Thrombosis Research 133 (2014) 826–836

Contents lists available at ScienceDirect

Thrombosis Research

j ourna l homepage: www.e lsev ie r .com/ locate / th romres

Regular Article

Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required foractivation and post-ligation signaling during clot retraction☆

Ronit Mor-Cohen a,c,⁎,1, Nurit Rosenberg a,c,1, Yulia Averbukh a,c, Uri Seligsohn a,c, Judith Lahav b,c

a The Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer, Israelb Hemostasis Laboratory, Rabin Medical Center, Petah Tiqva, Israelc Sackler Faculty of Medicine, Tel Aviv University, Israel

Abbreviations: BHK, baby hamster kidney; CHO, chdulbecco modified Eagle's medium; DTNB, dithiobisnitrobuffered saline; pCMBS, 4-(chloromercuri) benzenesulfoisomerase.☆ The data were presented at the XXIV InternationHaemostasis (ISTH), in Amsterdam, July 1–4, 2013.⁎ Corresponding author at: Amalia Biron Research

Hemostasis, Chaim Sheba Medical Center, Tel Hashom5307350; fax: +972 3 5351568.

E-mail address: ronit.cohen@sheba.health.gov.il (R. M1 The authors contributed equally to this paper.

0049-3848/$ – see front matter © 2014 Elsevier Ltd. All rihttp://dx.doi.org/10.1016/j.thromres.2014.02.001

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 24 October 2013Received in revised form 12 January 2014Accepted 3 February 2014Available online 7 February 2014

Keywords:IntegrinsαIIbβ3αvβ3Clot retractionDisulfide bondsThiols

Background: Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processesrequire activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 andαvβ3 activation.Methods: In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- andαvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb andWT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins.Time-course of fibrin clot retraction was examined.Results: Cells expressedWTormutated humanαIIbβ3 aswell as chimeric hamster/humanαvβ3. TheαIIbβ3mu-tants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their acti-vation state. WT cells retracted the clot and addition ofαvβ3 inhibitors decreased the retraction rate. The activemutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, partic-

ularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6.Conclusions: BothαIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activa-tion ofαIIbβ3, but not ofαvβ3 enhances clot retraction. BothαIIbβ3 activation and post-ligation signaling duringclot retraction require disulfide bond exchange.

© 2014 Elsevier Ltd. All rights reserved.

Introduction

Following vascular injury, the exposed subendothelial matrix acti-vates platelets and initiates, along with coagulation mechanisms, theproduction of fibrin from fibrinogen [1]. Platelet activation inducesinside-out signals that activate integrinαIIbβ3 resulting in ligand bindingto its large globular head. Engagement of αIIbβ3 by fibrinogen or fibringenerates outside-in signals that mediate cytoskeleton rearrangementwhich is required for platelet spreading and fibrin clot retraction. Clot re-traction enhances thrombus stability and wound healing by compressing

inese hamster ovary; DMEM,benzoic acid; PBS, phosphate-nic acid; PDI, protein disulfide

al Society on Thrombosis and

Institute of Thrombosis ander 52621, Israel. Tel.: +972 3

or-Cohen).

ghts reserved.

the clot and drawing the edges of the wound together [2,3]. IntegrinαIIbβ3 mediates clot retraction by transmitting contractile forces fromthe actin-myosin cytoskeleton to the fibrin polymers that are tetheredoutside the cells. Interestingly, several studies reported that αIIbβ3expressed in nucleated cells such as Chinese Hamster Ovary (CHO) cellsandM21melanoma cell linesmust be activated in order tomediate fibrinclot retraction and integrin activation by talin is required for platelet-mediated fibrin clot retraction [4,5]. Other studies indicated that αIIbβ3expressed in mammalian cell lines was able to mediate fibrin clot retrac-tion without prior activation of the integrin [6–9].

The vitronectin receptor,αvβ3, is anothermember of theβ3 integrinsubfamily. In contrast to αIIbβ3 which is restricted to platelets andmegakaryocytes, αvβ3 is widely expressed in various cell lines includ-ing endothelial cells and platelets. αvβ3 was also shown to mediate fi-brin clot retraction when expressed in mammalian cell lines [4,10–12].

Several studies report that activation of αIIbβ3 is induced by a cas-cade ofmultiple disulfide bond exchanges in the β3 subunit [13–15]. Al-though the precise disulfide bonds that participate in this disulfide bondshuffling have not been identified, they were shown to be located at acysteine rich region of β3 comprising four epidermal growth factor(EGF)-like domains [13,14]. Each EGF-like domain contains 3–4

827R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

disulfide bonds, of which one bond is unique for integrins and isnot conserved in other EGF domains [16–19]. We showed that disrup-tions of the unique disulfide bonds in the EGF domains of theβ3 subunitresulted in constitutively active αIIbβ3 and αvβ3 expressed in BabyHamster Kidney (BHK) cells and that these bonds play a primary func-tional role in αIIbβ3 and αvβ3 activation involving disulfide bond ex-change [20,21]. We also showed that disulfide bond exchange isnecessary for αIIbβ3 post-ligation signaling events including sustainedfibrinogen binding to the integrin and adhesion to immobilized fibrino-gen [22,23].

In the current study, we investigated the role of integrin activationand disulfide bond exchange in αIIbβ3-mediated fibrin-clot retractionby using BHK cells expressing WT αIIbβ3 or αIIbβ3 harboring cysteinesubstitutions in the β3 subunit. Since transfected BHK cells also expresschimeric hamster/human αvβ3 receptors, we also investigated the rel-ative contribution of αvβ3 to clot retraction. We found that disulfidebond exchanges are involved in both receptor activation and post-ligation signaling during β3 integrin-mediated clot retraction.

Materials and Methods

Reagents and Antibodies

Dulbecco modified Eagle's medium (DMEM), L-glutamine and fetalcalf serum were purchased from Biological Industries (Beit-Haemek,Israel), lipofectamine reagent and G418 were from Gibco BRL (Paisley,UK), hygromycin was from Roche Diagnosis Gmbh (Mannheim,Germany), human fibrinogen, ε-amino-n-caproic acid and the mem-brane impermeant free thiols blockers, dithiobisnitrobenzoic acid(DTNB) and 4-(chloromercuri) benzenesulfonic acid (pCMBS) werefrom Sigma (St Louis, MO) and Toronto Research Chemicals (Toronto,Canada), respectively. Bovine thrombin was purchased from Instru-mentation Laboratory, (MA, USA). FITC-conjugated monoclonal anti-body P2 against αIIbß3 was from Immunotech (Marseille, France). Themonoclonal antibody LM609 againstαvß3was fromMillipore (Temecula,California) and the FITC-conjugated monoclonal antibody 23C6against αvß3 was from eBioscience (San Diego, CA). FITC-conjugatedfibrinogen-mimetic murine monoclonal antibody PAC-1 was obtainedfrom BD Biosciences (San Jose, CA). The αvß3 inhibitor RO0655233-001was from Roche Applied Science (Basel, Switzerland). The activatingmonoclonal antibody anti-LIBS6 was kindly provided by Dr. MarkGinsberg (Department of Medicine, University of California, SanDiego, La Jolla, CA).

Construction of Expression Vectors for Wild Type (WT) and Mutant cDNAs

cDNAs of humanαIIb and ß3 in pCDNA3 vector were kindly provid-ed by Dr. Peter Newman from the Blood Center of Wisconsin, Milwau-kee, WI. cDNA of αIIb was subcloned into the PvuII site of pCEP4mammalian expression vector carrying the hygromycin resistancegene as a selectionmarker (Invitrogen, San Diego, CA) as previously de-scribed [24]. Substitutions of selected cysteine residues by serine orother residues were created in the pCDNA3/ß3 vector using theQuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA)using two overlapping oligonucleotide primers containing single basepair substitution (the primers are available upon request). Correct in-corporation of the mutations into the pCDNA3/ß3 vectors was verifiedby DNA sequencing. For creation of doublemutants containing two cys-teine substitutions together, we first introduced onemutation into nor-mal pCDNA3/ß3 vector and then used themutant pCDNA3/ß3 clone as atemplate for introducing the second mutation.

Co-Transfection of αIIb and ß3 cDNAs

Baby Hamster Kidney (BHK) cells were grown in DMEM supple-mented by 2 mg/ml L-glutamine and 5% fetal calf serum. The cells

were co-transfected with 1 μg of normal or mutated forms ofpCDNA3/ß3 and 1 μg of normal pCEP4/αIIb using lipofectamine reagent.The transfected cells were grown in a selection medium containing0.7 mg/ml G418 and 0.5 mg/ml hygromycin. Mock cells were producedby transfecting BHK cells with 1 μg of pCEP4 and 1 μg of pcDNA3 andwere selected in the same medium.

Flow Cytometry Analysis of Transfected BHK Cells

Transfected BHK cells were harvested with phosphate-buffered sa-line (PBS) supplemented with 1 mM EDTA, pelleted and incubated inDMEM for 30 min at room temperature. Then, cells were pelletedagain, resuspended in PBS supplemented with 1 mMMgCl2 and 1 mMCaCl2 (5 X 105 cells/100 μl) and incubated for 30 min at room tempera-turewith either 20 μl FITC-conjugated P2 antibody, 5 μl FITC-conjugated23C6 antibody or 20 μl FITC-conjugated PAC-1 antibody. To measurePAC-1 binding after activation of αIIbß3 by activating antibodies, 1 μlanti-LIBS6 was added to the cells suspended in PBS supplementedwith 0.25 mM MnCl2 (5 X 105 cells/100 μl) prior to the addition ofFITC-conjugated PAC-1. The cells were then diluted to 5 X 105 cells/600 μl and analyzed for surface fluorescence by flow cytometery(Becton Dickinson, NJ). To measure ligand binding after blocking freethiols, we repeated these experiments in the presence of 2.5 mMDTNB. Baseline for nonspecific binding of the antibodies was measuredin mock cells. PAC-1 binding to αIIbß3 was expressed as percent ofαIIbβ3 expression level measured by P2 binding. The effects of anti-LIBS6 and DTNB were compared by two tailed paired t-test.

Fibrin Clot Retraction

Transfected BHK cells were harvested with PBS/1 mM EDTA,pelleted and incubated in DMEM for 30 min at room temperature. 3.5x 106 cells were mixed with 250 μl DMEM supplemented with 25 mMHepes, 5 mM ε-amino-n-caproic acid, 15 mM CaCl2 and 0.5 units of bo-vine thrombin. The cells were placed in glass aggregometry cuvettesand 100 μg fibrinogen was added. The clots were allowed to retract at37 °C and photographed after 1, 3, 5 and 18 hours. All experimentswere performed in the presence or absence of 5 μM of the αvß3 inhib-itor RO0655233-001 and/or 2.5 mM DTNB that were added 5 minprior to the addition of thrombin. In some experiments, 3 μl of anti-LIBS6 antibody was added to WT cells either with or without DTNBadded 5 min before or after anti-LIBS6. Some experiments were alsoperformed in the presence or absence of the anti-αvß3 blocking anti-body LM609 at 1:50 dilution or in the presence or absence of 100 μMpCMBS. The images were processed by Adobe Photoshop CS softwareprogram to produce black and white images and were analyzed by theimageJ program to measure the percent of the retracted clot (black)from the whole clotting medium (black and white). At least 3 experi-ments for each WT and mutant were conducted. The differences be-tween the mutated and WT cells and the effects of the αvß3inhibitor and DTNB in each time point were analyzed by two tailedpaired t-test. Since the initial retraction curves appeared to be linearuntil 3 to 5 hours, we also assessed the differences in the initial rate ofclot retraction by the slope of the first 5 hours of the retraction using alinear regression analysis function of the GraphPad Prism 5 softwareprogram.

Results

Effect of Free Sulfhydryl Inhibitor (DTNB) on the Activation State ofWT andMutated αIIbß3

We previously showed that disruptions of the unique β3 EGF-3disulfide bond C523-C544 by substituting either Cys523 or Cys544for Serine (C523S or C544S, respectively) resulted in constitutivelyactive αIIbβ3 and αvβ3. The double mutant C523S/C544S, in which

828 R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

both cysteines of the disulfide bond were substituted for serine re-sulted in activation of αIIbβ3 but not of αvβ3 [21]. We thereforeused this unique disulfide bond as a model for studying the effectof disulfide bond disruptions in β3, with or without maintaining afree thiol, on αIIbβ3 and αvβ3 mediated clot retraction. To verifythat the results were not specific for this particular bond, we alsotested another β3 mutant, C560S that disrupts the unique C560-C583 disulfide bond in EGF-4 and displays constitutively activeαIIbβ3 andαvβ3 [20,21]. FACS analysis using theαIIbβ3-specific an-tibody P2 showed that all αIIbβ3 mutants were expressed to a levelof 35%-100% of WTαIIbβ3 as previously reported [21]. Measurementof the activation state of the mutant cells was carried out by PAC-1.

C523S/C544S

0

20

40

60

80

100

120

140

PAC

-1 b

ind

ing

, % o

f ααI

Ibβ3

exp

ress

ion

*

Co

un

t

Co

un

t

C523S

FL1 FL1

FL1 FL1

C560

WT + LIBS6

A

B

Co

un

t

Co

un

t

Fig. 1. The effect of the thiol blockerDTNB on the activation state ofαIIbβ3. (A)PAC-1 bindingwWT αIIbβ3 or αIIbβ3 harboring cysteine to serine substitutions disrupting the C523-C544 andanti-LIBS6 antibody, with orwithout DTNBwas also determined. The results are presented as pethree experiments. Statistical significancewas calculated by a two-tailed, unpaired t test. Asterising of PAC-1 antibody toαIIbβ3mutant orWTαIIbβ3 activated by anti-LIBS6 in the absence (bWT αIIbβ3 (filled gray histogram).

All mutants displayed a profound increase in PAC-1 binding com-pared to WT cells (7–14 fold increase, P b 0.05) in contrast to WTcells that bound PAC-1 only when treated with the activating anti-body anti-LIBS6 (Fig. 1). These results confirmed our previous resultsindicating that these mutants are constitutively active. Addition of2.5 mM DTNB did not significantly decrease PAC-1 binding to all sin-gle mutants and only slightly decreased PAC-1 binding to the doublemutant. In contrast, DTNB markedly reduced anti-LIBS6-induced ac-tivation of WT cells by 52% (p b 0.05) (Fig. 1). Thus, DTNB has no sig-nificant effect on the activation state of the constitutively activemutants but has a significant inhibitory effect on anti-LIBS6antibody-induced activation of WT αIIbß3.

PAC-1

PAC-1 + DTNB

*

Co

un

t

C544S

FL1

S

as assessed byflow cytometrywith orwithout 2.5mMDTNB in BHK cells expressing eitherthe C560-C583 disulfide bonds in β3. PAC-1 binding following activation of WT αIIbβ3 byrcent ofαIIbβ3 expressionmeasured by P2 antibody. Bars representmean± SE of at leastks denote a p value of b0.05. (B)Representative flow cytometry histograms showing bind-lack solid histogram) or presence (black dotted histogram) of DTNB, relative to inactivated

829R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

Surface Expression of the Chimeric αvβ3

Expression of humanβ3 inBHK cells gives rise to a chimeric complexwith the endogenous hamster αv. We assessed the surface expressionof the chimeric αvβ3 in cells transfected with both human β3 andαIIb by measuring the binding of the complex- specific 23C6 antibodythat recognizes human as well as chimeric αvβ3 [21] and comparedthe results with the expression of αIIbβ3 detected by P2 on the samecells. As shown in Fig. 2, cells transfectedwithWTαIIbβ3 demonstratedconsiderable expression of chimeric αvβ3 (33.6% positive cells) whichwas about 56% of WT αIIbβ3 surface expression. Transfection of theC523S, C544S, C523S/C544S and C560S mutants demonstrated lowerbut measurable amounts of chimeric αvβ3 expression (8.2-14.8% posi-tive cells), which was 36, 12.7, 39.7 and 31.8% of the respective αIIbβ3expression. Thus, all transfected BHK cells, with either WT or mutatedβ3, expressed some, though variable amounts of chimeric αvβ3 thatcould participate in fibrin clot retraction.

C523S/C544S C560

C52WT

Co

un

t

Co

un

t

Co

un

t

Co

un

t

FL1 FL

FL1 FL

A

B

0

5

10

15

20

25

30

35

40

% p

osi

tive

cel

ls

Fig. 2. Surface expression ofWT ormutated chimeric hamster/humanαvβ3 in BHK cells transfehuman αvβ3 was measured by flow cytometry using FITC-conjugated 23C6 antibody against αhistograms showing binding of 23C6 antibody to WT or mutated αIIbβ3 (solid black histogram

Clot Retraction of Cells Expressing WT αIIbß3 and Chimeric αvß3

The ability of αIIbß3 and the chimeric αvß3 to mediate clot retrac-tion without prior activation was examined by a time course measure-ments of thrombin-induced fibrin clot retraction of cells expressingthe WT integrins. To assess the relative contribution of αIIbβ3 and thechimeric αvβ3 to clot retraction, we measured clot retraction eitherwith or without RO0655233-001 that was shown to specifically inhibitαvβ3 [25,26].

Cells expressingWT αIIbß3 andαvß3 retracted the fibrin clot with-out prior activation as shown in Fig. 3A and B. Addition of the specificαvß3 inhibitor RO0655233-001 significantly decreased the extent of re-traction as compared to cells without the αvß3 inhibitor at 3, 5 and18 hours. Addition of the αvß3 inhibitor also decreased the initial rateof retraction, demonstrated by the significantly reduced slope com-pared to cells without the inhibitor. Mock cells that do not expressαIIbß3 and αvß3 did not retract the fibrin clot even after 18 hours

S

3S C544S

FL11

1

Co

un

t

ctedwith humanαIIb and β3. (A) Surface expression ofWT ormutated chimeric hamster/vβ3. Bars represent mean ± SE of three experiments. (B) Representative flow cytometry) compared to Mock cells (filled gray histogram).

+

1h

3h

5h

18h

WT MockRO

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

WT

WT+RO

Time (hour)

% C

lot

retr

acti

on

slopes18 hours5 hours3 hours1 hour

0.00850.04020.00080.00030.3335P value

WT+

1h

3h

5h

18h

LM609

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

WT

WT+609

Time (hours)

% C

lot

retr

acti

on

A

B

Fig. 3. The effect ofαvβ3 inhibitors onWTβ3 integrin-mediatedfibrin-clot retraction. (A) Representative images of afibrin-clot retraction experiment of BHK cells transfected eitherwith normalhumanαIIb andβ3 (WT) orwith the empty plasmids (Mock),with orwithout the specificαvβ3 inhibitor RO0655233-001 (RO) (left panel) and a time course of the average clot retraction ofWTcells, in the absence (black,●) or presence (grey, ) of theαvβ3 inhibitor RO,measured at 1, 3, 5 and18 hours (right panel). Eachpoint representsmean±SEof 14experiments. The slopes of thefirst 5 hours of retraction (blackbroken lines) and the statistical analyses of thedifferences in each timepointwere calculatedby theGraphPadPrism5program. (B)Representative imageandclotretraction time course of BHK cells expressingWT β3 integrins, measured at 1, 3, 5 and 18 hours, in the absence (black,●) or presence (grey, ) of LM609 antibody.

830 R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

(Fig. 3A). Very similar inhibitory effect on clot retraction was obtainedby the LM609 antibody, another known specific αvβ3 inhibitor, asshown by a representative experiment (Fig. 3B). Thus, blocking αvß3by different inhibitors decreased but did not eliminate clot retractionof cells expressing theWT ß3 integrins, indicating that both the chime-ricαvβ3 and the introduced humanαIIbβ3 contribute to clot retraction.

Blocking the free thiol groups with 2.5 mM DTNB significantly re-duced both the extent and the initial rate of clot retraction by cells ex-pressing WT αIIbß3 and αvß3, in either the absence (Fig. 4A) or thepresence (Fig. 4B) of the αvß3 inhibitor. This was demonstrable by astatistically significant inhibition of the retraction starting at 3 hoursand lasting until 18 hours and by a profoundly reduced slope in thepresence of DTNB compared to its absence (P b 0.0001). To show thatthe inhibition of clot retraction was related to the free thiols blockadeand was not specific to DTNB, we tested another free thiol blocker,pCMBS [22,23]. As shown in a representative experiment (Fig. 4),pCMBS caused a similar inhibitory effect on clot retraction by cells ex-pressing WT αIIbß3 and αvß3, in the absence or presence of the αvß3inhibitor RO0655233-001. These results imply that disulfide bond ex-change is necessary for ß3 integrins-mediated clot retraction.

Clot Retraction of Cells Expressing Mutated αIIbß3 and αvß3

Cells expressing αIIbß3 and αvß3 harboring the cysteine to serinesubstitutions C523S, C544S, C523S/C544S and C560S (Fig. 5, solidblack lines) retracted the clot faster than cells expressing the WTintegrins (solid grey lines) in the presence or in the absence of theαvß3 inhibitor. The most significant differences in the extent of clot re-traction between WT and mutated cells were found at 3 and 5 hoursand for some mutants the differences persisted for 18 hours (C523Sand C544S) (Table 1). In the C523S, C560S and C523S/C544S mutantsthese differences were more pronounced in the presence of the αvß3inhibitor reflected by more significant differences at 3, 5 or 18 hoursand by the significantly enhanced slope compared to WT cells only inthe presence of the αvß3 inhibitor (Table 1). Interestingly, these mu-tants expressed large amounts of αvß3 that were more than 30% oftheir respective αIIbβ3 expression. In contrast, C544S, in which αvß3expression constituted only about 10% of αIIbβ3 expression, was theonly mutant that displayed a significantly increased extent and rate ofretraction in both the presence and the absence of the αvß3 inhibitor(Table 1). Thus, the constitutive activation of αIIbß3 significantly

1h

3h

5h

ON

+DTNB

1h

3h

5h

ON

slopes18 hours5 hours3 hours1 hourWTvs.WT+DTNB

<0.0001<0.0001<0.0001<0.00010.2519Pvalue

slopes18 hours5 hours3 hours1 hourWTvs.WT+DTNB

<0.0001<0.0001<0.00010.02400.9121Pvalue

A

+DTNB

+pCMBS

1h

3h

5h

ON

+pCMBS

1h

3h

5h

ON

B With αvβ3 inhibitor

Fig. 4. The effect of thiol blockers onWT β3 integrin-mediated fibrin-clot retraction. A time course of the average clot retraction of BHK cells transfected with normal human αIIb and β3(WT),measured at 1, 3, 5 and 18 hours (ON), in the absence (black,●) or the presence of DTNB (black broken line,■) or pCMBS (gray broken line, ). The experimentswere done either inthe absence (A) or in the presence (B) ofαvβ3 inhibitor. Each point representsmean± SE of 14 experiments except for pCMBSwhere a representative experiment is shown. The slopes ofthe first 5 hours of retraction (black broken lines) and the statistical analysis of the differences in each time point were calculated by the GraphPad Prism 5 program. The images of a rep-resentative fibrin-clot retraction experiment are also shown.

831R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

enhanced fibrin clot retraction and the presence of substantial levels ofαvß3 reduced this enhancing effect.

We next studied the effect of the thiol-blocker DTNB on clot retrac-tion in cells expressing the constitutively active integrin mutants. Incontrast to cells expressing WT αIIbß3 and αvß3, addition of 2.5 mMDTNB to cells expressing a single cysteine to serine substitutions(C523S, C544S and C560S), yielding a free thiol in their unpaired cyste-inepartners, did not or onlyminimally inhibited clot retraction, either inthe presence or absence of the αvß3 inhibitor (Fig. 5A, B and C, dashedlines). Although some inhibition could be seen in the first 5 hours, itwasusually not statistically significant (Table 2). Moreover, unlike WT cells,the initial rate of retraction (slopes) by these mutants in the absence orpresence of DTNB did not differ significantly (Table 2).

Notably however, similar to cells expressing theWT integrins, DTNBstrongly inhibited the extent and the initial rate of clot retraction bycells expressing the double C523S/C544S mutation which contains nofree thiol (Fig. 5D). This was observed in the presence or absence of

the αvβ3 inhibitor. Statistical analyses established that significant inhi-bition of the extent of clot retraction in the presence of DTNBwas exhib-ited at 3 to 18 hours and the slopes of themutant with or withoutαvß3inhibitor were profoundly reduced (Table 2). These results suggest thatthe effect of DTNB depended on the presence or absence of free thiols.

The Effect of the Activating Antibody Anti-LIBS6 on Clot Retraction of CellsExpressing WT αIIbß3 and αvß3

Since cells expressing the constitutively activemutants retracted thefibrin clot faster than cells expressing WT αIIbß3 and αvβ3, we ad-dressed the question whether this effect was specific for the C523S,C544S, C523S/C544S and C560S mutations or, alternatively, activationof the integrins in general was responsible for this effect. We thereforemeasured clot retraction by cells expressing WT αIIbβ3 and αvß3 inthe presence of anti-LIBS6 antibody that activates both αIIbß3 andαvß3 [21,27].

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

C523S/C544SC523S/C544S+DTNB

Time (hours)

% c

lot

retr

acti

on

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

C523S/C544SC523S/C544S+DTNB

Time (hours)

% c

lot

retr

acti

on

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

C560SC560S+DTNB

Time (hours)

% c

lot

retr

acti

on

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

C560SC560S+DTNB

Time (hours)

% c

lot

retr

acti

on

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

C523S

WTWT+DTNB

WTWT+DTNB

WTWT+DTNB

WTWT+DTNB

WTWT+DTNB

WTWT+DTNB

WTWT+DTNB

WTWT+DTNB

C523S+DTNB

Time (hours)

% c

lot

retr

acti

on

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

C544SC544S+DTNB

Time (hours)

% c

lot

retr

acti

on

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

C523SC523S+DTNB

Time (hours)

% c

lot

retr

acti

on

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

C544SC544S+DTNB

Time (hours)%

clo

t re

trac

tio

n

With αvβ3 inhibitor

With αvβ3 inhibitor With αvβ3 inhibitor

With αvβ3 inhibitor

A B

C D

Fig. 5. Clot retraction of cells expressingmutated ß3 integrins in the presence or absence of DTNB. A time course of the average clot retraction of BHK cells transfectedwith normal humanαIIb and mutated β3 harboring the cysteine to serine substitutions C523S (A), C544S (B), C560S (C) and C523S/C544S (D), measured at 1, 3, 5 and 18 hours, in the absence (black,▼) orpresence (black broken line, ♦) of DTNB. The experiments were carried out either without or with a αvβ3 inhibitor as indicated. Each point represents mean ± SE of three experiments.The average clot retraction of BHK cells expressing the WT β3 integrins in the absence (grey) or the presence (dashed grey) of DTNB is also shown.

832 R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

As shown in Fig. 6, cells treated with anti-LIBS6 antibody (red line)displayed augmented retraction compared to non-treated cells (blueline), in both the absence (Fig. 6A) and the presence (Fig. 6B) of theαvß3 inhibitor. This effect was statistically significant at 3 and 5 hours(Table 1). Similar to the constitutively activemutants, the enhancing ef-fect was more pronounced in the presence of the αvß3 inhibitor, asdisplayed by a significantly increased slope in anti-LIBS6-treated cellscompared to non-treated cells, only in the presence of theαvß3 inhibi-tor (Table 1). Thus, the activation ofαIIbß3 but not ofαvß3 significantlyenhanced clot retraction.

Since DTNB inhibited anti-LIBS6-induced αIIbß3 activation (Fig. 1),we investigated whether DTNB will affect anti-LIBS6-enhanced clot

retraction, and if it does – whether its effect depends on whether itwas added before anti-LIBS6 or after it. The contribution of the chimericαvß3 in this settingwas also addressed.We found that in the absence ofαvß3 inhibitor (Fig. 6A), DTNB addition, either before anti-LIBS6 (or-ange line) or after it (green line), had only a minor inhibitory effect onanti-LIBS6-enhanced clot retraction. This minor effect was only signifi-cant at 1 hour, when DTNB was added before anti-LIBS6 (Table 2).Thus, the combined effect of activating αIIbß3 and αvß3 rendered clotretraction independent of disulfide bond exchange.

In the presence of theαvß3 inhibitor (Fig. 6B), however, DTNB had apronounced inhibitory effect and its effect varied depending on thetiming of DTNB addition: DTNB added before anti-LIBS6 (orange line)

Table 1Statistically significant differences in the extent and rate of clot retraction between WT and each mutant and WT + anti-LIBS6 antibody.

WT

C523S C554S C523S/544S C560S WT + L

αvβ3 inhibitor - + - + - + - + - +

1 hour T T T T * T T3 hours T ** * * T * * * ***5 hours * ** ** * *** * * *18 hours ** * * Tslope T *** ** * ** T ** *

T (trend), P b 0.1.*, P b 0.05.**, P b 0.01.***, P b 0.001.Non-significant P values which are N0.1 are not shown (empty boxes).

833R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

significantly inhibited clot retraction relative to that of anti-LIBS6 alone(red line). This was reflected by significantly decreased retraction at 3and 5 hours and the significantly reduced slope (Table 2). In contrast,DTNB added after anti-LIBS6 (green line) had a lesser inhibitory effecton clot retraction: a statistically significant difference was achievedonly at 3 hours and the slope was not significantly reduced relative toanti-LIBS6 alone (Table 2). These results imply that DTNB inhibition ofanti-LIBS6-enhanced clot retraction is more pronounced when it isadded before anti-LIBS6, i.e. when DTNB is present at the site of LIBS6activation. Thus, when we looked at the contribution to clot retractionby αIIbß3 alone, we observed that if DTNB was present at the time ofanti-LIBS6 activation, clot retraction was reduced to a level similar tothat without the activation. However if DTNB was added after anti-LIBS6 activation, clot retraction was reduced but was still significantlyenhanced relative to that without activation, implying that DTNB hadan inhibitory effect on post-ligation signaling.

Discussion

The aim of this studywas to investigate the roles ofαIIbβ3 andαvβ3activation and disulfide bond exchange in the process of fibrin clot re-traction. For this purpose we carried out a fibrin clot retraction assayusing BHK cells stably expressing either WT or constitutively activecysteine-mutated integrins. We examined the effect of the thiolblockers DTNB and pCMBS and the effect of anti-LIBS6 activating anti-body on αIIbβ3 and αvβ3 mediated clot retraction.

Mammalian cells expressing αIIbβ3 have been widely used as amodel system for studying integrin-specific functions in platelets suchas activation, adhesion and clot retraction. One of the advantages ofusing αIIbβ3 expressing cells rather than platelets is the possibility toisolate the effect of integrin αIIbβ3 without other platelet receptorsthat can bindαIIbβ3 ligands such as fibrinogen andfibrin and the abilityto express artificial mutants of the receptor. When mammalian cellssuch as HEK239 and CHO cells expressed human αIIbβ3, they were

Table 2Statistically significant differences in the extent and rate of clot retraction of each mutant or W

±DTNB

C523S C544S C523S/C54

αvβ3 inhibitor - + - + -

1 hour T T3 hours T T **5 hours T T * *18 hours T Tslope ***

T (trend), P b 0.1.*, P b 0.05.**, P b 0.01.***, P b 0.001.Non-significant P values which are N0.1 are not shown (empty boxes).

able to retract a fibrin clot and the retraction was considered to be me-diated by the expressed αIIbβ3 [6,7,9]. However, these cells harbor en-dogenic αv and can form a chimeric αvβ3 complex that can alsocontribute to clot retraction. In the work presented here, we used BHKcells, which harbor an endogenic hamster αv. We therefore measuredthe expression of the chimeric αvβ3 relative to αIIbβ3 and found thatin cells transfected with WT human αIIbβ3, αvβ3 was expressed toabout half of αIIbβ3 (Fig. 2). To assess the relative contribution ofhuman αIIbβ3 and the chimeric αvβ3 to clot retraction, we performedall our assays both in the presence and absence of a specificαvβ3 inhib-itor RO0655233-001. We observed that cells expressing the WT β3integrins retracted the fibrin clot over an 18 hours period both in thepresence and absence of the αvβ3 inhibitor. However, the presence ofthe inhibitor significantly decreased the extent and the initial rate ofclot retraction. A similar inhibitory effect was observed with anotherαvβ3 inhibitor, the LM609 antibody, reinforcing our conclusion thatthe chimeric αvβ3, as well as αIIbβ3, contribute to clot retraction(Fig. 3). Thus, it is very important to block the chimeric αvβ3 whenstudying the net effect of human αIIbβ3 expressed in mammaliancells on clot retraction.

It was previously reported that in nucleated cells expressing bothαIIbβ3 and αvβ3, only αvβ3 spontaneously supported fibrin clot re-traction while αIIbβ3 required prior activation [4]. In the presentstudy we show that prior activation is not a prerequisite for eitherαIIbβ3- or αvβ3- mediated clot retraction (Fig. 3). The conflicting re-sults probably stem from the difference in the time-frame of the exper-iments; in their study, Chen et al., measured clot retraction for twohours only [4]. At this time frame we also observed no substantial re-traction in the presence of the αvβ3 inhibitor (Fig. 3). However, over alonger time period of 3–18 hours, the cells with the inhibitor exhibiteda significant clot retraction, indicating that αIIbβ3 can mediate sponta-neous, although slower, clot retraction. One explanation to the ability ofαIIbβ3 to spontaneously retract the clot is that it can change its confor-mation from a resting to an active formwhen it is bound to a fibrin clot.

T + anti-LIBS6 antibody in the presence or absence of DTNB.

4S C560S WT + Lbefore WT + L after

+ - + - + - +

* T T* ** *

* * T***** *

A

B

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

WT

WT+DTNBWT+LIBS6WT+DTNB+LIBS6

WT+LIBS6+DTNB

Time (hours)

% c

lot

retr

acti

on

0 1 2 3 4 5 60

20

40

60

80

100

16 17 18

WT

WT+DTNBWT+LIBS6WT+DTNB+LIBS6

WT+LIBS6+DTNB

Time (hours)

% c

lot

retr

acti

on

With αvβ3 inhibitor

Fig. 6. The effect of the activating antibody anti-LIBS6 on clot retraction of cells expressingWT ß3 integrins in the presence or absence of DTNB. A time course of the average clot re-traction of BHK cells transfectedwith normal humanαIIb andβ3 (WT),measured at 1, 3, 5and 18 hours, in the absence (blue) or the presence (pale blue) of DTNB, in the presence ofanti-LIBS6 antibody (red), and in the presence of both anti-LIBS6 and DTNB that wasadded either before (orange) or after (green) anti-LIBS6. The experiments were done ei-ther without (A) or with (B) αvβ3 inhibitor as indicated. Each point represents mean ±SE of three experiments.

834 R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

This assumption was supported by the observation that prior activationof the receptor by the addition of anti-LIBS6 antibody significantly en-hanced clot retraction of cells expressing the WT integrins (Fig. 6). No-tably, the enhancing effect wasmore pronounced in the presence of theαvβ3 inhibitor, implying thatαIIbβ3 ismore affected byprior activationthan αvβ3.

Platelets and cells expressingαIIbβ3 adhere to and spread on fibrin-ogenwithout prior activation and integrin activation enhanced their ad-hesion [28,29]. Both αIIbβ3-mediated cell adhesion and fibrin clotretraction are considered to be post-ligation signaling events that in-duce tyrosine phosphorylation and cytoskeleton reorganization and inboth processes an initial step of ligand binding and conformationalchange of the integrin to an active conformer is required [28,29]. Ourdata show that similarly to αIIbβ3-mediated cell adhesion, αIIbβ3-mediated clot retraction occurs without prior integrin activation and isaugmented byprior activation suggesting that in bothprocesses, the ini-tial integrin activation is a rate limiting step.

We and others showed that disulfide bond exchange is required forαIIbβ3 mediated activation, aggregation and adhesion to immobilizedfibrinogen [13,14,20–23]. However, the role of disulfide exchange inαIIbβ3-mediated clot retraction has not been investigated. In thisstudy, we addressed the question whether disulfide bond exchange isinvolved in αIIbβ3 as well as αvβ3-mediated clot retraction. We dem-onstrated that addition of the thiol blocker DTNB to cells expressingthe WT β3 integrins significantly decreased the extent and rate of clotretraction, in either the presence or absence of the αvβ3 inhibitor(Fig. 4A). Similar inhibitory effect was observed with another knownthiol blocker, pCMBS (Fig. 4C), indicating that bothβ3 integrinsmediateclot retraction in a disulfide bond exchange-dependent manner.

We have previously shown that certain cysteinemutations in the β3subunit render the integrins constitutively active [21]. In the current

study, we showed that cells expressing these mutated integrinsretracted the clot faster than cells expressing the WT integrins (Fig. 5),just as did cells expressing WT integrins activated with anti-LIBS6(Fig. 6). This implies that the enhanced effect of integrin activation is ageneral mechanism, regardless of whether the activation was causedby anti-LIBS6 or by specific mutations.

In contrast to its effect on clot retraction by cells expressing WTintegrins, DTNB did not significantly decrease the extent and rate ofclot retraction by cells expressing single cysteine substitutions C523S,C544S and C560S (Fig. 5). This was consistent with the observationthat DTNB had no significant effect on the activation state of these mu-tants as they still bound high levels of PAC-1 in its presence (Fig. 1). Ineach of thesemutants, a disulfide bondwas disrupted by cysteinemuta-tion, leaving a free thiol in the unpaired cysteine partner. Conceivably,this free thiol initiates disulfide bond rearrangement that alters theintegrin conformation and converts it to the active form [13–15].These findings suggest that in these mutants, the process of disulfidebond rearrangementwas already completed and the integrins no longerdepended on disulfide bond exchange to be constitutively active and forpost-ligation signaling required for clot retraction.

Interestingly, DTNB significantly decreased the extent and rate of clotretraction by cells expressing the double cysteine substitution C523S/C544S. These mutated integrins resembled WT integrins since they didnot harbor free thiols. Thus, in the absence of free thiols, β3 integrin-mediated clot retraction depended on disulfide bond exchange, suggest-ing that new disulfide bond-shuffling occurred during this process. Itshould benoted that the C523S/C544SmutatedαIIbβ3was constitutivelyactive and its activation statewas hardly affected byDTNB (Fig. 1), imply-ing that when thismutantmediate fibrin clot retraction it depends on di-sulfide bond exchange during the post-ligation step.

Although the precise disulfide bonds involved in the disulfide bondexchange-mediated activation of αIIbβ3 and αvβ3 have not been iden-tified, our previouswork suggested that there is a regulatory role for theC560-C583 bond in αIIbβ3 and αvβ3 activation [20,21]. Moreover, theC560S substitution mimics two naturally occurring C560R and C560Fmutations that caused the severe bleeding disorder Glanzmannthrombasthenia and resulted in constitutive activation of αIIbβ3[20,30,31], suggesting that this bond has a physiologic role inαIIbβ3 ac-tivation. The C523-C544 bond has an allosteric configuration in theαvβ3 crystal structure, a configuration associated with a functionalrole of disulfide bonds [21,32]. Taken together, previous data and the re-sults presented here suggest that both the C560-C583 and the C523-C544 bonds can participate in the cascade of disulfide bond exchangein β3.

In contrast to the mutated integrins that were already expressed onthe cell surface in the active conformer, anti-LIBS6 antibody mediatedthe activation of integrins expressed on the cell surface in the restingsate. Indeed, addition of DTNB to cells expressing theWT integrins, pro-foundly inhibited anti-LIBS6-induced activation of αIIbβ3, as shown bya 52% reduction in PAC-1 binding compared to anti-LIBS6 activated cellsin the absence of DTNB (Fig. 1), confirming our previous studies whichshowed that disulfide bond exchange is important for the activation ofαIIbβ3 [20,21,23]. Thus, during β3-integrin mediated clot retraction,DTNB added to cells expressing the WT integrins prior to anti-LIBS6,inhibited the activation step and possibly the post-ligation step whileDTNB added after anti-LIBS6 inhibitedmainly the post-ligation step. No-tably, the significant inhibitory effect of DTNB on anti-LIBS6-enhancedclot retraction was only manifested in the presence of the αvβ3 inhibi-tor, indicating that disulfide bond exchange was necessary for anti-LIBS6-enhanced clot retraction by αIIbβ3 but not by αvβ3. However,in the presence of αvβ3 inhibitor, the inhibitory effect was muchmore pronounced when DTNB was added before anti-LIBS6, suggestingthat disulfide bond exchange was required for both αIIbβ3 activationand post-ligation signaling during fibrin clot retraction.

The thiol isomerases Protein Disulfide Isomerase (PDI) and ERp57,mediate disulfide bond exchanges and are important for platelet

835R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

aggregation and adhesion and for thrombus formation [22,33–36].Platelet αIIbβ3 and endothelial cells αvβ3 contribute to binding of PDIto vascular injury site [36]. Thus, it is reasonable to assume that PDIand ERp57 play a role in catalyzing disulfide exchange reaction leadingtoαIIbβ3- andαvβ3-mediated clot retraction upon vascular injury. Thespecific role of PDI and Erp57 in fibrin clot retraction mediated by β3integrins is of particular interest for further investigation.

In this study, we show that αvβ3 expressed in BHK cells did not de-pend on activation for mediating fibrin clot retraction and also was notaffected by prior activation. These results are consistent with evidencesuggesting that activation of αvβ3 is not mandatory for its function invarious cell lines and tissues [27,37–39]. Platelets express small amountsof αvβ3 on their surface, but its contribution to platelet mediated clotretraction is probably negligible since platelet αIIbβ3 expression ishigher than αvβ3 expression by two orders of magnitude. Moreover,LM609 antibody, which blocksαvβ3, did not prevent platelet clot retrac-tion [10]. In addition, platelets fromGlanzmann thrombasthenia patientsthat were homozygous for a mutation in the αIIb gene and thus did notexpress αIIbβ3 yet did express increased amounts of αvβ3, did not dis-play fibrin clot retraction [40,41], indicating that αvβ3 plays no role inplatelet clot retraction. In contrast to platelets, endothelial cells expresslarge amounts ofαvβ3 and upon vascular injury its expression increases[39]. Moreover, αvβ3 was shown to be involved in endothelial cell-mediated clot retraction [42]. Thus, αvβ3 can mediate fibrin clot retrac-tion by endothelial cells, especially at the edges of vascular injury, andpromote closure of the wound corners. Taken together, we suggest thatboth αIIbβ3 on platelets and αvβ3 on endothelial cells contribute toclot retraction following vascular injury and that platelet αIIbβ3 activa-tion at the site of injury enhances clot retraction.

In summary, we showed that both integrins αIIbβ3 and αvβ3 con-tribute to clot retraction without prior activation although αIIbβ3 acti-vation significantly enhances clot retraction rate. Both αIIbβ3activation and post-ligation signaling during clot retraction depend ondisulfide bond exchange, suggesting a regulatory role for disulfidebond exchange in this process.

Conflict of Interest Statement

The authors have no conflict of interest.

Acknowledgment

We thank Dr. Mark Ginsberg from the department of Medicine, Uni-versity of California, San Diego, La Jolla, CA for providing anti-LIBS6 an-tibody and Dr. Peter Newman from the Blood Center of Wisconsin,Milwaukee, WI for providing αIIb and β3 cDNAs.

References

[1] Jackson SP, NesbittWS, Kulkarni S. Signaling events underlying thrombus formation.J Thromb Haemost 2003;1:1602–12.

[2] Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelet paradigm. Blood1998;91:2645–57.

[3] Hynes RO. Integrins: bidirectional, allosteric signalingmachines. Cell 2002;110:673–87.[4] Chen YP, O'Toole TE, Leong L, Liu BQ, Diaz-Gonzalez F, GinsbergMH. Beta 3 integrin-

mediated fibrin clot retraction by nucleated cells: differing behavior of alpha IIb beta3 and alpha v beta 3. Blood 1995;86:2606–15.

[5] Haling JR, Monkley SJ, Critchley DR, Petrich BG. Talin-dependent integrin activationis required for fibrin clot retraction by platelets. Blood 2011;117:1719–22.

[6] Chen YP, O'Toole TE, Ylanne J, Rosa JP, Ginsberg MH. A point mutation in the integrinbeta 3 cytoplasmic domain (S752– N P) impairs bidirectional signaling throughalpha IIb beta 3 (platelet glycoprotein IIb-IIIa). Blood 1994;84:1857–65.

[7] Lyman S, Gilmore A, Burridge K, Gidwitz S, White II GC. Integrin-mediated activationof focal adhesion kinase is independent of focal adhesion formation or integrin acti-vation. Studies with activated and inhibitory beta3 cytoplasmic domain mutants.J Biol Chem 1997;272:22538–47.

[8] Hauschner H, Landau M, Seligsohn U, Rosenberg N. A unique interaction betweenalphaIIb and beta3 in the head region is essential for outside-in signaling-relatedfunctions of alphaIIbbeta3 integrin. Blood 2010;115:4542–50.

[9] Alrehani N, Pradhan S, Khatlani T, Kailasam L, Vijayan KV. Distinct roles for the alpha,beta and gamma1 isoforms of protein phosphatase 1 in the outside-in alphaIIbbeta3integrin signalling-dependent functions. Thromb Haemost 2013;109:118–26.

[10] Katagiri Y, Hiroyama T, Akamatsu N, Suzuki H, Yamazaki H, Tanoue K.Involvement of alpha v beta 3 integrin in mediating fibrin gel retraction. J BiolChem 1995;270:1785–90.

[11] Morel-Kopp MC, Melchior C, Chen P, Ammerlaan W, Lecompte T, Kaplan C, et al. Anaturally occurring point mutation in the beta3 integrin MIDAS-like domain affectsdifferently alphavbeta3 and alphaIIIbbeta3 receptor function. Thromb Haemost2001;86:1425–34.

[12] Schaffner-Reckinger E, Brons NH, Kieffer N. Evidence from site-directedmutagenesisthat the cytoplasmic domain of the beta3 subunit influences the conformationalstate of the alphaVbeta3 integrin ectodomain. Thromb Haemost 2001;85:716–23.

[13] Yan B, Smith JW. A redox site involved in integrin activation. J Biol Chem2000;275:39964–72.

[14] Yan B, Smith JW. Mechanism of integrin activation by disulfide bond reduction. Bio-chemistry 2001;40:8861–7.

[15] Essex DW, Li M, Miller A, Feinman RD. Protein disulfide isomerase and sulfhydryl-dependent pathways in platelet activation. Biochemistry 2001;40:6070–5.

[16] Xiong JP, Mahalingham B, Alonso JL, Borrelli LA, Rui X, Anand S, et al. Crystal struc-ture of the complete integrin alphaVbeta3 ectodomain plus an alpha/beta trans-membrane fragment. J Cell Biol 2009;186:589–600.

[17] Zhu J, Luo BH, Xiao T, Zhang C, Nishida N, Springer TA. Structure of a completeintegrin ectodomain in a physiologic resting state and activation and deactivationby applied forces. Mol Cell 2008;32:849–61.

[18] Beglova N, Blacklow SC, Takagi J, Springer TA. Cysteine-rich module structure revealsa fulcrum for integrin rearrangement upon activation. Nat Struct Biol 2002;9:282–7.

[19] Takagi J, Beglova N, Yalamanchili P, Blacklow SC, Springer TA. Definition of EGF-like,closely interacting modules that bear activation epitopes in integrin beta subunits.Proc Natl Acad Sci U S A 2001;98:11175–80.

[20] Mor-Cohen R, Rosenberg N, Landau M, Lahav J, Seligsohn U. Specific cysteines inbeta3 are involved in disulfide bond exchange-dependent and -independent activa-tion of alphaIIbbeta3. J Biol Chem 2008;283:19235–44.

[21] Mor-Cohen R, Rosenberg N, Einav Y, Zelzion E, Landau M, Mansour W, et al. Uniquedisulfide bonds in epidermal growth factor (EGF) domains of beta3 affect structureand function of alphaIIbbeta3 and alphavbeta3 integrins in different manner. J BiolChem 2012;287:8879–91.

[22] Lahav J, Gofer-Dadosh N, Luboshitz J, Hess O, Shaklai M. Protein disulfide isomerasemediates integrin-dependent adhesion. FEBS Lett 2000;475:89–92.

[23] Lahav J, Jurk K, Hess O, Barnes MJ, Farndale RW, Luboshitz J, et al. Sustained integrinligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfideexchange. Blood 2002;100:2472–8.

[24] Yatuv R, Rosenberg N, Zivelin A, Peretz H, Dardik R, Trakhtenbrot L, et al. Identificationof a region in glycoprotein IIIa involved in subunit association with glycoprotein IIb:further lessons from Iraqi-Jewish Glanzmann thrombasthenia. Blood 2001;98:1063–9.

[25] Ghevaert C, Salsmann A,Watkins NA, Schaffner-Reckinger E, Rankin A, Garner SF, et al.A nonsynonymous SNP in the ITGB3 gene disrupts the conservedmembrane-proximalcytoplasmic salt bridge in the alphaIIbbeta3 integrin and cosegregates dominantlywith abnormal proplatelet formation and macrothrombocytopenia. Blood2008;111:3407–14.

[26] Salsmann A, Schaffner-Reckinger E, Kabile F, Plancon S, Kieffer N. A new functionalrole of the fibrinogen RGD motif as the molecular switch that selectively triggersintegrin alphaIIbbeta3-dependent RhoA activation during cell spreading. J BiolChem 2005;280:33610–9.

[27] Pampori N, Hato T, Stupack DG, Aidoudi S, Cheresh DA, Nemerow GR, et al. Mecha-nisms and consequences of affinity modulation of integrin alpha(V)beta(3) detectedwith a novel patch-engineered monovalent ligand. J Biol Chem 1999;274:21609–16.

[28] Gartner TK, Amrani DL, Derrick JM, Kirschbaum NE, Matsueda GR, Taylor DB. Char-acterization of adhesion of "resting" and stimulated platelets to fibrinogen and itsfragments. Thromb Res 1993;71:47–60.

[29] Moroi M, Jung SM. Integrin-mediated platelet adhesion. Front Biosci 1998;3:d719–28.

[30] Ruiz C, Liu CY, Sun QH, Sigaud-Fiks M, Fressinaud E,Muller JY, et al. A point mutationin the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of aGPIIb-IIIa (alphaIIbbeta3) integrin receptor locked in a high-affinity state and aGlanzmann thrombasthenia-like phenotype. Blood 2001;98:2432–41.

[31] Ambo H, Kamata T, Handa M, Taki M, Kuwajima M, Kawai Y, et al. Three novelintegrin beta3 subunit missense mutations (H280P, C560F, and G579S) inthrombasthenia, including one (H280P) prevalent in Japanese patients. BiochemBiophys Res Commun 1998;251:763–8.

[32] Schmidt B, Ho L, Hogg PJ. Allosteric disulfide bonds. Biochemistry 2006;45:7429–33.[33] Lahav J, Wijnen EM, Hess O, Hamaia SW, Griffiths D, Makris M, et al. Enzymatically

catalyzed disulfide exchange is required for platelet adhesion to collagen via integrinalpha2beta1. Blood 2003;102:2085–92.

[34] Holbrook LM, Sasikumar P, Stanley RG, Simmonds AD, Bicknell AB, Gibbins JM. Theplatelet-surface thiol isomerase enzyme ERp57 modulates platelet function. JThromb Haemost 2012;10:278–88.

[35] Wang L, Wu Y, Zhou J, Ahmad SS, Mutus B, Garbi N, et al. Platelet-derived ERp57me-diates platelet incorporation into a growing thrombus by regulation of thealphaIIbbeta3 integrin. Blood 2013;122:3642–50.

[36] Cho J, Kennedy DR, Lin L, HuangM,Merrill-Skoloff G, Furie BC, et al. Protein disulfideisomerase capture during thrombus formation in vivo depends on the presence ofbeta3 integrins. Blood 2012;120:647–55.

[37] Kieffer N, Fitzgerald LA, Wolf D, Cheresh DA, Phillips DR. Adhesive properties of thebeta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individuallyexpressed in human melanoma cells. J Cell Biol 1991;113:451–61.

836 R. Mor-Cohen et al. / Thrombosis Research 133 (2014) 826–836

[38] Blystone SD, Williams MP, Slater SE, Brown EJ. Requirement of integrin beta3 tyro-sine 747 for beta3 tyrosine phosphorylation and regulation of alphavbeta3 avidity.J Biol Chem 1997;272:28757–61.

[39] Sajid M, Stouffer GA. The role of alpha(v)beta3 integrins in vascular healing. ThrombHaemost 2002;87:187–93.

[40] Coller BS, Seligsohn U, Zivelin A, Zwang E, Lusky A, ModanM. Immunologic and bio-chemical characterization of homozygous and heterozygous Glanzmannthrombasthenia in the Iraqi-Jewish and Arab populations of Israel: comparison oftechniques for carrier detection. Br J Haematol 1986;62:723–35.

[41] Coller BS, Cheresh DA, Asch E, Seligsohn U. Platelet vitronectin receptor expressiondifferentiates Iraqi-Jewish from Arab patients with Glanzmann thrombasthenia inIsrael. Blood 1991;77:75–83.

[42] Smith RA, Mosesson MW, Rooney MM, Lord ST, Daniels AU, Gartner TK. The role ofputative fibrinogen Aalpha-, Bbeta-, and GammaA-chain integrin binding sites in en-dothelial cell-mediated clot retraction. J Biol Chem 1997;272:22080–5.