Abstract
In a focal injury model, platelets adhere and activate under flow on a collagen-coated surface, creating a field of individual platelet aggregates. These aggregates exhibit distinct structural characteristics that are linked to the local flow conditions. By combining image analysis techniques and epifluorescence microscopy, we developed a robust strategy for quantifying the characteristic instantaneous width and length of a growing platelet deposit. We have confirmed the technique using model images consisting of ellipsoid objects and quantified the shear rate-dependent nature of aggregate morphology. Venous wall shear rate conditions (100 s−1) generated small, circular platelet deposits, whereas elevated arterial shear rates (500 and 1000 s−1) generated platelet masses elongated twofold in the direction of flow. At 2000 s−1, an important regime for von Willebrand Factor (vWF)-mediated recruitment, we observed sporadic platelet capture events on collagen that led to rapidly growing deposits. Furthermore, inter-donor differences were investigated with respect to aggregate growth rate. After perfusion at elevated shear rates (1000 s−1) for 5 min, we identified a twofold increase in aggregate size (81.5 ± 24.6 μm; p < 0.1) and a threefold increase in growth rate parallel to the flow (0.40 ± 0.09 μm/s; p < 0.01) for an individual donor. Suspecting a role for vWF, we found that this donor had a twofold increase in soluble vWF relative to the other donors and pooled plasma. Microfluidic devices in combination with automated morphology analysis offer new tools for characterizing clot development under flow.
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Duffy, D. C., J. C. McDonald, O. J. A. Schueller, and G. M. Whitesides. Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Anal. Chem. 70:4974–4984, 1998.
Folie, B. J., and L. V. McIntire. Mathematical analysis of mural thrombogenesis. Biophys. J. 56:1121–1141, 1989.
Gill, J. C., J. Endres-Brooks, P. J. Bauer, W. J. Marks, Jr., and R. R. Montgomery. The effect of ABO blood group on the diagnosis of von Willebrand disease. Blood 69:1691–1695, 1987.
Goldsmith, H. L., and V. T. Turitto. Rheological aspects of thrombosis and haemostasis: basic principles and applications. Thromb. Haemost. 55:415–435, 1986.
Hubbell, J. A., and L. V. McIntire. Technique for visualization and analysis of mural thrombogenesis. Rev. Sci. Instrum. 57:892–897, 1986.
Hubbell, J. A., and L. V. McIntire. Visualization and analysis of mural thrombogenesis on collagen, polyurethane, and nylon. Biomaterials 7:354–363, 1986.
Hubbell, J. A., and L. V. McIntire. Platelet active concentration profiles near growing aggregates. Biophys. J. 50:937–945, 1986.
Jackson, S. P., W. S. Nesbitt, and S. Kulkarni. Signaling events underlying aggregate formation. Thromb. Haemost. 1:1602–1612, 2003.
Kroll, M. H., J. D. Hellums, L. V. McIntire, A. I. Schafer, and J. L. Moake. Platelets and shear stress. Blood 88:1525–1541, 1996.
Kulkarni, S., S. M. Dopheide, C. L. Yap, C. Ravanat, M. Freund, P. Mangin, K. A. Heel, A. Street, I. S. Harper, F. Lanza, and S. P. Jackson. A revised model of platelet aggregation. J. Clin. Invest. 105:783–791, 2000.
Matsui, H., M. Sugimoto, S. Tsuji, S. Miyata, M. Matsuda, and A. Yoshioka. Distinct and concerted functions of von Willebrand factor and fibrinogen in mural aggregate growth under high shear flow. Blood 100:3604–3610, 2002.
Moake, J. L., N. A. Turner, N. A. Stathopoulos, L. Nolasco, and J. D. Hellums. Shear-induced platelet aggregation can be mediated by vWF released from platelets, as well as by exogenous large or unusually large vWF multimers, requires adenosine diphosphate, and is resistant to aspirin. Blood 71:1366–1374, 1988.
Neeves, K. B., and S. L. Diamond. A membrane-based microfluidic device for controlling the flux of platelet agonists into flowing blood. Lab Chip 8:701–709, 2008.
Neeves, K. B., S. F. Maloney, K. P. Fong, A. A. Schmaier, M. L. Kahn, L. F. Brass, and S. L. Diamond. Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear-resistance of platelet aggregates. Thromb. Haemost. 6:2193–2201, 2008.
O’Donnell, J., and M. A. Laffan. The relationship between ABO histo-blood group, factor VIII and von Willebrand factor. Transfus. Med. 11:343–351, 2001.
Ruggeri, Z. M. The role of von Willebrand factor in aggregate formation. Thromb. Res. 120:S5–S9, 2007.
Ruggeri, Z. M., J. A. Dent, and E. Saldivar. Contribution of distinct adhesive interactions to platelet adhesion under flow. Blood 94:172–178, 1999.
Savage, B., M. H. Ginsberg, and Z. M. Ruggeri. Influence of fibrillar collagen structure on the mechanisms of platelet aggregate formation under flow. Blood 94:2704–2715, 1999.
Savage, B., S. J. Shattil, and Z. M. Ruggeri. Modulation of platelet function through adhesion receptors. Circulation 86:11300–11306, 1992.
Shankaran, H., P. Alexandridis, and S. Neelamegham. Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension. Blood 101:2637–2645, 2003.
Shattil, S. J., and P. J. Newman. Integrins: dynamic scaffolds for adhesion and signaling in platelets. Blood 104:1606–1615, 2004.
Skipwith, C. G., W. Cao, and X. L. Zheng. Factor VIII and platelets synergistically accelerate cleavage of von Willebrand factor by ADAMTS13 under fluid shear stress. J. Biol. Chem. 285:28596–28603, 2010.
Sokal, R. R. Spatial autocorrelation in biology. Biol. J. Linn. Soc. 10:199–228, 1978.
Sternberg, S. R. Biomedical image processing. Computer 16:22–34, 1983.
Tsuji, S., M. Sugimoto, S. Miyata, M. Kuwahara, S. Kinoshita, and A. Yoshioka. Real-time analysis of mural aggregates formation in various platelet aggregation disorders: distinct shear-dependent roles of platelet receptors and adhesive proteins under flow. Blood 94:968–975, 1999.
Yoo, J. C., and T. H. Han. Fast normalized cross-correlation. Circ. Syst. Signal Process. 28:819–843, 2009.
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Associate Editor Konstantinos Konstantopoulos oversaw the review of this article.
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Colace, T., Falls, E., Zheng, X.L. et al. Analysis of Morphology of Platelet Aggregates Formed on Collagen Under Laminar Blood Flow. Ann Biomed Eng 39, 922–929 (2011). https://doi.org/10.1007/s10439-010-0182-4
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DOI: https://doi.org/10.1007/s10439-010-0182-4