Factor VIII is inactivated by limited proteolytic cleavages at Arg336 and Lys36 by plasmin (Abst #1017 and 1018,

BLOOD 106, 2005
). We recently identified a plasmin-interactive site in the A2 domain responsible for cleavage at Arg336, and also reported that the cleavage at Lys36 was selectively regulated by the light chain that might interact with its protease. In the current study, several approaches were employed to examine the localization of plasmin-interactive site(s) responsible for cleavage at Lys36. Rate constant of plasmin-catalyzed factor VIIIa inactivation by the addition of the 1649A3-C1-C2 (intact light chain) or 1690A3-C1-C2 fragment was reduced by ~2-folds in a dose-dependent manner using one-stage clotting assay, but not by the 1722A3-C1-C2. SDS-PAGE analysis revealed that Lys36 cleavage in A1/1722A3-C1-C2 dimer by plasmin was markedly slower than that in the A1/1649A3-C1-C2 or A1/1690A3-C1-C2 dimer. Surface plasmon resonance-based assay using anhydro-plasmin (Ah-plasmin), catalytically inactive derivative of plasmin in which the active-site serine was converted to dehydroalanine, showed that the 1722A3-C1-C2 bound to Ah-plasmin (Kd; 191 nM) with an ~3-fold lower affinity compared with 1649A3-C1-C2 or 1690A3-C1-C2 (68 or 83 nM, respectively). Recombinant A3 domain, expressed in E.Coli, also bound to Ah-plasmin with similar affinity (Kd; 44 nM), but the C2 failed to bind, suggesting the presence of a plasmin-binding site within N-terminus of the A3 domain. On the other hand, Glu-Gly-Arg-active-site modified factor IXa blocked the light chain binding to Ah-plasmin by ~60% (Ki: 10.5 nM), supporting that another plasmin-binding site juxtaposes or overlaps with a factor IXa-binding site (residues 1804–1818) in the A3 domain. The 6-aminohexanoic acid, a competitor of lysine-binding site of plasmin, blocked by >90% the light chain and Ah-plasmin interaction (Ki; 6.8 microM). Based on the major contribution of lysine-binding site(s) of plasmin for interaction with the light chain, evaluation of the A3 sequence revealed two regions involving clustered lysine residues of 1690–1705 and 1804–1818. Two synthetic peptides corresponding to these regions blocked the light chain binding to Ah-plasmin by ~50% (Ki: ~20 microM). We conclude that an extended surface centered the 1690–1705 and 1804–1818 regions within the A3 domain interacts with plasmin for the cleavage at Lys36 within the A1 domain.

Topics:
antigens, cd98 light chains, cytokinesis, factor viii, plasmin, proteolysis, hypertension, mechlorethamine, lysine, 6-aminocaproic acid, endopeptidases

Disclosure: No relevant conflicts of interest to declare.

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2006, The American Society of Hematology
2006
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