Identification of plasmin-interactive sites in the light chain of factor VIII responsible for proteolytic cleavage at Lys36

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Abstract

We have recently reported that plasmin likely associates with the factor VIII light chain to proteolyze at Lys36 within the A1 domain. In this study, we determined that the rate of plasmin-catalyzed inactivation on the forms of factor VIIIa containing A1-(1-336) and 1722A3C1C2, reflecting Lys36 cleavage, was reduced by ∼60%, compared with those containing 1649A3C1C2 and 1690A3C1C2. SDS-PAGE analysis revealed that Lys36 cleavage of factor VIIIa with 1722A3C1C2 was markedly slower than those with 1649A3C1C2 and 1690A3C1C2. Surface plasmon resonance-based assays, using active site-modified anhydro-plasmin (Ah-plasmin) showed that 1722A3C1C2 bound to Ah-plasmin with an ∼3-fold lower affinity than 1649A3C1C2 or 1690A3C1C2 (Kd, 176, 68.2, and 60.3 nM, respectively). Recombinant A3 bound to Ah-plasmin (Kd, 44.2 nM), whereas C2 failed to bind, confirming the presence of a plasmin-binding site within N terminus of A3. Furthermore, the Glu-Gly-Arg active site-modified factor IXa also blocked 1722A3C1C2 binding to Ah-plasmin by ∼95%, supporting the presence of another plasmin-binding site overlapping the factor IXa-binding site in A3. In keeping with a major contribution of the lysine-binding sites in plasmin for interaction with the factor VIII light chain, analysis of the A3 sequence revealed two regions involving clustered lysine residues in 1690-1705 and 1804-1818. Two peptides based on these regions blocked 1649A3C1C2 binding to Ah-plasmin by ∼60% and plasmin-catalyzed Lys36 cleavage of factor VIIIa with A1-(1-336) by ∼80%. Our findings indicate that an extended surface, centered on residues 1690-1705 and 1804-1818 within the A3 domain, contributes to a unique plasmin-interactive site that promotes plasmin docking during cofactor inactivation by cleavage at Lys36. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Nogami, K., Nishiya, K., Saenko, E. L., Takeyama, M., Ogiwara, K., Yoshioka, A., & Shima, M. (2009). Identification of plasmin-interactive sites in the light chain of factor VIII responsible for proteolytic cleavage at Lys36. Journal of Biological Chemistry, 284(11), 6934–6945. https://doi.org/10.1074/jbc.M802224200

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