Localization of Factor VIII Interactive Site within Plasmin/Plasminogen Which Is Responsible for Plasmin-Catalyzed Activation/Inactivation of Factor VIII.

Plasmin (Plm), an active form of plasminogen (Plg), not only functions as a key enzyme in the fibrinolytic system, but also directly inactivates several coagulation factors. Especially, factor VIII is inactivated by Plm immediately after the activation by limited proteolytic cleavage at Lys³⁶, Arg³³⁶, Arg³⁷², and Arg⁷⁴⁰ in the heavy chain, and at Arg¹⁶⁸⁹ and Arg¹⁷²¹ in the light chain (Nogami et al. J. Biol. Chem. 2007, 282, 5287). We recently have identified the plasmin-interactive sites on the A2 domain responsible for cleavages at Arg³³⁶ and Arg³⁷², and on the light chain responsible for cleavage at Lys³⁶ (Abst #1991/1709, BLOOD 102/108, 2005/2006). In the present study, we attempted to localize a factor VIII-interactive site on Plm (and Plg). Competitive binding assay using 6-aminohexanoic acid (6-AHA), a competitor of lysine-binding site (LBS) of Plm/Plg, showed that 6-AHA markedly inhibited (by >90%) the light chain binding to active-site modified Plm (anhydro-Plm), whilst inhibited weakly the A2 binding (by ∼30%). These results suggested that the light chain interaction with Plm was mainly dependent upon LBS, but the A2 interaction was independent. The addition of monoclonal antibody (mAb) against Plg kringle 5-catalytic domain (K5-CD) significantly inhibited Plm-catalyzed activation/inactivation of factor VIII or VIIIa with an ∼4-fold lower rate constant. On the other hand, anti-K1-3 and anti-K4 mAbs any little affected. SDS-PAGE analysis revealed that only anti-K5-CD mAb blocked Plm-catalyzed cleavages at Arg³³⁶ and Arg³⁷² by ∼90% in dose-dependent manners (IC₅₀: ∼20 nM). Surface plasmon resonance-based assays showed that the isolated K5-CD bound to factor VIII with an ∼50-fold higher affinity (K_d: 3 nM) compared to the K1-3 and K4, similar to the affinity obtained with anhydro-Plm (K_d: 4 nM). In particular, the K5-CD bound to the A2 domain with an ∼5-fold higher affinity (K_d: 42 nM) than those obtained with the K1-3 and K4. In contrast, both the K1-3 and K4 bound to the light chain predominantly (K_d: 43 and 87 nM, respectively), whilst the K5-CD failed to bind. Furthermore, the addition of a goat antibody against the CD (C-14; Santa Cruz Biotechnology) completely blocked the A2 and K5-CD interaction (by ∼95%). These findings suggest that the CD of Plm (and Plg) interacts with the factor VIII A2 domain through the LBS-independent mechanisms, whilst the K1-3 (and/or K4) interacts with the light chain through the LBS-dependent mechanisms. Furthermore, the CD and A2 interaction would regulate the activation/inactivation of factor VIII by proteolytic cleavages of Arg³³⁶ and Arg³⁷².