known to induce integrin activation via
shifting the receptor conformation from an inactive to an
active state [4] In the next part of our work, we studied
the influence of Mn 2+ on the ERK2 activation in
fibrino-gen-adherent CHO cells Moreover, we assessed how the
Leu33/Pro33 polymorphism modulates this effect To
an-alyze whether Mn 2+ alone induces outside-in signaling to
ERK2, we examined the effect of 0.5 m M Mn 2+ on ERK2
activation in both Leu33 and Pro33 CHO cell suspensions
over BSA surface As shown in Figure 2 , Mn 2+ slightly
stimulated ERK2 activation, but the extent of the
stimula-tion was considerably less than in cells adhering to 100
μg/mL immobilized fibrinogen Higher concentrations of
Mn 2+ (1 or 2 m M ) exhibited a similar effect as 0.5 m M
Mn 2+ (data not shown) The combination of Mn 2+ and
immobilized fibrinogen resulted in a synergism of ERK
activation Mn 2+ concentrations of 0.5 and 1 m M induced
significantly greater ERK2 phosphorylation in Pro33 cells
than in Leu33 cells Using a concentration of 2 m M Mn 2+ ,
both HPA-1 isoforms showed approximately equal ERK
activation ( Fig. 2 )
To analyze the kinetics of the ERK2 phosphorylation
as a consequence of the immobilized fibrinogen-Mn 2+
combination, we allowed Leu33 and Pro33 CHO cells to
adhere to fibrinogen surfaces in the presence of 0.5 m M
Mn 2+ for various periods of time As shown in Figure 3 ,
both Leu33 and Pro33 cells exhibited a maximal ERK2
activity after 10 min of incubation with a subsequent
de-crease after 20 min adhesion
Src Family Kinases and Rho Kinases in ERK Signaling
ERK2 activation is mediated by dual phosphorylation
on threonine 185 and tyrosine 187 residues [23] There-fore, Src tyrosine kinase and Rho kinase (ROCK) are sug-gested to be involved in ERK activation Following fibrin-ogen engagement, Src pY418 activity was enhanced in both Leu33 and Pro33 cells ( Fig. 4 a) To examine the role
of Src tyrosine kinases in ERK2 signaling, we incubated Leu33 and Pro33 cells with the selective Src family kinase inhibitor PP1 and subsequently allowed them to adhere
to 100 μg/mL fibrinogen Cell suspensions over BSA sur-faces were used as a control As shown in Figure 4 , PP1 completely blocked the ERK2 phosphorylation in both isoforms indicating an Src kinase-dependent ERK2
acti-64
0
20
0
Fluorescence intensity
Fluorescence intensity
HPA-1a (Leu33) HPA-1b (Pro33)
HPA-1b
a
b
Fig 1. Characterization of stable αIIbβ3 expression of a CHO cell line; expression levels in HPA-1a (Leu33) and HPA-1b (Pro33) CHO cells were determined by flow-cytometric analysis with the
HPA-1a variant, antibody SZ21, a HPA-1a-specific antibody, was
Trang 4vation PP3, an inactive analogue of PP1, did not exhibit
an inhibitory effect
To examine the potential role of ROCK in the
αIIbβ3-dependent ERK2 activation, we performed adhesion
ex-periments on fibrinogen in the presence of either Y27632
or HA1077, 2 pharmacologically distinct specific
inhibi-tors of ROCK Both inhibiinhibi-tors completely blocked the
ERK activation ( Fig. 5 ) These observations suggest an
in-volvement of the Src family kinases and ROCK in the
fi-brinogen-mediated αIIbβ3 outside-in signaling to ERK2
Discussion
The platelet integrin αIIbβ3 plays a crucial role in
platelet aggregation and thrombus formation by binding
to fibrinogen initiating fibrinogen-dependent
platelet-crosslinking [24, 25] The fibrinogen engagement of the
integrin activates a great variety of outside-in signals
leading to elevated intracellular Ca 2+ flux and cytoskeletal
rearrangement [2, 10] Several polymorphisms in the
in-tegrin β3 subunit have been associated with platelet
dys-function Among them, the Leu33Pro substitution of
αIIbβ3 has been reported to exhibit prothrombotic
char-acteristics in several works [4, 7, 20]
ERK1 and ERK2 are involved in cell growth,
prolifera-tion, and adhesion, megakaryocyte differentiaprolifera-tion,
pro-platelet formation [26, 27] , and the release of stored Ca 2+
Total ERK1/2 pERK2
2.0
Leu33 1.5
1.0 0.5
0 Fibrinogen
BSA + 0.5 m M
Mn 2+
+ 2 m M
Mn 2+
+ 1 m M
Mn 2+
+ 0.5 m M
Mn 2+
*
*
*
Pro33
Fig 2. pERK2 activity in αIIbβ3-expressing
CHO cells adhering to immobilized
fibrin-ogen in the presence of various
the absence or presence of the indicated
sub-sequently allowed to adhere to 100 μg/mL
fibrinogen or maintained in suspension
over 1% BSA After a 10-min incubation at
were processed as described in Materials
and Methods The enhanced ERK2
activa-tion in Pro33 compared to Leu33 cells was
significant at concentrations of 0, 0.5, and
3 experiments * p < 0.05, evaluated by
un-paired t test
Total ERK1/2 pERK2
1.5 1.0 0.5 0 2.5
20 10
Adhesion time, min5
*
*
*
* Pro33
Fig 3. Activation of ERK2 in αIIbβ3-expressing CHO cells
min and subsequently allowed to adhere to 100 μg/mL fibrinogen
or maintained in suspension over 1% BSA for 2.5, 5, 10, and 20 min
lysates were analyzed for pERK2 activity The enhanced ERK2 ac-tivation in Pro33 compared to Leu33 cells was significant at 5, 10,
and 20 min Results are representative of 4 experiments * p < 0.05, evaluated by unpaired t test
Trang 5Total Src
Src pY418
3.0
2.0
2.5
1.5
1.0
0.5
0
HPA-1a (Leu33)
a
Fibrinogen
HPA-1b (Pro33)
*
*
Total ERK1/2 pERK2
2.0
1.0 1.2 1.4 1.6 1.8
0.2 0.4 0.6 0.8
0
HPA-1a (Leu33)
b
Fibrinogen
BSA + 10 μ M
PP1 + 10 μPP3M
HPA-1b (Pro33)
*
*
*
*
Fibrinogen
BSA + 10 μ M
PP1 + 10 μPP3M
Fig 4. Src pY418 activity ( a ) and effect of PP1 on ERK2 activity ( b )
in αIIbβ3-expressing CHO cells adhering to immobilized
fibrino-gen CHO cells were maintained in suspension over 1% BSA or
allowed to adhere to 100 μg/mL fibrinogen To study the effect of
30 min and subsequently allowed to adhere to fibrinogen After 10
min of incubation, cells were solubilized, and equal aliquots of samples containing 50 μg of protein were separated by 10% SDS-PAGE gel The blots were probed with anti-Src pY418, anti-v-Src, anti-pERK2, or anti-ERK antibodies and quantified by
densitom-etry * p < 0.05, evaluated by unpaired t test Results are
representa-tive of 3 experiments
Total ERK1/2 pERK2
1.0 1.2
0.2 0.4 0.6 0.8
0
HPA-1a (Leu33)
Y27632 HA107720 μM
HPA-1b (Pro33)
*
*
Y27632 HA107720 μM
Fig 5. Effect of Rho kinase inhibition on
ERK2 activity in αIIbβ3-expressing CHO
cells adhering to immobilized fibrinogen
CHO cells preincubated either with PBS or
to adhere to 100 μg/mL fibrinogen After 10
solu-bilized, and equal aliquots of samples
con-taining 50 μg of protein were separated by
10% SDS-PAGE gel The blots were probed
with antiphospho-antibody (pERK2) or
anti-ERK antibody and quantified by
den-sitometry (ratio of pERK2 to total ERK in
arbitrary units) * p < 0.05, evaluated by
un-paired t test Results are representative of 2
experiments
Trang 6in platelets [15] Fibrinogen-adherent Pro33 CHO cells
exhibit enhanced αIIbβ3-mediated outside-in signaling
to ERK2 and MLC [18] , suggesting a role of ERK2
signal-ing in prothrombotic characteristics of this isoform in
platelets Our aim was to further assess which signaling
pathways are involved in ERK activation
One of the possibilities to activate integrins is
trigger-ing an active conformation in their extracellular domains
by divalent cations [28, 29] We raised the following
ques-tion: to what extent do Mn 2+ ions alone regulate
αIIbβ3-mediated outside-in signaling in comparison to ligand
engagement? To analyze how this distinct manner of
ac-tivation is reflected in receptor signaling, we examined
outside-in signaling induced by immobilized fibrinogen
or Mn 2+ alone and by a combination of both
Further-more, we analyzed how these processes are modified by
the Leu33/Pro33 polymorphism Our observations that
Mn 2+ cations alone elevate the ERK2 activity only to a
small extent when compared to immobilized fibrinogen
suggest a less important role of Mn 2+ in regulating ERK2
signaling ( Fig. 2 ) Previous studies have shown that Mn 2+
increases the binding affinity of αIIbβ3 to ligands, but this
activation is not maximal and depends on the integrin
isoform type as well as the context [30] Based on previous
studies and our data, it is hypothesized that Mn 2+ alone
cannot induce ERK signaling of αIIbβ3 In contrast, when
combined with immobilized fibrinogen, Mn 2+ induces a
synergistic effect leading to maximal ERK activity after 10
min of adhesion In general, upon the whole incubation
time, Pro33 cells exhibited a significantly higher ERK
ac-tivity than Leu33 cells
In our work, we showed that the Leu33/Pro33
poly-morphism modulates the αIIbβ3-mediated outside-in
signaling to Src ( Fig. 4 a) This tyrosine kinase plays an
essential role in integrin signaling and is directly
associ-ated with αIIbβ3 integrin [13, 14, 31] Src kinase has been
reported to play a central role in the regulation of various
pathways, including the MAP kinase cascade [32] On the
one hand, it has been shown that in adherent chick
em-bryo fibroblast cells phosphorylated ERK is targeted after
integrin engagement or upon v-Src activation to newly
forming cell-matrix adhesion [33] On the other hand, in
thrombactivated human platelets, the Src kinase
in-hibitor PP1 did not block ERK activation [34] , indicating
Src-independent ERK signaling It seems that
integrin-mediated ERK activation can occur through several, from
each other independent, signaling cascades Therefore,
we raised the question whether Src kinases participate in
the regulation of ERK2 signaling in fibrinogen-adherent
CHO cells Our observation that the Src kinase family
in-hibitor PP1 entirely blocked ERK2 activation in both iso-forms ( Fig. 4 ) provides evidence that the ERK2 activation
in fibrinogen-adhering CHO cells is mediated via Src ki-nases It has been reported that Src family kinases are also involved in the regulation of the small GTPases [32] These signal proteins are essential for cytoskeleton reor-ganization, and ROCK is an effector protein of the Rho GTPase with a regulatory function Moreover, ROCK is proposed to be included in MLC phosphorylation [35]
As the Thr696 phosphorylation of the PP1-myosine phosphatase regulatory subunit is modulated by the Leu33/Pro33 polymorphism in thrombin-treated plate-lets [21] and this phosphorylation is regulated by ROCK,
we investigated the role of ROCK in the αIIbβ3-mediated outside-in signaling to ERK2 Both Y27632 and HA1077,
2 pharmacologically distinct, specific inhibitors of ROCK, completely blocked ERK2 activation, indicating an essen-tial role of ROCK in αIIbβ3-mediated outside-in signal-ing to ERK2 ( Fig. 5 )
In conclusion, we provided evidence that the αIIbβ3-associated outside-in signaling to ERK is mediated via the Src kinase-ROCK signaling pathway in fibrinogen-ad-herent CHO cells Although Mn 2+ alone only slightly ac-tivates ERK, it synergizes the effect of adhesive fibrinogen
on ERK activation in both genotypes, showing a signifi-cantly higher ERK2 activation in the Pro33 isoform
Acknowledgments
We are grateful to Mrs Bianka Maaßen-Weingart und to Mrs Elisabeth Kirchhoff for their excellent technical assistance This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 612, TP B2, and grant No 1161/QĐ-ĐHQG-KHCN of the Vietnam National Universities Ho Chi Minh City.
References 1 Shattil SJ, Newman PJ: Integrins: dynamic
scaffolds for adhesion and signaling in plate-lets Blood 2004; 104: 1606–1615
2 Stegner D, Nieswandt B: Platelet receptor sig-naling in thrombus formation J Mol Med (Berl) 2011; 89: 109–121
3 Varga-Szabo D, Pleines I, Nieswandt B: Cell adhesion mechanisms in platelets Arterio-scler Thromb Vasc Biol 2008; 28: 403–412
4 Michelson AD, Furman MI, Goldschmidt-Clermont P, Mascelli MA, Hendrix C, Cole-man L, Hamlington J, Barnard MR, Kickler T, Christie DJ, Kundu S, Bray PF: Platelet GP IIIa Pl A polymorphisms display different sen-sitivities to agonists Circulation 2000; 101: 1013–1018
Trang 714 Arias-Salgado EG, Lizano S, Sarkar S, Brugge
JS, Ginsberg MH, Shattil SJ: Src kinase activa-tion by direct interacactiva-tion with the integrin beta cytoplasmic domain Proc Natl Acad Sci USA 2003; 100: 13298–13302
15 Rosado JA, Sage SO: Phosphoinositides are required for store-mediated calcium entry in human platelets J Biol Chem 2000; 275: 9110–
9113
16 Zhu X, Assoian RK: Integrin-dependent acti-vation of MAP kinase: a link to shape-depen-dent cell proliferation Mol Biol Cell 1995; 6:
273–282
17 Klemke RL, Cai S, Giannini AL, Gallagher PJ,
de Lanerolle P, Cheresh DA: Regulation of cell motility by mitogen-activated protein ki-nase J Cell Biol 1997; 137: 481–492
18 Vijayan KV, Liu Y, Dong JF, Bray PF: En-hanced activation of mitogen-activated pro-tein kinase and myosin light chain kinase by the Pro33 polymorphism of integrin beta 3 J Biol Chem 2003; 278: 3860–3867
19 Kamm KE, Stull JT: Dedicated myosin light chain kinases with diverse cellular functions
J Biol Chem 2001; 276: 4527–4530
20 Vijayan KV, Bray PF: Molecular mechanisms
of prothrombotic risk due to genetic varia-tions in platelet genes: enhanced outside-in signaling through the Pro33 variant of integ-rin β3 Exp Biol Med (Maywood) 2006; 231:
505–513
21 Vijayan KV, Liu Y, Sun W, Ito M, Bray PF:
The Pro33 isoform of integrin β3 enhances outside-in signaling in human platelets by regulating the activation of serine/threonine phosphatases J Biol Chem 2005; 280: 21756–
21762
22 Stoldt VR, Berendes S, Scharf RE: The HPA-1b (Pro33) variant integrin αIIbβ3 increases the resistance of adherent platelets and trans-fected CHO cells upon exposure to shear stress 54th Annu Meet Soc Thromb Hemost, Nuremberg, 2010, A93
23 Buscà R, Pouyssegur J, Lenormand P: ERK1 and ERK2 map kinases: specific roles or func-tional redundancy? Front Cell Dev Biol 2016;
4: 53
24 Calvete JJ: Clues for understanding the struc-ture and function of a prototypic human inte-grin: the platelet glycoprotein IIb/IIIa com-plex Thromb Haemost 1994; 72: 1–15
25 Ruggeri ZM: Platelets in atherothrombosis Nat Med 2002; 8: 1227–1234
26 Whalen AM, Galasinski SC, Shapiro PS, Nah-reini TS, Ahn NG: Megakaryocytic differen-tiation induced by constitutive activation of mitogen-activated protein kinase kinase Mol Cell Biol 1997; 17: 1947–1958
27 Jiang F, Jia Y, Cohen I: Fibronectin- and pro-tein kinase C-mediated activation of ERK/ MAPK are essential for proplateletlike forma-tion Blood 2002; 99: 3579–3584
28 Bazzoni G, Hemler ME: Are changes in inte-grin affinity and conformation overempha-sized? Trends Biochem Sci 1998; 23: 30–34
29 Plow EF, Haas TA, Zhang L, Loftus J, Smith JW: Ligand binding to integrins J Biol Chem 2000; 275: 21785–21788
30 Smith JW, Piotrowicz RS, Mathis D: A mech-anism for divalent cation regulation of beta 3-integrins J Biol Chem 1994; 269: 960–967
31 Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS, Lowell CA, Shattil SJ: Coordinate interactions of Csk, Src, and Syk kinases with αIIbβ3 initiate integrin signaling
to the cytoskeleton J Cell Biol 2002; 157: 265–
275
32 Lee JW, Juliano R: Mitogenic signal transduc-tion by integrin- and growth factor receptor-mediated pathways Mol Cells 2004; 17: 188–
202
33 Fincham VJ, James M, Frame MC, Winder SJ: Active ERK/MAP kinase is targeted to newly forming cell-matrix adhesions by integrin en-gagement and v-Src EMBO J 2000; 19: 2911–
2923
34 Tulasne D, Bori T, Watson SP: Regulation of RAS in human platelets Evidence that activa-tion of RAS is not sufficient to lead to ERK1-2 phosphorylation Eur J Biochem 2002; 269: 1511–1517
35 Schoenwaelder SM, Hughan SC, Boniface K, Fernando S, Holdsworth M, Thompson PE, Salem HH, Jackson SP: RhoA sustains integ-rin α IIb β 3 adhesion contacts under high shear
J Biol Chem 2002; 277: 14738–14746
5 Feng D, Lindpaintner K, Larson MG, Rao VS,
O’Donnell CJ, Lipinska I, Schmitz C,
Suther-land PA, Silbershatz H, D’Agostino RB,
Muller JE, Myers RH, Levy D, Tofler GH:
In-creased platelet aggregability associated with
platelet GPIIIa PlA2 polymorphism: the
Framingham Offspring Study Arterioscler
Thromb Vasc Biol 1999; 19: 1142–1147
6 Vijayan KV, Goldschmidt-Clermont PJ, Roos
C, Bray PF: The Pl A2 polymorphism of
integ-rin beta 3 enhances outside-in signaling and
adhesive functions J Clin Invest 2000; 105:
793–802
7 Loncar R, Stoldt V, Hellmig S, Zotz RB, Mihalj
M, Scharf RE: HPA-1 polymorphism of
αIIbβ3 modulates platelet adhesion onto
im-mobilized fibrinogen in an in-vitro flow
sys-tem Thromb J 2007; 5: 2
8 Williams MS, Bray PF: Genetics of arterial
prothrombotic risk states Exp Biol Med
(Maywood) 2001; 226: 409–419
9 Zotz RB, Winkelmann BR, Müller C, Boehm
BO, März W, Scharf RE: Association of
poly-morphisms of platelet membrane integrins
α IIb β 3 (HPA-1b/Pl A2 ) and α 2 β 1 (α 2 807TT)
with premature myocardial infarction J
Thromb Haemost 2005; 3: 1522–1529
10 Litvinov RI, Nagaswami C, Vilaire G, Shuman
H, Bennett JS, Weisel JW: Functional and
structural correlations of individual αIIbβ3
molecules Blood 2004; 104: 3979–3985
11 Petrich BG, Fogelstrand P, Partridge AW,
Yousefi N, Ablooglu AJ, Shattil SJ, Ginsberg
MH: The antithrombotic potential of
selec-tive blockade of talin-dependent integrin
α IIb β 3 (platelet GPIIb–IIIa) activation J Clin
Invest 2007; 117: 2250–2259
12 Shattil SJ: Signaling through platelet integrin
αIIbβ3: inside-out, outside-in, and sideways
Thromb Haemost 1999; 82: 318–325
13 Shattil SJ: Integrins and Src: dynamic duo of
adhesion signaling Trends Cell Biol 2005; 15:
399–403