Abstract

Plasmin (Plm), an active form of plasminogen (Plg), functions as a key enzyme in the fibrinolytic system. Furthermore, this enzyme directly inactivates various coagulation factors such as factor V (FV) and factor VIII (FVIII) by limited proteolysis, suggesting another role of Plm in the regulation of the coagulation system. We recently reported that Plm/Plg interacts with FVIII and its active form (FVIIIa), both dependently and independently of lysine-binding site (LBS) (

Blood
2007
;
110
,
522a
). In this study, we attempted to localize a factor Va (FVa)-interactive region on Plm (and Plg) using Plm/Plg kringle fragments. Surface plasmon resonance-based assays showed that FVa directly bound to active-site modified Plm (anhydro-Plm) with an ~2-fold higher affinity, compared to Plg (Kd; 97 and 198 nM, respectively). In particular, FVa bound to the immobilized-Plg fragment consisting of kringle 1-2-3 domains (K1-3) (Kd: 706 nM), whilst FVa failed to bind both the kringle 4 domain (K4) and Plg fragment consisting of kringle 5 and catalytic domains (K5-CD). A similar experiment using immobilized FVa also revealed that the K1-3 solely bound to FVa. These results were quite different from those obtained by FVIII and Plm/Plg binding experiment that the K5-CD bound to FVIII(a) more preferably. Competitive binding assay using 6-aminohexanoic acid (6-AHA), a competitor of LBS of Plm/Plg, showed that 6-AHA markedly inhibited (by >90%) the K1-3 binding to FVa (IC50; ~25 μM), suggesting that interaction of FVa with Plm is mostly dependent upon LBS. According to the one stage-clotting assay, 6-AHA inhibited (>90%) Plm-catalyzed inactivation of FVa in a dose-dependent manner (IC50; ~10 μM). Furthermore, Plm-catalyzed inactivation of FVa was blocked by an anti-K1-3 monoclonal antibody (mAb), not by either anti-K4 or anti-K5-CD mAb, although Plm-catalyzed inactivation of FVIII was blocked by anti-K5-CD mAb. In order to confirm that the inhibitory effect of 6-AHA on the Plm-catalyzed inactivation, we performed SDS-PAGE experiment. Plm cleaves FVa at Lys309 and Arg348 in the heavy chain, and at Arg1752 in the light chain. SDS-PAGE analysis revealed that 6-AHA blocked Plm-catalyzed cleavages of the light chain more prominently than that of the heavy chain (IC50; ~10 and ~>100 μM, respectively). These findings suggest that the K1-3 of Plm (and Plg) interacts with the FVa through the LBS-dependent mechanisms, and these interactions likely contribute to the FVa-catalyzed inactivation by proteolytic cleavages at Arg1752 in the light chain. Present study indicated that plasmin-catalyzed protelytic inactivation of coagulation factor is complicatedly regulated by the LBS dependency in the protein and protein interaction.

Disclosures: No relevant conflicts of interest to declare.

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