Tissue factor (TF) pathway inhibitor alpha (TFPIα), a secreted isoform of TFPI, regulates coagulation primarily by inhibiting the initiation phase of the coagulation cascade. TFPIα consists of three Kunitz-type protease inhibitor domains (K1, K2, and K3) and a basic C-terminal domain, each contributing to its anticoagulant function. The basic C-terminal domain of TFPIα interacts with the exposed acidic region (AR) of either the factor V (FV) intermediate form, generated by limited proteolysis of FV at Arg737 and Arg1046 during the initiation phase, or the natural splice FV isoform (FV-short), which is secreted at low concentrations and is constitutively active (Wood JP et al., 2013; Mast AE 2016; Petrillo T et al., 2021). Both FV intermediate and FV-short lack the basic region of the B domain, enabling high-affinity interaction between their AR and the basic C-terminal tail of TFPIα (Wood JP et al., 2013; Mast AE, 2015; Petrillo T et al., 2021; Mohammed BM et al., 2024). This interaction with TFPIα is thought to prevent cleavage of these FV species at Arg1573 (van Doorn P et al., 2017), thereby inhibiting their prothrombinase cofactor activity and limiting the assembly of the prothrombinase complex (FXa:FVa) during the initiation phase of coagulation. Fully activated FV (FVa), produced by proteolysis at Arg737, Arg1046, and Arg1573, lacks AR and is thus no longer subject to TFPIα-mediated inhibition (Bunce MW et al., 2013; Bos MHA & Camire RM, 2012; Mohammed BM et al., 2023, 2024). In addition to regulating prothrombinase activity, TFPIα inhibits the activity of factor VIIa (FVIIa) and factor Xa (FXa) 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 K1 and K2 domains of TFPIα bind and inhibit the protease activities of FVIIa and FXa, respectively (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 inhibition of FXa (and FVIIa), TFPIα relies on cofactors such as protein S (PROS1) and FV-short (and/or FV intermediate), which synergistically enhance TFPIα's anticoagulant function by forming a trimolecular complex TFPIα:FV intermediate:PROS1 that stabilizes TFPIα in plasma (Hackeng TM et al., 2006; Wood JP et al., 2014; Santamaria S et al., 2017; Dahlbäck B & Tran S 2022; Dahlbäck B et al., 2017, 2024; reviewed by Dahlbäck B 2023; Ahnström J et al., 2024). Protein S (PROS1), a vitamin K-dependent glycoprotein, interacts with the K3 domain of TFPIα (Ndonwi M et al., 2010; Reglińska-Matveyev N et al., 2013). The TFPIα:PROS1 interaction is significantly enhanced upon binding to phospholipid surfaces, which is mediated via the Gla domain of PROS1, facilitating the recruitment of TFPIα to the surfaces (Hackeng TM et al., 2006; Dahlbäck B et al., 2018).