Study of methods for platelet function testing in the perspective of lab-on-chip applications

Research goals Platelets are reactive cells with the main function to maintain blood vessel integrity. Altered platelet function can lead to cardiovascular diseases such as thrombosis or bleeding disorders and platelets can also play a role in atherosclerosis. Current methods to quantify platelet function are laboratory techniques such as flow cytometry, aggregometry or luminescent assays. However, these techniques are complex and time consuming. Therefore we are interested in novel methods suited for future application in a lab-on-a-chip format. In our research we have focused on the use of well-established biomarkers, such as membrane marker expression, cytosolic calcium signaling and secretion markers, to quantify platelet activation as well as responsiveness. We report studies on platelet function using these read-out parameters and we discuss the relevance of the results for lab-on-a-chip biosensor research. Measurement of platelet membrane markers using antibody coated magnetic beads Magnetic beads are convenient carriers for rapid capture and manipulation of biological cells in a miniaturized system. We have studied the use of antibody coated magnetic beads to measure platelet function via membrane markers expressed upon activation. We used anti-P-selectin coated beads to capture activated platelets from samples stimulated with Thrombin Receptor Activator Peptide (TRAP). The responsiveness of the platelets was analyzed via the remaining unbound platelets in solution and compared to a reference method in which the number of activated platelets is analyzed via fluorescent labeling. The effective concentration for platelet responsiveness found with the bead capture assay (17.9 ± 4.9 µM) was in good agreement with the effective concentration found with the reference assay (23.5 ± 0.4 µM) in buffer. In 10% plasma the effective concentrations were 14.0 ± 4.4 µM and 13.8 ± 0.3 µM respectively, proving that platelet responsiveness can be quantified using antibody coated magnetic beads. In addition, we showed that we were able to discriminate between non-specific and specific interactions between functionalized beads and immobilized platelet with the use of magnetic actuation. The demonstrated read-out method is an interesting first step toward a lab-on-a-chip system for platelet function testing. Measurement of calcium signaling to study platelet-surface interactions Most lab-on-chip biosensor concepts are based on the use of a substrate for immobilization and subsequent detection of the biological system of interest. So for lab-on-chip platelet function testing it is important to understand the influence that a substrate can have on the platelets. We studied the interaction between platelets and various surfaces with calcium signaling in platelets. We used a calcium indicator and studied the response of individual platelets upon adhesion to Bovine Serum Albumin (BSA), Poly-L-lysine (PLL), mouse immunoglobulin (IgG), anti-GPIb and collagen coated surfaces. We recorded the fraction of cells that increase their cytosolic calcium upon binding to these surfaces. Furthermore, we recorded the delay time between the moments of platelet adhesion or chemical stimulation and the increase of cytosolic calcium. The experiments showed binding of platelets to PLL, IgG, anti-GP1b and collagen, but not to BSA coated surfaces. We found that under static conditions the number of cells that respond upon binding to an IgG coated surface (40 ± 7 % with Fc-receptor blocker; 41 ± 6 % without Fcreceptor blocker) was the lowest, followed by adhesion on a PLL coated surface (74 ± 7 %). On an anti-GPIb (88 ± 3 %) or a collagen coated surface (89 ± 4 %), the percentage of responding cells was similar. In addition, we found that the percentage of responding immobilized cells on a chemical stimulus was the lowest on anti-GPIb (7 ± 4 %) or collagen (6 ± 4 %), followed by an IgG coated surface with Fc-receptor blocker (39 ± 3 %) or without Fc-receptor blocker (35 ± 2 %). The percentage of cells responding to chemical stimulation was the highest on a BSA coated surface (95 ± 4 %). These measurements showed that there is a negative correlation between the percentage of cells that respond upon binding and that respond to chemical stimulation after binding. The delay times found upon binding ranged from 11 ± 8 s for platelets on PLL, followed by an anti-GPIb (29 ± 19 s) or collagen (24 ± 21 s) coated surface, and IgG coated surfaces with Fc-receptor blocker (62 ± 19 s) and without Fc-receptor blocker (51 ± 26 s). The delay times recorded on chemical stimulation were probably not sensitive enough to measure the physiological processes in adhered platelets. From the data we conclude that BSA is the most quiescent surface for platelet immobilization, while an anti-GPIb coated surface showed to be as thrombogenic as a collagen coated surface. Our experiments demonstrate that the analysis of the responding cells upon binding and chemical stimulation can discriminate between different types of platelet-surface interactions. We expect that the discrimination can be further improved e.g. by automated data processing. In conclusion, we find that the measurement of the responding fraction and the response delay time by calcium signaling are convenient methods to study the time-dependent interaction of platelets with surfaces relevant for lab-on-chip applications. Measurement of exocytosis to study platelet-surface interactions In our second approach to investigate the interaction between platelets and surfaces, we have studied the secretion process of dense granules of a cell ensemble, as well as single cell exocytosis. Adenosine triphosphate (ATP) secretion was quantified by the luminescent luciferin/luciferase reaction. Platelets were allowed to interact with BSA, PLL, mouse IgG and anti-GPIb coated surfaces, and after incubation the amount of secreted ATP was analyzed. The baseline signal was given by ATP secretion from resting platelets in suspension. ATP levels secreted from platelets immobilized on BSA were approximately 2 times as high as the baseline. The immobilization of platelets on PLL showed about 4 times baseline ATP concentrations. On IgG as well as anti-GPIb the maximum amount of ATP was secreted after 1 hour of platelet incubation. Again, we can conclude that the most quiescent surface for platelet immobilization is BSA, followed by PLL, mouse IgG and anti-GPIb. We have also studied the immobilization of the enzyme luciferase on a surface, in order to enable the detection of ATP direct at a surface. We have demonstrated that luciferase adsorbed onto PLL is indeed active and generates luminescence in the presence of ATP. However, the assay still has a low sensitivity and needs further optimization. Finally, we have investigated an exploratory method to resolve exocytosis in single platelet cells, using the fluorescent staining of the dense granules by the quinacrine dye. In our experiments we have observed that the exocytosis events are induced by the fluorescence excitation light. Very probably this is caused by the production of reactive oxygen, which disintegrates the vesicle membrane thereby releasing the vesicle content. Further studies may focus on the use of lower quinacrine concentrations, the use of lower light intensities, or on ways to scavenge reactive oxygen. It will be interesting to further develop the exocytosis assay, as a quantitative method for research on biosensor assays and surfaces suitable for biosensor applications. Conclusions and outlook The scope of the research presented in this thesis was to develop knowledge to support future technological developments, aiming at the measurement of platelet activation or responsiveness in a lab-on-chip device. We found that magnetic particles functionalized with specific antibodies can be used to measure platelet responsiveness. We have also studied the interaction of platelet with different surfaces, by an intracellular calcium signaling assay and by an ATP exocytosis assay using luciferin/luciferase. The most quiescent surface for platelets was BSA, but this surface has a low binding affinity for platelets. Anti-GPIb and collagen coated surfaces show stronger binding, but these surfaces change the activation status of the platelets. In view of these results, we have proposed a new concept to measure platelet function in a lab-on- chip device, namely by labelling platelet activation markers prior to platelet immobilization on a surface. We envision that this design will enable the sensitive labelling of platelets as well as the accurate readout of the platelets at a detection surface in the lab-on-chip device.