Abstract

Integrins are membrane receptors that mediate cell adhesion and mechanosensing. The structure–function relationship of integrins remains incompletely understood, despite the extensive studies carried out because of its importance to basic cell biology and translational medicine. Using a fluorescence dual biomembrane force probe, microfluidics and cone-and-plate rheometry, we applied precisely controlled mechanical stimulations to platelets and identified an intermediate state of integrin αIIbβ3 that is characterized by an ectodomain conformation, ligand affinity and bond lifetimes that are all intermediate between the well-known inactive and active states. This intermediate state is induced by ligand engagement of glycoprotein (GP) Ibα via a mechanosignalling pathway and potentiates the outside-in mechanosignalling of αIIbβ3 for further transition to the active state during integrin mechanical affinity maturation. Our work reveals distinct αIIbβ3 state transitions in response to biomechanical and biochemical stimuli, and identifies a role for the αIIbβ3 intermediate state in promoting biomechanical platelet aggregation.

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The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank A. Garcia (Georgia Tech), Z. Ruggeri (The Scripps Research Institute), B. Coller (Rockefeller University), P. Newman, R. Aster, J. Zhu and D. Bougie (BloodCenter of Wisconsin), W. Lam (Emory University) and F. Tovar Lopez (RMIT University) for providing precious reagents. The authors thank Y. Sakurai, D. Myer, Y. Qiu, R. Tran and J. Ciciliano from W. Lam lab (Georgia Tech) for the blood collection; R. Darbousset for support with platelet isolation and flow cytometry; A. Samson (WEHI) and J. MaClean (HRI) for cone-and-plate rheometry training; I. Alwis (USYD) for support with confocal microscopy; N. Court and E. Ilagan (USYD ANFF Research & Prototype Foundry) for advice on stenosis microchannel fabrication and characterization; S. Schoenwaelder (USYD), J. McFadyen (Baker Institute) and Z. Li (QUT) for helpful discussion. This work was supported by grants from the NIH (HL1320194, to C.Z.; R21EB020424, to H.L.), the NSF (DMS-1505256 and DMS-1811552, to L.X.), the NIDA (P50 DA039838, to L.X.), the NHMRC (APP1028564 and APP1048574, to S.P.J.), the Australian Research Council (LE120100043, to S.P.J.), the University of Technology Sydney’s Grant for IBMD (to Q.P.S.), the Diabetes Australia Research Program General Grant (G179720), the University of Sydney Early-Career Researcher Kickstart Grant and Cardiovascular Initiative Catalyst Grant for Precision CV Medicine, the Royal College of Pathologists of Australasia Kanematsu research award and the Cardiac Society of Australia and New Zealand BAYER Young Investigator Research Grant (to L.A.J.). S.P.J. is an NHMRC Senior Principal Research Fellow. L.A.J. is an Australian Research Council DECRA fellow (DE190100609) and a former National Heart Foundation of Australia postdoctoral fellow (101798).

Author information

Author notes

  1. These authors contributed equally: Yunfeng Chen, Lining Arnold Ju.

Affiliations

  1. Woodruff School of Mechanical Engineering and Georgia Institute of Technology, Atlanta, GA, USA

    • Yunfeng Chen
    • , Fangyuan Zhou
    •  & Cheng Zhu
  2. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA, USA

    • Yunfeng Chen
    • , Lining Arnold Ju
    • , Fangyuan Zhou
    • , Jiexi Liao
    •  & Cheng Zhu
  3. Department of Molecular Medicine, MERU-Roon Research Center on Vascular Biology, The Scripps Research Institute, La Jolla, CA, USA

    • Yunfeng Chen
    •  & Shaun P. Jackson
  4. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA

    • Lining Arnold Ju
    • , Jiexi Liao
    •  & Cheng Zhu
  5. Heart Research Institute, The University of Sydney, Camperdown, New South Wales, Australia

    • Lining Arnold Ju
    • , Yuping Yuan
    • , Shaun P. Jackson
    •  & Cheng Zhu
  6. Charles Perkins Centre, The University of Sydney, Camperdown, New South Wales, Australia

    • Lining Arnold Ju
    • , Yuping Yuan
    •  & Shaun P. Jackson
  7. School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, Camperdown, New South Wales, Australia

    • Lining Arnold Ju
  8. Department of Statistics, Pennsylvania State University, University Park, PA, USA

    • Lingzhou Xue
  9. Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney, Sydney, New South Wales, Australia

    • Qian Peter Su
    •  & Dayong Jin
  10. School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA

    • Hang Lu

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Contributions

Y.C. and L.A.J. designed and performed experiments, analysed data and co-wrote the paper. F.Z., J.L. and Q.P.S. performed experiments and analysed data. L.X. analysed data and co-wrote the paper. Y.Y. provided critical suggestions and co-wrote the paper. D.J. co-supervised studies and H.L. provided critical devices and reagents. S.P.J. co-wrote the paper and co-supervised studies. C.Z. supervised the study, designed experiments and wrote the paper. Research activities related to this work complied with relevant ethical regulations.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Shaun P. Jackson or Cheng Zhu.

Supplementary information

  1. Supplementary Information

    Supplementary Notes 1–3, Supplementary Figures 1–11, Supplementary Tables 1–3, Supplementary Video Legends 1–6, Supplementary References 56–69

  2. Reporting Summary

  3. Supplementary Video 1

    A fraction of integrin αIIbβ3 undergoes extension on biomechanical platelet aggregates at stenosis.

  4. Supplementary Video 2

    Integrin αIIbβ3 hybrid domain remains swing-in on biomechanical platelet aggregates at stenosis.

  5. Supplementary Video 3

    Ligand binding site of integrin αIIbβ3 is not fully activated on biomechanical platelet aggregates at stenosis.

  6. Supplementary Video 4

    A fraction of integrin αIIbβ3 undergoes ectodomain extension on agonist-induced platelet aggregates at stenosis.

  7. Supplementary Video 5

    A fraction of integrin αIIbβ3 swings out hybrid domain on agonist-induced platelet aggregates at stenosis.

  8. Supplementary Video 6

    Ligand binding site of a fraction of integrin αIIbβ3 becomes activated on agonist induced platelet aggregates at stenosis.

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DOI

https://doi.org/10.1038/s41563-019-0323-6