Biochemical insights into coagulation Factor XII: To explore and exploit its functions

Abstract

Blood is essential for transporting oxygen and nutrients throughout the body, so it must keep flowing while also being able to stop bleeding when vessels are damaged. This balance is maintained by hemostasis. Activation of the extrinsic coagulation cascade leads to a fibrin-rich clot, but overactivation can lead to thrombosis. Many anticoagulant drugs target this system to prevent thrombosis, but because these pathways are also vital for normal clotting, such drugs increase the risk of bleeding. Clot formation can also occur through the intrinsic coagulation cascade, which is activated when blood comes into contact with negatively charged surfaces such as bacteria or foreign materials. This pathway begins with coagulation Factor XII (FXII), which, upon activation, triggers Factor XI and prekallikrein. Together, these proteins form the “contact system.” Activation of Factor XI promotes clot formation, while prekallikrein activation induces inflammation to fight pathogens—a process known as thromboinflammation. However, FXII can also react with artificial surfaces like medical devices, leading to unwanted clot formation that impairs their function. Targeting FXII is a promising strategy for safer anticoagulants because, unlike other clotting factors, inhibiting FXII does not increase bleeding risk. Individuals lacking FXII do not show bleeding disorders, indicating that normal clotting can still occur without it. Despite this potential, no FXII-targeting drugs are currently available. This thesis aims to better understand FXII to support the development of safe and effective anticoagulants. Because the experimental structure of FXII has not been determined, the research relies on an AI-predicted model from AlphaFold. This model suggests that the heavy chain of FXII shields a critical cleavage site (R353) through interdomain interactions, keeping the protein inactive. It also identifies positively-charged amino acid clusters the EGF-1 domain which are supposedly important for binding negatively charged surfaces. Further investigation shows that interactions between distant domains in the heavy chain help maintain FXII in its inactive (zymogen) state. Mutating specific amino acids that disrupt these electrostatic interactions leads to faster activation, confirming their stabilizing role. Additionally, a nanobody was developed to recognize an “open” conformation of FXII, enabling precise monitoring of its activation. To selectively inhibit FXII mediated clotting without affecting its inflammatory role, the research focuses on preventing its surface binding. Neutralizing positively charged regions in the EGF-1 domain reduces FXII binding and delays clot formation. A nanobody targeting these regions effectively inhibits FXII’s procoagulant activity without interfering with its other functions. Finally, the thesis explores enhancing natural inhibitors (SERPINs) by fusing them with the EGF-1 domain. This fusion directs the inhibitor to procoagulant surfaces, improving its ability to suppress the contact system on both natural and artificial materials. Overall, this work provides new structural and functional insights into FXII, experimentally validating predictions from AI models. These findings contribute to the development of selective FXII inhibitors, which could offer safer anticoagulant therapies without increasing bleeding risk.