Coagulation Factor IX (FIX) is synthesized by hepatocytes (Yoshitake et al., 1985; Kurachi & Kurachi, 1995). The nascent FIX protein consists of a pre-sequence (28 amino acids), a pro-sequence (18 amino acids), and a mature peptide (415 amino acids), resulting in a total length of 461 amino acids (Yoshitake et al., 1985; Kurachi & Kurachi, 1995; Andersson et al., 1975; Anson et al., 1984). The pre-sequence (signal sequence) facilitates FIX secretion, while the pro-sequence serves as a binding domain for the vitamin K-dependent (VKD) γ-glutamyl carboxylase (GGCX) (Fryklund et al., 1976; Galeffi & Brownlee, 1987; Lingenfelter & Berkner, 1996; Stanley et al., 1998). GGCX, an integral membrane protein in the endoplasmic reticulum (ER) of hepatocytes, carboxylates specific glutamic acid residues within the adjacent Gla domain of FIX (Fryklund et al., 1976; Galeffi & Brownlee, 1987; Hao et al., 2020). During γ-carboxylation, vitamin K hydroquinone is oxidized to vitamin K 2,3-epoxide, and a carboxyl group is added to a glutamic acid residue (Wallin et al., 2002). In its native state, the Gla domain of FIX contains 12 glutamic acid residues; 10 of these residues are conserved across all VKD proteins, while the remaining two are unique to FIX (Gillis et al., 1997). FIX undergoes additional post-translational modifications, including N- and O-linked glycosylation, sulfation, phosphorylation, and hydroxylation, primarily in the ER and Golgi apparatus (reviewed by Zacchi et al., 2021). In the ER, a coordinated network of molecular chaperones and lectins, such as calreticulin (CRT) and calnexin (CNX), ensures proper protein folding and quality control. The unfolded protein response (UPR) induces chaperones like glucose-regulated protein GRP78/BiP to prevent protein aggregation. GRP78/BiP and CRT were found to co-immunoprecipitate with FIX in human hepatocellular carcinoma (HepG2) cell lysates expressing FIX (Enjolras et al., 2004). In the Golgi apparatus, the propeptide (amino acids 29–46) of FIX is cleaved by paired basic amino acid cleaving enzyme (PACE) (Wasley et al., 1993). This cleavage facilitates the formation of (Ca2+)-induced secondary and tertiary structures in the Gla domain, which are essential for FIX activity (Pipe, 2008). The mature FIX is secreted as an inactive 57kDa zymogen form (47-461). Its domains are defined by structure and function: the Gla domain mediates interaction with phospholipid surfaces, two epidermal growth factor (EGF)-like domains are critical for binding factor VIIIa, the activation peptide is released during proteolytic activation, and the serine protease domain enables catalytic activity (Pipe, 2008; Yoshitake et al., 1985; Di Scipio et al., 1977; Rees et al., 1988; Freedman et al., 1995). Secreted factor IX (FIX) exists in two primary pools: plasma-circulating FIX and extravascular FIX. Plasma FIX contributes to the coagulation cascade upon activation at sites of vascular injury. Extravascular FIX, found outside the bloodstream, binds specifically and reversibly to type IV collagen, a major structural component of the endothelial basement membrane (reviewed by Feng et al., 2013; Mann et al., 2021). This interaction is mediated by the N-terminal region of the Gla domain of FIX and occurs with high affinity (~5 nM) (Cheung et al., 1992; 1996; Wolberg et al., 1997; Gui et al., 2002). The binding of FIX to type IV collagen is believed to create an extravascular reservoir, ensuring the availability of FIX at hemostatically active sites (Cloesmeijer et al., 2024). Evidence from in vivo models of hemostasis and thrombosis further supports the critical role of extravascular FIX in hemostatic processes (Machado et al., 2023; Leuci et al., 2024)

FIX activation involves proteolytic cleavage at two sites: R191 (α-cleavage) and R226 (β-cleavage) (Di Scipio et al., 1978; Zögg & Brandstetter, 2009). Activated FIX comprises an N-terminal light chain and a C-terminal heavy chain held together by a disulfide bond between C178 and C335 (Di Scipio et al., 1978; Zögg & Brandstetter, 2009).