(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 (Ti) 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. Ti 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 Ti 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 Ti 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.