(H-294) The effect of tissue factor concentration in hemophilia patients: computational prediction of thrombin generation.

Introduction:: Hemophilia causes spontaneous reoccurring bleeding episodes associated with long term joint degeneration and early death. 10-30% of patients with severe hemophilia will develop inhibitors to factor replacement therapy, the clinical gold standard in treating the disease. Lower blood concentrations of FIX (hemophilia B) or FVIII (hemophilia A) are associated with higher bleeding risk; however, severity of FIX or FVIII deficiency does not always correlate directly to symptom severity. Thrombin generation assays (TGAs) are gaining popularity in predicting bleeding risk of hemophilia patients, as low thrombin generation shows better correlation with bleeding events than FXI or FVIII blood concentration. Clinical hemophilia management is moving towards diagnostic tools that consider the entire blood microenvironment, as evidence suggests that factor deficiency or malfunction is the root of a bleeding phenotype influenced by many factors.


Materials and Methods:: The thrombin generation curves in simulations with reduced blood concentrations of FIX or FVIII were generated in an extended Hockin-Mann model of coagulation based on initiating clotting with TF. This computational model was initially validated with in vitro TGA data generated with normal physiological coagulation factor concentrations in platelet-poor plasma. The effect of varying TF concentration (0.125-90 pM) on thrombin generation was modeled within a simulated hemophilia environment over time. Hemophilia environments were simulated by altering the initial concentrations of FIX or FVIII from 0-100% of normal physiological coagulation factor concentrations. Endogenous thrombin potential (ETP) and peak thrombin concentration were analyzed.

Results, Conclusions, and Discussions:: Computational thrombin generation steadily decreased as FVIII or FIX concentrations were reduced, recapitulating clinical trends in hemophilia. To evaluate the effect of FIX concentration in the context of hemophilia, TGA simulations of mild, moderate, and severe hemophilia were performed while changing TF concentrations (Fig. 1a,b). A simulation of normal physiological conditions (90 nM FIX, 0.7 nM FVIII), with low TF concentration (0.125 pM), exhibited a modeled ETP of 2764 units and peak thrombin of 153 nM. In contrast, a simulation of mild hemophilia B (40% FIX concentration) resulted in an ETP of 680 units and a peak thrombin of 31 nM. Simulations of moderate to severe hemophilia failed to generate thrombin at low TF concentrations. Interestingly, TF concentration rescued thrombin generation, with higher TF concentration required to rescue lower coagulation factor concentrations (Fig. 1c,d). A model with 1% FIX, for example, had an ETP of 2308 units and a peak thrombin of 87 nM, when 50 pM of TF was present. This trend suggests that TF levels play a critical role in successful clotting in severe hemophilia cases. Our model predicts decreasing thrombin generation in hemophilia patients and shows higher sensitivity to factor deficiency at low TF concentrations. Our findings are consistent with literature studies that shows a similar trend in TGAs of hemophilia patient blood. Ongoing experiments will compare the in silico results to functional in vitro assays to validate observed behavior in modeled hemophilia A and B.

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