Figure 1. High throughput 384-well plate measurement of thrombin production in human blood.

(A) Experimental protocol. Calcium and the fluorogenic thrombin substrate (S_IIa) were added to the 384-well plate on a Thermo Multidrop. The plate was placed on a Perkin-Elmer Janus where various concentrations of each individual species were added to each well. After the blood was drawn, the plate was moved to a Perkin-Elmer Evolution P³ where the blood was added to all wells simultaneously (t = 0). The plate was read in a Thermo-Electron Fluoroskan where the fluorescence was measured for 4 hr. The time from vein to first measurement was under 5 min. (B) Initiation Time. The time required to reach 5% conversion of the fluorogenic substrate was set as the initiation time (T _i). This metric correlated well with ∼10 nM TAT and preceded a burst of thrombin and a maximization of the second derivative of fluorecense. Relative prolongation or reductions in T_i were used to quantify coagulation initiation.