Bi-KTI were similar to the activity of Bi-VSP alone (Figure 4). Whereas both Bi-VSP and plasmin were inhibited by Bi-KTI, the activity of Bi-VSP in the presence of plasmin was not significantly affected by Bi-KTI. These results show that Bi-KTI strongly inhibited plasmin during fibrinolysis, indicating that Bi-KTI specifically targets plasmin, as has been demonstrated for textilinin-1, a Kunitz-type inhibitor from Pseudonaja textilis venom [7–10]. This result further defines a specific role for Bi-KTI as a plasmin inhibitor. Given that each bee venom component must be in balance with its own function, Bi-VSP and Bi-KTI appear to play important roles in an efficient process because Bi-VSP acts as a fibrin(ogen)- olytic agent and Bi-KTI acts as an antifibrinolytic agent. The findings that Bi-VSP activates prothrombin and that Bi-VSP also acts as a fibrin(ogen)olytic protease [13] suggest that Bi-VSP is used to facilitate the spread of bee venom throughout the bloodstream, as has been demonstrated for snake venom fibrin(ogen)olytic enzymes, which remove fibrinogen effectively and, thereby, reduce blood viscosity [23,24]. Given the similarity in plasmin targeting with textilinin-1, an anti-bleeding agent [7–9], Bi-KTI is likely to be an antifibrinolytic agent that reduces bleeding at the sting site of victims. Taken together, our results suggest that prothrombin activation by and the fibrin(ogen)olytic activity of Bi-VSP and the inhibition of plasmin by Bi-KTI may act in a cooperative manner to promote the spread of bee venom under anti-bleeding conditions. Conclusion Our results reveal that bumblebee venom affects the hemostatic system through plasmin inhibition by Bi-KTI and through prothrombin activation and Bi-VSP-mediated thrombin- and plasmin-like protease activities (Figure 5). These findings may have therapeutic significance. Although the antifibrinolytic activity of Bi-KTI must be investigated in animal models and human studies, we propose that this protein can function as a clotting factor and may represent a potential clinical agent. Materials and Methods Gene cloning and sequence analysis A clone encoding Bi-KTI was selected from the ESTs generated from a cDNA library produced using the venom glands of B. ignitus worker bees [13]. Plasmid DNA was extracted using the Wizard Mini-Prep Kit (Promega, Madison, WI) and sequenced using an ABI 310 automated DNA sequencer (Perkin-Elmer Applied Bee Venom Serine Protease Inhibitor Figure 5. The proposed mechanism for Bi-VSP- and Bi-KTI- mediated fibrin(ogen)olytic and antifibrinolytic activities. Bi- VSP activates prothrombin and degrades fibrinogen into FDPs [13]. Bi- KTI inhibits plasmin, which degrades fibrin into FDPs. doi:10.1371/journal.pone.0032269.g005 Biosystems, Foster City, CA). The sequences were compared using DNASIS and BLAST (http://www.ncbi.nlm.nih.gov/ BLAST). Protein expression and purification A baculovirus/Sf9 insect cell expression system [13] was used for the production of recombinant Bi-KTI. A Bi-KTI cDNA fragment containing the full-length open reading frame was inserted into the pBAC1 vector (Clontech, Palo Alto, CA) to generate an expression vector that drives the expression of the recombinant protein under the control of the Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin promoter. Recombi- nant baculoviruses were propagated in Sf9 cells cultured in TC100 medium (Gibco BRL, Gaithersburg, MD) at 27uC. The recombinant proteins were purified using the MagneHisTM Protein Purification System (Promega, Madison, WI). The protein concentrations were determined using a Bio-Rad Protein Assay Kit. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were performed as described previously [13] using an Enhanced Chemiluminescence Western Blotting Analysis System (Amersham Biosciences, Piscataway, NJ). Measurement of protease and inhibitor activity Trypsin (400 ng, Sigma) or a-chymotrypsin (400 ng, Sigma) was incubated in 100 mM Tris-HCl (pH 8.0) containing 20 mM CaCl2 and 0.05% Triton X-100 with increasing amounts of Bi- KTI at 37uC for 30 min. The residual enzyme activity was determined at 405 nm or 410 nm using the following substrates: Figure 4. Fibrin(ogen)olytic and antifibrinolytic activities of Bi-VSP and Bi-KTI. The fibrin(ogen)olytic activities of Bi-VSP and plasmin in the presence of Bi-KTI were assayed. Human fibrinogen was incubated with the following: plasmin; plasmin and Bi-KTI; Bi-VSP; Bi-VSP and plasmin; or Bi- VSP, plasmin, and Bi-KTI. The fibrin(ogen)olytic activity was then determined after various periods of time. doi:10.1371/journal.pone.0032269.g004 PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32269