During the initiation phase of coagulation, the secreted isoform of tissue factor (TF) pathway inhibitor (TFPI), TFPIα, inhibits the activities of factor Xa (FXa) and factor VIIa (FVIIa) within the heterotetrameric TFPIα:FXa:TF:FVIIa complex at the endothelial surface (reviewed by Broze GJ Jr. & Girard TJ, 2012; Maroney SA et al., 2013; Wood JP et al., 2014; Mast AE & Ruf W, 2022; Ahnström J et al., 2024). In this complex, the Kunitz-type protease inhibitor domain 2 (K2) of TFPIα initially binds and inhibits FXa through the formation of an encounter complex, which is subsequently stabilized via structural isomerization. Subsequently, the K1 domain of TFPIα binds to FVIIa, inhibiting the activity of the TF:FVIIa complex. Inhibition of TF:FVIIa requires the prior formation of the TFPI:FXa complex (Baugh RJ et al., 1998; Peraramelli S et al., 2013, 2014; reviewed by Broze GJ Jr. & Girard TJ, 2012; Ahnström J et al., 2024). For optimal inhibitory effects on FXa (and FVIIa), TFPIα relies on cofactors such as protein S (PROS1) and factor V (FV)-short (and/or FV intermediate). Both the FV intermediate form, generated by limited proteolysis of FV at Arg737 and Arg1046 during the initiation phase, and the natural splice FV isoform (FV-short), secreted at low concentrations, possess an exposed acidic region (AR) that interacts with the basic C-terminal tail of TFPIα (Wood JP et al., 2013). This interaction not only stabilizes TFPIα protein levels in plasma, but also inhibits the prothrombinase cofactor activity of FV, essential for the conversion of prothrombin to thrombin. Protein S (PROS1), a vitamin K-dependent glycoprotein, interacts with the K3 domain of TFPIα. The TFPIα:PROS1 interaction is significantly enhanced upon binding to phospholipid surfaces. The surface binding is mediated by the Gla domain of PROS1, facilitating the recruitment of TFPIα to these surfaces. Together, TFPIα, protein S (PROS1) and truncated FV species form a trimolecular inhibitory complex, TFPIα:FV intermediate:PROS1 (or TFPIα:FV-short:PROS1), which significantly enhances TFPIα's anticoagulant function. In vivo studies confirm the essential roles of protein S (PROS1) and FV-short as cofactors for optimal TFPIα function.

This Reactome event shows the binding of the inhibitory TFPIα:FV intermediate:PROS1 complex to the membrane-bound TF:FVIIa:FXa complex where TFPIα interacts with FXa and FVIIa to limit their procoagulant activities.

Concizumab, a humanized monoclonal antibody, targets the K2 domain of TFPI, preventing its interaction with FXa and thereby restoring thrombin generation (Hilden I et al., 2012; reviewed by Petersen LC, 2012).