Pairwise Agonist Screening and Microfluidics Reveal Platelet Function Phenotype

Pairwise agonist screening and microfluidics reveal platelet function phenotype

T. Colace, M. Chatterjee, and S.L. Diamond

In this study we consider the relationship between a high-throughput approach designed to investigate platelet activation via calcium mobilization and a microfluidic model used to study platelet function. Chatterjee et al. (2010) have demonstrated that calcium responses to a variety of platelet agonists can serve as a platelet activation phenotype in an assay called Pairwise Agonist Scanning (PAS), but did not illustrate a connection to platelet function. Using PAS data collected for 3 healthy donors we attempted to predict changes in platelet function in the presence of the anti-platelet agent Indomethacin, a non-selective COX inhibitor, and to confirm them in a microfluidic model of platelet aggregation on a collagen surface. The combination of these techniques represents a link between the complex and unique activating environment experienced by a platelet under flow conditions and the traditional methods of measuring platelet activation in a static, well mixed setting.

Whole blood, anticoagulated with PPACK (100 μM), was treated with Indomethacin (140 μM), Iloprost (a stable prostacyclin analog that globally inhibits platelet activation, 1 μM), both agents together, or was untreated (control). Each condition was loaded in duplicate into a separate inlet of an 8 channel microfluidic device with a single outlet [Figure 1A]. The samples were perfused over a fibrillar collagen surface at a shear rate of 200 s^-1 for 8 min [Figure 1B]. Platelet accumulation at the collagen strip was measured in each channel simultaneously in 15 sec intervals. To perform the PAS study platelet rich plasma from the same blood draw was loaded with a calcium sensitive dye and Indomethacin (to prevent autocrine activation via TXA₂). Samples were treated with all pairwise combinations of 4 concentrations (zero, low, medium, high) of the platelet agonists convulxin (a collagen mimetic), ADP, and U46619 (a stable TXA₂ mimetic), in the presence or absence of Iloprost. Platelet calcium mobilization was measured via fluorescence for 200 sec.

The platelet collagen response was recorded for 4 trials per donor on two separate days, with corresponding PAS data. While Iloprost was effective for all donors (mean aggregation inhibition of 70%), Indomethacin showed effectiveness only for donors 1 and 2 (35% and 20%, respectively), and caused a significant increase in platelet accumulation for donor 3 (20%) [Figure 1C]. Platelet calcium mobilization in response to U46619 alone illustrated a reduced sensitivity to the TXA₂ analog for donor 2 as compared to donor 1 (~44% reduction in Ca²⁺ mobilization) and for donor 3 as compared to donor 2 (~80% reduction) [Figure 1D]. In order to confirm an activating role for high doses of Indomethacin, the study was repeated at 14 μM Indomethacin. At this concentration donor 2 showed a 34% reduction in platelet adhesion while donor 3 showed at 13% reduction (donor 1 has yet to be investigated). PAS data was not affected by the change in Indomethacin concentration. Furthermore, we have observed a 2-fold increase in platelet aggregation for donor 1 as compared to donors 2 and 3. We believe this result is reflective of a universal increase in Ca²⁺ mobilization as measured under all conditions in the PAS output.

These data reveal that donor sensitivity to anti-platelet agents can be predicted using PAS and confirmed in a microfluidic model of platelet adhesion. The combination of these techniques bridges the gap between traditional measures of platelet activation (Ca²⁺ mobilization) and studies of platelet function under physiologic flow conditions. We believe these methods hold promise as a novel diagnostic tool for evaluating platelet function and efficacy of anti-platelet therapy.  

Figure 1 (A) Schematic of the 8 channel microfluidic device. 8 separate channels converge on one patterned collagen strip (blue) that can be imaged in a 2x field of view. (B) Whole blood (PPACK 100 μM), treated with the indicated anti-platelet agents, is perfused at 200 s-1 and platelet accumulation on the collagen surface is measured in 15 sec intervals.  (C) Normalized platelet accumulation for three separate donors (n=16). Iloprost shows effectiveness for all 3 donors while Indomethacin shows effectiveness for donors 1 and 2 and an activating effect for donor 3.  (* p < .01) (D) Calcium responses to 4 doses of U46619 (green:zero, pink:low, red:medium, blue:high) are presented for 3 donors in duplicate.