Autonomous conformational regulation of β3 integrin and the ...Autonomous conformational regulation...

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Autonomous conformational regulation of β 3 integrin and the conformation-dependent property of HPA-1a alloantibodies Aye Myat Myat Thinn a,b , Zhengli Wang a , Dongwen Zhou a , Yan Zhao a,c , Brian R. Curtis a , and Jieqing Zhu a,b,1 a Blood Research Institute, BloodCenter of Wisconsin, Part of Versiti, Milwaukee, WI 53226; b Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226; and c Department of Physiology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Edited by Barry S. Coller, The Rockefeller University, New York, NY, and approved August 16, 2018 (received for review April 10, 2018) Integrin α/β heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale con- formational rearrangement. During the inside-out activation, sig- nals impinging on the cytoplasmic tail of β subunit induce the α/β separation at the transmembrane and cytoplasmic domains, lead- ing to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrange- ment in the inside-out activation. The β 3 integrins, including α IIb β 3 and α V β 3 , are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the β 3 PSI do- main defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically im- portant bleeding disorders. Some, but not all, antiHPA-1a alloan- tibodies can distinguish the α IIb β 3 from α V β 3 and affect their functions with unknown mechanisms. Here we designed a single-chain β 3 subunit that mimics a separation of α/β hetero- dimer on inside-out activation. Our crystallographic and functional studies show that the single-chain β 3 integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the β 3 subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain β 3 integrin, we identified the conformation-dependent property of antiHPA-1a alloantibodies, which enables them to differently recognize the β 3 in the bent state vs. the extended state and in the complex with α IIb vs. α V . This study provides deeper understandings of integrin conformational activation on the cell surface. integrin structure | integrin activation | conformational change | human platelet alloantigen 1 | alloimmune thrombocytopenia I ntegrins are cell adhesion molecules containing α and β sub- units. The combination of 18 α and 8 β subunits forms 24 integrin α/β heterodimers in humans, playing important roles in numerous physiological and pathophysiological events (1). Each subunit contains a large ectodomain composed of head- piece and leg domains, a single-pass transmembrane (TM) do- main, and usually a short cytoplasmic tail (CT) (SI Appendix, Fig. S1 AF). The β subunit is composed of βI, hybrid, PSI, I-EGF14, β-tail, TM, and CT domains. The α subunit is composed of β-propeller, thigh, calf-12, TM, and CT domains (SI Appendix, Fig. S1 AF). A subset of α subunits contains an αI domain inserted into the β-propeller domain (SI Appendix, Fig. S1 DF). The αI and βI are structural homologs that share a similar fea- ture of conformational change (2, 3) (SI Appendix, Fig. S1 AF). The αI itself has the full ligand binding capability, while the βI requires the combination with the β-propeller domain of α sub- unit to bind ligand (SI Appendix, Fig. S1). Crystallographic and EM studies revealed the large-scale conformational rearrangements of both α and β subunits, which are one of the key processes of integrin activation (4) (SI Appendix, Fig. S1). Crystal structures of the ectodomains of platelet integrin α IIb β 3 and endothelial integrin α V β 3 revealed the large interfaces between the α-β head and leg domains, and between the headpiece and leg domains (58). The interface between the headpiece and leg domains is largely contributed by the β subunit (SI Appendix, Fig. S1A). Disulfide crosslinking and NMR studies of α IIb β 3 demonstrate the associations of α-β at the TM and CT domains (912). It has been suggested that these α-β interactions at the leg, TM, and CT domains help maintain integrin in the bent resting state (SI Appendix, Fig. S1 A and D). Activating signals inside the cell (i.e., the binding of talin and kindlin to the β CT) (1315), induce the α-β separation at the TM and CT domains, leading to the headpiece extension and opening, resulting in an extended high-affinity conformation competent for binding ligand (4) (SI Appendix, Fig. S1 C and F). However, it remains unknown what drives the large-scale con- formational rearrangement on the cell surface after the α-β TM-CT separation. The relative contributions of the α and β subunits to the bent and extended conformation of integrin are not well defined. Moreover, the integrin ectodomains adopt the bent conformation in solution even in the absence of TM-CT domains (5, 16). The integrin headpiece fragments were crystallized in the closed Significance Integrin inside-out activation is initiated by the α/β separation at the cytoplasmic and transmembrane domains. Such separa- tion leads to complicated local and global conformational rearrangements of β subunit, while the conformational change of α subunit is relatively simple. It is unclear whether the global structural changes of β subunit depend on the α subunit. Using a single-chain β 3 construct to mimic an extreme condition of fully separated α/β heterodimer, we show that the β 3 subunit autonomously drives the membrane-dependent bent-to- extended conformational rearrangement in the absence of the α subunit. In addition, we show that the clinically important antiHPA-1a (β 3 -L33) alloantibodies perform the conformation- dependent property when interfering with the β 3 integrin, which may correlate with the pathogenesis of β 3 -mediated alloimmune thrombocytopenia. Author contributions: J.Z. designed research; A.M.M.T., Z.W., D.Z., Y.Z., and J.Z. per- formed research; B.R.C. contributed new reagents/analytic tools; A.M.M.T., Z.W., D.Z., Y.Z., B.R.C., and J.Z. analyzed data; and A.M.M.T. and J.Z. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Published under the PNAS license. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID code 6BXJ). 1 To whom correspondence should be addressed. Email: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1806205115/-/DCSupplemental. Published online September 12, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1806205115 PNAS | vol. 115 | no. 39 | E9105E9114 CELL BIOLOGY Downloaded by guest on May 24, 2021

Transcript of Autonomous conformational regulation of β3 integrin and the ...Autonomous conformational regulation...

Page 1: Autonomous conformational regulation of β3 integrin and the ...Autonomous conformational regulation of β3 integrin and the conformation-dependent property of HPA-1a alloantibodies

Autonomous conformational regulation of β3 integrinand the conformation-dependent property ofHPA-1a alloantibodiesAye Myat Myat Thinna,b, Zhengli Wanga, Dongwen Zhoua, Yan Zhaoa,c, Brian R. Curtisa, and Jieqing Zhua,b,1

aBlood Research Institute, BloodCenter of Wisconsin, Part of Versiti, Milwaukee, WI 53226; bDepartment of Biochemistry, Medical College of Wisconsin,Milwaukee, WI 53226; and cDepartment of Physiology, School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai201203, China

Edited by Barry S. Coller, The Rockefeller University, New York, NY, and approved August 16, 2018 (received for review April 10, 2018)

Integrin α/β heterodimer adopts a compact bent conformation inthe resting state, and upon activation undergoes a large-scale con-formational rearrangement. During the inside-out activation, sig-nals impinging on the cytoplasmic tail of β subunit induce the α/βseparation at the transmembrane and cytoplasmic domains, lead-ing to the extended conformation of the ectodomain with theseparated leg and the opening headpiece that is required for thehigh-affinity ligand binding. It remains enigmatic which integrinsubunit drives the bent-to-extended conformational rearrange-ment in the inside-out activation. The β3 integrins, including αIIbβ3and αVβ3, are the prototypes for understanding integrin structuralregulation. The Leu33Pro polymorphism located at the β3 PSI do-main defines the human platelet-specific alloantigen (HPA) 1a/b,which provokes the alloimmune response leading to clinically im-portant bleeding disorders. Some, but not all, anti–HPA-1a alloan-tibodies can distinguish the αIIbβ3 from αVβ3 and affect theirfunctions with unknown mechanisms. Here we designed asingle-chain β3 subunit that mimics a separation of α/β hetero-dimer on inside-out activation. Our crystallographic and functionalstudies show that the single-chain β3 integrin folds into a bentconformation in solution but spontaneously extends on the cellsurface. This demonstrates that the β3 subunit autonomouslydrives the membrane-dependent conformational rearrangementduring integrin activation. Using the single-chain β3 integrin, weidentified the conformation-dependent property of anti–HPA-1aalloantibodies, which enables them to differently recognize theβ3 in the bent state vs. the extended state and in the complex withαIIb vs. αV. This study provides deeper understandings of integrinconformational activation on the cell surface.

integrin structure | integrin activation | conformational change | humanplatelet alloantigen 1 | alloimmune thrombocytopenia

Integrins are cell adhesion molecules containing α and β sub-units. The combination of 18 α and 8 β subunits forms

24 integrin α/β heterodimers in humans, playing important rolesin numerous physiological and pathophysiological events (1).Each subunit contains a large ectodomain composed of head-piece and leg domains, a single-pass transmembrane (TM) do-main, and usually a short cytoplasmic tail (CT) (SI Appendix, Fig.S1 A–F). The β subunit is composed of βI, hybrid, PSI, I-EGF1–4, β-tail, TM, and CT domains. The α subunit is composed ofβ-propeller, thigh, calf-1–2, TM, and CT domains (SI Appendix,Fig. S1 A–F). A subset of α subunits contains an αI domaininserted into the β-propeller domain (SI Appendix, Fig. S1 D–F).The αI and βI are structural homologs that share a similar fea-ture of conformational change (2, 3) (SI Appendix, Fig. S1 A–F).The αI itself has the full ligand binding capability, while the βIrequires the combination with the β-propeller domain of α sub-unit to bind ligand (SI Appendix, Fig. S1).Crystallographic and EM studies revealed the large-scale

conformational rearrangements of both α and β subunits,which are one of the key processes of integrin activation (4)

(SI Appendix, Fig. S1). Crystal structures of the ectodomains ofplatelet integrin αIIbβ3 and endothelial integrin αVβ3 revealed thelarge interfaces between the α-β head and leg domains, andbetween the headpiece and leg domains (5–8). The interfacebetween the headpiece and leg domains is largely contributed bythe β subunit (SI Appendix, Fig. S1A). Disulfide crosslinking andNMR studies of αIIbβ3 demonstrate the associations of α-β at theTM and CT domains (9–12). It has been suggested that theseα-β interactions at the leg, TM, and CT domains help maintainintegrin in the bent resting state (SI Appendix, Fig. S1 A and D).Activating signals inside the cell (i.e., the binding of talin andkindlin to the β CT) (13–15), induce the α-β separation at theTM and CT domains, leading to the headpiece extension andopening, resulting in an extended high-affinity conformationcompetent for binding ligand (4) (SI Appendix, Fig. S1 C and F).However, it remains unknown what drives the large-scale con-formational rearrangement on the cell surface after the α-β TM-CTseparation. The relative contributions of the α and β subunits to thebent and extended conformation of integrin are not well defined.Moreover, the integrin ectodomains adopt the bent conformationin solution even in the absence of TM-CT domains (5, 16).The integrin headpiece fragments were crystallized in the closed

Significance

Integrin inside-out activation is initiated by the α/β separationat the cytoplasmic and transmembrane domains. Such separa-tion leads to complicated local and global conformationalrearrangements of β subunit, while the conformational changeof α subunit is relatively simple. It is unclear whether the globalstructural changes of β subunit depend on the α subunit. Usinga single-chain β3 construct to mimic an extreme condition offully separated α/β heterodimer, we show that the β3 subunitautonomously drives the membrane-dependent bent-to-extended conformational rearrangement in the absence ofthe α subunit. In addition, we show that the clinically importantanti–HPA-1a (β3-L33) alloantibodies perform the conformation-dependent property when interfering with the β3 integrin,which may correlate with the pathogenesis of β3-mediatedalloimmune thrombocytopenia.

Author contributions: J.Z. designed research; A.M.M.T., Z.W., D.Z., Y.Z., and J.Z. per-formed research; B.R.C. contributed new reagents/analytic tools; A.M.M.T., Z.W., D.Z.,Y.Z., B.R.C., and J.Z. analyzed data; and A.M.M.T. and J.Z. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.wwpdb.org (PDB ID code 6BXJ).1To whom correspondence should be addressed. Email: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1806205115/-/DCSupplemental.

Published online September 12, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1806205115 PNAS | vol. 115 | no. 39 | E9105–E9114

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low-affinity conformation in the absence of the leg domains andligand (17–19). Thus, integrin may undergo different conforma-tional regulation in solution than on the cell surface.A Leu33Pro polymorphism at the β3 PSI domain, forming

human platelet antigen-1a/1b (HPA-1a/1b), can induce alloan-tibodies against incompatible HPA-1a during pregnancy or afterblood transfusion, leading to two severe bleeding disorders, fetal/neonatal alloimmune thrombocytopenia (F/NAIT) and post-transfusion purpura (PTP) (20). The severe risk of F/NAIT isintracranial hemorrhage (ICH), often leading to death or life-long neurological disability (21, 22). Recent studies suggest astrong connection between the ICH and the presence of a sub-type of anti-HPA-1a that is specific to the αVβ3 on endothelialcells (23). Some anti-HPA-1a subtypes are specific to αIIbβ3 andaffect the αIIbβ3 function in platelets (24). The αVβ3-specific anti-HPA-1a can interfere with endothelial cell function by inhibitingthe αVβ3-mediated cell adhesion and angiogenesis, which may beresponsible for the development of ICH (23). Certain anti–HPA-1a antibodies were also found to affect αIIbβ3-mediated plateletfunction (24). It is unknown how the anti–HPA-1a alloanti-bodies, which bind to the β3 subunit, can distinguish the β3 be-tween αVβ3 and αIIbβ3, and why their binding can differentlyaffect the function of β3 integrins.In this study, we engineered a chimeric β3 (cβ3) integrin that can

be expressed as a single-chain without the association of α subunitto mimic a fully separated state of a β subunit. Surprisingly, ourcrystallographic and biophysical assays revealed a bent confor-mation of the single-chain cβ3 in solution. Strikingly, the full-length cβ3 spontaneously adopts an extended conformation onthe cell surface. These data demonstrate a membrane-dependentautonomous conformational regulation of the β3 subunit, sug-gesting that the β subunit may be the major driving force inintegrin extension. Using the single-chain β3 construct, we char-acterized the complex-dependent anti–HPA-1a alloantibodies. Wedemonstrated that the conformation-dependent property of anti–HPA-1a alloantibodies enabled them to distinguish the differentconformations of β3 in the context of the bent state vs. the ex-tended state and of the αIIbβ3 complex vs. the αVβ3 complex. Wefound that the bent conformation-dependent anti–HPA-1a allo-antibodies could stabilize the β3 integrin in the bent resting state.Our study provides insight into the integrin conformational acti-vation on the cell surface and has important implications for thetreatment and diagnosis of F/NAIT.

Resultsβ3 Integrin Autonomously Adopts a Bent Conformation in SolutionIndependent of α Subunit. To mimic a fully separated state of anintegrin β subunit, we created a strategy to express the β subunitas a single-chain protein without the association of an α subunit.Given that the α β-propeller and the β βI domains form thelargest α-β interface, which is maintained during the large-scaleconformational rearrangement (SI Appendix, Fig. S1 A–F), werationalized that disrupting this interface might facilitate theautonomous expression of a β subunit. A chimeric β integrin (cβ)was engineered by replacing the βI domain with its structuralhomolog, the αL integrin αI domain (Fig. 1A and SI Appendix,Fig. S1G). Unlike the βI domain, the αI domain can be expressedautonomously and is solely responsible for binding ligands (4,25). We tested this design on β1, β3, β6, and β7 integrins.All the ectodomains of cβ1, cβ3, cβ6, and cβ7 integrins were

successfully expressed without their α partners in the secretedform in HEK293S GnTI− cells. All purified proteins appeared ata molecular weight of approximately 75 kDa after endoglycosi-dase H (EndoH) treatment (Fig. 1B). Analysis with gel filtrationchromatography showed similar retention volumes between cβ1and cβ6 and between cβ3 and cβ7 (Fig. 1C). Consistently, dynamiclight scattering (DLS) measurements showed the similar hydro-dynamic radii between cβ1 (5.47 nm) and cβ6 (5.44 nm), and

between cβ3 (4.82 nm) and cβ7 (4.90 nm) (Fig. 1D). These hy-drodynamic radii are close to that of the bent (5.58 nm), but notto that of the extended (6.37 nm) αVβ3 ectodomain measuredpreviously (16). These data suggest that the isolated β ectodo-main adopts a compact conformation in solution.We determined the crystal structure of cβ3 ectodomain that

was refined to a resolution of 2.09 Å with an Rfree value of 0.234(SI Appendix, Table S1). Remarkably, the cβ3 crystal structuredemonstrates a compact bent conformation (Fig. 2A). Thestructure of each domain, as well as the bound metal ion, is verywell defined, except that the C1-C2 loop of I-EGF2 is in-completely resolved due to the lack of electron density (Fig. 2A).Structure superimpositions show that the overall cβ3 structureresembles the bent β3 structure in either αIIbβ3 (Fig. 2B) or αVβ3(Fig. 2C). Most interdomain interfaces are formed in the sameway as in the bent native β3 structure. Interestingly, in the crystallattice, the β3-hybrid domain interacts with the αI domain as apseudoligand (SI Appendix, Fig. S2A). Unlike the direct co-ordination between a Glu residue and Mg2+ in the ICAM/αIinteraction (SI Appendix, Fig. S2B), the Asp-66 of β3-hybriddomain coordinates with the Mg2+ of αI through two in-termediate water molecules (SI Appendix, Fig. S2A). Such in-direct coordination does not induce the active conformation of αIdomain. The αI domain in the cβ3 is in the resting rather thanthe active conformation (Fig. 2D). The cβ3 headpiece is in aclosed conformation (Fig. 2E). This result demonstrates that thesingle-chain β3 ectodomain autonomously adopts a bent con-formation independent of the α subunit.Small-angle X-ray scattering (SAXS) was performed to ana-

lyze the structure of cβ7 ectodomain in solution. The cβ7 proteinwas not treated with EndoH and thus retained the high-mannosetype N-glycans. Three different concentrations gave very similarpatterns of X-ray scattering curves (Fig. 2F). The distance dis-tribution profile showed a length of approximately 15 nm (Fig.

Fig. 1. Purification and characterization of the soluble single-chain βintegrin ectodomains. (A) Construct design of a cβ integrin ectodomain. Theectodomain of the cβ subunit was designed by replacing the βI domain withthe αL integrin I domain. (B) SDS/PAGE of the purified cβ integrin ectodo-mains. The indicated cβ integrin ectodomains were purified from the cellculture supernatant and treated with HRV 3C protease to remove the C-terminal tags and with EndoH to trim the N-glycans. (C) Overlaid size ex-clusion chromatography (Superdex 200; GE Healthcare) of the purified chi-meric ectodomains of β1, β3, β6, and β7 integrins. The elution volumes areshown in parentheses. (D) Hydrodynamic radii of the chimeric integrinectodomains measured by DLS. The DLS measurements were performed inthree independent experiments.

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2F), which is close to the length measured based on the cβ3crystal structure (Fig. 2A). The bead model of the cβ7 ectodo-main generated using the SAXS data shows a compact confor-mation. It has a larger volume than the cβ3 crystal structure (Fig.2G), due at least in part to the differences in N-linked glycans.

Structural Plasticity of the β3-Ankle Region Regulates IntegrinActivation. The structural comparison between the cβ3 and the β3revealed a nearly 90° rotation of the entire β-tail domain whensuperimposed on the I-EGF4 domain (Fig. 3A). This may be due tothe flexibility of the β3-ankle region (Fig. 3B). The β3-ankle connectsthe I-EGF4 and β-tail through a short loop cyclized by a conservedC601-C604 disulfide bond (Fig. 3 A and B). In the bent αIIbβ3integrin, the β-tail interacts with the αIIb calf-2 domain (Fig. 3C).The rotation of the β-tail may exert steric clashes with the calf-2 domain and thus facilitate the separation of αIIb and β3 legdomains (Fig. 3C), leading to integrin activation. To test this hy-pothesis, we mutated the C601 and C604 or the P605 to glycine,aiming to increase the flexibility of the β3 ankle. The activation ofαIIbβ3 in HEK293FT cells was measured by the binding of ligand-mimetic mAb PAC-1. The β3-C601G-C604G mutation dramaticallyenhanced the PAC-1 binding in either the physiological Ca2+/Mg2+

or Mn2+ condition (Fig. 3D). The αIIb-R995A mutation was used tomimic the inside-out activation of αIIbβ3 integrin (26). The β3-C601G-C604G mutation greatly enhanced the αIIb-R995A–

mediated αIIbβ3 activation in both metal ion conditions (Fig.3D). The β3-P605G mutation also slightly enhanced the PAC-1binding to both αIIbβ3 wild type (WT) and the αIIb-R995A mutant(Fig. 3D). These data demonstrate that the structural plasticity of theβ3 ankle contributes to the bidirectional activation of αIIbβ3 integrin.

Single-Chain cβ3 Adopts an Extended Active Conformation on the CellSurface. We next examined the cell surface expression of cβ3containing the TM and CT domains (SI Appendix, Fig. S3A). The

surface expression of both αIIb and β3 requires α-β hetero-dimerization (SI Appendix, Fig. S3B). A similar level of β3 wasdetected in the cell lysates by Western blot analysis with orwithout αIIb cotransfection (SI Appendix, Fig. S3C), suggestingthat the cell surface transportation of β3 requires the associationwith αIIb. However, only very low levels of αIIb were detected inthe cell lysate without β3 coexpression (SI Appendix, Fig. S3C).In contrast, high levels of cβ3 were detected both on the cellsurface and in the cell lysates independent of the αIIb subunit(SI Appendix, Fig. S3 B and C). The cotransfection of αIIb withcβ3 did not enable the surface expression of αIIb (SI Appendix,Fig. S3B).We then analyzed the cβ3 conformation on the cell surface

using the conformation-specific mAbs, LIBS-1, and 319.4 thatreport β3 extension. When transiently expressed in HEK293FTcells, the αIIbβ3 bound low levels of LIBS-1 and 319.4, which wasenhanced by the ligand-mimetic antagonist eptifibatide (Fig.4A). In contrast, the cβ3 on the surface constitutively bound highlevels of both LIBS-1 and 319.4 (Fig. 4 B and C), indicating anextended conformation.The presence of the αL αI domain enabled us to examine the

ligand-binding capability of cβ3 using the αL ligand ICAM-1. Thecβ3 on the surface of HEK293FT cells strongly bound humanICAM-1 under a physiological concentration of Mg2+ (Fig. 4D).Such spontaneous ICAM-1 binding was greatly inhibited by anαL-specific inhibitor (Fig. 4E). Furthermore, ICAM-1 bindingwas completely abolished by the alanine substitutions of twoserine residues (cβ3-SSAA) that coordinate with the Mg2+ at themetal ion-dependent adhesion site of the αL αI domain (Fig. 4 Dand E), demonstrating the metal ion-dependent interaction be-tween ICAM-1 and cβ3. The cβ3 was also stably expressed inCHO-k1 cells (Fig. 4F) and HEK293 cells (Fig. 4G). The surfaceexpression of cβ3 was detected by the anti-β3 mAb AP3 or

Fig. 2. Crystal structure of the single-chain cβ3 ectodomain. (A) The overall structure of the cβ3 full-length ectodomain with the subdomains shown indifferent colors. Disulfide bonds are shown as blue sticks. Glycans are shown as sticks with green carbons, red oxygens, and blue nitrogens. Mg2+ ion is shownas an orange sphere. (B) Structure superposition of the cβ3 (in red) with the β3 structure (in green) obtained in complex with αIIb (PDB ID code 3FCS). (C)Structure superposition of the cβ3 (in red) with the β3 structure (in green) obtained in complex with αV (PDB ID code 4G1E). (D) Structure superimposition ofthe αL-I domain in the cβ3 crystal structure (in red) onto the αL-I domain structures in the resting (in green; PDB ID code 3F74) and in the active state (in cyan;PDB ID code 1T0P). The conformations of Mg2+-binding site, α1-helix, α6-helix, and α7-helix are identical to the resting rather than the active conformationobserved in the crystal structure of an isolated αL αI domain. (E) Structure superposition at the headpiece region of the cβ3 (in red) with the β3 structure (ingreen; PDB ID code 3FCS). (F) SAXS measurement of purified cβ7 at different concentrations. The inset plot shows the distance distribution based on the SAXSdata of 1 mg/mL. (G) SAXS ab initial model of cβ7. The bead model was generated with DAMMIF using the SAXS data of cβ7 measured at 1 mg/mL. The crystalstructure of cβ3 (cartoon in red) was superimposed with the bead model of cβ7 using SUPCOMB.

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SZ21 and by the anti–αL-αI mAb MHM24 (Fig. 4 F and G). Thecβ3 in these stable cell lines bound ICAM-1 at a high level inMg2+ or Mn2+, which was completely blocked by the αL-specificinhibitor (Fig. 4 F and G). Taken together, these data providecompelling evidence that the single-chain cβ3 autonomouslyadopts an extended active conformation on the cell surface.

cβ3 Can Be Locked in the Bent Conformation by a Disulfide Bond onthe Cell Surface. It is intriguing that the cβ3 ectodomain bends insolution but extends on the cell surface. We asked whether themembrane-bound cβ3 could also adopt the bent conformation. Inthe bent cβ3 structure, the hybrid domain folds onto the I-EGF3 domain to form a close contact, which may enable adisulfide bond formation between the β3-S367 and β3-S551 whenmutated to cysteine (Fig. 5A). A previous study showed that thisputative disulfide bond blocked αIIbβ3 activation by locking the β3in the bent conformation (27). When introduced into our cβ3construct, the double cβ3-S367C-S551C, but not single cysteinemutations, dampened ICAM-1 and LIBS-1 binding by nearly50% (Fig. 5 B and C), suggesting that cβ3 was locked in a bentconformation. This is consistent with our previous study showingthat the bent conformation of β3 is not accessible to bind themacromolecular ligands (28). The incomplete inhibition ofICAM-1 or LIBS-1 binding by the double cysteine mutation wasdue to the incomplete disulfide bond formation, as indicated bythe labeling of free cysteines with the maleimide-PEG2-biotin

(Fig. 5D). These results demonstrate that the cβ3 on the cellsurface can adopt both the bent and extended conformations,but with the extended conformation favored in the equilibriumof conformational change.

Effect of N-Glycans on the ICAM-1 Binding of cβ3. The cβ3 proteinsexpressed in HEK293S GnTI− cells contain the high-mannosetype but lack the complex N-linked glycans. We asked whetherthe full-length cβ3 expressed on the surface of HEK293S GnTI−

cells could bind ICAM-1 using the competition assay. Pre-incubation of ICAM-1 with the full-length cβ3-WT cells, but notthe cβ3-SSAA–transfected HEK293FT cells, significantly inhibi-ted the ICAM-1 binding to the cβ3-transfected HEK293FT cells(Fig. 5E). Similarly, compared with the cβ3-SSAA transfectedHEK293S GnTI− cells, the HEK293S GnTI− cells carrying cβ3significantly blocked ICAM-1 binding (Fig. 5E), suggesting thatthe complexity of N-glycans does not contribute to the activeconformation of cβ3 on the cell surface. In contrast, the solublecβ3 ectodomain with or without EndoH treatment did not sig-nificantly block ICAM-1 binding (Fig. 5E), consistent with thepresence of a resting conformation of the αI domain (Fig. 2D).

Application of the cβ3 Construct in Identifying the Conformation-Dependent Property of Anti–HPA-1a Alloantibodies. Some anti–HPA-1a alloantibodies were found to be able to distinguish theαIIbβ3 from αVβ3 (23), although they all require the Leu33 residing

Fig. 3. Structure plasticity of the β3 ankle region and effect on integrin activation. (A and B) Structure superimposition based on the I-EGF4 domain (A) and on theβ-tail domain (B). The structures of the cβ3 and the native β3 are shown in red and green, respectively. Disulfide bonds are shown as yellow sticks, except the one at theankle region is shown in blue. (C) Structure superimposition of the cβ3 (in red) on the structure of the αIIbβ3 complex (β3 in green). (D) Ligand-mimetic mAb PAC-1 binding of the HEK293FT cells transfectedwith the indicated αIIbβ3 constructs. The bindingwas done in the presence of 1mMCa2+/Mg2+ (Ca/Mg) or 0.2 mMCa2+ plus2 mMMn2+ (Ca/Mn) and measured by flow cytometry. Data are presented as MFI normalized to integrin expression. Data are shown as mean ± SEM (n = 6). Studentt tests were used to calculate differences between the β3 mutations and β3-WT in the same metal ion conditions. *P < 0.05; **P < 0.01; ***P < 0.001.

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at the β3 PSI domain (Figs. 6A and 7A). We sought to use oursingle-chain cβ3 to characterize the complex-dependent property ofthe anti–HPA-1a alloantibodies. We examined 3 PTP samplesand 13 NAIT samples by flow cytometry using HEK293FT cellstransiently transfected with αIIbβ3, cβ3, or αVβ3 (SI Appendix, Fig. S4A and B). Although the anti–HPA-1a alloantibodies could be de-tected in most of the samples, they showed substantial heteroge-neities in binding with αIIbβ3, cβ3, and αVβ3. Some samples such asPTP-I, NAIT-45, and NAIT-51 bound equally well to the threetypes of β3 integrins (SI Appendix, Fig. S4 A and B). Interestingly,some samples, such as PTP-c, NAIT-III, and NAIT-b, bound wellonly to αIIbβ3 and not to cβ3 and αVβ3 (SI Appendix, Fig. S4 A andB). The other samples, such as NAIT-46, NAIT-50, and NAIT-52,bound to both αIIbβ3 and αVβ3, but bound better to αVβ3 and poorlyto cβ3 (SI Appendix, Fig. S4B). These data demonstrate the presenceof complex-dependent and complex-independent anti–HPA-1a al-loantibodies. We used PTP-I, NAIT-III, and NAIT-52 as repre-sentative samples for further characterization since these samplescontain high levels of complex-independent (PTP-I), αIIbβ3-specific(NAIT-III), and αVβ3-specific (NAIT-52) anti–HPA-1a alloanti-bodies (Fig. 6 B and C).Given that the cβ3 is in an extended active conformation on

the cell surface and the NAIT-III and NAIT-52 bind poorly to

the surface cβ3, we speculated that the complex-dependent anti–HPA-1a alloantibodies might be specific to the bent conforma-tion of β3. To test this hypothesis, we used the β3-N305T mu-tation, which stabilizes β3 in the extended conformation byintroducing a glycan wedge in between the hybrid and βI do-mains (29). Compared with the WT αIIbβ3, the αIIbβ3-N305Tmutant reacted poorly to the NAIT-III serum (Fig. 6B), sug-gesting that the NAIT-III anti–HPA-1a alloantibodies are spe-cific to the bent conformation of αIIbβ3. In a sharp contrast, thePTP-I anti–HPA-1a alloantibodies bound well to all the β3 con-structs harboring L33 but not P33 (Fig. 6B). In addition, we usedMn2+ and GRGDSP peptide (Mn/RGD) to induce the extensionof WT αIIbβ3 (Fig. 6D) and αVβ3 (Fig. 6E). Compared with theCa2+/Mg2+ condition, Mn/RGD increased the binding of LIBS-1mAb to both αIIbβ3 (Fig. 6D) and αVβ3 (Fig. 6E), indicating theβ3 extension. In contrast, the binding of NAIT-III anti–HPA-1ato αIIbβ3 (Fig. 6D) and the binding of NAIT-52 anti–HPA-1a toαVβ3 (Fig. 6E) were deceased under the Mn/RGD condition,

Fig. 4. Conformation and ligand binding of the full-length cβ3 integrin onthe cell surface detected by flow cytometry. (A and B) Binding ofconformation-specific anti-β3 mAb LIBS-1 or 319.4 to HEK293FT cells tran-siently transfected with αIIb plus β3 or cβ3 alone. For the αIIbβ3 transfection,mAb binding was also performed in the presence of ligand-mimetic drugeptifibatide (Ept). Integrin expression was reported with the conformation-independent mAb AP3. (C) Quantitation of the conformation-specific mAbbinding presented as a percentage of AP3 binding. (D) ICAM-1 binding inbuffer containing 1 mM Mg2+ to HEK293FT cells transiently transfected withcβ3 WT or the metal ion-binding defective mutant cβ3-SSAA. (E) Quantitationof the ICAM-1 binding in 1 mMMg2+ with or without the αL-specific inhibitorpresented as a percentage of AP3 binding. (F and G) Expression and ligandbinding of cβ3 in stably transfected CHO-k1 or HEK293 cells. The cβ3 ex-pression was detected by the anti-β3 mAbs AP3 and SZ21 or the anti–αL-Idomain mAb MHM24. Human ICAM-1 binding was measured in 1 mM Mg2+

(Mg) or 2 mM Mn2+ (Mn) with or without the αL-specific inhibitor.

Fig. 5. Correlation of the cβ3 conformation with the binding of ligand. (A)The structure of cβ3 with indicated cysteine mutations. The putative disulfidebond formed between S367C and S551C is shown as a red stick, and nativedisulfide bonds are shown as blue sticks. (B) Binding of ICAM-1 to HEK293FT cellstransiently transfected with the indicated cβ3 cysteine mutants with or withoutαL integrin inhibitors. (C) LIBS-1 mAb binding to HEK293FT cells transientlytransfected with the indicated cβ3 cysteine mutants. (D) Biotin-maleimide-PEG (BM) labeling of the cell surface cβ3 cysteine mutants. The membranewas scanned with the LI-COR Odyssey infrared imaging system. The WT cβ3could not be labeled by maleimide, indicating that all the native cysteines aredisulfide-bonded. In contrast, the single cysteine mutations were obviouslylabeled by maleimide. The double cysteine mutation was still labeled bymaleimide, suggesting that the disulfide bond was not completely formed.(E) Competition of ICAM-1 binding by the cβ3 expressed on the cell surface orin solution. The ICAM-1 ligand was first incubated with the indicated full-length cβ3-transfected cells or the purified soluble cβ3 ectodomain and thenused for the binding with the cβ3-expressing HEK293FT cells. Data are pre-sented as mean ± SEM. *P < 0.05; ***P < 0.001.

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suggesting that the complex-dependent anti–HPA-1a alloanti-bodies favor the bent conformation of β3 integrin.We next tested whether the complex-dependent anti–HPA-1a

alloantibodies in the NAIT sera react with the bent cβ3 either onthe cell surface or in solution. The cβ3-S367C-S551C mutationpartially maintains the cβ3 in the bent conformation on the cellsurface (Fig. 4 A–C). Compared with the cβ3-WT, the cβ3-S367C-S551C mutation increased the binding of NAIT-III and NAIT-52 to cβ3 by twofold to sixfold (Fig. 6F). In contrast, no increasewas observed for the PTP-I serum (Fig. 6F). The soluble cβ3ectodomain is in the bent conformation in solution (Figs. 1 and2). As shown by a competition assay, the soluble cβ3 blocked thebinding of NAIT-III to αIIbβ3 (Fig. 6G and SI Appendix, Fig.S4C) and the binding of NAIT-52 to αVβ3, αIIbβ3, and cβ3 onthe cell surface (Fig. 6H). Similar results were obtained forthe NAIT-50 sample (SI Appendix, Fig. S4D). As a control, thesoluble cβ3, but not cβ7, blocked the binding of PTP-I toHEK293 cells stably expressing αIIbβ3 (SI Appendix, Fig. S4E).Taken together, these data reveal a subtype of anti–HPA-1aalloantibodies that specifically recognize the bent conforma-tion of β3.Given the bent conformation-specific character of anti–HPA-

1a alloantibodies, we asked whether such antibodies could sta-bilize the bent conformation of β3 integrin. As shown by the

Mn2+-stimulated PAC-1 binding assay, the NAIT-III serum, butnot the normal and PTP-I sera, significantly blocked PAC-1 binding to αIIbβ3 expressed in HEK293FT cells (Fig. 6I). Asimilar result was obtained using the purified IgG from theNAIT-III serum (SI Appendix, Fig. S4F). Two active conformation-dependent mAbs, 370.3 and LIBS-1, which bind to αIIb and β3,respectively (3, 30) (Fig. 6A), were used to report αIIbβ3 extension.Compared with the buffer and normal serum controls, the NAIT-IIIserum significantly reduced the Mn2+-stimulated binding of both370.3 and LIBS-1 to αIIbβ3 (Fig. 6 J and K). Similarly, the NAIT-52 serum, but not the normal and NAIT-III sera (containingonly high levels of αIIbβ3-specific anti–HPA-1a), reduced theLIBS-1 binding to αVβ3 integrin (Fig. 6L). Thus, these datademonstrate that the bent conformation-specific anti–HPA-1aalloantibodies can stabilize the bent inactive conformation ofβ3 integrin.

Structural Analysis Suggests the Conformational Diversities of thePSI/I-EGF1-2 Domains May Contribute to the Heterogeneity of Anti–HPA-1a Binding. It is striking that the anti-HPA-1a alloantibodiescan distinguish the αIIbβ3 vs. αVβ3 as well as the bent vs. extendedβ3 integrin. It is unlikely that the α subunit can directly partici-pate in the alloantibody binding, due to the distant location ofthe central Leu-33 residue of the HPA-1a epitope from the αsubunit (SI Appendix, Fig. S5). In addition, the single-chain cβ3

Fig. 6. Identification of the bent conformation-dependent anti–HPA-1a alloantibodies. (A) Cartoon model of the bent and the extended αIIbβ3 integrin. Theepitopes recognized by anti–HPA-1a antibody and the extended conformation-specific mAbs are indicated. (B and C) Binding of the anti–HPA-1a antibodiesfrom the representative PTP or NAIT sera to HEK293FT cells transiently transfected with the indicated integrin constructs. The presence of anti–HPA-1a al-loantibodies in the sera was confirmed by the reaction with β3 harboring L33 (HPA-1a) but not P33 (HPA-1b). (D) Effect of αIIbβ3 extension on the binding ofanti–HPA-1a in NAIT-III. (E) Effect of αVβ3 extension on the binding of anti–HPA-1a in NAIT-52. Integrin extension was induced by GRGDSP plus Mn2+. Data arepresented as percentage of β3 expression after subtracting the background binding of the sera or mAbs to mock transfected HEK293FT cells. (F) Increasedbinding of the NAIT-III and NAIT-52 anti–HPA-1a alloantibodies to the cβ3-S367C-S551C HEK293FT transfections. Data are presented as a ratio of binding tocβ3-S367C-S551C vs. cβ3-WT. Data are mean ± SD (n ≥ 3). (G and H) Inhibition by the soluble cβ3 protein of the binding of the NAIT-III purified IgG (G) or NAIT-52 serum (H) to HEK293FT cells transfected with αIIbβ3, cβ3, or αVβ3. (I–K) Effect of NAIT-III anti–HPA-1a on the binding of PAC-1, 370.3, and LIBS-1 to αIIbβ3expressed in HEK293FT cells. (L) Effect of NAIT-52 anti–HPA-1a on the binding of LIBS-1 to αVβ3 expressed in HEK293FT cells. The binding of PAC-1, 370.3, orLIBS-1 mAbs to integrin was induced by Mn2+. For I–L, data are mean ± SEM (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001.

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gained binding to the complex-specific anti–HPA-1a alloanti-bodies when adopting the bent conformation on the cell surfaceor in solution (Fig. 6 F–H), suggesting indirect involvement ofthe α subunit with the HPA-1a epitope. The conformation-dependent property of anti–HPA-1a antibodies suggests thatthe β3 may adopt different conformations around the PSI do-main in the bent state vs. the extended state and when pairingwith αIIb vs. with αV. We analyzed the structural flexibility asindicated by the B factors. Compared with the domains such ashybrid, I, and β-tail, the PSI (particularly the C26-C38 loop), I-EGF1, and I-EGF2 domains exhibit high flexibility among thereported β3 crystal structures (Fig. 7 A–E). Using our cβ3 struc-ture as a reference, the structural superimpositions revealed theconformational diversity at the PSI/I-EGF1-2 domains amongthe bent β3 structures when superimposed based on the hybrid,PSI, or I-EGF1 domain (Fig. 7 F and G and SI Appendix, Fig. S6A–D). The conformations of PSI/I-EGF1 domains in the bent cβ3crystal structure resemble those in the αIIbβ3 crystal structure(Fig. 7F and SI Appendix, Fig. S6 A and B). In contrast, theconformations of β3 PSI/I-EGF1 as well as I-EGF2 domains in

the αVβ3 show substantial differences from those in the cβ3structure (Fig. 7G and SI Appendix, Fig. S6 C and D). The ob-vious conformational differences at the PSI/I-EGF1 domainswere also observed when superimposing the β3 structures at thebent and extended states (Fig. 7H and SI Appendix, Fig. S6 E andF). Some anti–HPA-1a alloantibodies are known to require bothPSI and I-EGF1 domains (31–33). The structural diversity of thePSI/I-EGF1 domains may account for the conformational se-lectivity of anti–HPA-1a binding. The I-EGF2 domain may alsocontribute to the HPA-1a epitope due to the close proximity(Fig. 7H). During β3 extension, the PSI/I-EGF1 domains moveaway from the I-EGF2 domain (34) (Fig. 6A and SI Appendix,Fig. S6G). Binding of the bent conformation-specific anti–HPA-1a alloantibodies may restrain the extension of PSI/I-EGF1 domains and thus stabilize the bent conformation.

DiscussionOur design of a single-chain β subunit with the ligand-binding ca-pability provides a unique approach to understanding the structuraland functional regulation of integrin. Using a single-chain β3

Fig. 7. Structural diversity of the β3 PSI and I-EGF1-2 domains. (A–E) Structure comparison of the β3 crystal structures of the cβ3 (A; PDB ID code 6BXJ), theαIIbβ3 complex (B; PDB ID code 3FCS), the αVβ3 complex (C; PDB ID code 4G1M), the αIIbβ3 headpiece (HP) at the closed conformation (D; PDB ID code 3T3P), andthe αIIbβ3 HP at the open conformation (E; PDB ID code 2VDO). The structures were superimposed on the hybrid domain and are shown separately in the sameorientation with the color and cartoon putty radius scaled based on the B factors. High B factors indicate high structural flexibility. The PSI domains are circled.The Leu33 is shown as a sphere of Cα atom. To make the B factors comparable, all the structures were refined using the same version of the refinementprogram in the PHENIX package. (F–H) Structure superposition of the αIIbβ3 (F, in green), αVβ3 (G, in green), or β3 at the extended conformation (H, in green;PDB ID code 6BXB) onto the cβ3 (in red) based on the hybrid domain. Disulfide bonds are shown as blue sticks. The αIIb and αV subunits are shown as thesolvent-accessible surface in gray. Leu33 residues are shown as spheres of Cα atoms. In H, the potential HPA-1a epitopes, including PSI, I-EGF1, and I-EGF2 domains, are circled at a radius of 15 Å centered at Leu33.

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subunit, we were able to answer the question of whether the long-range conformational change of β subunit depends on the α sub-unit after α/β separation at the cytoplasmic and TM domains.Remarkably, we found that the β3 subunit independently adoptsthe bent conformation that resembles the bent β subunits observedin the crystal structures of αVβ3, αIIbβ3, and αXβ2 (5, 7, 8, 35),suggesting that the close contacts between the headpiece and legdomains are sufficient to maintain the β3 in the bent state. Sur-prisingly, the single-chain β3 undergoes extension on the cell sur-face independently of the α subunit, demonstrating its autonomousconformational regulation.One caveat of this study is the chimeric feature of our single-

chain β3 subunit. However, our crystal structure clearly demon-strates that replacing the βI with αI does not affect the overallconformation of β3 integrin. The αI and βI share many commonstructural features despite αI being smaller than βI and lackingtwo additional metal ion-binding sites. The αI domain resemblesall of the crucial conformational changes of βI, including inwardmovements of the β1-α1 loop and α1-helix and downward move-ments of the β6-α7 loop and α7-helix (SI Appendix, Fig. S1), all ofwhich are required for the headpiece opening and high-affinityligand binding (3, 36). A previous study suggested that deletionof the specificity-determining loop of βI domain facilitates au-tonomous expression of the integrin β subunit (37); however, thesingle-chain β subunit is incompetent for binding ligand due to theincompleteness of the ligand-binding site that requires both the αand β subunits. Our design facilitates the autonomous expressionof β subunit while maintaining the ligand-binding capability.In addition to the conformation-dependent mAbs, we used

ICAM-1–binding assay to assess the conformation and activationstatus of single-chain cβ3 on the cell surface. Since integrin ex-tension is required for the binding of macromolecular ligands onthe cell surface (28), the spontaneous binding of ICAM-1 to cellsurface cβ3 indicates the accessibility of αI ligand-binding sites asa result of the headpiece extension. One limitation of our assay isthe multivalent features of ICAM-1 binding, which also involvesthe avidity/clustering effect of integrin on the cell surface. Suchan effect was not present when measuring the binding of ICAM-1 and soluble cβ3, due to the free diffusion of molecules in so-lution; however, ICAM-1 binding requires the up-regulation ofintegrin affinity (36, 38, 39), which is associated with integrinextension. Our studies suggest that cβ3 undergoes the transitionbetween the bent conformation and extended conformationwithout assistance of the α subunit, and the conformationalequilibrium could be shifted to the extended conformation onthe cell surface by the binding of ICAM-1 and conformation-dependent antibodies.It is striking that the single-chain β3 ectodomain can bend in

solution but spontaneously extend on the cell surface. The cur-rent model suggests that integrin activation is triggered by sep-aration at the α/β cytoplasmic and TM domains induced by thebinding of talin and kindlin to the β CT (4, 9–12, 40, 41). In-terestingly, the soluble integrin ectodomains adopt a bent con-formation in solution even in the absence of TM and CTdomains (5, 7, 8, 35, 42, 43). In particular, both EM and crystalstructures revealed a bent conformation of the αVβ3 ectodomaineven in the absence of a C-terminal clasp that mimics TM as-sociation (5, 16). Moreover, all the headpieces of αIIbβ3, α5β1,α4β7, αVβ6, and αLβ2 were crystallized in a closed resting con-formation even without the leg, TM, and CT domains (17–19,44–46). Thus, the absence of TM and CT domains does not leadto the active integrin conformation in solution, which can beinduced by binding of the ligands or activating antibodies (16, 47,48). In contrast, the α/β separation at the TM and CT domains,for example, induced by deletion of the α or β CT can lead to theactive integrin conformation on the cell surface (41). With oursingle-chain β3 that mimics a fully separated state, we demon-strate that the separation of α and β subunits results in a spon-

taneous conformational change of β3 on the cell surface but notin solution, suggesting that the cell membrane contributes toprovoking integrin extension. This membrane-dependent con-formational change seems to be an intrinsic property of the β3subunit. Recent EM studies of αIIbβ3 embedded in the lipidnanodiscs revealed distinct conformations than the αIIbβ3 in so-lution or in detergent (8, 49–51), indicating the effect of the lipidbilayer on integrin conformation. Clearly, how integrin confor-mations are regulated by the cell membrane is an intriguing andsignificant question meriting further investigation.Our study demonstrates the autonomous conformational regu-

lation of the β3 subunit, suggesting its major role in the large-scaleconformational changes within an α/β heterodimer. This is con-sistent with the central function of the β subunit in integrin sig-naling (4, 52). Most β subunits can pair with more than one αsubunit; for example, β3 pairs with αIIb and αV, while β1 pairs with12 α subunits. Such combinations may require a loose structurallinkage between α and β subunits. The β subunit contains morecomplicated domain organization and undergoes much morecomplicated conformational rearrangements than the α subunit(SI Appendix, Fig. S1). The α subunit may passively participate inthis process through the association with the β subunit. The αsubunit contributes to the specificity of ligand recognition anddownstream signaling and participates in the integrin inside-outactivation via its CT (1, 41, 53, 54). Furthermore, the αIIb subunithas been suggested to help the β3 subunit fold into a bent con-formation during biosynthesis (55). A structural study on the αVβ8ectodomain by single-particle electron cryomicroscopy suggestedthat the αV subunit might contribute to stabilizing the extendedintegrin conformation (56). The β subunit contains several flexiblehinge regions, such as the I-EGF1/2 junction and the β-ankle, thataccommodate the conformational flexibility. In contrast, the αsubunit is relatively rigid. Accumulating evidence demonstratesthat integrin structure and function are regulated by the cellularforces applied by the cytoskeleton to the β CT and by the extra-cellular ligands to the head domain (8, 57–61). The flexibility ofthe β subunit might be important for the sensing of force, while therigidity of α subunit may help the force resistance of the β subunit(60, 62). Our unique design of the single-chain β subunit providesan α-subunit “knockout” approach to deduce the function of αsubunit in the α/β heterodimer.The most serious risk of F/NAIT is intracranial hemorrhage

(ICH), often leading to death or life-long neurologic disability(21, 22). ICH is not caused simply by thrombocytopenia, but islinked with the anti–HPA-1a alloantibodies (63), in which theanti-αVβ3–specific subtype may play a major role by interferingwith the αVβ3 function in endothelial cells (23). It is striking thatthe anti–HPA-1a alloantibodies can distinguish αIIbβ3 and αVβ3,since the alloantibodies bind only to the β3 subunit. Our studysuggests that the complex-specific feature of anti–HPA-1a allo-antibodies is determined by their conformation-dependentproperty. We observed the substantial conformational differ-ences of β3 PSI/I-EGF1-2 domains between αIIbβ3 and αVβ3. Ourcrystal structure of extended β3 integrin also revealed a differentconformation of PSI and I-EGF1 domains than seen in the bentstate (34). Remarkably, these conformational differences can bedistinguished by the anti–HPA-1a alloantibodies. More impor-tantly, we demonstrated that the bent conformation-dependentanti–HPA-1a alloantibodies could stabilize the bent conformationof β3 integrin and thus exert functional inhibition of αIIbβ3 and αVβ3(23, 24), which may contribute to the severity of bleeding andpathogenesis of ICH. Our single-chain β3 construct provides aunique tool to distinguish the complex/conformation-dependentanti–HPA-1a alloantibodies, which may aid the diagnosis andproposed treatment of F/NAIT. Many active conformation-specificmAbs have been generated that serve as useful tools for studyingintegrin conformational regulation (4, 64). Our study demonstratesthe feasibility of developing the bent conformation-specific anti-β3

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antibodies, which may be used to modulate integrin function bystabilizing the bent integrin conformation for therapeutic andresearch purposes.

Materials and MethodsDNA Constructs. Human αIIb and β3 constructs were as described previously (3).The ectodomain constructs of β3, β1, β6, and β7 integrins with the βI domainsreplaced by the αL αI domain (residues G128–K304) were cloned into the XhoIand MluI sites of the ExpressTag 1 (ET1) vector (65). The cβ integrin was taggedwith an HRV 3C protease cleavage site, a protein C epitope, a hexahistidine,and a streptavidin-binding protein peptide. The full-length cβ3 was cloned intothe pCDNA3.1/Myc-His (+) A vector using the HindIII and XbaI sites.

Protein Expression, Purification, and DLS. The cβ integrin ectodomainswere stably expressed in HEK293S GnTI− cells that are deficient inN-acetylglucosaminyltransferase I (66). Proteins produced in these cellscontain only the high-mannose type of N-glycans, which can be removed byEndoH. The proteins were purified from the cell culture supernatant withStrep-Tactin Superflow Plus beads (Qiagen) and then treated with HRV 3Cprotease to remove the C-terminal tags and with EndoH to trim the N-glycans. The untagged proteins were finally purified by gel filtration usinga Superdex 200 column (GE Healthcare) equilibrated with 20 mM Tris·HCl(pH 7.5), 150 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2. The purified proteinsat 1 mg/mL were subjected to DLS measurement at 25 °C with the ZetasizerμV (Malvern Panalytical).

Protein Crystallography. Crystallization was performed at 4 °C by the hanging-drop vapor diffusion method with 1:1 vol/vol of protein and mixture solutions.The cβ3 ectodomain proteins were crystallized in 10% PEG 8000, 20% 1,5-pen-tanediol, 4 mM alkalis, 66 mM Gly-Gly, and 33 mM AMPD, pH 8.5. A 5–15% finalconcentration of glycerol was added as an additive. Crystals were grown at 4 °Cfor at least 7 d and then directly flash-frozen in liquid nitrogen. Crystal X-raydiffraction data were collected at the APS LS-CAT beamline 21-ID-F and pro-cessed with iMosflm (67). The structure was solved by molecular replacementwith PHASER (68) using the αL αI and the β3 hybrid domain structures as thesearch models. The structures were built manually in Coot (69) and refined usingPHENIX.REFINE (70). The atomic coordinates and structure factors have beendeposited in the Protein Data Bank (PDB) under ID code 6BXJ.

SAXS. The untagged purified cβ7 ectodomain without EndoH treatment wasconcentrated to 1, 3, and 4.82 mg/mL in 20 mM Tris·HCl (pH 7.5), 150 mMNaCl, 1 mM CaCl2, and 1 mM MgCl2. The SAXS data were collected at theAPS beamline 12-ID-B. The datasets obtained at three different concentra-tions were processed with the PRIMUS program in the ATSAS package (71).The ab initial bead model was generated with DAMMIF. The crystal structureof cβ3 was superimposed with the bead model of cβ7 using SUPCOMB.

Cell Surface Expression, Conformation-Specific Antibody, and Ligand-BindingAssay. The binding of ligand-mimetic mAb PAC-1 (BD Biosciences) to αIIbβ3-transfected HEK293FT cells was as described previously (41, 72). Integrin cellsurface expression was measured by flow cytometry using the anti-β3 mAbAP3 (73) or SZ21 (Santa Cruz Biotechnology), anti-αIIb mAb 10E5 (74, 75), theanti-αVβ3 complex-specific mAb LM609 (EMD Millipore), and the anti-αL Idomain mAb MHM24 (Developmental Studies Hybridoma Bank). The αIIband β3 subunits in the cell lysates were detected by Western blot analysisusing mAb 314.5 and H-96 (Santa Cruz Biotechnology), respectively.

LIBS-1 and 319.4 are active conformation-specific mAbs that bind to theI-EGF/β-tail domains of β3 (3, 30). 370.3 is an active conformation-specific mAbthat binds to the calf-1 domain of αIIb subunit (3). Binding of the biotinylatedLIBS-1 or 319.4 to the HEK293FT transfectants with or without the αIIbβ3-spe-cific ligand-mimetic inhibitor eptifibatide (Santa Cruz Biotechnology) was asdescribed before (3).

Binding of human ICAM-1 with a C-terminal human IgG1-Fc tag (ICAM-1-Fc) was as described previously (36, 54). The binding was performed in thebuffer containing 1 mM Mg2+ or 2 mM Mn2+ with or without 50 μM of αLβ2-specific inhibitor A286982 (Santa Cruz Biotechnology). Bound ICAM-1 wasdetected by flow cytometry and presented as the mean fluorescence in-tensity (MFI) normalized to integrin expression. For the competition assay,the mixtures of ICAM-1-Fc (at 4 μg/mL) and anti-Fc were first incubatedwith the purified cβ3 ectodomain or cells transfected with full-length cβ3.Then the cells, but not the soluble cβ3, were removed by centrifugation andthe supernatant was used for the ICAM-1 binding assay as described above.

Maleimide Labeling of Free Cysteine. HEK293FT cells transfected with the cβ3constructs were washed once with ice-cold PBS buffer and incubated with10 mM maleimide-PEG2-biotin on ice for 15 min. The cells were thenwashed, lysed, and subjected to immunoprecipitation using mAb AP3. Theimmunoprecipitated cβ3 were detected by Western blot analysis using IRDye800CW-conjugated streptavidin (LI-COR Biosciences) for the maleimide-labeled cβ3. Total cβ3 was detected by H-96 and IRDye 680LT-conjugatedanti-rabbit IgG (LI-COR Biosciences).

Detection of Anti–HPA-1a Alloantibodies by Flow Cytometry. Normal humansera or the sera from the PTP patients and the mothers of NAIT patients werepreabsorbed by the HEK293FT cells. Alternatively, total IgG proteins werepurified from the sera using the Melon gel IgG spin purification kit (ThermoFisher Scientific). HEK293FT cells transfectedwith EGFP alone or EGFP plus theintegrin constructs were incubatedwith the sera at 1:10 or 1:20 dilution in thebuffer containing 1 mM Ca2+/Mg2+ or 0.2 mM Ca2+/2 mM Mn2+ plus 30 μMGRGDSP peptide at room temperature for 30 min, and then incubated withAlexa Fluor 647-conjugated goat anti-human IgG. For detecting the surfaceexpression of β3 integrin, the same transfected cells were stained separatelywith AP3, followed by staining with Alexa Fluor 647-conjugated goat anti-mouse IgG. The EGFP-positive cells were acquired by flow cytometry tomeasure the MFI of bound anti–HPA-1a or AP3. The MFI measured for thecells transfected with EGFP only was used for background subtraction.Binding of anti–HPA-1a alloantibodies was assessed by the MFI normalizedto β3 surface expression. For the competition assay, the sera were pre-incubated with the purified ectodomain of cβ3 or cβ7 before incubation withHEK293-αIIbβ3 stable cells or transiently transfected HEK293FT cells. The cellswere then incubated with Alexa Fluor 647-conjugated goat anti-human IgGand subjected to flow cytometry as described above.

Ligand- and Conformation-Specific mAb Binding Assay in the Presence of Anti–HPA-1a Alloantibodies. For PAC-1 binding to HEK293FT cells transfected withαIIbβ3 plus EGFP, the cells were preincubated with PTP, NAIT, or normal seraor purified IgG at room temperature for 30 min, followed by the addition of2.5 μg/mL PAC-1 plus 0.2 mM Ca2+/2 mM Mn2+ and incubated for another30 min. For the LIBS-1 and 370.3 binding assay, the transfected cells werepreincubated with NAIT or normal sera at room temperature for 30 minbefore adding 2.5 μg/mL 370.3 (for αIIbβ3) or LIBS-1 (for αIIbβ3 and αVβ3) plus0.2 mM Ca2+/2 mM Mn2+ and incubated for another 30 min. The cells werealso stained separately with AP3. The EGFP-positive cells were analyzed byflow cytometry after staining with Alexa Fluor 647-conjugated goat anti-mouse IgM (for PAC-1) or Alexa Fluor 647-conjugated goat anti-mouse IgG(for 370.3, LIBS-1, and AP3). The binding data are presented as MFI nor-malized to β3 expression detected by AP3.

ACKNOWLEDGMENTS. We thank Drs. Peter Newman and Richard Aster forhelpful discussions, Drs. Daniel Bougie and Richard Aster for providingantibodies, and Dr. Xiaobing Zuo for helping with SAXS data collection. Thiswork was supported by National Heart, Lung, and Blood Institute GrantsHL122985 and HL131836 (to J.Z.).

1. Hynes RO (2002) Integrins: Bidirectional, allosteric signaling machines. Cell 110:673–687.

2. Springer TA (2006) Complement and the multifaceted functions of VWA and integrinI domains. Structure 14:1611–1616.

3. Zhang C, et al. (2013) Modulation of integrin activation and signaling by α1/α1′-helixunbending at the junction. J Cell Sci 126:5735–5747.

4. Springer TA, Dustin ML (2012) Integrin inside-out signaling and the immunologicalsynapse. Curr Opin Cell Biol 24:107–115.

5. Xiong J-P, et al. (2001) Crystal structure of the extracellular segment of integrin αVbeta3. Science 294:339–345.

6. Xiong JP, et al. (2009) Crystal structure of the complete integrin alphaVbeta3 ectodomainplus an α/β transmembrane fragment. J Cell Biol 186:589–600.

7. Dong X, et al. (2012) α(V)β(3) integrin crystal structures and their functional implica-tions. Biochemistry 51:8814–8828.

8. Zhu J, et al. (2008) Structure of a complete integrin ectodomain in a physiologicresting state and activation and deactivation by applied forces. Mol Cell 32:849–861.

9. Luo B-H, Springer TA, Takagi J (2004) A specific interface between integrin trans-membrane helices and affinity for ligand. PLoS Biol 2:e153.

10. Zhu J, et al. (2009) The structure of a receptor with two associating transmembranedomains on the cell surface: Integrin alphaIIbbeta3. Mol Cell 34:234–249.

11. Yang J, et al. (2009) Structure of an integrin alphaIIb β3 transmembrane-cytoplasmicheterocomplex provides insight into integrin activation. Proc Natl Acad Sci USA 106:17729–17734.

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Page 10: Autonomous conformational regulation of β3 integrin and the ...Autonomous conformational regulation of β3 integrin and the conformation-dependent property of HPA-1a alloantibodies

12. Lau TL, Kim C, Ginsberg MH, Ulmer TS (2009) The structure of the integrinalphaIIbbeta3 transmembrane complex explains integrin transmembrane signalling.EMBO J 28:1351–1361.

13. Shattil SJ, Kim C, Ginsberg MH (2010) The final steps of integrin activation: The endgame. Nat Rev Mol Cell Biol 11:288–300.

14. Calderwood DA, Campbell ID, Critchley DR (2013) Talins and kindlins: Partners inintegrin-mediated adhesion. Nat Rev Mol Cell Biol 14:503–517.

15. Das M, Ithychanda S, Qin J, Plow EF (2014) Mechanisms of talin-dependent integrinsignaling and crosstalk. Biochim Biophys Acta 1838:579–588.

16. Takagi J, Petre BM, Walz T, Springer TA (2002) Global conformational rearrange-ments in integrin extracellular domains in outside-in and inside-out signaling. Cell110:599–11.

17. Zhu J, et al. (2010) Closed headpiece of integrin αIIbβ3 and its complex with an αIIbβ3-specific antagonist that does not induce opening. Blood 116:5050–5059.

18. Sen M, Springer TA (2016) Leukocyte integrin αLβ2 headpiece structures: The αI do-main, the pocket for the internal ligand, and concerted movements of its loops. ProcNatl Acad Sci USA 113:2940–2945.

19. Xia W, Springer TA (2014) Metal ion and ligand binding of integrin α5β1. Proc NatlAcad Sci USA 111:17863–17868.

20. Newman PJ, Derbes RS, Aster RH (1989) The human platelet alloantigens, PlA1 andPlA2, are associated with a leucine33/proline33 amino acid polymorphism in mem-brane glycoprotein IIIa, and are distinguishable by DNA typing. J Clin Invest 83:1778–1781.

21. Sachs UJ (2013) Fetal/neonatal alloimmune thrombocytopenia. Thromb Res 131:S42–S46.

22. Curtis BR (2015) Recent progress in understanding the pathogenesis of fetal andneonatal alloimmune thrombocytopenia. Br J Haematol 171:671–682.

23. Santoso S, et al. (2016) Antiendothelial αVβ3 antibodies are a major cause of in-tracranial bleeding in fetal/neonatal alloimmune thrombocytopenia. ArteriosclerThromb Vasc Biol 36:1517–1524.

24. Kroll H, Penke G, Santoso S (2005) Functional heterogeneity of alloantibodies againstthe human platelet antigen (HPA)-1a. Thromb Haemost 94:1224–1229.

25. Lee J-O, Rieu P, Arnaout MA, Liddington R (1995) Crystal structure of the A domain fromthe α subunit of integrin CR3 (CD11b/CD18). Cell 80:631–638.

26. Hughes PE, et al. (1996) Breaking the integrin hinge: A defined structural constraintregulates integrin signaling. J Biol Chem 271:6571–6574.

27. Kamata T, et al. (2010) Structural requirements for activation in alphaIIb β3 integrin.J Biol Chem 285:38428–38437.

28. Zhu J, Boylan B, Luo B-H, Newman PJ, Springer TA (2007) Tests of the extension anddeadbolt models of integrin activation. J Biol Chem 282:11914–11920.

29. Luo B-H, Springer TA, Takagi J (2003) Stabilizing the open conformation of the in-tegrin headpiece with a glycan wedge increases affinity for ligand. Proc Natl Acad SciUSA 100:2403–2408.

30. Lin FY, Zhu J, Eng ET, Hudson NE, Springer TA (2016) β-Subunit binding is sufficient forligands to open the integrin αIIbβ3 Headpiece. J Biol Chem 291:4537–4546.

31. Honda S, Honda Y, Bauer B, Ruan C, Kunicki TJ (1995) The impact of three-dimensional structure on the expression of PlA alloantigens on human integrin β 3.Blood 86:234–242.

32. Stafford P, et al. (2008) Immunologic and structural analysis of eight novel domain-deletion β3 integrin peptides designed for detection of HPA-1 antibodies. J ThrombHaemost 6:366–375.

33. Sun Q-H, Liu CY, Wang R, Paddock C, Newman PJ (2002) Disruption of the long-rangeGPIIIa Cys(5)-Cys(435) disulfide bond results in the production of constitutively activeGPIIb-IIIa (α(IIb)β(3)) integrin complexes. Blood 100:2094–2101.

34. Zhou D, Thinn AMM, Zhao Y, Wang Z, Zhu J (2018) Structure of an extended β3 in-tegrin. Blood, 10.1182/blood-2018-01-829572.

35. Xie C, et al. (2010) Structure of an integrin with an alphaI domain, complement re-ceptor type 4. EMBO J 29:666–679.

36. Wang Z, Thinn AMM, Zhu J (2017) A pivotal role for a conserved bulky residue at theα1-helix of the αI integrin domain in ligand binding. J Biol Chem 292:20756–20768.

37. Takagi J, Debottis DP, Erickson HP, Springer TA (2002) The role of specificity-determining loop of the integrin β-subunit I-like domain in folding, associationwith the α subunit, and ligand binding. Biochemistry 41:4339–4347.

38. Kim M, Carman CV, Yang W, Salas A, Springer TA (2004) The primacy of affinity overclustering in regulation of adhesiveness of the integrin αLβ2. J Cell Biol 167:1241–1253.

39. Schürpf T, Springer TA (2011) Regulation of integrin affinity on cell surfaces. EMBO J30:4712–4727.

40. Kim C, Ye F, Ginsberg MH (2011) Regulation of integrin activation. Annu Rev Cell DevBiol 27:321–345.

41. Liu J, Wang Z, Thinn AM, Ma YQ, Zhu J (2015) The dual structural roles of themembrane distal region of the α-integrin cytoplasmic tail during integrin inside-outactivation. J Cell Sci 128:1718–1731.

42. Su Y, et al. (2016) Relating conformation to function in integrin α5β1. Proc Natl AcadSci USA 113:E3872–E3881.

43. Nishida N, et al. (2006) Activation of leukocyte β2 integrins by conversion from bent toextended conformations. Immunity 25:583–594.

44. Yu Y, et al. (2012) Structural specializations of α(4)β(7), an integrin that mediatesrolling adhesion. J Cell Biol 196:131–146.

45. Dong X, Hudson NE, Lu C, Springer TA (2014) Structural determinants of integrinβ-subunit specificity for latent TGF-β. Nat Struct Mol Biol 21:1091–1096.

46. Nagae M, et al. (2012) Crystal structure of α5β1 integrin ectodomain: Atomic details ofthe fibronectin receptor. J Cell Biol 197:131–140.

47. Zhu J, Zhu J, Springer TA (2013) Complete integrin headpiece opening in eight steps.J Cell Biol 201:1053–1068.

48. Li J, et al. (2017) Conformational equilibria and intrinsic affinities define integrinactivation. EMBO J 36:629–645.

49. Eng ET, Smagghe BJ, Walz T, Springer TA (2011) Intact alphaIIbbeta3 integrin is ex-tended after activation as measured by solution X-ray scattering and electron mi-croscopy. J Biol Chem 286:35218–35226.

50. Choi WS, Rice WJ, Stokes DL, Coller BS (2013) Three-dimensional reconstruction ofintact human integrin αIIbβ3: New implications for activation-dependent ligandbinding. Blood 122:4165–4171.

51. Xu XP, et al. (2016) Three-dimensional structures of full-length, membrane-embedded human αIIbβ3 integrin complexes. Biophys J 110:798–809.

52. Morse EM, Brahme NN, Calderwood DA (2014) Integrin cytoplasmic tail interactions.Biochemistry 53:810–820.

53. Li A, Guo Q, Kim C, Hu W, Ye F (2014) Integrin αIIb tail distal of GFFKR participates ininside-out αIIbβ3 activation. J Thromb Haemost 12:1145–1155.

54. Thinn AMM, Wang Z, Zhu J (2018) The membrane-distal regions of integrin α cyto-plasmic domains contribute differently to integrin inside-out activation. Sci Rep 8:5067.

55. Mitchell WB, et al. (2007) Mapping early conformational changes in alphaIIb andβ3 during biogenesis reveals a potential mechanism for alphaIIbbeta3 adopting itsbent conformation. Blood 109:3725–3732.

56. Cormier A, et al. (2018) Cryo-EM structure of the αvβ8 integrin reveals a mechanism forstabilizing integrin extension. Nat Struct Mol Biol 25:698–704.

57. Sun Z, Guo SS, Fässler R (2016) Integrin-mediated mechanotransduction. J Cell Biol215:445–456.

58. Nordenfelt P, Elliott HL, Springer TA (2016) Coordinated integrin activation by actin-dependent force during T-cell migration. Nat Commun 7:13119.

59. Li J, Springer TA (2017) Integrin extension enables ultrasensitive regulation by cyto-skeletal force. Proc Natl Acad Sci USA 114:4685–4690.

60. Swaminathan V, et al. (2017) Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions. Proc Natl Acad Sci USA 114:10648–10653.

61. Case LB, Waterman CM (2015) Integration of actin dynamics and cell adhesion by athree-dimensional, mechanosensitive molecular clutch. Nat Cell Biol 17:955–963.

62. Nordenfelt P, et al. (2017) Direction of actin flow dictates integrin LFA-1 orientationduring leukocyte migration. Nat Commun 8:2047.

63. Yougbaré I, et al. (2015) Maternal anti-platelet β3 integrins impair angiogenesis andcause intracranial hemorrhage. J Clin Invest 125:1545–1556.

64. Byron A, et al. (2009) Anti-integrin monoclonal antibodies. J Cell Sci 122:4009–4011.65. Mi LZ, et al. (2008) Functional and structural stability of the epidermal growth factor

receptor in detergent micelles and phospholipid nanodiscs. Biochemistry 47:10314–10323.

66. Reeves PJ, Callewaert N, Contreras R, Khorana HG (2002) Structure and function inrhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negativeHEK293S stable mammalian cell line. Proc Natl Acad Sci USA 99:13419–13424.

67. Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG (2011) iMOSFLM: A newgraphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr DBiol Crystallogr 67:271–281.

68. McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Cryst 40:658–674.69. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot.

Acta Crystallogr D Biol Crystallogr 66:486–501.70. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro-

molecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221.71. Franke D, et al. (2017) ATSAS 2.8: A comprehensive data analysis suite for small-angle

scattering from macromolecular solutions. J Appl Cryst 50:1212–1225.72. Cai X, Thinn AMM, Wang Z, Shan H, Zhu J (2017) The importance of N-glycosylation

on β3 integrin ligand binding and conformational regulation. Sci Rep 7:4656.73. Newman PJ, Allen RW, Kahn RA, Kunicki TJ (1985) Quantitation of membrane gly-

coprotein IIIa on intact human platelets using the monoclonal antibody, AP-3. Blood65:227–232.

74. Coller BS, Peerschke EI, Scudder LE, Sullivan CA (1983) A murine monoclonal antibodythat completely blocks the binding of fibrinogen to platelets produces athrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/orIIIa. J Clin Invest 72:325–338.

75. Xiao T, Takagi J, Coller BS, Wang JH, Springer TA (2004) Structural basis for allosteryin integrins and binding to fibrinogen-mimetic therapeutics. Nature 432:59–67.

E9114 | www.pnas.org/cgi/doi/10.1073/pnas.1806205115 Thinn et al.

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