Mechanistic Studies of Thrombin Generation Assay to Evaluate Procoagulant Potential of Fitusiran

Introduction: Fitusiran is a once-monthly subcutaneously administered RNA interference therapeutic targeting antithrombin III (AT) to rebalance hemostasis in patients with hemophilia A and B with or without inhibitors. As for other non-FVIII therapies in development, there is a need to better assess the procoagulant potential of fitusiran.

Using thrombin generation assays (TGA), we studied the hemostatic activity in hemA plasma with low AT levels in the presence and absence of varying factor VIII (FVIII) levels to mimic conditions associated with breakthrough bleeds. To better understand the impact of AT depletion in the assay, we monitored multiple TGA parameters as well as the levels of several coagulation components potentially influencing the reactions.

Methods: Pooled HemA plasma was applied to an AT affinity resin to remove AT. Residual AT activity was 5% and additional AT was spiked back to produce HemA plasmas with various amounts of AT (5, 10, 20 and 100%). FVIII was spiked into the 4 plasmas at 0, 5, 10, 20, 30, 50, and 100 IU/dL and all samples were analyzed by Calibrated Automated Thrombography (Stago). Thrombin generation parameters were calculated by thrombinoscope software.After the TGA reaction, samples were further analyzed by immunoblot for evaluation of thrombin-AT (TAT) and alpha2-macroglobulin-thrombin (a2M:T) complex formation, as well as remaining (pro)thrombin, AT and a2M levels . In parallel, a2M:T complex levels were assessed by ELISA capture of a2M followed by addition of thrombin substrate to directly measure the thrombin activity stemming from a2M-inhibited thrombin. .

Results: TGA parameters for FVIII titrations in HemA plasmas with reduced AT were calculated and compared to the FVIII standard curves generated in HemA plasma with normal AT levels for each TGA parameter (lag time, time to peak, peak height, ETP and velocity index). As expected, a greater reduction of AT activity in hemA plasma correlated with increased thrombin generation compared to hemA plasma (with 100% AT). The apparent FVIII-like activity observed at low AT conditions in HemA plasma was dependent on the TGA parameter evaluated, with velocity index suggesting the lowest and ETP the highest FVIII-like activity. Furthermore, particularly at lower starting levels of AT, AT is depleted during the TGA reaction and leads to enhanced levels of a2M:T complex as observed by immunoblot or a2M:T chromocapture assays. Importantly, the a2M:T complex is capable of cleaving the assay substrate and contributing to the TGA signal. Typically, the a2M:T generated signal is calculated and substracted by the thrombinoscope software. However, under low AT conditions, increased levels of a2M-T lead to substrate consumption at later stages of the reaction, resulting in erroneous underestimation of the a2M:T activity that does not agree with direct measurement of a2M: T levels. These limitations need to be taken into consideration when interpreting TGA data under conditions of low AT activity. While this remains an area of investigation, parameters derived earlier in the reaction prior to AT depletion and significant levels of a2M:T complex formation may better correlate with the true hemostatic potential associated with AT inhibition.

Conclusion: Our results show that TGA can be a valuable tool to measure the hemostatic potential associated with fitusiran therapy. We demonstrate that reduced levels of AT are associated with enhanced thrombin generation, but that activity comparison to FVIII varies significantly depending on the TGA parameter analyzed. In addition we show that under low AT conditions, AT can become depleted leading to a2M:T complex accumulation which may lead to overestimation of some TGA parameters. This overestimation is likely an artifact of the TGA method as it is a closed in vitro system that can be influenced by the depletion of components or accumulation of products. Our results suggest that TGA parameters calculated early in the reaction are less influenced by some of these limitations and may be better predictors of the true hemostatic activity of fitusiran. Furthermore, a2M may play a more active role during fitusiran treatment, ensuring free thrombin does not migrate outside of the site of injury.