Fitusiran treatment modulates the ratio between alpha‐ and beta‐antithrombin isoforms

Abstract Antithrombin (AT) circulates as two distinct isoforms, alpha‐ and beta‐AT, which differ in their glycosylation profiles; alpha‐AT is fully glycosylated at positions Asn 128 , Asn 167 , Asn 187 , and Asn 224 , whereas beta‐AT lacks Asn 167 glycosylation. The ratio of alpha‐AT/beta‐AT is approximately 9:1 in plasma, with beta‐AT being a stronger inhibitor due to its increased affinity for heparin. Post‐transcriptional silencing of AT via fitusiran has been shown to efficiently ameliorate the hemostatic balance in hemophilia. In this study, we analyzed if and how fitusiran affected the distribution of alpha‐AT and beta‐AT. Using different experimental approaches (isoform‐specific activity, antigen, and immunoprecipitation assays), we were able to distinguish beta‐AT and alpha‐AT. Fitusiran treatment reduced the total AT activity to less than 20% of normal in both F8 −/− ‐mice and hemophilia A patients. Compared to controls, a 3.8‐ and 3.3‐fold increase in the amount of beta‐AT activity relative to residual total AT activity was detected in F8 −/− ‐mice and fitusiran‐treated patients, respectively (P < 0.0001). Furthermore, the ratio of beta‐AT/total AT antigen levels increased 1.8‐fold in the human patient samples (from 0.09 ± 0.03 to 0.16 ± 0.01; P = 0.003), which coincided with an increased intensity of the beta‐AT band detected in immunoprecipitation assays. It is noteworthy that this increase in the beta‐AT/total AT ratio significantly increased its anticoagulant potential. Finally, we measured beta‐AT/total AT ratios also in unrelated pathologies: congenital AT deficiency and advanced liver cirrhosis. Both conditions were also associated with an up to twofold higher ratio of beta‐AT/total AT. Altogether, our results demonstrate that reduced AT production modulates the ratio between beta‐AT and alpha‐AT.