Abstract 16547: Thrombin-Thrombomodulin Fusion Proteins as New Anticoagulants

Introduction: Thrombin is a trypsin-like protease that plays opposing functional roles in blood coagulation due to the interaction with numerous macromolecular substrates, receptors and inhibitors. The specific binding of fibrinogen and PAR-1 to the exosite-1 leads to the conversion of fibrinogen to fibrin, platelet activation and clot formation. Binding of the cofactor thrombomodulin to the same exosite-1 hinders docking of the procoagulant substrates and accelerates the activation of protein C by 1500-fold thereby enabling the most powerful enzymatic anticoagulant system that exists in vivo . Down-regulation and washout of thrombomodulin from the endothelium have been documented in pathological conditions such as atherosclerotic lesions, sepsis and disseminated intravascular coagulation leading to impaired activation of protein C and thrombosis. Hypothesis: A solution to this problem would be the administration of a thrombomodulin-independent protein C activator. Results: In this study we report the design, expression and functional characterization of a new family of fusion proteins that specifically activate protein C without the need of endogenous thrombomodulin. In our molecules, the enzyme thrombin is genetically fused via artificial linkers to the soluble extracellular portion of the cofactor thrombomodulin. A total of 6 fusion proteins were expressed in mammalian cells at high yield and high purify. As expected, their anticoagulant profile varies with the length of the artificial linker. An optimal anticoagulant activity is achieved with a linker composed of 31 and 41 amino acids. Specifically, the activity of the L31 and L41 fusion proteins towards procoagulant substrates such as fibrinogen and PAR-1 was reduced >100-fold while protein C activation was minimally affected (2-fold) compared to the endogenous complex. Conclusions: Similar values of specificity for protein C activation constitute evidence of productive docking between the subunits of thrombin and thrombomodulin in vitro . However, the partial blockade of the procoagulant substrate suggests that further optimization of length, composition and localization of the linker and/or of the protein partners is necessary to achieve an exquisite anticoagulant profile.