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

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.

Rupture Forces among Human Blood Platelets at different Degrees of Activation

“…Platelet-surface interactions have been quantified in real-time using calcium mobilization assays, showing that the lag time until platelets activate depends on the physico-chemical properties of the surface 27 . It has been shown that platelets are activated nearly instantaneously upon coming in contact with immobilized fibrinogen and completing their contraction after 15 min 28 .…”

Microfludic platforms for the evaluation of anti-platelet agent efficacy under hyper-shear conditions associated with ventricular assist devices

“…A fluorescence-based approach could be utilized, such as monitoring of platelet Ca 2+ influx by using Ca 2+ fluorescent probe incorporated in the platelet sample and performing fluorescence acquisition on chip using fluorescence microscopy. Similar approaches have been utilized in previous studies [55–57] and allowed to discriminate different levels of platelet response. Fluorescence-based assays are one of the main approaches for microfluidic on chip tests as they allow for targeting specific molecules or biological products and due to their non-destructive characteristic, which allows acquisitions at different time points of the experimental procedure.…”

Differences in intracellular calcium dynamics cause differences in α-granule secretion and phosphatidylserine expression in platelets adhering on glass and TiO2

“…Others have studied intracellular calcium dynamics in adhering platelets. [53][54][55][56][57][58] Ikeda et al observed a sustained increase in the intracellular calcium level on poly(lysine)and fibrinogen coated surfaces but not on glass, where a peak followed by a drop to a level somewhat above the baseline was reported. 54 Those authors used the same calcium dye as we did, but did not control for the changes in the intensity due to platelet evolution with another dye.…”