Protein S As a Potential Treatment for FIX-Derived Deep Vein Thrombosis

Background: Every year, 0.1-0.2% of the USA population experiences deep vein thrombosis (DVT). Two causes of DVT are increased Factor IX (FIX) levels and hyperactivating mutations in FIX (FIX Padua variant- R338L and Malmo variant T148A). In principle, inhibition of activated FIX (FIXa) should alleviate DVT. Previous in vitro studies demonstrated that the anticoagulant Protein S (PS) inhibits the intrinsic pathway mediated by wild type FIXa, making PS an attractive candidate to treat DVT.

Aims: To establish Protein S as a remedy for FIX-mediated DVT/Padua/Malmo

Methods: Anisotropy, clotting assays, thrombin generation assays, co-localization, co-immunoprecipitation, and bleeding assays.

Results: We further explored the physiological relevance of the PS-FIXa interaction and PS-mediated inhibition of FIXa by ex vivo (co-immunoprecipitation) and in vivo (co-localization) studies. Because PS can inhibit FIXa in vivo, we used competitive, direct anisotropy assays and co-immunoprecipitation assays to measure the efficiency PS and hyperactive FIXa (R338L) interaction. Interestingly, the results demonstrated that FIXa R338L has lost its affinity towards PS compared with wild type FIXa. The same finding was obtained by ex vivo thrombin generation assays and FXa generation assays supplemented with various concentrations of PS. Thus, to be inhibited, hyperactive FIX requires a greater amount of PS compared with wild type FIXa. We are further confirming this finding with mouse models.

Conclusion: Addition of PS to plasma inhibits both wild type and R338L FIXa and extends clotting time. Previous studies showed that the addition of PS has no significant negative effects. Thus, we conclude that PS supplementation potentially constitutes a novel and effective treatment for FIX-mediated DVT.