Determination of a factor VIII-interactive region within plasmin responsible for plasmin-catalysed activation and inactivation of factor VIII(a)^[*]

Summary

Plasmin, an active form of plasminogen, activates and inactivates factor VIII (FVIII) by limited proteolysis. We have previously identifiedly sine-binding site-independent plasmin-interactive sites on the FVIII A2 domain responsible for cleavages at Arg³³⁶ and Arg³⁷², together with lysine-binding site-dependent plasmin sites on the light chain responsible for cleavage at Lys³⁶. We have now characterised FVIII-interactive regions on plasmin. SDS-PAGE analysis demonstrated that a monoclon al antibody (mAb) against kringle (K)5-catalytic domain (K5-CD) of plasmin significantly blocked plasmin-catalysed cleavages at Arg³³⁶ and Arg³⁷². K5-CD fragment and this mAb blocked plasmincatalysed activation and inactivation of FVIII(a). Anti-K1–2–3 and anti-K4 mAbs blocked plasmin-catalysed cleavages at Lys³⁶, and K1–2–3 and K4 fragments inhibited plasmin-catalysed inactivation of A1^1–336 FVIIIa. The K5-CD preferentially bound to the A2 domain (K_d ^app ; 52 nM), whilst the K1–2–3 and K4 bound to the light chain (K_d ^app; 75 and 106 nM, respectively) in ELISA. Binding was attributed to the A2 484–509 region and A3 1690–1705/1804–1818 region, respectively. 6-aminohexanoic acid, a lysine analogue, significantly inhibited the light chain/K1–2–3 (and K4) binding by ∼90%, whilst A2/K5-CD binding was moderated by only ∼35%. Furthermore, an anti-CD antibody blocked plasmin-catalysed cleavage by inhibiting the A2/K5-CD interaction. These data demon strate that the K5-CD of plasmin (and plasminogen) interacts with the A2 domain independent of lysine-binding site, whilst interactions of K1–2–3 and K4 with the light chain are lysine-binding site-dependent. Interactions between the K5-CD and A2 likely constitute the major regulatory mechanism for activation and inactivation of FVIII(a) mediated by cleavage at Arg³⁷² and Arg³³⁶.

Keywords

Activation/inactivation - binding-site - factor VIII(a) - kringle domain - plasmin(ogen)

^* An account of this work was presented at the 49th annual meeting of the American Society of Hematology, 2007, Atlanta, GA. This work was partly supported by grants for MEXT KAKENHI 21591370 and The Mother and Child Health Foundation in Japan.

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