Blood coagulation kinetics: high throughput method for real-time reaction monitoring

Summary

A high throughput 384-well plate assay of blood function in 60 μl 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 (T_max) from 45 min to 11 min. Over this range of TF,T_max 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 T_max 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 T_max. Addition of up to 0.67 nM reVIIa reduced T_max by up to 53% in the range of 0.7 to 7 pM TF. Interestingly, platelet factor 4 (2.7 μM) caused a prolongation of T_max from 45 min to 78 min at 0 TF, while protamine (1.8 μM) reduced T_max 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.

Keywords

Fluorescence - thrombin - kinetics - platelet factor 4

• References

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