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

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.