Abstract
During thrombosis, thrombin generates fibrin, however fibrin reversibly binds thrombin with low affinity E-domain sites (KD = 2.8 μM) and high affinity γ’-fibrin sites (KD = 0.1 μM). For blood clotting on collagen/tissue factor (1 TF-molecule/μm2) at 200 s-1 wall shear rate, high μM-levels of intraclot thrombin suggest robust prothrombin penetration into clots. Setting intraclot zymogen concentrations to plasma levels (and neglecting cofactor rate limitations) allowed the linearization of 7 Michaelis-Menton reactions between 6 species to simulate intraclot generation of: Factors FXa (via TF/VIIa or FIXa), FIXa (via TF/FVIIa or FXIa), thrombin, fibrin, and FXIa. This reduced model [7 rates, 2 KD’s, enzyme half-lives~1 min] predicted the measured clot elution rate of thrombin-antithrombin (TAT) and fragment F1.2 in the presence and absence of the fibrin inhibitor Gly-Pro-Arg-Pro. To predict intraclot fibrin reaching 30 mg/mL by 15 min, the model required fibrinogen penetration into the clot to be strongly diffusion-limited (actual rate/ideal rate = 0.05). The model required free thrombin in the clot (~100 nM) to have an elution half-life of ~2 sec, consistent with measured albumin elution, with most thrombin (>99%) being fibrin-bound. Thrombin-feedback activation of FXIa became prominent and reached 5 pM FXIa at >500 sec in the simulation, consistent with anti-FXIa experiments. In predicting intrathrombus thrombin and fibrin during 15-min microfluidic experiments, the model revealed “cascade amplification” from 30 pM levels of intrinsic tenase to 15 nM prothrombinase to 15 μM thrombin to 90 μM fibrin. Especially useful for multiscale simulation, this reduced model predicts thrombin and fibrin co-regulation during thrombosis under flow.
Highlights
The reaction network and kinetics of human blood clotting impact diseases such as coronary thrombosis, stroke, deep vein thrombosis, hemophilia, disseminated intravascular coagulopathy (DIC), and traumatic bleeding
Reduced model to predict thrombin and fibrin under flow under flow with parameters from literature, with only 3 adjusted in order to fit the experimental data. This model gave insights into the dynamics of the species involved, and the roles of γ’-fibrin and thrombin feedback activation. This reduced model may be useful in further multiscale simulations
We present a reduced model where key assumptions are supported by direct experiment measurements
Summary
The reaction network and kinetics of human blood clotting impact diseases such as coronary thrombosis, stroke, deep vein thrombosis, hemophilia, disseminated intravascular coagulopathy (DIC), and traumatic bleeding. Excluding platelet metabolism other than the availability of anionic phospholipid, isotropic kinetic models of plasma coagulation in a closed system can include 50 to 100 reactions, 1 to 3 kinetic rate coefficients per reaction, and about 10 initial conditions for zymogen or cofactor concentrations [3,4,5]. These large ODE models can be parameterized and solved with minor computational expense. Calibrated automated thrombinography (CAT) assay reports these dynamics for platelet-poor or platelet-rich plasma with typical time lags of 3.1 and 8.1 min, peak thrombin levels of 458 and 118 nM at 10 min, and reaction completion by 25 min [6]
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