Abstract

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 (Kd: 3 nM) compared to the K1-3 and K4, similar to the affinity obtained with anhydro-Plm (Kd: 4 nM). In particular, the K5-CD bound to the A2 domain with an ∼5-fold higher affinity (Kd: 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 (Kd: 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.

Author notes

Disclosure: No relevant conflicts of interest to declare.