ABSTRACT Integrins have a critical role in thrombosis and haemostasis1. Antagonists of the platelet integrin αIIbβ3 are potent anti-thrombotic drugs, but also have the life-threatening

adverse effect of causing bleeding2,3. It is therefore desirable to develop new antagonists that do not cause bleeding. Integrins transmit signals bidirectionally4,5. Inside-out signalling

activates integrins through a talin-dependent mechanism6,7. Integrin ligation mediates thrombus formation and outside-in signalling8,9, which requires Gα13 and greatly expands thrombi. Here

we show that Gα13 and talin bind to mutually exclusive but distinct sites within the integrin β3 cytoplasmic domain in opposing waves. The first talin-binding wave mediates inside-out

EXE-motif-based inhibitor of Gα13–integrin interaction selectively abolishes outside-in signalling without affecting integrin ligation, and suppresses occlusive arterial thrombosis without

affecting bleeding time. Thus, we have discovered a new mechanism for the directional switch of integrin signalling and, on the basis of this mechanism, designed a potent new anti-thrombotic

drug that does not cause bleeding. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your

institution Subscribe to this journal Receive 51 print issues and online access $199.00 per year only $3.90 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access

to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read

our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS INTEGRIN Β3 DIRECTLY INHIBITS THE GΑ13-P115RHOGEF INTERACTION TO REGULATE G PROTEIN SIGNALING AND PLATELET

EXOCYTOSIS Article Open access 16 August 2023 TARGETING INTEGRIN PATHWAYS: MECHANISMS AND ADVANCES IN THERAPY Article Open access 02 January 2023 PLATELET INTEGRIN ΑIIBΒ3 PLAYS A KEY ROLE IN

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labeling reagent for liquid chromatographic determination of carboxylic acids. _Anal. Chem._ 60, 2067–2070 (1988) Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank

T. Kozasa, B. Kreutz and C. Chow for providing purified recombinant Gα13 protein; and B. Petrich and D. Critchley for providing talin−/− mice. We acknowledge that H. Gong performed

experiments for this project. This work is supported by grants from National Heart, Lung, and Blood Institute (HL080264, HL062350 (X.D.) and HL109439 (J.C.)). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Department of Pharmacology, University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, Illinois 60612, USA, Bo Shen, Xiaojuan Zhao, Kelly A. O’Brien, Aleksandra

Stojanovic-Terpo, M. Keegan Delaney, Kyungho Kim, Jaehyung Cho, Stephen C.-T. Lam & Xiaoping Du Authors * Bo Shen View author publications You can also search for this author inPubMed 

Google Scholar * Xiaojuan Zhao View author publications You can also search for this author inPubMed Google Scholar * Kelly A. O’Brien View author publications You can also search for this

author inPubMed Google Scholar * Aleksandra Stojanovic-Terpo View author publications You can also search for this author inPubMed Google Scholar * M. Keegan Delaney View author publications

You can also search for this author inPubMed Google Scholar * Kyungho Kim View author publications You can also search for this author inPubMed Google Scholar * Jaehyung Cho View author

publications You can also search for this author inPubMed Google Scholar * Stephen C.-T. Lam View author publications You can also search for this author inPubMed Google Scholar * Xiaoping

Du View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS B.S. performed most of the experiments and participated in experimental design, data

analysis and manuscript writing. X.Z., K.A.O., A.S.-T. and M.K.D. each performed parts of the experiments and participated in aspects of data analyses and manuscript writing. K.K. and J.C.

performed laser-induced thrombosis experiments and data analysis; S.C.-T.L. provided talin constructs and purified proteins, and participated in discussions and data analyses; X.D. designed

and directed the research, analysed data and wrote the paper. CORRESPONDING AUTHOR Correspondence to Xiaoping Du. ETHICS DECLARATIONS COMPETING INTERESTS X.D. holds pending patents on the

EXE-motif-containing peptide inhibitors. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 A SCHEMATIC SHOWING HOW SELECTIVE INHIBITORS OF INTEGRIN OUTSIDE-IN SIGNALLING WORK AS

ANTI-THROMBOTICS. Blue arrows indicate steps that are inhibited. EXTENDED DATA FIGURE 2 THE IMPORTANCE OF THE CONSERVED EXE MOTIF IN INTEGRIN–GΑ13 INTERACTIONS. A, B, Lysates from CHO cells

expressing similar levels of wild-type (WT) αIIbβ3, β3 C-terminal truncation mutants (Δ759, Δ741, Δ728 and Δ715) complexed with wild-type αIIb (A), and β3 EXE motif mutants (E731A, E732A,

E733A and EEE to AAA) complexed with wild-type αIIb (B) were immunoprecipitated with anti-Gα13 antibody or equal amount of control rabbit IgG. Immunoprecipitates and lysates (equivalent of

10% used for immunoprecipitation) were immunoblotted with anti-Gα13 and anti-β3 antibodies. C, D, GST–β3CD (WT) or GST–β3(AAA)CD (AAA mutant) proteins immobilized in glutathione-coated

microtitre wells were incubated with increasing concentrations of Gα13 (C), or increasing concentrations of THD (D). After washing, bound Gα13 and THD were respectively detected using

anti-Gα13 or anti-talin (mouse IgG was used as a specificity control) followed by secondary horseradish peroxidase-labelled anti-IgG antibody. E, In addition to the AAA mutation, conserved

mutations of EEE to DED and EEE to QSE (as found in β5) were introduced to the β3 cytoplasmic domain. These mutants were co-transfected with wild-type αIIb into CHO cells, which were sorted

to achieve comparable expression levels with wild-type-αIIbβ3-expressing cells (as shown in Extended Data Fig. 4e). Lysates from these cells were immunoprecipitated with anti-β3 or equal

amount of pre-immune rabbit serum. Lysates (10%) and immunoprecipitates were immunoblotted with anti-Gα13 or anti-β3. F, Lysates from human platelets (with or without stimulation with 0.025 

U ml−1thrombin) were immunoprecipitated with anti-Gα13 antibody or equal amount of control rabbit IgG. Immunoprecipitates were immunoblotted with anti-Gα13 and anti-β1 antibodies. Gα13 is

associated with β1, which is increased after thrombin stimulation. EXTENDED DATA FIGURE 3 LIGAND OCCUPANCY INDUCES SWITCH OF INTEGRIN ΑIIBΒ3 FROM THE TALIN-BOUND TO THE GΑ13-BOUND STATE. A,

To determine the effect of integrin activation and ligand occupancy on Gα13–β3 association, human platelets were incubated with or without 1 mM MnCl2 and 30 μg ml−1 fibrinogen for 5 min at

22 °C. Platelet lysates were then immunoprecipitated with anti-β3 or pre-immune rabbit serum. Lysates (10%) and immunoprecipitates were immunoblotted with anti-β3 or anti-Gα13. B, C, Washed

human platelets were stimulated with 0.025 U ml−1 α-thrombin with or without adding 2 mM EDTA (an inhibitor of the ligand binding function of integrins), stirred (1,000 r.p.m.) at 37 °C,

solubilized at various time points, and immunoprecipitated with anti-β3 or equal amounts of pre-immune rabbit serum. Lysates (10%) and immunoprecipitates were immunoblotted with anti-Gα13,

anti-talin or anti-β3 antibodies. B, Western blot results. C, Turbidity changes in platelet suspension indicating integrin-dependent platelet aggregation. Note the inhibitory effect of EDTA

on talin dissociation and Gα13 binding to β3. D, As additional controls for Fig. 2a to exclude the possibility of loss of talin and β3 in platelet lysates to insoluble fraction during

integrin signalling, washed human platelets were stimulated with 0.025 U ml−1 α-thrombin in the absence or presence of 2 mM integrin inhibitor RGDS, stirred (1,000 r.p.m.) at 37 °C, and then

solubilized at various time points as in Fig. 2a. Solubilized platelets were centrifuged at 14,000_g_ for 10 min to separate lysates from insoluble pellets. Pellets were dissolved in SDS

sample buffer to the same volume as the lysates after diluting them 1:1 with 2× SDS sample buffer, and both were immunoblotted with anti-β3 and anti-talin antibodies. Note that the levels of

talin and β3 in platelet lysates kept essentially constant during the course of platelet aggregation and, with low concentrations of thrombin used to stimulate platelets, very little

insoluble β3 and talin were present in the pellet, which were detectable only after prolonged exposure (5-min exposure compared to 10 s of normal exposure time) and with no obvious variation

during the course of platelet aggregation. EXTENDED DATA FIGURE 4 EFFECTS OF SHRNA-INDUCED TALIN KNOCKDOWN AND TALIN KNOCKOUT ON INTEGRIN SIGNALLING. A, Western blot comparison of talin 1

expression levels in mouse platelets derived from control shRNA- or talin-shRNA-transfected bone marrow stem cells. Western blots of Gα13, and integrin β1 and β3 are also shown. B, Adhesion

of unstimulated mouse platelets to immobilized fibrinogen for 1 h. Adherent platelets were quantified as percentage of total platelets loaded (mean ± s.d., _n_ = 4). C, Turbidity changes in

mouse platelet suspension stimulated with 5 μM ADP in the presence of 20 μg ml−1 fibrinogen, with or without 1 mM MnCl2, as detected using an aggregometer. D, Fluorescence microscopy images

of phalloidin-stained mouse platelet spreading on fibrinogen for 1 h, with or without 1 mM MnCl2. E, Quantification of surface areas of individual adherent platelets as shown in Fig. 2g

(mean ± s.e.m.). EXTENDED DATA FIGURE 5 EFFECTS OF AAA MUTATION ON INTEGRIN OUTSIDE-IN SIGNALLING IN PLATELETS. A, Flow cytometric analysis of integrin αIIbβ3 expression levels in β3−/−

mouse platelets transfected with wild-type or AAA mutant β3 using bone marrow stem cell transplantation technology in comparison with C57BL/6 mouse platelets. β3−/− platelets were used as a

negative control. αIIbβ3 complex was detected using an anti-mouse αIIb antibody. B, Mouse platelets expressing recombinant wild-type or AAA mutant β3 as in A were lysed and

immunoprecipitated with anti-β3 or equal amounts of pre-immune rabbit serum. Lysates (10%) and immunoprecipitates were immunoblotted with anti-SRC or anti-β3 antibodies. C, D, Spreading of

phalloidin-stained wild-type platelets (EEE), AAA mutant platelets and AAA mutant platelets incubated with mP6Scr or mP6 on immobilized fibrinogen for 1 h. C, Typical fluorescence microscopy

images. D, Quantification of surface areas of individual platelets (mean ± s.e.m.). EXTENDED DATA FIGURE 6 EFFECTS OF MUTATIONAL DISRUPTION OF THE EXE MOTIF ON INTEGRIN OUTSIDE-IN

SIGNALLING. A, Expression levels of wild-type or the EXE motif (QSE, DED or AAA) mutants of β3 in complex with wild-type αIIb in CHO cells, as determined by flow cytometry. Mouse IgG was

used as a negative control. B, C, Spreading of CHO-1b9 cells expressing wild-type αIIbβ3, and QSE, DED or AAA mutant αIIbβ3 on fibrinogen for 1 h. B, Quantification of surface areas of

individual cells (mean ± s.e.m.). C, Typical microscopy images. D, Flow cytometric analysis of wild-type αIIbβ3, AAA or Y747A mutant αIIbβ3 expression in CHO cells. Mouse IgG was used as a

control. E, CHO cells expressing wild-type, AAA or Y747A β3 without (top panels) or with (bottom panels) co-expression of recombinant THD were solubilized and immunoprecipitated with anti-β3

or pre-immune serum. 10% lysates and immunoprecipitates were immunoblotted with anti-talin, anti-Gα13 or anti-β3 antibodies. F, Typical western blots for Fig. 3g. Wild-type or

AAA-mutant-αIIbβ3-expressing CHO-1b9 cells were allowed to adhere to immobilized fibrinogen, solubilized at various time points, and analysed for RHOA activation and SRC Tyr 416

phosphorylation. EXTENDED DATA FIGURE 7 MP6 SELECTIVELY INHIBITS INTEGRIN OUTSIDE-IN SIGNALLING WITHOUT AFFECTING INSIDE-OUT SIGNALLING. A–D, Washed human platelets were stimulated with

0.025 U ml−1 α-thrombin in the absence or presence of 250 μM myristoylated peptides, mP13 (A, B) and mP6 (C, D) with stirring (1,000 r.p.m.) at 37 °C, and then solubilized at various time

points. Lysates were immunoprecipitated with anti-β3 rabbit serum or equal amounts of pre-immune serum. Lysates (10%) and immunoprecipitates were immunoblotted with anti-Gα13, anti-talin or

anti-β3 antibodies. A, C, Typical western blot results. B, D, Typical turbidity changes in platelet suspension indicating integrin-dependent platelet aggregation. E, Quantification of human

platelet spreading on immobilized fibrinogen for 1 h, without or with treatment with DMSO, mP6Scr, or mP6 as shown in Fig. 4b (mean surface area ± s.e.m.). F, Flow cytometric analysis of

PAR4-AP-induced Oregon Green-labelled soluble fibrinogen binding to human platelets pre-treated with 100 μM mP6Scr or 100 μM mP6 stimulated with increasing concentrations of PAR4-AP.

Integrilin-treated platelets were used as a negative control. G, Flow cytometric analysis of 100 μM PAR4-AP-induced PAC1 binding to human platelets pre-treated with 100 μM mP6Scr or mP6.

Integrilin-treated platelets were used as negative control. H, Flow cytometric analysis of PAR4-AP-induced Oregon Green-labelled soluble fibrinogen binding to human platelets pre-treated

with solvent DMSO, mP13Scr or mP13. Resting platelets were used as a negative control. EXTENDED DATA FIGURE 8 THE _IN VIVO_ EFFECT OF MP6: SELECTIVE INHIBITION OF THROMBOSIS BUT NOT

HAEMOSTASIS. A, Representative images of laser-induced mouse cremaster arteriolar thrombosis (red) in the context of the bright-field microvascular histology, visualized by infusion of

nonblocking rat anti-mouse GPIbβ antibody conjugated to DyLight 649. The C57BL/6 mice were injected with 5 μmol kg−1micellar formulated mP6 or mP6Src (negative control), 12 μmol 

kg−1Integrillin or buffer, 3 min before laser-induced arteriolar wall injury. White arrows indicate the directions of the blood flow. B, The mean platelet fluorescence intensity for 30

thrombi (performed in three mice) for each treatment at selected time points (mean ± s.e.m., _n_ = 30, _t_-test). Fluorescence in mP6- and Integrilin-treated mice is minimal. C, Comparison

of mP6 (5 μmol kg−1) with the same dose of Integrilin and their respective controls in occlusion time of FeCl3-induced carotid artery thrombosis in mice. Typical arterial blood flow charts

of FeCl3-induced occlusive thrombosis are shown. D, Comparison of mP6 (5 μmol kg−1) with the same dose of Integrilin and controls in mouse tail bleeding analysis. Released haemoglobin levels

were used as a parameter to assess blood loss (mean ± s.d., _n_ = 10). EXTENDED DATA FIGURE 9 PLATELET UPTAKE OF MP6 AND MP6SCR, AND NO EFFECT OF MP6 ON HEMOGRAM. A, Estimation of

intracellular levels of 1-pyrenyldiazomethane (PDAM)-conjugated mP6 and mP6Scr following incubation with platelets for 5 min. Platelets were pelleted by centrifugation, and the amounts of

PDAM-conjugated peptides in platelet lysates were estimated (mean ± s.d., _n_ = 3). B, Haemogram of mouse whole blood before or 1 h after injection of mP6 or mP6Scr (5 μmol kg−1), showing no

significant differences. SUPPLEMENTARY INFORMATION ARTERIOLAR THROMBOSIS: BUFFER CONTROL This file shows Intravital videomicroscopy of the cremaster muscle arteriolar circulation and

platelet thrombus formation (red) after laser injury, with HEPES buffer injection. (MOV 8136 kb) ARTERIOLAR THROMBOSIS: INTEGRILIN TREATMENT This file shows Intravital videomicroscopy of the

cremaster muscle arteriolar circulation and thrombus formation (red) after laser injury, with Integrilin (12 µM per kg) injection. (MOV 9312 kb) ARTERIOLAR THROMBOSIS: MP6SRC CONTROL This

file shows Intravital videomicroscopy of the cremaster muscle arteriolar circulation and thrombus formation (red) after laser injury, with mP6Scr micelle (5 µM per kg) injection. (MOV 8138

kb) ARTERIOLAR THROMBOSIS: MP6SRC CONTROL This file shows Intravital videomicroscopy of the cremaster muscle arteriolar circulation and thrombus formation (red) after laser injury, with mP6

micelle (5 µM per kg) injection. (MOV 7981 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Shen, B., Zhao, X., O’Brien, K. _et al._ A directional switch of integrin signalling and a new anti-thrombotic

strategy. _Nature_ 503, 131–135 (2013). https://doi.org/10.1038/nature12613 Download citation * Received: 08 November 2012 * Accepted: 28 August 2013 * Published: 27 October 2013 * Issue

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