Modulating serine proteases to counteract bleeding

Abstract

This thesis investigates novel strategies to restore hemostasis by engineering key enzymes in the coagulation cascade and focuses on two major clinical challenges: bleeding in hemophilia A (FVIII deficiency) and bleeding associated with direct oral anticoagulants. Central to both causes of bleeding is the delicate balance of serine protease activity that governs thrombin generation and clot formation.

Chapter 2 discusses targeted modifications of factor IX that impair active site maturations. Paradoxically, these variants exhibit reduced susceptibility to inhibition by antithrombin, thereby enhancing overall clotting eNiciency despite decreased intrinsic enzymatic activity. This demonstrates that modulating regulatory interaction can improve hemostasis.

Chapters 3 focuses on the developments of factor IX variants with reduced dependence on factor VIII. Through structure-guided engineering, modifications primarily located around the S4 subsite enhance intrinsic catalytic function, enabling partial bypass of the intrinsic tenase complex. This works identifies FIX-FIAV as a promising cofactorindependent variants. Chapter 4 evaluates this FIX variant in hemophilia A patient plasma, demonstrating that it can restore thrombin generation in a factor-VIII-independent manner, thereby partially mitigating the hemophilia A phenotype. Chapter 5 extends this approach to thrombin, showing that targeted modifications in the S4 subsite can confer resistance to direct thrombin inhibitors while preserving enzymatic activity. Overall, this thesis demonstrates that rational engineering of serine proteases can shift the hemostatic balance toward eNective clot formation, oNering a promising framework for developing next-generation therapies for bleeding disorders.