Introduction: Fitusiran is being evaluated in phase 3 clinical studies as a once monthly subcutaneously administered RNA interference therapy designed to decrease endogenous expression of the anticoagulant antithrombin (AT). Reducing the anticoagulant potential of AT in the plasma of Hemophilia A and B patients, with or without inhibitors, restores the hemostatic balance and improves thrombin generation potential.

Fitusiran and other non-Factor replacement molecules, such as emicizumab, use novel mechanisms to restore hemostasis. Consequently, some standard coagulation assays typically used to measure factor levels, such as the chromogenic, one-stage, and APTT assays have been shown to be affected by emicizumab. In order to assess the potential impact of fitusiran treatment on our ability to measure clotting factor activity in individuals with hemophilia A (HemA), we investigated the effect of decreased AT levels on standard coagulation laboratory assays. The following four assays were evaluated: one stage (OSA) and chromogenic (CSA) factor VIII (FVIII) activity assays, activated partial thromboplastin time (aPTT), and the prothrombin clotting time assay (PT). These assays are relevant to determine the FVIII plasma levels in patients dosed with FVIII replacement factors while treating potential breakthrough bleeds or undergoing surgery.

Method and Results: Pooled human HemA plasma was used directly or after AT depletion using an affinity-based method to remove 95% AT activity, resulting in HemA plasma with 5% AT activity. Additional batches of HemA/AT levels were created by AT reconstitution to 10 and 20% AT plasma activity. Subsequently, the 4 HemA plasma pools (one with normal -100 %- AT and three with reduced levels of AT-5, 10, 20%) were prepared with FVIII spiked to 0, 5, 10, 20, 30, 50, and 100% . FVIII plasma activity was measured by OSA and CSA against Siemens normal standard plasma. Compared to FVIII plasma activity for all FVIII spikes in normal HemA plasma containing 100% AT, respective FVIII spikes in HemA/AT depleted plasma samples (5, 10, and 20% AT) did not show therapeutic significant effects of AT levels on measured FVIII activity by OSA and chromogenic FVIII activity assay.

Additionally, we evaluated the effect of the various AT levels and FVIII spikes on clotting assays, aPTT and PT, among the HemA plasma with varying (5-100%) AT levels. AT level did not affect the aPTT for respective FVIII spikes in HemA and Hem/AT depleted plasma. Furthermore, no therapeutic relevant effect of ATIII and FVIII level was observed in PT values between HemA and Hem/AT depleted plasma.

Conclusions: Our results demonstrate that a reduction of AT levels in plasma does not affect standard coagulation assays, suggesting these assays can be utilized to assess hemostasis and factor levels in individuals treated with fitusiran. The ability to monitor FVIII levels in patients treated with non-replacement therapies is important as these patients require factor to treat breakthrough bleeds and during some surgeries.

Disclosures

Wang:sanofi: Current Employment, Current equity holder in publicly-traded company. Kattula:Sanofi: Current Employment, Current equity holder in publicly-traded company. Ismail:Sanofi: Current Employment, Current equity holder in publicly-traded company. Leksa:Sanofi: Current Employment, Current equity holder in publicly-traded company. van Der Flier:sanofi: Current Employment, Current equity holder in publicly-traded company. Salas:sanofi: Current Employment, Current equity holder in publicly-traded company.

Author notes

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Asterisk with author names denotes non-ASH members.