Reaction dynamics of human blood coagulation
We developed a high throughput 384 well plate assay for blood function and a stochastic Monte Carlo simulation of blood coagulation to jointly explore the coagulation cascade reaction space. The assay used a liquid handling system to combine 60 μL reaction volumes with 3-fold diluted whole blood. Using a high-speed microplate fluorimeter, thrombin generation was monitored in real-time under a diverse set of reaction conditions using a fluorogenic substrate, boc-VPR-MCA. Concurrently, a kinetic Monte Carlo simulation was developed using the deterministic reaction network developed by the Mann laboratory for tissue factor (TF)-initiated blood coagulation. Experimentally, 0.28 pM of TF was required to initiate coagulation. Addition of convulxin, a GPVI platelet activator, decreased the required TF concentration to 0.07 pM. It was hypothesized that maximal thrombin production rate was a function of platelet activation, as only convulxin produced a 30% increase, while added TF, Va, or reVIIa did not. Combinatorial reaction studies with ADP, histamine, fMLP, indomethacin, anti-CD 18, and fibrinogen revealed no unusual synergies amongst the agents, but fibrinogen demonstrated striking procoagulant activity due to protease contaminants in “purified” fibrinogen: Titrations of Factor Xa and IXa showed initiating concentrations (324 pM Xa and 500 pM IXa) that were considerably higher than TF (0.28 pM). The model matched thrombin data in whole blood (3-fold diluted) pretreated with convulxin and TF (1 to 14 pM) collected by the assay. The model did not accurately predict coagulation times at low TF (0 to 0.7 pM). The simulation revealed that ∼0.2 pM TF was the critical concentration to cause a 50% probability of 3-fold diluted whole blood reaching a clotting threshold of 0.05 U/mL thrombin by 1 hr. The high throughput approach allowed automated profiling of blood (50 reactions/ml of blood), generating large data sets for testing cellular-proteomic kinetic models and for screening drug interactions. By elucidating the fundamental balance of procoagulant and anticoagulant factors in blood and how slight alterations to this equilibrium can lead to thrombosic events, this research offers both model and method that provide fundamental insight relevant to stroke, myocardial infarction, and deep vein thrombosis. ^
Biology, Animal Physiology|Engineering, Biomedical
"Reaction dynamics of human blood coagulation"
(January 1, 2005).
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