Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Single-platelet nanomechanics measured by high-throughput cytometry

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

  1. Jen, C. J. & McIntire, L. V. The structural properties and contractile force of a clot. Cell Motil. 2, 445–455 (1982).

    CAS  Article  Google Scholar 

  2. Hvas, A.-M. et al. Tranexamic acid combined with recombinant factor VIII increases clot resistance to accelerated fibrinolysis in severe hemophilia A. J. Thromb. Haemost. 5, 2408–2412 (2007).

    CAS  Article  Google Scholar 

  3. Collet, J. P. et al. Altered fibrin architecture is associated with hypofibrinolysis and premature coronary atherothrombosis. Arterioscler. Thromb. Vasc. Biol. 26, 2567–2573 (2006).

    CAS  Article  Google Scholar 

  4. Carr, M. E. Development of platelet contractile force as a research and clinical measure of platelet function. Cell Biochem. Biophys. 38, 55–78 (2003).

    CAS  Article  Google Scholar 

  5. Cohen, I. & De Vries, A. Platelet contractile regulation in an isometric system. Nature 246, 36–37 (1973).

    CAS  Article  Google Scholar 

  6. Young, G. et al. Thrombin generation and whole blood viscoelastic assays in the management of hemophilia: current state of art and future perspectives. Blood 121, 1944–1950 (2013).

    CAS  Article  Google Scholar 

  7. Qiu, Y. et al. Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation. Proc. Natl Acad. Sci. USA 111, 14430–14435 (2014).

    CAS  Article  Google Scholar 

  8. Lam, W. A. et al. Mechanics and contraction dynamics of single platelets and implications for clot stiffening. Nat. Mater. 10, 61–66 (2011).

    CAS  Article  Google Scholar 

  9. Kita, A. et al. Microenvironmental geometry guides platelet adhesion and spreading: a quantitative analysis at the single cell level. PLoS ONE 6, e26437 (2011).

    CAS  Article  Google Scholar 

  10. Stalker, T. J. et al. Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood 121, 1875–1885 (2013).

    CAS  Article  Google Scholar 

  11. Nesbitt, W. S. et al. A shear gradient-dependent platelet aggregation mechanism drives thrombus formation. Nat. Med. 15, 665–673 (2009).

    CAS  Article  Google Scholar 

  12. Kroll, M. H., Hellums, J. D., McIntire, L. V., Schafer, A. I. & Moake, J. L. Platelets and shear stress. Blood 88, 1525–1541 (1996).

    CAS  Article  Google Scholar 

  13. Weisel, J. W. Biophysics. Enigmas of blood clot elasticity. Science 320, 456–457 (2008).

    CAS  Article  Google Scholar 

  14. Schwarz Henriques, S., Sandmann, R., Strate, A. & Köster, S. Force field evolution during human blood platelet activation. J. Cell Sci. 125, 3914–3920 (2012).

    Article  Google Scholar 

  15. Liang, X. M., Han, S. J., Reems, J.-A. A., Gao, D. & Sniadecki, N. J. Platelet retraction force measurements using flexible post force sensors. Lab Chip 10, 991–998 (2010).

    CAS  Article  Google Scholar 

  16. Suzuki-Inoue, K. et al. Involvement of Src kinases and PLCγ2 in clot retraction. Thromb. Res. 120, 251–258 (2007).

    CAS  Article  Google Scholar 

  17. Litvinov, R. I., Gorkun, O. V., Owen, S. F. & Shuman, H. Polymerization of fibrin: specificity, strength, and stability of knob-hole interactions studied at the single-molecule level. Blood 106, 2944–2951 (2005).

    CAS  Article  Google Scholar 

  18. Litvinov, R. I. et al. Polymerization of fibrin: direct observation and quantification of individual B:b knob–hole interactions. Blood 109, 130–138 (2007).

    CAS  Article  Google Scholar 

  19. Engler, A. et al. Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating. J. Cell Sci. 121, 3794–3802 (2008).

    CAS  Article  Google Scholar 

  20. Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241–254 (2005).

    CAS  Article  Google Scholar 

  21. Burridge, K. & Wittchen, E. The tension mounts: Stress fibers as force-generating mechanotransducers. J. Cell Biol. 200, 9–19 (2013).

    CAS  Article  Google Scholar 

  22. De Gennes, P.-G. Scaling Concepts in Polymer Physics (Cornell University Press).

  23. Shah, J. & Janmey, P. Strain hardening of fibrin gels and plasma clots. Rheol. Acta 36, 262–268 (1997).

    CAS  Article  Google Scholar 

  24. Godwin, H. & Ginsburg, D. May–Hegglin anomaly: a defect in megakaryocyte fragmentation? Br. J. Haematol. 26, 117–127 (1974).

    CAS  Article  Google Scholar 

  25. Shcherbina, A. et al. WASP plays a novel role in regulating platelet responses dependent on αIIbβ3 integrin outside-in signalling. Br. J. Haematol. 148, 416–427 (2010).

    CAS  Article  Google Scholar 

  26. Pedersen, J. A. & Swartz, M. A. Mechanobiology in the third dimension. Ann. Biomed. Eng. 33, 1469–1490 (2005).

    Article  Google Scholar 

  27. Von Philipsborn, A. C. et al. Microcontact printing of axon guidance molecules for generation of graded patterns. Nat. Protoc. 1, 1322–13228 (2006).

    CAS  Article  Google Scholar 

  28. Jirousková, M., Jaiswal, J. K. & Coller, B. S. Ligand density dramatically affects integrin αIIbβ3-mediated platelet signaling and spreading. Blood 109, 5260–5269 (2007).

    Article  Google Scholar 

  29. Polio, S. R., Rothenberg, K. E., Stamenović, D. & Smith, M. L. A micropatterning and image processing approach to simplify measurement of cellular traction forces. Acta Biomater 8, 82–88 (2012).

    CAS  Article  Google Scholar 

  30. Tse, J. R. & Engler, A. J. Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate. PLoS ONE 6, e15978 (2011).

    CAS  Article  Google Scholar 

  31. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    CAS  Article  Google Scholar 

  32. Maloney, J. M., Walton, E. B., Bruce, C. M. & VanVliet, K. J. Influence of finite thickness and stiffness on cellular adhesion-induced deformation of compliant substrata. Phys. Rev. E 78, 041923 (2008).

    Article  Google Scholar 

  33. Tse, J. R. & Engler, A. J. Preparation of hydrogel substrates with tunable mechanical properties. Curr. Protoc. Cell Biol. (2010) Ch. 10 Unit 10.16.

  34. Sabass, B., Gardel, M., Waterman, C. & Schwarz, U. High resolution traction force microscopy based on experimental and computational advances. Biophys. J. 94, 207–220 (2008).

    CAS  Article  Google Scholar 

  35. McCabe White, M. & Jennings, L. K. Platelet Protocols: Research and Clinical Laboratory Procedures (Academic).

  36. Gersh, K. C., Nagaswami, C. & Weisel, J. W. Fibrin network structure and clot mechanical properties are altered by incorporation of erythrocytes. Thromb. Haemost. 102, 1169–1175 (2009).

    CAS  Article  Google Scholar 

  37. Collet, J.-P. P., Shuman, H., Ledger, R. E., Lee, S. & Weisel, J. W. The elasticity of an individual fibrin fiber in a clot. Proc. Natl Acad. Sci. USA 102, 9133–9137 (2005).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Wilbur A. Lam.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2845 kb)

Supplementary Information

Supplementary movie 1 (AVI 415 kb)

Supplementary Information

Supplementary movie 2 (AVI 440 kb)

Supplementary Information

Supplementary movie 3 (AVI 407 kb)

Supplementary Information

Supplementary movie 4 (AVI 131940 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmat4772

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing