Structure of heavy and light chains of blood coagulation factor VIII (FVIII) involved in the activation of FVIII

ABSTRACT

Structure of human blood coagulation factor VIII (FVIII) in relation to its activation process was investigated. FVIII was purified from a commercial FVIII concentrate by immunoaffinity chromatography and its dissociated subunits, heavy and light chains were isolated. The light chain (FVIII-L) was treated with thrombin or factor Xa (FXa) in order to cleave the peptide at Aug(1689) or Arg(1721), respectively, Reassociation of FVIII-H with either of FVIII-L derivatives, FVIII-L-72 (72 kDa) and FVIII-L-65 (65 kDa) brought about the formation of heterodimers which have similar cofactor activity. The association constant of FVIII-H with FVIII-L-72 was about two-fold faster than that with FVIII-L-65, Cleavage of major FVIII-H with thrombin generated two peptides with molecular weights of 50 kDa (A(1)) and 40 kDa (A(2)) Formation of heterotrimer by reassociation of A(1), A(2) and FVIII-L-72-generated FVIII cofactor activity, while the dimers formed from A(1) or A(2) with FVIII-L-72 had no activity, suggesting that both A(1) and A(2) are required for FVIII activity, Heterotrimers formed from A(1) and A(2) with either of FVIII-L-72 or FVIII-L-65 in the presence of CaCl2 (10 mM) revealed cofactor activity, and they were dissociated into subunits with the loss of activity when EDTA (10 mM) was added, indicating that the formation of heterotrimer, the functional unit of FVIII, from A1, A(2) and FVIII-L is calcium dependent and that the cleavage of FVIII-L by FXa does not inactivate FVIII.