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Quantification of platelet-surface interactions in real-time using intracellular calcium signaling

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Abstract

Platelets get easily activated when in contact with a surface. Therefore in the design of microfluidic blood analysis devices surface activation effects have to be taken into account. So far, platelet-surface interactions have been quantified by morphology changes, membrane marker expression or secretion marker release. In this paper we present a simple and effective method that allows quantification of platelet-surface interactions in real-time. A calcium indicator was used to visualize intracellular calcium variations during platelet adhesion. We designated cells that showed a significant increase in cytosolic calcium as responding cells. The fraction of responding cells upon binding was analyzed for different types of surfaces. Thereafter, the immobilized platelets were chemically stimulated and the fraction of responding cells was analyzed. Furthermore, the time between the binding or chemical stimulation and the increased cytosolic calcium level (i.e. the response delay time) was measured. We used surface coatings relevant for platelet-function testing including Poly-L-lysine (PLL), anti-GPIb and collagen as well as control coatings such as Bovine Serum Albumin (BSA) and mouse immunoglobulin (IgG). We found that a lower percentage of responding cells upon binding, results in a higher percentage of responding cells upon chemical stimulation after binding. The measured delay time between platelet binding under sedimentation and calcium response was the lowest on a PLL-coated surface, followed by an anti-GPIb and collagen-coated surface and IgG-coated surface. The presented method provides real-time information of platelet-surface interactions on a single cell as well as on a cell ensemble level. For future in-vitro diagnostic tests, this real-time single-cell function analysis can reveal heterogeneities in the biological processes of a cell population.

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Acknowledgments

This research was performed within the framework of CTMM, the Center for Translational Molecular Medicine (www.ctmm.nl), project CIRCULATING CELLS (grant 01C-102), and supported by the Dutch Heart Foundation.

Conflict of interest

M.W.J. Prins is an employee of Philips Research (Eindhoven, the Netherlands)

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Authors and Affiliations

  1. Department of Applied Physics, Eindhoven University of Technology, Eindhoven, The Netherlands

    H. M. van Zijp, J. Riegman, J. M. H. Goudsmits, A. M. de Jong, H. Kress & M. W. J. Prins

  2. Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands

    A. D. Barendrecht

  3. Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands

    J. M. H. Goudsmits

  4. Experimental Physics I, University of Bayreuth, Bayreuth, Germany

    H. Kress

  5. Philips Research Laboratories, Eindhoven, The Netherlands

    M. W. J. Prins

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  1. H. M. van Zijp
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  3. J. Riegman
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Correspondence to M. W. J. Prins.

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Fig. S1

The relation between two different methods to quantify the surface induced activation of platelets. On the horizontal axis the relative amount of secreted ATP from platelets immobilized at different types of surfaces is presented. The ATP concentration was quantified with the use of the luminescent luciferin/luciferase reaction. Each value is the average of three samples; the error bars represent the standard deviation. The percentage of immobilized platelets showing a calcium response upon chemical stimulation (chem. stim.) with TRAP is plotted on the vertical axis. This percentage was quantified by the measurement of the fluorescence intensity of the platelets that were loaded with a calcium indicator as described in section 2.3. Since both the ATP secretion and TRAP stimulation introduce irreversible activation in platelets, it is expected that there is an inverse relation between ATP release and response on a TRAP-stimulation. FcRbl = Fc-receptor blocker, aGPIb = anti-GPIb (JPEG 175 kb)

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van Zijp, H.M., Barendrecht, A.D., Riegman, J. et al. Quantification of platelet-surface interactions in real-time using intracellular calcium signaling. Biomed Microdevices 16, 217–227 (2014). https://doi.org/10.1007/s10544-013-9825-1

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