In plasma, factor XII adopts a zymogen "closed" conformation maintained by intramolecular interactions between heavy-chain domains (fibronectin type 2 (FN2) and kringle (KNG)) and the catalytic domain, protecting the activation site from cleavage (Clark CC et al., 2020; Shamanaev A, Ivanov I et al., 2022; Shamanaev A, Ma Y et al., 2025; reviewed by Shamanaev A, Litvak M, Gailani D, 2022). It is believed that binding of the FXII heavy chain to artificial negatively charged surfaces induces a conformational change that exposes the active site arginine (Arg), thereby permitting proteolytic cleavage. A similar mechanism may occur on biological membranes.

FXII activation occurs through cleavage of a single peptide bond after Arg372, generating activated factor XII (FXIIa), a serine protease composed of a heavy chain (residues 20-372) and a light chain (residues 373-615) held together by disulfide bonds (Fujikawa K and McMullen BA 1983; McMullen BA and Fujikawa K 1985). In vitro studies demonstrate that this cleavage can occur through several mechanisms including autoactivation of factor XII or activation by PKa (encoded by the KLKB1 gene). These reactions require the presence of negatively charged surfaces or cell membranes and are accelerated in the presence of high-molecular-weight kininogen (HK, encoded by the KNG1 gene) (Griffin JH and Cochrane CG 1976; Meier HL et al. 1977; Silverberg M et al. 1980).

Studies suggest that factor XII activation in vivo occurs primarily on endothelial cell surfaces and, as in vitro, is accelerated by association with HK (Mahdi F et al. 2002; Schmaier AH 2004). FXII binds endothelial cells in a specific, saturable, and reversible manner. In the presence of Zn²?, FXII binds endothelial receptors including complement C1q binding protein (C1QBP, also known as globular C1q receptor or gC1qR), urokinase plasminogen activator receptor (uPAR, encoded by the PLAUR gene), and cytokeratin 1 (CK1, encoded by the KRT1 gene) (Mahdi F et al., 2002; Joseph K et al., 2001; Kaira BG et al., 2020; Stavrou EX et al., 2018). Alternative mechanisms for factor XII association with the cell surface have not been excluded (Joseph K et al. 2001). FXII binding alone does not result in FXII activation (Merkulova AA et al., 2023). FXII can remain bound to cultured endothelial cells for up to 2 hours without being activated. However, in the presence of HK and PK on endothelial surfaces, FXII is rapidly converted to FXIIa, establishing a reciprocal amplification loop in which FXIIa generates PKa and PKa further activates FXII (Merkulova AA et al., 2023).

FXII also binds artificial anionic materials such as kaolin and silica (Griffin and Cochrane 1976; Silverberg M et al., 1980), as well as biological surfaces including polyphosphate (Müller F et al., 2009; Engel R et al., 2014), extracellular DNA and NETs (Oehmcke S et al., 2009; Englert H et al., 2021), misfolded proteins (Maas C et al., 2008; Hardy LJ et al., 2023), collagen (van der Meijden PEJ et al., 2009), hemin (Becker CG et al., 1985), extracellular vesicles, and microbial products (Frick IM et al., 2007). Artificial surface binding sites have been identified within the non-catalytic heavy chain of FXII, particularly in the FN2 domain (Citarella F et al., 2000), first epidermal growth factor like (EGF1) (Shamanaev A, Litvak M, Ivanov I et al., 2023; Shamanaev A, Litvak M, Cheng Q et al., 2023), FN1 and EGF2 domains (Beringer DX & Kroon-Batenburg LMJ 2013), KNG domain (Ravon DM et al.,1995), and the proline rich region (PRR) (Heestermans M et al., 2021). However, the importance of each of these domains? in FXII surface binding and activation require further clarification (Clark CC et al., 2020; Shamanaev A, Litvak M, Cheng Q et al., 2023).

The cell surface binding sites for FXII have been identified and largely correspond to regions within the FXII heavy chain that mediate binding to artificial, negatively charged surfaces. Peptides derived from the FN2 region block FXII binding to endothelial cells (Madhi et al, 2002). FXII binds gC1qR at a different region than high-molecular-weight kininogen (HK), allowing formation of a trimolecular complex (Kaira BG et al. 2020). Additional cell surface binding regions (reviewed by Silbak S & Schmaier AH, 2024) include the EGF1 domain (Shamanaev A et al., 2022) and the proline-rich region (Heestermans M et al., 2021) that were also an artificial surface binding sites as well. Of note, two additional peptides on FXII?s kringle region block FXII binding to cells (Elwakiel A et al, 2024). Collectively, these findings suggest that multiple (at least five) regions on FXII participate in cell surface interactions. It is believed that binding of the heavy chain of FXII to artificial negatively charged surfaces or cell membranes induces a conformational shift, unmasking the active site Arg for plasma kallikrein-mediated cleavage (Clark CC et al., 2020; Shamanaev A, Ivanov I et al., 2022; Shamanaev A, Ma Y et al., 2025; reviewed by Shamanaev A, Litvak M, Gailani D, 2022). Mutations affecting the structure of FXII can result in abnormal FXII activation, as observed in carriers of the FXII W287R variant (Hofman ZLM et al., 2020; Scheffel J et al., 2020) which shows increased susceptibility to cleavage by plasmin and plasma kallikrein (Hofman ZLM et al., 2020).

Zn²? promotes the activation of FXII on surfaces (Samuel M et al., 1992; de Maat S et al., 2019; Clark CC et al., 2020; Shamanaev A, Ivanov I et al., 2022; Shamanaev A, Ma Y et al., 2025; reviewed by Shamanaev A, Litvak M, Gailani D 2022; Shamanaev A, Litvak M, Ivanov I et al., 2023) and is essential for FXII to bind to endothelial cell and neutrophil membranes (Mahdi F et al. 2002; Stavrou EX et al., 2018).

FXII (and PK) can be proteolytically cleaved by bacterial enzymes (Blair KM et al., 2025; reviewed by Frick IM et al., 2007), and by endogenous serine proteases. For example, factor XIa, have been reported to activate FXII (reviewed by Gailani D et al., 2015). Furthermore, the endothelial cell protease prolylcarboxypeptidase (PRCP) cleaves PK to PKa independently of FXIIa. PRCP-generated PKa can subsequently activate FXII (Shariat-Madar Z et al., 2002; Merkulova AA et al., 2023).

Activated form of FXII contributes to complement activation, coagulation, fibrinolysis, and along with PKa, to conversion of prorenin to renin (reviewed by Renne T et al., 2012; Maas C & Renne T., 2018; Schmaier AH 2016; Long AT et al., 2016; Shamanaev A, Litvak M et al., 2023; Motta G et al., 2023).

Additionally, FXII has non-canonical signaling activity as a growth factor independent of its serine protease function. Structurally, FXII shares homology with hepatocyte growth factor (HGF), tissue-type plasminogen activator (tPA), and single-chain urokinase plasminogen activator (scuPA) (Miyazawa K et al., 1998). FXII signals through uPAR (PLAUR) via ?-integrins, promoting endothelial cell growth, proliferation, and neoangiogenesis (LaRusch GA et al., 2010), and neutrophil adhesion, migration, chemotaxis, and NETosis (Stavrou E et al., 2018). In lung, smooth muscle, and kidney tissues, this pathway has been associated with cell proliferation and fibrosis (Kalina D et al., 2025). FXII expression in tissues is regulated by SMAD proteins and TGF-? signaling (Jablonska E et al., 2010). Further, FXII-driven fibrotic signaling has been linked to cellular senescence pathways (SenMayo) in tissues after injury, e.g., diabetic kidney disease (Elwakiel A et al., 2024).