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Ken Lo, Scott L. Diamond
Blood coagulation kinetics: high throughput method for real-time reaction monitoring
A high throughput 384-well plate assay of blood function in 60 l reactions with the fluorogenic thrombin substrate, boc-VPR-MCA, allowed for real-time monitoring of coagulation under a diverse set of reaction conditions. Using recalcified, citrated whole blood diluted 3-fold with corn trypsin inhibitor (to block Factor XIIa), addition of 0 to 13.8 pM of tissue factor (TF) reduced the time of maximal rate of thrombin production (Tmax) from 45 min to 11 min. Over this range of TF,Tmax was reduced from 35 min to 6 min by co-addition of 10 nM convulxin to activate platelets via GPVI. The maximal rate of thrombin production at Tmax was not a function of exogenously-added TF,Va, or reVIIa, but increased 30% with added convulxin. Addition of 0.07 to 0.7 pM TF along with convulxin produced small, but detectable reductions in Tmax. Addition of up to 0.67 nM reVIIa reduced Tmax by up to 53% in the range of 0.7 to 7 pM TF. Interestingly, platelet factor 4 (2.7 M) caused a prolongation of Tmax from 45 min to 78 min at 0 TF, while protamine (1.8 M) reduced Tmax to 30 min at 0 TF. Finally, combinatorial reaction studies with exogenously-added ADP, histamine, fMLP, indomethacin, anti-CD18, and fibrinogen revealed no unusual synergies amongst the agents, but demonstrated a striking procoagulant activity of added fibrinogen, due to protease contaminants in the purified fibrinogen. This high throughput approach allowed automated profiling of blood (50 reactions/ml of blood) to generate large data sets for testing cellular-proteomic kinetic models, screening drug interactions, and potentially monitoring subtle changes in the functional phenotype of a patient blood sample.
Thrombosis and Haemostasis, Schattauer
Print ISSN: 0340-6245
Volume: 92, 10/2004
Pages: 874 - 882