(516u) Reaction Dynamics Of Human Blood Coagulation Initiation

To study clotting dynamics, citrated human blood (contact pathway blocked with corn trypsin inhibitor) was evaluated in 384-well plate assay with boc-VPR-MCA and Ca²⁺ to measure thrombin production. Using buffer dilution to vary the initial concentration of all blood species, the coagulation initiation time (T_i) with no added activators doubled from 40 min to 80 min as blood was diluted up to 100-fold. Remarkably, the maximal rate of thrombin formation was unchanged at 100-fold dilution. T_i slowed to 240 min at up to 400-fold dilution and blood failed to clot at >500-fold dilution. Addition of Factor Xa revealed a switch-like, 10 to 100 pM dynamic range to move from no effect to saturation. In contrast, lipidated tissue factor (TF) or Factor IXa addition revealed broad dynamic ranges of activity over 3 orders of magnitude of concentration. The Hockin-Mann model was only accurate at > 1 pM of added TF. TF titration bounded the maximal endogenous TF activity detectable in static assay to ~40 fM, depending on donor. While 5 nM Factor VIIa or individual platelet agonists (ADP, convulxin, SFLLRN) reduced T_i by half (equivalent to adding ~100 fM TF), the platelet agonists did not reveal synergism with factor VIIa. Also, the ribosome inhibitor puromycin or Clk1 kinase inhibitor Tg003 (to regulate pre-mRNA splicing) had no effect on T_i in the presence or absence of platelet agonists. These observations support an ?engine-running? model of blood with < 10 pM Factor Xa, < 1 pM Factor IXa, and < ~20 to 100 fM of pre-synthesized TF that become kinetically significant with platelet activation when observed in vitro.