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- PDB-9dao: AlphaIIbbeta3 in fully-extended conformation in complex with R6H8 Fab -

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Basic information

Entry
Database: PDB / ID: 9dao
TitleAlphaIIbbeta3 in fully-extended conformation in complex with R6H8 Fab
Components
  • Integrin alpha-IIb
  • Integrin beta-3
  • R6H8 Fab heavy chain
  • R6H8 Fab light chain
KeywordsBLOOD CLOTTING / alphaIIbbeta3 integrin / platelet aggregation / clot retraction
Function / homology
Function and homology information


tube development / regulation of serotonin uptake / positive regulation of adenylate cyclase-inhibiting opioid receptor signaling pathway / alpha9-beta1 integrin-ADAM8 complex / regulation of trophoblast cell migration / integrin alphaIIb-beta3 complex / regulation of postsynaptic neurotransmitter receptor diffusion trapping / maintenance of postsynaptic specialization structure / alphav-beta3 integrin-vitronectin complex / regulation of extracellular matrix organization ...tube development / regulation of serotonin uptake / positive regulation of adenylate cyclase-inhibiting opioid receptor signaling pathway / alpha9-beta1 integrin-ADAM8 complex / regulation of trophoblast cell migration / integrin alphaIIb-beta3 complex / regulation of postsynaptic neurotransmitter receptor diffusion trapping / maintenance of postsynaptic specialization structure / alphav-beta3 integrin-vitronectin complex / regulation of extracellular matrix organization / positive regulation of glomerular mesangial cell proliferation / platelet alpha granule membrane / negative regulation of lipoprotein metabolic process / integrin alphav-beta3 complex / alphav-beta3 integrin-PKCalpha complex / fibrinogen binding / alphav-beta3 integrin-HMGB1 complex / blood coagulation, fibrin clot formation / glycinergic synapse / vascular endothelial growth factor receptor 2 binding / negative regulation of lipid transport / positive regulation of vascular endothelial growth factor signaling pathway / : / regulation of release of sequestered calcium ion into cytosol / Elastic fibre formation / mesodermal cell differentiation / cell-substrate junction assembly / alphav-beta3 integrin-IGF-1-IGF1R complex / platelet-derived growth factor receptor binding / angiogenesis involved in wound healing / filopodium membrane / extracellular matrix binding / positive regulation of fibroblast migration / positive regulation of vascular endothelial growth factor receptor signaling pathway / regulation of bone resorption / apolipoprotein A-I-mediated signaling pathway / positive regulation of cell adhesion mediated by integrin / positive regulation of leukocyte migration / apoptotic cell clearance / wound healing, spreading of epidermal cells / integrin complex / heterotypic cell-cell adhesion / smooth muscle cell migration / Molecules associated with elastic fibres / Mechanical load activates signaling by PIEZO1 and integrins in osteocytes / positive regulation of cell-matrix adhesion / negative chemotaxis / cell adhesion mediated by integrin / cellular response to insulin-like growth factor stimulus / Syndecan interactions / microvillus membrane / p130Cas linkage to MAPK signaling for integrins / positive regulation of smooth muscle cell migration / regulation of postsynaptic neurotransmitter receptor internalization / protein disulfide isomerase activity / cell-substrate adhesion / positive regulation of osteoblast proliferation / PECAM1 interactions / TGF-beta receptor signaling activates SMADs / GRB2:SOS provides linkage to MAPK signaling for Integrins / lamellipodium membrane / platelet-derived growth factor receptor signaling pathway / fibronectin binding / negative regulation of macrophage derived foam cell differentiation / negative regulation of lipid storage / ECM proteoglycans / Integrin cell surface interactions / positive regulation of bone resorption / positive regulation of T cell migration / negative regulation of endothelial cell apoptotic process / coreceptor activity / cell adhesion molecule binding / cellular response to platelet-derived growth factor stimulus / positive regulation of endothelial cell proliferation / positive regulation of substrate adhesion-dependent cell spreading / positive regulation of endothelial cell migration / Integrin signaling / embryo implantation / substrate adhesion-dependent cell spreading / protein kinase C binding / Turbulent (oscillatory, disturbed) flow shear stress activates signaling by PIEZO1 and integrins in endothelial cells / cell-matrix adhesion / Signal transduction by L1 / response to activity / integrin-mediated signaling pathway / regulation of actin cytoskeleton organization / positive regulation of smooth muscle cell proliferation / wound healing / RUNX1 regulates genes involved in megakaryocyte differentiation and platelet function / Signaling by high-kinase activity BRAF mutants / MAP2K and MAPK activation / cell-cell adhesion / platelet activation / VEGFA-VEGFR2 Pathway / platelet aggregation / ruffle membrane / cellular response to mechanical stimulus / positive regulation of fibroblast proliferation / positive regulation of angiogenesis / Signaling by RAF1 mutants
Similarity search - Function
Integrin beta, epidermal growth factor-like domain 1 / Integrin beta epidermal growth factor like domain 1 / Integrin beta subunit, cytoplasmic domain / Integrin beta cytoplasmic domain / Integrin_b_cyt / Integrin beta tail domain / : / Integrin alpha Ig-like domain 3 / Integrin EGF domain / Integrin beta subunit, tail ...Integrin beta, epidermal growth factor-like domain 1 / Integrin beta epidermal growth factor like domain 1 / Integrin beta subunit, cytoplasmic domain / Integrin beta cytoplasmic domain / Integrin_b_cyt / Integrin beta tail domain / : / Integrin alpha Ig-like domain 3 / Integrin EGF domain / Integrin beta subunit, tail / Integrin beta tail domain superfamily / Integrin_B_tail / EGF-like domain, extracellular / EGF-like domain / Integrins beta chain EGF (I-EGF) domain profile. / Integrin alpha cytoplasmic region / Integrin beta subunit, VWA domain / Integrin beta subunit / Integrin beta N-terminal / Integrin beta chain VWA domain / Integrin plexin domain / Integrins beta chain EGF (I-EGF) domain signature. / Integrin beta subunits (N-terminal portion of extracellular region) / Integrin alpha-2 / Integrin alpha Ig-like domain 1 / Integrin alpha chain / Integrin alpha beta-propellor / Integrin alpha chain, C-terminal cytoplasmic region, conserved site / : / Integrin alpha Ig-like domain 2 / Integrins alpha chain signature. / FG-GAP repeat profile. / Integrin alpha (beta-propellor repeats). / FG-GAP repeat / FG-GAP repeat / Integrin domain superfamily / Integrin alpha, N-terminal / PSI domain / domain found in Plexins, Semaphorins and Integrins / von Willebrand factor A-like domain superfamily / EGF-like domain signature 1. / EGF-like domain signature 2.
Similarity search - Domain/homology
Integrin beta-3 / Integrin alpha-IIb
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsWalz, T. / Coller, B.S. / Wang, J.L. / Buitrago, L. / Wang, L. / Li, J.H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)5R01HL019278-48 United States
CitationJournal: Blood Adv / Year: 2025
Title: An αIIbβ3 Ligand-Mimetic Murine Monoclonal Antibody That Produces Platelet Activation by Engaging the FcγIIa Receptor.
Authors: Jialing Wang / Lorena Buitrago / Lu Wang / Jihong Li / Thomas Walz / Barry S Coller /
Abstract: To produce a murine monoclonal antibody that binds to αIIbβ3 and inhibits clot retraction (CR) we immunized mice with human platelets and tested hybridoma supernatants for their ability to bind to ...To produce a murine monoclonal antibody that binds to αIIbβ3 and inhibits clot retraction (CR) we immunized mice with human platelets and tested hybridoma supernatants for their ability to bind to αIIbβ3 and inhibit CR. The IgG1 mAb R6H8 completely inhibited CR at 20 µg/ml. Paradoxically, 5 µg/ml R6H8 initiated platelet aggregation and induced P-selectin expression, fibrinogen binding, and PAC-1 binding. At 20 µg/ml, however, R6H8 completely inhibited aggregation induced by peptide SFLLRN (25 µg/ml; T6) and T6-induced fibrinogen and PAC-1 binding to platelets. Platelet aggregation induced by R6H8 was inhibited by mAb IV.3, which blocks the FcγIIa receptor (FcγRIIa), and the Fab fragment of R6H8 did not induce platelet aggregation, suggesting that R6H8 binds to both αIIbβ3 and FcγRIIa. Cryo-EM analysis of the R6H8 Fab-αIIbβ3 complex revealed that R6H8: 1) Binds to the αIIbβ3 RGD binding pocket via an RYD sequence in its heavy chain CDR3, 2) Interacts with β3 Asp126, producing a reorientation of Asp126 and loss of the ADMIDAS Ca2+, and 3) Initiates swing-out of the β3 hybrid domain. We conclude that R6H8 is an αIIbβ3 ligand-mimetic mAb that activates platelets via FcγRIIa at low concentrations and potently inhibits platelet aggregation and clot retraction at high concentrations. R6H8 simulates the actions of a number of pathological antibodies, including platelet-activating antibodies developed after therapy with glycoprotein IIb/IIIa (αIIbβ3) inhibitors and platelet-activating antibodies in heparin-induced thrombocytopenia and vaccine-induced immune thrombotic thrombocytopenia. As such, it may be a valuable reagent for better understanding these disorders and identifying potential therapies.
History
DepositionAug 22, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
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Assembly

Deposited unit
A: Integrin alpha-IIb
B: Integrin beta-3
H: R6H8 Fab heavy chain
L: R6H8 Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)243,06210
Polymers242,8384
Non-polymers2256
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Integrin alpha-IIb / GPalpha IIb / GPIIb / Platelet membrane glycoprotein IIb


Mass: 110106.391 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P08514
#2: Protein Integrin beta-3 / Platelet membrane glycoprotein IIIa / GPIIIa


Mass: 84478.500 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P05106

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Antibody , 2 types, 2 molecules HL

#3: Antibody R6H8 Fab heavy chain


Mass: 24535.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#4: Antibody R6H8 Fab light chain


Mass: 23717.123 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Non-polymers , 2 types, 6 molecules

#5: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1AlphaIIbbeta3 in fully-extended conformation in complex with R6H8 FabCOMPLEXAlphaIIbbeta3 is platelet-derived and R6H8 fab is hybridoma cell-derived#1-#40MULTIPLE SOURCES
2AlphaIIbbeta3 integrinCOMPLEX#1-#21NATURAL
3R6H8 FabCOMPLEX#3-#41RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.25 MDaYES
210.2 MDaYES
310.05 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Homo sapiens (human)9606
43Mus musculus (house mouse)10090
Source (recombinant)Organism: Mus musculus (house mouse)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 595163 / Num. of class averages: 4 / Symmetry type: POINT

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