Kinetic model facilitates analysis of fibrin generation and its modulation by clotting factors: implications for hemostasis-enhancing therapies

Abstract

Current mechanistic knowledge of protein interactions driving blood coagulation has come largely from experiments with simple synthetic systems, which only partially represent the molecular composition of human blood plasma. Here, we investigate the ability of the suggested molecular mechanisms to account for fibrin generation and degradation kinetics in diverse, physiologically relevant in vitro systems. We represented the protein interaction network responsible for thrombin generation, fibrin formation, and fibrinolysis as a computational kinetic model and benchmarked it against published and newly generated data reflecting diverse experimental conditions. We then applied the model to investigate the ability of fibrinogen and a recently proposed prothrombin complex concentrate composition, PCC-AT (a combination of the clotting factors II, IX, X, and antithrombin), to restore normal thrombin and fibrin generation in diluted plasma. The kinetic model captured essential features of empirically detected effects of prothrombin, fibrinogen, and thrombin-activatable fibrinolysis inhibitor titrations on fibrin formation and degradation kinetics. Moreover, the model qualitatively predicted the impact of tissue factor and tPA/tenecteplase level variations on the fibrin output. In the majority of considered cases, PCC-AT combined with fibrinogen accurately approximated both normal thrombin and fibrin generation in diluted plasma, which could not be accomplished by fibrinogen or PCC-AT acting alone. We conclude that a common network of protein interactions can account for key kinetic features characterizing fibrin accumulation and degradation in human blood plasma under diverse experimental conditions. Combined PCC-AT/fibrinogen supplementation is a promising strategy to reverse the deleterious effects of dilution-induced coagulopathy associated with traumatic bleeding.

Fig. 1

Protein interaction network responsible for tissue-factor-activated thrombin generation, fibrin formation, and fibrinolysis. The schematic shows protein interactions reflected in the kinetic model. Arrows represent molecular conversion, enzymatic catalysis, or binding/unbinding; T-shaped lines designate inhibition. Thrombin (FIIa) generation is initiated when, in the course of hemorrhage, blood comes in contact with the extravascular protein tissue factor (TF).^, The formation or activity of thrombin is inhibited by the three natural anticoagulant systems: TF pathway inhibitor (TFPI), antithrombin (AT), and protein C. While active, thrombin converts fibrinogen (Fg) monomers into fibrin I (FnI) monomers. FnI is cleaved further and converted to fibrin II (FnII).^, Fibrin degradation is catalyzed by the enzyme plasmin (Pn), whose precursor, plasminogen (Pg), is activated by tissue-type plasminogen activator (tPA). This degradation is inhibited at different levels by α₂-antiplasmin (AP), plasminogen activator inhibitor 1 (PAI), and thrombin-activatable fibrinolysis inhibitor (TAFI, which, like the protein C pathway, requires thrombomodulin for full activity). For the sake of a convenient visual presentation, some intermediate complexes and reactions were omitted in the schematic. For a full list of the protein interactions in the model, see Table S1 (ESI†). Green: thrombin generation; blue: fibrin formation; orange: fibrin degradation. Further abbreviations: APC, activated protein C; FDP, fibrin degradation products; FPA, fibrinopeptide A; FPB, fibrinopeptide B; FII, prothrombin. The letter “F” followed by a Roman numeral designates a clotting factor, and if the Roman numeral is followed by the letter “a,” then the clotting factor is in its active form.

Fig. 2

Kinetic model captures fibrin formation modulation by prothrombin and fibrinogen. Solid lines, model output; square markers, turbidimetric measurements. A: Prothrombin titration. The colors represent different levels of initial prothrombin: black, 1% of the normal average value (i.e., 1.40 μM); blue, 10%; green, 50%; and red, 100%. Experimental data were extracted from Fig. 2 of Ref. . To facilitate comparisons with model predictions, the absorbance baseline was set to 0 and the data were normalized to the maximum absorbance level achieved for 100% prothrombin. Model output was normalized to the maximum concentration achieved in the simulation for 100% prothrombin. While in the experiment the TF concentration was not determined, we used the default TF concentration in the simulation. B: Fibrinogen titration. The colors represent different levels of initial fibrinogen: black, 8.82 μM; blue, 13.24 μM; green, 17.65 μM; and red, 22.06 μM. Experimental data were extracted from Fig. 5D in Ref. . The absorbance baseline was set to 0 and the experimental data were normalized to the maximum absorbance level. The model output was normalized to the maximum fibrin concentration achieved for 22.06 μM fibrinogen. In the model, thrombin generation was initiated with 1 pM TF and fibrinolysis was activated by adding external tPA (at time 0) at a concentration of 3.50 nM; these values were chosen to match the experimental conditions.

Fig. 3

Effects of thrombin-activatable fibrinolysis inhibitor (TAFI) on fibrinolysis kinetics. A: Model-generated fibrinolysis kinetic curves for preformed clots for different concentrations of added TAFI. In the simulations, clots were initially formed for 30 min in the absence of TAFI. Fibrinolysis was initiated by 3.5 nM external tPA added at time 0. Left to right, TAFI was added at time 0 at a concentration of 0, 0.20, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, and 200 nM. B: Experimentally determined dependency of relative lysis time for preformed clots on the concentration of added TAFI (square markers) is approximated by a model-generated curve (solid line). Experimental data for clots formed in TAFI-deficient normal human plasma were extracted from Fig. 6 in Ref. . In the simulations, the lysis times were computed directly from simulated fibrinolysis kinetic curves, which looked similar to the curves shown in panel A.

Fig. 4

Triggers of thrombin generation and fibrinolysis impact fibrin kinetics. A: Model-predicted fibrin generation at different TF levels. B: Baseline-subtracted turbidimetric data (squares) extracted from Fig. 7B in Ref. . To enhance the visual perception of the experimentally detected patterns, the data points are connected by thin dashed lines. The colors represent three types of reagents added to the cells supporting thrombin generation (i.e., human umbilical vein endothelial cells). “anti-TF” denotes anti-TF antibody. AU, arbitrary units. C: Model-predicted fibrin levels for a tissue-type plasminogen activator (tPA) titration. Thrombin generation was initiated with 1 pM TF. The initial fibrinogen concentration was 8.82 μM. D: Baseline-subtracted turbidimetric data (squares) for a tenecteplase titration performed as described (see Materials and Methods). AU, arbitrary units.

Fig. 5

Model-predicted restoration of normal thrombin and fibrin generation in diluted plasma by clotting factor supplementation. “Fg” stands for supplementation with fibrinogen, “PCC” denotes supplementation with PCC-AT (a combination of the factors II, IX, X, and antithrombin), and “Fg/PCC” denotes simultaneous supplementation with fibrinogen and PCC-AT. A: Clotting was initiated with 2 pM TF. B: Clotting was initiated with 5 pM TF. C: Clotting was initiated with 10 pM TF. Here, the blue and red lines for fibrin generation practically coincide, as do the orange and red lines for thrombin generation.

Fig. 6

Model-predicted effects of supplementing diluted plasma with the three procoagulant PCC components (i.e., factors II, IX, and X) with and without fibrinogen; no antithrombin was added. The PCC component supplementation is denoted “PCC(No AT)” without fibrinogen and “Fg/PCC(No AT)” with fibrinogen. A: Clotting was initiated with 2 pM TF. B: Clotting was initiated with 5 pM TF. C: Clotting was initiated with 10 pM TF.