Thromb Haemost 2021; 121(10): 1274-1288
DOI: 10.1055/s-0041-1723996
Coagulation and Fibrinolysis

Acidic Region Residues 1680–1684 in the A3 Domain of Factor VIII Contain a Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689

Yuto Nakajima
1  Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
Hiroaki Minami
1  Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
Keiji Nogami
1  Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
› Author Affiliations
Funding This work was partly supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) to K.N. (18K07885) and by a research grant for health science, Health and Labor Sciences Research Grants for Research on HIV/AIDS, and Japan Agency for Medical Research and Development (AMED) under grant number JP20fk0410017.


Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg372, Arg740, and Arg1689. Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg1689. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg1689. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54–65 residues). Two synthetic peptides, spanning residues 1659–1669 with sulfated Tyr1664 and residues 1675–1685 with sulfated Try1680, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg1689. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664–1666 and 1683–1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg1689 in the alanine-substituted rFVIII mutants within 1663–1666 residues were similar to those of wild type (WT). Similar studies of 1680–1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg1689 in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680–1684 residues containing sulfated Tyr1680 in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg1689.


An account of this work was presented at the ASH 2019 annual meeting and exposition, Orland, Florida, United States, December 9, 2019.

Authors' Contributions

Y.N. performed the experiments, analyzed the data, made the figures, and wrote the paper; H.M. performed the experiments; K.N. designed the research, interpreted the data, wrote the paper, edit the manuscript, and approved the final version to be published.

Supplementary Material

Publication History

Received: 27 August 2020

Accepted: 02 January 2021

Publication Date:
16 February 2021 (online)

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