Conversion of factor IX (FIX) to FIXa requires proteolytic cleavages after Arg191 and Arg226, releasing an activation peptide (Ala192-Arg226) (Geng Y et al. 2012; Vadivel K & Bajaj SP 2012). This calcium-dependent reaction is catalyzed by factor VIIa (FVIIa) in the presence of tissue factor (TF) and phosphatidylserine-rich phospholipid (Osterud B, Rapaport SI 1977; Banner DW et al. 1996; Bajaj SP et al. 2006). In this reaction, FVIIa and FIX anchor to the phospholipid bilayer through their Gla domains for optimal rates of FIXa formation (Vadivel K & Bajaj SP 2012). Further, the N-terminal Gla and epidermal growth factor-like (EGF1) domains of FIX represent the primary recognition determinants in binding to FVIIa & TF and formation of the ternary complex (Zhong D et al. 2002; Vadivel K & Bajaj SP 2012). In the formed ternary complex, the scissile peptide bond sequence in FIX or FX then approaches the active site cleft in FVIIa and induces the formation of the oxyanion hole for efficient proteolysis (Vadivel K & Bajaj SP 2012). FVIIa, bound to TF at the endothelial cell surface, cleaves FIX first after Arg191, forming the inactive intermediate which is released from FVIIa. The intermediate form of FIX must rebind to the protease to be cleaved after Arg226 to form an activated FIXa. As the second cleavage is rate-limiting, the inactive intermediate accumulates during FIX activation by FVIIa. The proteolytic cleavage of FIX results in a two-chain protein consisting of a light chain (Gla-EGF1-EGF2 domains) and a heavy chain (protease domain with the catalytic center) held together by a single disulfide bond (Yoshitake S et al. 1985). The released activation peptide FIX (192-226) has no known function.