Skip to main content

A Multimode-TIRFM and Microfluidic Technique to Examine Platelet Adhesion Dynamics

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1046))

Abstract

Fluorescence microscopy techniques have provided important insights into the structural and signalling events occurring during platelet adhesion under both static and blood flow conditions. However, due to limitations in sectioning ability and sensitivity these techniques are restricted in their capacity to precisely image the adhesion footprint of spreading platelets. In particular, investigation of platelet adhesion under hemodynamic shear stress requires an imaging platform with high spatial discrimination and sensitivity and rapid temporal resolution. This chapter describes in detail a multimode imaging approach combining total internal reflection fluorescence microscopy (TIRFM) with high speed epifluorescence and differential interference contrast (DIC) microscopy along with a novel microfluidic perfusion system developed in our laboratory to examine platelet membrane adhesion dynamics under static and flow conditions.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Dosquet C, Weill D, Wautier JL (1992) Molecular mechanism of blood monocyte adhesion to vascular endothelial cells. Nouv Rev Fr Hematol 34(Suppl):S55–S59

    PubMed  CAS  Google Scholar 

  2. Zarbock A, Ley K (2009) Neutrophil adhesion and activation under flow. Microcirculation 16:31–42

    Article  PubMed  CAS  Google Scholar 

  3. Simon SI, Sarantos MR, Green CE et al (2009) Leucocyte recruitment under fluid shear: mechanical and molecular regulation within the inflammatory synapse. Clin Exp Pharmacol Physiol 36:217–224

    Article  PubMed  CAS  Google Scholar 

  4. Simon SI, Goldsmith HL (2002) Leukocyte adhesion dynamics in shear flow. Ann Biomed Eng 30:315–332

    Article  PubMed  Google Scholar 

  5. Jackson SP, Nesbitt WS, Westein E (2009) Dynamics of platelet thrombus formation. J Thromb Haemost 7(Suppl 1):17–20

    Article  PubMed  CAS  Google Scholar 

  6. Ruggeri ZM (2009) Platelet adhesion under flow. Microcirculation 16:58–83

    Article  PubMed  CAS  Google Scholar 

  7. Makino A, Shin HY, Komai Y et al (2007) Mechanotransduction in leukocyte activation: a review. Biorheology 44:221–249

    PubMed  CAS  Google Scholar 

  8. Sriramarao P, Broide DH (1996) Differential regulation of eosinophil adhesion under conditions of flow in vivo. Ann N Y Acad Sci 796:218–225

    Article  PubMed  CAS  Google Scholar 

  9. Jones DA, Smith CW, McIntire LV (1996) Leucocyte adhesion under flow conditions: principles important in tissue engineering. Biomaterials 17:337–347

    Article  PubMed  CAS  Google Scholar 

  10. Nesbitt WS, Mangin P, Salem HH et al (2006) The impact of blood rheology on the molecular and cellular events underlying arterial thrombosis. J Mol Med 84:989–995

    Article  PubMed  Google Scholar 

  11. Nesbitt WS, Westein E, Tovar-Lopez FJ et al (2009) A shear gradient-dependent platelet aggregation mechanism drives thrombus formation. Nat Med 15:665–673

    Article  PubMed  CAS  Google Scholar 

  12. Dopheide SM, Maxwell MJ, Jackson SP (2002) Shear-dependent tether formation during platelet translocation on von Willebrand factor. Blood 99:159–167

    Article  PubMed  CAS  Google Scholar 

  13. Maxwell MJ, Dopheide SM, Turner SJ et al (2006) Shear induces a unique series of morphological changes in translocating platelets: effects of morphology on translocation dynamics. Arterioscler Thromb Vasc Biol 26:663–669

    Article  PubMed  CAS  Google Scholar 

  14. Maxwell MJ, Westein E, Nesbitt WS et al (2007) Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation. Blood 109:566–576

    Article  PubMed  CAS  Google Scholar 

  15. Nesbitt WS, Kulkarni S, Giuliano S et al (2002) Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow. J Biol Chem 277:2965–2972

    Article  PubMed  CAS  Google Scholar 

  16. Nesbitt WS, Giuliano S, Kulkarni S et al (2003) Intercellular calcium communication regulates platelet aggregation and thrombus growth. J Cell Biol 160:1151–1161

    Article  PubMed  CAS  Google Scholar 

  17. Goncalves I, Nesbitt WS, Yuan Y et al (2005) Importance of temporal flow gradients and integrin alphaIIbbeta3 mechanotransduction for shear activation of platelets. J Biol Chem 280:15430–15437

    Article  PubMed  CAS  Google Scholar 

  18. Nesbitt WS, Jackson SP (2006) Imaging signaling processes in platelets. Blood Cells Mol Dis 36:139–144

    Article  PubMed  CAS  Google Scholar 

  19. Reininger AJ, Heijnen HF, Schumann H et al (2006) Mechanism of platelet adhesion to von Willebrand factor and microparticle formation under high shear stress. Blood 107:3537–3545

    Article  PubMed  CAS  Google Scholar 

  20. Molnar J, Lorand L (1961) Studies on apyrases. Arch Biochem Biophys 93:353–363

    Article  PubMed  CAS  Google Scholar 

  21. Tovar-Lopez FJ, Rosengarten G, Westein E et al (2010) A microfluidics device to monitor platelet aggregation dynamics in response to strain rate micro-gradients in flowing blood. Lab Chip 10:291–302

    Article  PubMed  CAS  Google Scholar 

  22. White F (1991) Viscous fluid flow. McGraw-Hill, New York

    Google Scholar 

  23. Truskey GA, Burmeister JS, Grapa E et al (1992) Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances. J Cell Sci 103:491–499

    PubMed  Google Scholar 

  24. Allen RD, Zacharski LR, Widirstky ST et al (1979) Transformation and motility of human platelets: details of the shape change and release reaction observed by optical and electron microscopy. J Cell Biol 83:126–142

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Nesbitt, W.S., Tovar-Lopez, F.J., Westein, E., Harper, I.S., Jackson, S.P. (2013). A Multimode-TIRFM and Microfluidic Technique to Examine Platelet Adhesion Dynamics. In: Coutts, A. (eds) Adhesion Protein Protocols. Methods in Molecular Biology, vol 1046. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-538-5_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-538-5_3

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-537-8

  • Online ISBN: 978-1-62703-538-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics