The agonists in this assay were chosen as representative of the major signaling cues a platelet will encounter during a thrombotic event. They are also the same agonists used in the calcium assay previously developed by our lab to allow for direct comparison of results (2). Convulxin (CVX; Centerchem, Norwalk, CT) is a rattlesnake venom protein that directly binds and activates glycoprotein VI (GPVI), the primary collagen signaling receptor on platelets (16). This is used because soluble monomeric collagen only binds to the integrin α2β1, which is an adhesive receptor and has little direct effect on signaling (17), and fibrillar collagen is not soluble, making it unsuitable for use in flow cytometry (18). Thrombin acts on the two Gq coupled protease activated receptors (PARs) in humans, PAR1 and PAR4, which signal differentially (19, 20). As such, we used the individual PAR agonist peptides, SFLLRN and AYPGKF (Bachem, King of Prussia, PA), to investigate PAR1 and PAR4 signaling individually. This also removes the need for inhibitors of fibrin polymerization such as Gly-Pro-Arg-Pro, which would be required in the presence of thrombin to maintain sample viscosity. The use of U46619 (Sigma-Aldrich, St. Louis, MO) in place of the physiological thromboxane A2 (TXA2) was required due to the short (∼30 s) half-life of TXA2 in solution (21). Similarly, prostacyclin is a very short-lived molecule, so the more chemically stable prostaglandin E2 (PGE2; Sigma-Aldrich, St. Louis, MO) was chosen. In both cases, the more stable analog signals through the same receptor as the physiological ligand (8, 22). The only physiologic agonist that could be directly utilized in this assay was adenosine diphosphate (ADP; Sigma-Aldrich, St. Louis, MO).96 well plate preparation
Each sample well of a white, flat-bottomed 96 well plate (Corning, Corning, NY) was loaded with 10 µL 10% v/v PRP, as well as 2 µL each FITC PAC-1, PE anti-CD62P (AK-4 clone), and Cy5 annexin V (BD Biosciences, San Jose, CA) as shown in Figure 1. In addition, 64 µL HBS was added to wells that would receive a pair of agonists, while 74 µL was added to wells for single agonist controls. Ten minutes prior to flow cytometry analysis, 10 µL of a 10× stock of the appropriate agonist was added, giving a final volume of 100 µL in each well. This gives a final concentration of 1% v/v PRP, which eliminates autocrine and paracrine signaling that could affect responses in a platelet concentration dependent manner (23).
This assay utilized an Accuri C6 flow cytometer with CSampler (BD Biosciences, San Jose, CA) to automate well plate handling. The sample flow rate was set to low (14 µL/min with a 10 µm core), and samples were analyzed for 60 s following 10 min of incubation with agonist(s). The time required for movement of the CSampler arm and suction of each sample into the flow cytometer meant it was possible to analyze one sample every two minutes. Compensation was set such that 7.5% of FL1 was subtracted from FL2 and 4.0% FL2 from FL1 to account for emission spectra overlap between FITC and PE.Results and discussion Determining the dynamic range of each agonist
In order to determine the concentration range over which each of the six agonists affects integrin activation, degranulation, and PS exposure, each one was tested individually. No single agonist led to significant PS exposure, so EC50s could not be calculated for that response. Sigmoidal dose-response curves were constructed based on the mean fluorescence of PAC-1 and anti-CD62P binding at each dose. The inhibitory effects of PGE2 were studied by simultaneous stimulation with 15 µM SFLLRN. This allowed us to define an EC50 for each agonist for both integrin activation and degranulation (Table 1). Addition of U46619 did not elicit an increase in either signal, but it does exhibit signaling in the pairwise conditions. The EC50 concentrations of each agonist calculated for PAC-1 and anti-CD62P binding are within a factor of three of those seen in the previous calcium assay (2). Therefore, we chose to use the concentrations from the calcium assay so that data from both assays would be directly comparable.
le> Pairwise agonist combinations and donor specificity
Each of the 15 pairwise combinations of the 6 agonists at their EC50 concentrations was tested. For each sample, the mean fluorescence of PAC-1 and anti-CD62P binding was determined, as well as the percentage of PS exposing platelets. To determine whether the donor specificity seen in the pairwise calcium responses was maintained through the signaling cascades to integrin activation, degranulation, and PS exposure, PAS-FC was carried out on blood draws no more than 2 weeks apart for each of 10 healthy male donors. The 45 data points from each experiment (15 agonist pairs × 3 colors for αIIbβ3 activation, P-selectin display, and PS exposure) were converted into a vector that became a column in the pairwise response matrix (Figure 2). Donor specificity was determined by generating a hierarchical cluster tree based on the Euclidean distance between each possible pair of vectors as calculated by the linkage function in MATLAB. Of the 10 donors, 4 exhibited self-clustering, where their 2 vectors clustered together, indicating the possibility of donor specificity. To determine the significance of 4 out of 10 donors self-clustering, in silico cohorts of 10 hypothetical donors were constructed by randomly choosing values from the 10 original donors for each of the 45 data points. Simulating 10 million such cohorts showed that 4 self-clustering donors is highly significant (P < 0.001), and no cohorts with 8 or more self-clustering donors ever occurred.