Identification Of The Thrombin-Binding Site On Factor VIII Regulating Arg372 Cleavage In The Factor VIII Heavy Chain
Authors: Minami Hiroaki , Nogami Keiji , Yada Koji , Shima Midori 
Published: October 2019

Abstract
Factor VIII is activated by cleavage at Arg372, Arg740, and Arg1689 by thrombin. Activated factor VIII (VIIIa) forms the tenase complex and markedly amplifies the activation of factor X as a cofactor of factor IX. We had demonstrated that thrombin interacts with factor VIII through the residues 392-394 and 484-509 in the A2 domain and the C2 domain, and each association regulates cleavage at Arg740, Arg372, and Arg1689, respectively (Nogami K, JBC 2000, 2005; BJH 2008). The A2 residues 484-509 partially contribute to cleavage at Arg372 by thrombin, however, the major thrombin binding-site(s) regulating cleavage at Arg372 is unclear. Thrombin recognizes macromolecular substrates and cofactors through either or both of two anion-binding exosite I and II (ABE-I and -II), which are characterized by a high density of solvent-exposed basic residues. ABE-I binds to fibrinogen and hirudin (residues 54-65), whilst ABE-II is primarily characterized as the heparin-binding exosite. The A1 domain of factor VIII also binds to thrombin through the ABE-I (Nogami K. JBC 2005). In this study, we attempted to identify the thrombin-binding region on A1, and focused on the A1 residues 340-350, involving the clustered acidic residues and similar sequences of hirudin (residues 54-65). A synthetic peptide corresponding to the A1 residues 340-350 with sulfated Tyr346 (340-350-S(+)) was prepared to investigate factor VIII interaction with thrombin. Activation of factor VIII (100 nM) by thrombin (0.4 nM) with various concentrations of peptide was evaluated by measurement of factor VIIIa activity in a one-stage clotting assay. A 340-350-S(+) peptide showed a dose-dependent inhibition (by ∼60%) of thrombin-catalyzed activation, and the IC50 was 75 µM. A non-sulfated peptide also showed a modest inhibition by ∼40% (IC50 >400 µM), however. An experiment using thrombin substrate S-2238 demonstrated that P340-350-S(+) did not affect the thrombin activity. The effect of 340-350-S(+) peptide on the thrombin-catalyzed cleavage of heavy chain was further examined by SDS-PAGE/western blotting.The peptide significantly blocked the cleavage at Arg372 in a timed- and dose-dependent manner (IC50; 150 µM), whilst of interest the cleavage at Arg740 was little affected. A non-sulfated peptide also delayed the cleavage at Arg372, with a modest fast cleavage compared to sulfated one. The peptide did not inhibit factor FXa-catalyzed reaction to factor VIII. Direct binding of 340-350-S(+) peptide to thrombin was examined by a surface resonance plasmon (SPR)-based assay and by the zero-length cross-linking reagent EDC. In SPR-based solid phase assay, thrombin bound to immobilized 340-350-S(+) peptide with high affinity (Kd; 1.13 nM). EDC cross-linking fluid phase assay similarly revealed that formation of EDC cross-linking product between the biotinylated 337-350-S(+) peptide and thrombin were observed, and this cross-linking was completely inhibited by non-labeled 340-350-S(+) peptide (IC50; 1.0 µM). Taken together, we demonstrated that the A1 residues 340-350 (NEEAED(sY)DDDL) involving sulfated Tyr346 contained the thrombin binding-site responsible for the proteolytic cleavage at Arg372 in factor VIII.

Disclosures:
No relevant conflicts of interest to declare.