Abstract
In addition to its procoagulant and proinflammatory functions mediated by cleavage of fibrinogen and PAR1, the trypsin-like protease thrombin activates the anticoagulant protein C in a reaction that requires the cofactor thrombomodulin and the endothelial protein C receptor. Once in the circulation, activated protein C functions as an anticoagulant, anti-inflammatory and regenerative factor. Hence, availability of a protein C activator would afford a therapeutic for patients suffering from thrombotic disorders and a diagnostic tool for monitoring the level of protein C in plasma. Here, we present a fusion protein where thrombin and the EGF456 domain of thrombomodulin are connected through a peptide linker. The fusion protein recapitulates the functional and structural properties of the thrombin-thrombomodulin complex, prolongs the clotting time by generating pharmacological quantities of activated protein C and effectively diagnoses protein C deficiency in human plasma. Notably, these functions do not require exogenous thrombomodulin, unlike other anticoagulant thrombin derivatives engineered to date. These features make the fusion protein an innovative step toward the development of protein C activators of clinical and diagnostic relevance.
Highlights
Exogenous administration of Activated protein C (aPC) include reduced risk of bleeding[30,33], unique antiplatelet activity via inhibition of the GPIb-von Willebrand factor interaction[34] and enhanced cytoprotective signaling at the level of the endothelium[35]
A total of six fusion proteins (FPs) with a linker composed of 7, 17, 31, 41, 55 and 69 amino acids were produced in baby hamster kidney cells to identify the optimal length that would allow proper intramolecular complex formation
Thrombin wild-type was fused to TM456 and the kinetics of cleavage of PC, fibrinogen and an extracellular fragment of PAR1 were used to establish if FPs recapitulate the properties of thrombin bound to TM
Summary
Exogenous administration of aPC include reduced risk of bleeding[30,33], unique antiplatelet activity via inhibition of the GPIb-von Willebrand factor (vWF) interaction[34] and enhanced cytoprotective signaling at the level of the endothelium[35] Though promising, these existing PC activators have a major weakness insofar as they fail to generate pharmacological quantities of aPC in clinical settings where TM is down-regulated or inactivated by proteases[13,14,15,16]. The absolute requirement for TM precludes use of such variants as diagnostic tools alternative to Protac To overcome these limitations, we engineered proteins where the N-terminal of the EGF456 domain of TM is fused to the C-terminal of thrombin through a peptide linker of variable length. These innovative thrombin-thrombomodulin fusion proteins (FPs) feature efficient PC activation with only modest activity toward procoagulant and proinflammatory substrates and hold much promise as clinical and diagnostic tools
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