Engineered Soluble Protein C Pathway Receptors as Novel Pro-Haemostatic Agents

Haemophilia is a serious bleeding disorder caused by a deficiency in the components of the intrinsic tenase complex. Prophylactic replacement of the missing clotting factor is the current treatment for haemophilia, yet there remains a significant risk of inhibitor development to replacement factors. Moreover, there are also many other acquired bleeding disorders for which haemostatic therapies remain sub-optimal. Existing pro-haemostatic therapies improve thrombin generation in haemophilia patients by boosting levels or activity of existing procoagulant factors, however, an alternative approach is to instead inhibit natural endogenous anticoagulant pathways such that thrombin generation can proceed unhindered. Notably, defects in activated protein C (APC) anticoagulant activity predisposes affected individuals towards thrombosis, but can reduce bleeding tendency when co-inherited with haemophilia A (HA). Selective inhibition of APC generation or activity may therefore be a viable alternative option to reduce bleeding in affected individuals. Furthermore, individuals with severe HA have increased fibrinolytic activity, indicating that novel treatments that promote thrombin generation but inhibit fibrinolysis are also likely to be advantageous for individuals at high risk of bleeding. In this study, I demonstrated that soluble endothelial protein C receptor (sEPCR) can act as a decoy receptor to inhibit APC generation on endothelial cells and can limit anticoagulant activity in normal and haemophilia plasma. In addition, I showed that a recombinant non-anticoagulant soluble thrombomodulin (sTM) variants retains anti-fibrinolytic activity, using a novel plasmin generation assay. Furthermore, given the co-incidence of diminished thrombin generation and increased fibrinolytic activity observed in many individuals with bleeding disorders, I explored a novel strategy to fuse the pro-coagulant sEPCR with the non-anticoagulant and anti-fibrinolytic properties of the sTM variant using a protein ligation strategy. Creation of a new multifunctional fusion protein with diverse pro-haemostatic activities has significant potential to correct bleeding in people with bleeding disorders.