A shear gradient–dependent platelet aggregation mechanism drives thrombus formation

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.

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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.

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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.

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Authors

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)

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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

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