Bee Venom Serine Protease Inhibitor inhibitory constants (Ki) of Bi-KTI and aprotinin against plasmin were 3.6 nM and 1.6 nM, respectively (Table 1). To study the mechanism of plasmin inhibition by Bi-KTI, we assessed the formation of plasmin-Bi-KTI complexes via native gel electro- phoresis followed by western blotting (Figure 3C). The electro- phoretic mobility shift assay showed that Bi-KTI binds to plasmin, indicating the formation of a plasmin-Bi-KTI complex. The antifibrinolytic activity of Bi-KTI due to its ability to inhibit plasmin could help alleviate the bleeding caused by plasmin- mediated fibrin clot digestion [7–10,22]. Consequently, the ability of Bi-KTI to inhibit plasmin suggests that Bi-KTI is an antifibrinolytic agent. Antifibrinolytic role of Bi-KTI Bee venom also contains a Bi-VSP that acts as a fibrin(ogen)- olytic serine protease [13]. Thus, we investigated whether Bi-KTI Table 1. The inhibitory activities of Bi-KTI and aprotinin against plasmin. Aprotinin Bi-KTI doi:10.1371/journal.pone.0032269.t001 IC50 (nM) 21.66 43.53 Ki (nM) 1.6 3.6 affects Bi-VSP, plasmin, or both during fibrin(ogen)olysis by determining the activity of Bi-VSP and plasmin in the presence of Bi-KTI. When Bi-VSP and plasmin were not treated with Bi-KTI, the fibrinogen was converted into fibrin by Bi-VSP and the fibrin was degraded into FDPs by both Bi-VSP and plasmin (Figure 4). However, the activities of Bi-VSP and plasmin in the presence of Figure 3. Antifibrinolytic activity of Bi-KTI. (A) Inhibitory activity of Bi-KTI. Factor Xa, thrombin, or tPA was incubated with increasing amounts of Bi-KTI, and the residual enzyme activity was determined (n = 3). (B) Comparison of the inhibitory ability of Bi-KTI with that of aprotinin. Plasmin was incubated with increasing amounts of Bi-KTI or aprotinin, and the residual enzyme activity was determined (n = 3). (C) Western blot analysis of plasmin-Bi-KTI complex formation via native gel electrophoresis. Three micrograms of plasmin were incubated with 1 mg of Bi-KTI, and the samples (plasmin, Bi-KTI, or plasmin-Bi-KTI mixture) were resolved on a 10% polyacrylamide gel. After electrophoresis, the protein samples were incubated with antiserum against plasmin (left) or His-tag (right). The plasmin, Bi-KTI, or plasmin-Bi-KTI complexes are shown. doi:10.1371/journal.pone.0032269.g003 PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e32269