Bee Venom Serine Protease Inhibitor Figure 1. Bi-KTI is a Kunitz-type serine protease inhibitor. (A) The alignment of the amino acid sequences for Bi-KTI and known Kunitz-type serine protease inhibitors. Identical residues are shown in solid boxes. The characteristic cysteine residues are indicated by solid circles. The P1 position is marked with an asterisk. The sources for the aligned sequences were B. ignitus (this study, GenBank accession no. JN381496), Rhinoplocephalus nigrescens (GenBank accession no. B5KL37), P. textilis textilinin-4 (GenBank accession no. Q90W98), P. textilis textilinin-1 (GenBank accession no. AF402324), Hoplocephalus stephensii (GenBank accession no. B5L5R7), and Bos taurus aprotinin (GenBank accession no. P00974). The Bi- KTI sequence was used as a reference for the identity/similarity (Id/Si) values. (B) SDS-PAGE (left) and western blot analysis (right) of purified recombinant Bi-KTI expressed in baculovirus-infected Sf9 insect cells. Recombinant Bi-KTI was identified using an anti-Bi-KTI antibody. (C) Enzyme inhibition by Bi-KTI. Trypsin or chymotrypsin was incubated with increasing amounts of Bi-KTI, and the residual enzyme activity was then determined (n = 3). doi:10.1371/journal.pone.0032269.g001 evidence that Bi-KTI inhibits plasmin, we assayed the fibrinolytic activity of this inhibitor on a fibrin plate. Our results showed that the addition of Bi-KTI led to the inhibition of the formation of a clear area (Figure 2B), indicating that Bi-KTI inhibits plasmin by inhibiting the degradation of fibrin into FDPs, which suggests that Bi-KTI has an antifibrinolytic function. We next assayed the ability of Bi-KTI to inhibit important enzymes that belong to the hemostatic system. The results indicate that Bi-KTI has no detectable inhibitory effect on factor Xa, thrombin, or tPA (Figure 3A); however, Bi-KTI strongly inhibited plasmin (Figure 3B), indicating that Bi-KTI has a role as a plasmin inhibitor. We also compared the inhibitory ability of Bi-KTI with that of aprotinin, which is widely used as a plasmin inhibitor [20,21]. In this experiment, the inhibitory activity of Bi-KTI (IC50: 43.53 nM) against plasmin was approximately two-fold weaker than that of aprotinin (IC50: 21.66 nM) (Table 1). Similarly, the Figure 2. Bi-KTI inhibits plasmin. (A) Bi-KTI-mediated plasmin inhibition assay. The number indicates the time (in min) that fibrin was incubated with plasmin or both plasmin and Bi-KTI. The FDPs are shown. (B) The antifibrinolytic activity of Bi-KTI. Plasmin was dropped onto fibrin plates along with different amounts of Bi-KTI, and the plates were then incubated at 37uC for various periods of time. doi:10.1371/journal.pone.0032269.g002 PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32269