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.

A shear gradient–dependent platelet aggregation mechanism drives thrombus formation

Nature Medicine volume 15, pages 665–673 (2009)Cite this article

Abstract

Platelet aggregation at sites of vascular injury is essential for hemostasis and arterial thrombosis. It has long been assumed that platelet aggregation and thrombus growth are initiated by soluble agonists generated at sites of vascular injury. By using high-resolution intravital imaging techniques and hydrodynamic analyses, we show that platelet aggregation is primarily driven by changes in blood flow parameters (rheology), with soluble agonists having a secondary role, stabilizing formed aggregates. We find that in response to vascular injury, thrombi initially develop through the progressive stabilization of discoid platelet aggregates. Analysis of blood flow dynamics revealed that discoid platelets preferentially adhere in low-shear zones at the downstream face of forming thrombi, with stabilization of aggregates dependent on the dynamic restructuring of membrane tethers. These findings provide insight into the prothrombotic effects of disturbed blood flow parameters and suggest a fundamental reinterpretation of the mechanisms driving platelet aggregation and thrombus growth.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Shear microgradients promote platelet aggregate formation in vivo.
Figure 2: Platelet aggregation induced shear microgradients occurs independently of ADP, TXA2 and thrombin.
Figure 3: Stabilized discoid platelet aggregation is a general feature of thrombus development.
Figure 4: The magnitude and spatial distribution of the shear microgradient directly affects platelet aggregate size.
Figure 5: Stabilized discoid platelet aggregation occurs via restructuring of membrane tethers.
Figure 6: Working model of shear microgradient platelet aggregation.

References

  1. Trip, M.D., Cats, V.M., van Capelle, F.J. & Vreeken, J. Platelet hyperreactivity and prognosis in survivors of myocardial infarction. N. Engl. J. Med. 322, 1549–1554 (1990).

    Article  CAS  Google Scholar 

  2. Ruggeri, Z.M. Platelets in atherothrombosis. Nat. Med. 8, 1227–1234 (2002).

    Article  CAS  Google Scholar 

  3. Bhatt, D.L. & Topol, E.J. Scientific and therapeutic advances in antiplatelet therapy. Nat. Rev. Drug Discov. 2, 15–28 (2003).

    Article  CAS  Google Scholar 

  4. Virchow, R. Gesammelte abhandlungen zur wissenschaftlichen medicin. Medsinger Sohn & Co. 219–732 (1856).

  5. Savage, B., Saldivar, E. & Ruggeri, Z.M. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 84, 289–297 (1996).

    Article  CAS  Google Scholar 

  6. Savage, B., Almus-Jacobs, F. & Ruggeri, Z.M. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 94, 657–666 (1998).

    Article  CAS  Google Scholar 

  7. Nesbitt, W.S. et al. Distinct glycoprotein Ib/V/IX and integrin αIIbβ3-dependent calcium signals cooperatively regulate platelet adhesion under flow. J. Biol. Chem. 277, 2965–2972 (2002).

    Article  CAS  Google Scholar 

  8. Nesbitt, W.S. et al. Intercellular calcium communication regulates platelet aggregation and thrombus growth. J. Cell Biol. 160, 1151–1161 (2003).

    Article  CAS  PubMed Central  Google Scholar 

  9. Giuliano, S., Nesbitt, W.S., Rooney, M. & Jackson, S.P. Bidirectional integrin αIIbβ3 signalling regulating platelet adhesion under flow: contribution of protein kinase C. Biochem. J. 372, 163–172 (2003).

    Article  CAS  PubMed Central  Google Scholar 

  10. Goncalves, I., Nesbitt, W.S., Yuan, Y. & Jackson, S.P. Importance of temporal flow gradients and integrin αIIbβ3 mechanotransduction for shear activation of platelets. J. Biol. Chem. 280, 15430–15437 (2005).

    Article  CAS  Google Scholar 

  11. Ruggeri, Z.M. & Mendolicchio, G.L. Adhesion mechanisms in platelet function. Circ. Res. 100, 1673–1685 (2007).

    Article  CAS  Google Scholar 

  12. Ruggeri, Z.M. The role of von Willebrand factor in thrombus formation. Thromb. Res. 120 Suppl 1, S5–S9 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  13. Jackson, S.P. The growing complexity of platelet aggregation. Blood 109, 5087–5095 (2007).

    Article  CAS  Google Scholar 

  14. Dubois, C., Panicot-Dubois, L., Gainor, J.F., Furie, B.C. & Furie, B. Thrombin-initiated platelet activation in vivo is vWF independent during thrombus formation in a laser injury model. J. Clin. Invest. 117, 953–960 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  15. van Gestel, M.A. et al. Real-time detection of activation patterns in individual platelets during thromboembolism in vivo: differences between thrombus growth and embolus formation. J. Vasc. Res. 39, 534–543 (2002).

    Article  Google Scholar 

  16. Maxwell, M.J. et al. Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation. Blood 109, 566–576 (2007).

    Article  CAS  Google Scholar 

  17. Furie, B. & Furie, B.C. Thrombus formation in vivo. J. Clin. Invest. 115, 3355–3362 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  18. Ruggeri, Z.M., Orje, J.N., Habermann, R., Federici, A.B. & Reininger, A.J. Activation-independent platelet adhesion and aggregation under elevated shear stress. Blood 108, 1903–1910 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  19. Mustard, J.F., Jorgensen, L., Hovig, T., Glynn, M.F. & Rowsell, H.C. Role of platelets in thrombosis. Thromb. Diath. Haemorrh. Suppl. 21, 131–158 (1966).

    CAS  PubMed  Google Scholar 

  20. Mustard, J.F., Murphy, E.A., Rowsell, H.C. & Downie, H.G. Factors influencing thrombus formation in vivo. Am. J. Med. 33, 621–647 (1962).

    Article  CAS  Google Scholar 

  21. Dopheide, S.M., Maxwell, M.J. & Jackson, S.P. Shear-dependent tether formation during platelet translocation on von Willebrand factor. Blood 99, 159–167 (2002).

    Article  CAS  Google Scholar 

  22. Antithrombotic Trialists' Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction and stroke in high risk patients. Br. Med. J. 324, 71–86 (2002).

  23. Dintenfass, L. Rheologic approach to thrombosis and atherosclerosis. Angiology 15, 333–343 (1964).

    Article  CAS  Google Scholar 

  24. Stein, P.D. & Sabbah, H.N. Measured turbulence and its effect on thrombus formation. Circ. Res. 35, 608–614 (1974).

    Article  CAS  Google Scholar 

  25. Yoganathan, A.P., Corcoran, W.H., Harrison, E.C. & Carl, J.R. The Bjork-Shiley aortic prosthesis: flow characteristics, thrombus formation and tissue overgrowth. Circulation 58, 70–76 (1978).

    Article  CAS  Google Scholar 

  26. Schoephoerster, R.T., Oynes, F., Nunez, G., Kapadvanjwala, M. & Dewanjee, M.K. Effects of local geometry and fluid dynamics on regional platelet deposition on artificial surfaces. Arterioscler. Thromb. 13, 1806–1813 (1993).

    Article  CAS  Google Scholar 

  27. Eckstein, E.C., Tilles, A.W. & Millero, F.J. III. Conditions for the occurrence of large near-wall excesses of small particles during blood flow. Microvasc. Res. 36, 31–39 (1988).

    Article  CAS  Google Scholar 

  28. Wang, S.K. & Hwang, N.H. On transport of suspended particulates in tube flow. Biorheology 29, 353–377 (1992).

    Article  CAS  Google Scholar 

  29. Kulkarni, S. et al. A revised model of platelet aggregation. J. Clin. Invest. 105, 783–791 (2000).

    Article  CAS  PubMed Central  Google Scholar 

  30. Schoenwaelder, S.M. et al. Identification of a unique co-operative phosphoinositide 3-kinase signaling mechanism regulating integrin α3 adhesive function in platelets. J. Biol. Chem. 282, 28648–28658 (2007).

    Article  CAS  Google Scholar 

  31. Denis, C. et al. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis. Proc. Natl. Acad. Sci. USA 95, 9524–9529 (1998).

    Article  CAS  Google Scholar 

  32. Kulkarni, S. et al. Techniques to examine platelet adhesive interactions under flow. Methods Mol. Biol. 272, 165–186 (2004).

    CAS  PubMed  Google Scholar 

  33. Léon, C. et al. Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y(1) receptor-null mice. J. Clin. Invest. 104, 1731–1737 (1999).

    Article  PubMed Central  Google Scholar 

  34. Mayadas, T.N., Johnson, R.C., Rayburn, H., Hynes, R.O. & Wagner, D.D. Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice. Cell 74, 541–554 (1993).

    Article  CAS  Google Scholar 

  35. Rosen, E.D. et al. Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi. Am. J. Pathol. 158, 1613–1622 (2001).

    Article  CAS  PubMed Central  Google Scholar 

  36. Kurz, K.D., Main, B.W. & Sandusky, G.E. Rat model of arterial thrombosis induced by ferric chloride. Thromb. Res. 60, 269–280 (1990).

    Article  CAS  Google Scholar 

  37. Sturgeon, S.A., Jones, C., Angus, J.A. & Wright, C.E. Advantages of a selective β-isoform phosphoinositide 3-kinase antagonist, an anti-thrombotic agent devoid of other cardiovascular actions in the rat. Eur. J. Pharmacol. 587, 209–215 (2008).

    Article  CAS  Google Scholar 

  38. Mangin, P. et al. Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRgamma deficiency. Blood 107, 4346–4353 (2006).

    Article  CAS  Google Scholar 

  39. Weibel, D.B., Diluzio, W.R. & Whitesides, G.M. Microfabrication meets microbiology. Nat. Rev. Microbiol. 5, 209–218 (2007).

    Article  CAS  Google Scholar 

  40. Fouras, A., Lo Jacono, D. & Hourigan, K. Target-free stereo PIV: a novel technique with inherent error estimation and improved accuracy. Exp. Fluids 44, 317–329 (2008).

    Article  Google Scholar 

  41. Fouras, A.S.J. Accuaracy of out-of-plane vorticity measurements derived from in-plane velocity field data. Exp. Fluids 25, 409–430 (1998).

    Article  Google Scholar 

  42. Edmondson, K.E., Denney, W.S. & Diamond, S.L. Neutrophil-bead collision assay: pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime. Biophys. J. 89, 3603–3614 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  43. Yap, C.L. et al. Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin alpha IIbbeta 3. Studies in human platelets and transfected Chinese hamster ovary cells. J. Biol. Chem. 275, 41377–41388 (2000).

    Article  CAS  Google Scholar 

  44. Versteeg, H. & Malalasekrera, W. An Introduction to Computational Fluid Dynamics: The Finite Volume Method (Longman Scientific & Technical, Harlow, UK, 1995).

    Google Scholar 

Download references

Acknowledgements

We thank Z. Ruggeri, H.H. Salem, R. Andrews and B. Kile for helpful feedback on the work; H. Blackburn and G. Rosengarten for advice and input on the CFD analysis; C. Nguyen for technical assistance in the analysis of in vivo blood flow rates; SciTech Proprietary Ltd. and Andor Proprietary Ltd. for the generous loan of a DV897CS EMCCD camera; M. Hickey (Monash University Department of Medicine, Monash, Australia) and A. Issekutz (Dalhousie University, Halifax, Canada) for providing the P-selectin–specific antibody; and C. Gachet (INSERM, Strasbourg, France) for P2Y1−/− mice. This work was supported by project funding from the National Health and Medical Research Council of Australia and the Australian Research Council. E.W. was supported by the National Heart Foundation of Australia.

Author information

Author notes

  1. Warwick S Nesbitt and Erik Westein: These authors contributed equally to this work.

Authors and Affiliations

  1. The Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Educational Precinct, Melbourne, Victoria, Australia

    Warwick S Nesbitt, Erik Westein, Jia Fu & Shaun P Jackson

  2. Microelectronics and Materials Technology Centre, School of Electrical and Computer Engineering, RMIT University, Melbourne, Victoria, Australia

    Francisco Javier Tovar-Lopez & Arnan Mitchell

  3. Department of Mechanical Engineering, Monash University, Clayton, Victoria, Australia

    Elham Tolouei & Josie Carberry

  4. Division of Biological Engineering, Monash University, Clayton, Victoria, Australia

    Andreas Fouras

Authors
  1. Warwick S Nesbitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Erik Westein
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Francisco Javier Tovar-Lopez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elham Tolouei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Arnan Mitchell
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jia Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Josie Carberry
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Andreas Fouras
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shaun P Jackson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.S.N. designed the study and experiments, performed the in vitro platelet and micro-PIV experiments, codesigned vascular mimetics, performed microchannel perfusion experiments, analyzed data, supervised the study and cowrote the manuscript. E.W. designed experiments, performed the in vivo and in vitro experiments, analyzed data and assisted with manuscript preparation. F.J.T.-L. codesigned and fabricated vascular mimetics (microchannels), performed microchannel perfusion experiments and performed CFD simulations. E.T. performed and analyzed the micro-PIV experiments. J.F. performed in vivo experiments. A.M. codesigned the vascular mimetics and supervised their fabrication. J.C. supervised the micro-PIV analysis. A.F. designed and supervised the micro-PIV analysis and formulated and coded the micro-PIV analysis software. S.P.J. supervised the study and cowrote the manuscript.

Corresponding authors

Correspondence to Warwick S Nesbitt or Shaun P Jackson.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–6 and Supplementary Methods (PDF 975 kb)

Supplementary Video 1

Rheology-driven platelet aggregation in vivo. (MOV 2808 kb)

Supplementary Video 2

Shear microgradient-induced platelet aggregation in vitro. (MOV 2976 kb)

Supplementary Video 3

Discoid aggregate formation independent of ADP, TXA2 and thrombin. (MOV 3197 kb)

Supplementary Video 4

Discoid platelet aggregation and aggregate consolidation in vivo. (MOV 3433 kb)

Supplementary Video 5

Platelet tethering interactions in vivo and in vitro. (MOV 2814 kb)

Supplementary Video 6

Intracellular calcium flux during platelet tether restructuring. (MOV 3110 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Nesbitt, W., Westein, E., Tovar-Lopez, F. et al. A shear gradient–dependent platelet aggregation mechanism drives thrombus formation. Nat Med 15, 665–673 (2009). https://doi.org/10.1038/nm.1955

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.1955

This article is cited by

Search

Advanced 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