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Single-platelet nanomechanics measured by high-throughput cytometry

Nature Materials volume 16, pages 230–235 (2017)Cite this article

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Abstract

Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot’s capability to stem haemorrhage are its changing mechanical properties, the major drivers of which are the contractile forces exerted by platelets against the fibrin scaffold1. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding2 and thrombotic disorders3. Here, we report a high-throughput hydrogel-based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.

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Figure 1: The fundamental unit driving clot stiffening, a single platelet pulling against a fibrin/ogen substrate, is established by recapitulating the mechanical and biological microenvironment of the platelet.
Figure 2: Platelet-contraction cytometer—hydrogels with microprinted arrays of fibrinogen microdots are encapsulated in separate microchannels, enabling the biochemical, mechanical, and shear microenvironments to be precisely controlled and varied simultaneously.
Figure 3: Biochemical and mechanical cues synergistically mediate platelet-contraction force.
Figure 4: Mechanotransductive platelet contraction is mediated by the Rho-associated protein kinase (ROCK) pathway, as measured with platelet-contraction cytometry and standard bulk clot contraction and bulk clot rheology.
Figure 5: Patients with phenotypic bleeding lack highly contractile platelets associated with clot contraction and force generation.

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Acknowledgements

The authors wish to thank A. Shaw of the Parker H. Petit Institute for Bioengineering and Bioscience at the Georgia Institute of Technology (GT); N. Anthony and the Emory University Integrated Cellular Imaging Microscopy Core of the Children’s Pediatric Research Center; and the GT Institute for Electronics and Nanotechnology (IEN) cleanroom. Financial support provided by NIH R01 (HL121264), NIH U54 (HL112309), and NSF CAREER (1150235) to W.A.L., as well as an AHA Postdoctoral Fellowship to D.R.M. are acknowledged. D.R.M. thanks Christy R. Dillon and Gabriel A. Kwong for comments and discussion.

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Authors and Affiliations

  1. Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children’s Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia 30322, USA

    David R. Myers, Yongzhi Qiu, Meredith E. Fay, Yumiko Sakurai, Jong Baek, Reginald Tran, Jordan C. Ciciliano, Byungwook Ahn, Robert G. Mannino, Carolyn Bennett, Michael Briones & Wilbur A. Lam

  2. The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, Georgia 30332, USA

    David R. Myers, Yongzhi Qiu, Meredith E. Fay, Yumiko Sakurai, Jong Baek, Reginald Tran, Byungwook Ahn, Robert G. Mannino & Wilbur A. Lam

  3. Winship Cancer Institute of Emory University, Atlanta, Georgia 30322, USA

    David R. Myers, Yongzhi Qiu, Meredith E. Fay, Yumiko Sakurai, Jong Baek, Reginald Tran, Jordan C. Ciciliano, Byungwook Ahn, Robert G. Mannino & Wilbur A. Lam

  4. Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

    David R. Myers, Yongzhi Qiu, Meredith E. Fay, Yumiko Sakurai, Jong Baek, Reginald Tran, Jordan C. Ciciliano, Byungwook Ahn, Robert G. Mannino, Alberto Fernandez-Nieves & Wilbur A. Lam

  5. Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

    David R. Myers, Yongzhi Qiu, Meredith E. Fay, Yumiko Sakurai, Jong Baek, Reginald Tran, Jordan C. Ciciliano, Byungwook Ahn, Robert G. Mannino & Wilbur A. Lam

  6. School of Physics, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

    Michael Tennenbaum, Jonas Cuadrado & Alberto Fernandez-Nieves

  7. Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill, Raleigh, North Carolina 27695, USA

    Daniel Chester & Ashley C. Brown

  8. Comparative Medicine Institute at North Carolina State University, Raleigh, North Carolina 27695, USA

    Daniel Chester & Ashley C. Brown

  9. George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

    Jordan C. Ciciliano & Todd Sulchek

  10. Department of Pathology, Children’s Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia 30322, USA

    Silvia T. Bunting

  11. Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA

    Michael L. Smith

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  1. David R. Myers
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Contributions

D.R.M. and W.A.L. conceived of and designed the platelet-contraction experiments. D.R.M., Y.Q., A.C.B., J.C.C., B.A., M.L.S., T.S. and W.A.L. designed and tested the platelet-contraction cytometer. D.R.M., M.T., D.C., J.C., Y.S., J.B., R.T., R.G.M., S.T.B., C.B., M.B. and A.F.-N. performed experiments. M.E.F. designed and wrote image analysis algorithms. D.R.M. and W.A.L. analysed data and wrote the manuscript.

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Correspondence to Wilbur A. Lam.

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The authors declare no competing financial interests.

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Myers, D., Qiu, Y., Fay, M. et al. Single-platelet nanomechanics measured by high-throughput cytometry. Nature Mater 16, 230–235 (2017). https://doi.org/10.1038/nmat4772

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