A Human Platelet Calcium Calculator Trained by Pairwise Agonist Scanning

Since platelet intracellular calcium mobilization [Ca(t)](i) controls granule release, cyclooxygenase-1 and integrin activation, and phosphatidylserine exposure, blood clotting simulations require prediction of platelet [Ca(t)](i) in response to combinatorial agonists. Pairwise Agonist Scanning (PAS) deployed all single and pairwise combinations of six agonists (ADP, convulxin, thrombin, U46619, iloprost and GSNO used at 0.1, 1, and 10xEC(50); 154 conditions including a null condition) to stimulate platelet P(2)Y(1)/P2Y(12) GPVI, PAR1/PAR4, TP, IP receptors, and guanylate cyclase, respectively, in Factor Xa-inhibited (250 nM apixaban), diluted platelet rich plasma that had been loaded with the calcium dye Fluo-4 NW. PAS of 10 healthy donors provided [Ca(t)](i) data for training 10 neural networks (NN, 2-layer/12-nodes) per donor. Trinary stimulations were then conducted at all 0.1x and 1xEC(50) doses (160 conditions) as was a sampling of 45 higher ordered combinations (four to six agonists). The NN-ensemble average was a calcium calculator that accurately predicted [Ca (t)](i) beyond the single and binary training set for trinary stimulations (R = 0.924). The 160 trinary synergy scores, a normalized metric of signaling crosstalk, were also well predicted (R = 0.850) as were the calcium dynamics (R = 0.871) and high-dimensional synergy scores (R = 0.695) for the 45 higher ordered conditions. The calculator even predicted sequential addition experiments (n = 54 conditions, R = 0.921). NN-ensemble is a fast calcium calculator, ideal for multiscale clotting simulations that include spatiotemporal concentrations of ADP, collagen, thrombin, thromboxane, prostacyclin, and nitric oxide.