Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemical and Biomolecular Engineering

First Advisor

Scott L. Diamond

Abstract

Platelet intracellular calcium mobilization [Ca(t)]i is a measure of platelet activation and controls important events downstream that contribute to hemostasis such as granule release, cyclooxygenase-1 and integrin activation, and phosphatidylserine exposure. Accurate simulations of blood clotting events require prediction of platelet [Ca(t)]i in response to combinatorial agonists. Therefore, a data-driven human platelet calcium calculator was developed using neural network (NN) ensemble trained on pairwise agonist scanning (PAS) data. PAS deployed all single and pairwise combinations of six important agonists (ADP, convulxin, thrombin, U46619, iloprost and GSNO used at 0.1, 1, and 10xEC50 to stimulate platelet P2Y1/P2Y12, GPVI, PAR1/PAR4, TP, IP receptors, and guanylate cyclase, respectively, in Factor Xa-inhibited (250 nM apixaban), diluted platelet rich plasma. PAS of 10 healthy donors (5 male, 5 female) 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 1xEC50 doses (160 conditions) as was a sampling of 45 higher ordered combinations (four to six agonists). The NN-ensemble average 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 that proved to be useful for multiscale clotting simulations that include spatiotemporal concentrations of ADP, collagen, thrombin, thromboxane, prostacyclin, and nitric oxide.

From sequential addition experiments done in PAS, it was discovered that activating platelets with thrombin in platelet-rich plasma (PRP) caused an attenuation of convulxin-induced, GPVI platelet receptor-mediated, calcium mobilization when convulxin was added to PRP approximately six minutes later. This attenuation effect was not observed when ADP and thromboxane analog, U46619 was used in place of thrombin. When PAR-1 and PAR-4 receptor agonists (AYPGKF and SFLLRN) were used instead of thrombin for the initial dispense, the subsequent convulxin-induced calcium response was also unaffected, demonstrating thrombin’s unique role in causing attenuation of subsequent convulxin-induced calcium mobilization. Thrombin, unlike ADP, U46619 or the PAR-1 and PAR-4 receptor agonists, is able to polymerize fibrinogen into fibrin. When GPRP was added to prevent polymerization of fibrin, initial platelet activation by thrombin did not result in attenuation of convulxin- induced calcium mobilization. This experiment was repeated using a mixture of washed platelets and fibrinogen monomers instead of PRP and yielded similar results. The presence of polymerized fibrin also reduced platelet deposition in a microfluidic assay on a collagen surface. These results suggest that polymerized fibrin binds to and downregulates platelet GPVI, a platelet receptor that is important to thrombus growth and is central to mediating hemostasis.

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