title: Determination of a factor VIII-interactive region within plasmin responsible for plasmin-catalysed activation and inactivation of factor VIII(a).
abstract: Plasmin, an active form of plasminogen, activates and inactivates factor VIII (FVIII) by limited proteolysis. We have previously identified lysine-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 monoclonal 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 plasmin-catalysed 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 A11-336FVIIIa. The K5-CD preferentially bound to the A2 domain (Kdapp; 52 nM), whilst the K1-2-3 and K4 bound to the light chain (Kdapp; 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 approximately 90%, whilst A2/K5-CD binding was moderated by only approximately 35%. Furthermore, an anti-CD antibody blocked plasmin-catalysed cleavage by inhibiting the A2/K5-CD interaction. These data demonstrate 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.
authors.affiliation: Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara, Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Nishiya
authors.forename: Keiji
authors.initials: K
authors.lastname: Nogami
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Okada
authors.forename: Osamu
authors.initials: O
authors.lastname: Matsuo
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeyama
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ogiwara
authors.forename: Midori
authors.initials: M
authors.lastname: Shima
mesh_terms: D019138:Antibodies, Blocking / Q000378:metabolism; D055162:Biocatalysis; D001777:Blood Coagulation; D004789:Enzyme Activation; D004794:Enzyme Repression; D005176:Factor XIII / Q000378:metabolism*; D005341:Fibrinolysin / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D011485:Protein Binding
chemical_list: D019138:Antibodies, Blocking; D005176:Factor XIII; D005341:Fibrinolysin