Following limited processing, factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (A1-A2-B) and a light chain (A3-C1-C2), linked by calcium ions (reviewed by Stoilova-McPhie S 2021; Childers KC et al., 2022). FVIII circulates in the bloodstream bound to von Willebrand factor (VWF), which stabilizes it and prevents FVIII degradation (Lollar P et al. 1988; Hill-Eubanks DC et al. 1989). During coagulation, FVIII is cleaved at R391, R759 and R1708, forming activated FVIII (FVIIIa), a heterotrimer of A1, A2, and A3-C1-C2 domains (Pittman DD & Kaufman RJ 1988; Regan LM & Fay PJ 1995; reviewed by Fay PJ 2004, 2006; Shen BW et al., 2008; Camire RM & Bos MHA 2009; Childers KC et al., 2022). Structural studies reveal a trimeric arrangement of A domains atop juxtaposed C domains (Ngo JCK et al., 2008; Shen BW et al., 2008; reviewed by Childers KC et al., 2022). While several proteases are capable of catalyzing these cleavages in vitro, only thrombin is generally considered the primary physiological activator responsible for cleavage of FVIII:VWF complexes (Eaton D et al. 1986; Hill-Eubanks DC et al. 1989; Lollar P et al. 1988; Pieters J et al. 1989).

This Reactome event describes thrombin-catalyzed cleavage of FVIII at R391, R759 and R1708. The polypeptide FVIII (760-1332) of the C-terminal B domain of the heavy chain is released. The a3 acidic polypeptide on the N-terminal side of the A3 domain of the light chain is also released, however, and as this acidic region mediates the association of FVIII with VWF, the activated FVIIIa is released from VWF (Lollar P et al. 1988; Dagil L et al., 2019). The cleavage of FVIII and its dissociation from VWF allows FVIIIa to form the intrinsic tenase complex with activated factor IX (FIXa) on the surfaces of activated platelets (Regan LM & Fay PJ 1995; Fay PJ et al., 2001; Ngo JCK et al., 2008). This complex enhances the conversion of factor X (FX) to FXa, amplifying the coagulation response (van Dieijen G et al., 1981).

FVIIIa is kinetically unstable, with a half-life of approximately six minutes due to the weak attachment of the A2 domain, which can dissociate or degrade via protein C or antithrombin III (Regan LM et al., 1996; Lenting PJ et al., 1998; reviewed by Stoilova-McPhie S 2021). Some direct oral anticoagulant (DOAC) drugs are potent, competitive direct thrombin inhibitors (DTIs). They reversibly and specifically bind both clot-bound and free thrombin (unlike warfarin or heparin), as well as inhibiting thrombin-induced platelet aggregation. These drugs can be synthetic organic compounds (dabigatran, argatroban) or recombinant peptides (lepirudin, bivalirudin, desirudin). Dabigatran (brand name Pradexa) is formulated as a lipophilic prodrug, dabigatran etexilate, to promote gastrointestinal absorption before it is metabolised to the active drug. The kidneys excrete the majority (80%) of unchanged drug (Stangier J et al. 2007). Argatroban is a synthetic inhibitor of thrombin derived from L-arginine, which has a relatively short period of binding only to thrombin’s active site (Hursting MJ et al. 1997). It is given intravenously and is metabolised in the liver. Because of its hepatic metabolism, it may be used in patients with renal dysfunction. Lepirudin (brand name Refludan) is a recombinant hirudin derived from yeast cells (Weitz JI et al. 1990). Hirudin is a naturally occurring anticoagulant produced by the salivary glands of medicinal leeches. Bivalirudin (brand name Angiomax, Angiox) is a synthetic analog of hirudin, with a shorter period of binding to thrombin (Gladwell TD 2002). Desirudin (brand name Iprivask) is another recombinant hirudin derivative that directly inhibits free and fibrin-bound thrombin (Graetz TJ et al. 2011). Melagatran is the active drug formed from the prodrug ximelagatran and is a competitive and rapid inhibitor of thrombin (Gustafsson D et al. 1998). DuP 714 is a potent and specific thrombin inhibitor (Chiu AT et al. 1991).