Systems Biology of Coagulation Initiation: Kinetics of Thrombin Generation in Resting and Activated Human Blood

Blood function defines bleeding and clotting risks and dictates approaches for clinical intervention. Independent of adding exogenous tissue factor (TF), human blood treated in vitro with corn trypsin inhibitor (CTI, to block Factor XIIa) will generate thrombin after an initiation time (T-i) of 1 to 2 hours (depending on donor), while activation of platelets with the GPVI-activator convulxin reduces T-i to similar to 20 minutes. Since current kinetic models fail to generate thrombin in the absence of added TF, we implemented a Platelet-Plasma ODE model accounting for: the Hockin-Mann protease reaction network, thrombin-dependent display of platelet phosphatidylserine, VIIa function on activated platelets, XIIa and XIa generation and function, competitive thrombin substrates (fluorogenic detector and fibrinogen), and thrombin consumption during fibrin polymerization. The kinetic model consisting of 76 ordinary differential equations (76 species, 57 reactions, 105 kinetic parameters) predicted the clotting of resting and convulxin-activated human blood as well as predicted T-i of human blood under 50 different initial conditions that titrated increasing levels of TF, Xa, Va, XIa, IXa, and VIIa. Experiments with combined anti-XI and anti-XII antibodies prevented thrombin production, demonstrating that a leak of XIIa past saturating amounts of CTI (and not blood-borne TF'' alone) was responsible for in vitro initiation without added TF. Clotting was not blocked by antibodies used individually against TF, VII/VIIa, P-selectin, GPIb, protein disulfide isomerase, cathepsin G, nor blocked by the ribosome inhibitor puromycin, the Clk1 kinase inhibitor Tg003, or inhibited VIIa (VIIai). This is the first model to predict the observed behavior of CTI-treated human blood, either resting or stimulated with platelet activators. CTI-treated human blood will clot in vitro due to the combined activity of XIIa and XIa, a process enhanced by platelet activators and which proceeds in the absence of any evidence for kinetically significant blood borne tissue factor.