The Acidic Region of Factor VIII Light Chain Contains the Thrombin-Binding Site(s) Responsible for the Cleavage at Arg1689
Authors: Minami Hiroaki , Nogami Keiji , Shima Midori 
Published: October 2019

Thrombin-catalyzed activation of factor (F)VIII by cleavages at Arg372, Arg740, and Arg1689 is essential for the propagation phase of blood coagulation cascade. Activated FVIII (FVIIIa) forms the tenase complex and markedly amplifies the activation of FX as a cofactor of FIX. We had already demonstrated that thrombin interacts with FVIII 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), and recently reported that the A1 acidic clustered region 340-350 involving the sulfated tyrosine regulate the cleavage of Arg372 (Minami et al. 55th ASH). On the other hand, Fay and colleague suggested that recombinant FVIII lacking the C2 domain retains greater than 50% cofactor activity (JBC 2010), supporting the presence of other thrombin-binding region responsible for cleavage at Arg1689 of the light chain. In this study, we attempted to identify this thrombin-binding site(s). We focused on the acidic residues 1659-1669 and 1675-1685 within the light chain, which had similar sequence to the A1 residues 340-350 in terms of involving the clustered acidic residues and sulfated tyrosine as well as hirugen residues 54-65. We prepared four of synthetic peptides corresponding to the residues 1659-1669 and 1675-1685 with sulfated tyrosine, P(1659-69s) and P(1675-85s), and with non-sulfated tyrosine, P(1659-69) and P(1675-85). The inhibitory effect on the thrombin-catalyzed FVIII activation by each peptide was evaluated in a one-stage clotting assay. Each peptide showed a dose-dependent inhibition on thrombin-catalyzed activation. These inhibitory effects were greater in order of P1675-85s, P1659-69s, P1675-85, P1659-69, and the IC50 were 25, 67, 71 and 225 µM, respectively. The peptides with sulfated tyrosine had approximately 3-fold greater inhibition of the FVIII activation by thrombin than with non-sulfated tyrosine. The IC50 in the presence of mixture of P1675-85s and P1659-69s was 30.4 µM, suggesting that these peptides had no an additive effect. The impacts of P1659-69s and P1675-85s on the thrombin-catalyzed cleavage at Arg1689 were examined by SDS-PAGE/western blotting. These peptides blocked the cleavage at Arg1689 in dose-dependent fashions. In timed-course assay, the presence of P1659-69s and P1675-85s decreased the cleavage rate of Arg1689 by 61.3 % and 81.8 %, respectively compared to its absence. The direct binding of P1659-69s and P1675-85s to thrombin was examined by surface resonance plasmon (SPR)-based assay and by the zero-length cross-linking reagent EDC. In SPR-based assay using a Biacore T200TM, thrombin bound to immobilized P1659-69s and P1675-85s directly with high affinity. The Kd values adjusted to 1:1 binding model of global fitting were 203 nM and 94 nM, respectively. EDC cross-linking in fluid-phase assay revealed that formation of EDC cross-linking products between biotinylated P1659-69s or P1675-85s and thrombin were observed in dose-dependent fashions. The products between the biotinylated peptides (800 nM) and thrombin were competitively reduced by the addition of non-biotinylated peptides. Moreover, N-terminal sequence analysis of cross-linking products between both peptides-thrombin indicated that thrombin bound to the residues 1664-1669 and 1683-1684. Taken together, we demonstrated that the A3 residues 1659-1669 (QEEIDYDDTIS) and residues 1675-1685 (EDFDIYDEDEN) contained the thrombin binding-sites responsible for proteolytic cleavage at Arg1689 of the A3 domain.

Disclosures

Nogami: Bayer, NovoNordisk, Baxalta, Chugai, Kaketsuken, Pfizer, Biogen: Honoraria; Chugai: Membership on an entity's Board of Directors or advisory committees; Bayer, Novo Nordisk, Baxalta. Biogen: Research Funding. Shima:Chugai Pharmaceutical Co., Ltd. and F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.