Antifibrinolytic Role of a Bee Venom Serine Protease Inhibitor That Acts as a Plasmin Inhibitor Young Moo Choo1., Kwang Sik Lee1., Hyung Joo Yoon 2 , Yuling Qiu1, Hu Wan1, Mi Ri Sohn 1 , Hung Dae Sohn 1 , Byung Rae Jin1* 1 College of Natural Resources and Life Science, Dong-A University, Busan, Republic of Korea, 2 Department of Agricultural Biology, National Academy of Agricultural Science, Suwon, Republic of Korea Abstract Bee venom is a rich source of pharmacologically active substances. In this study, we identified a bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) that acts as a plasmin inhibitor. Bi-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tissue plasminogen activator. In contrast, Bi-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent; however, this inhibitory ability was two-fold weaker than that of aprotinin. The fibrin(ogen)olytic activities of B. ignitus venom serine protease (Bi-VSP) and plasmin in the presence of Bi-KTI indicate that Bi- KTI targets plasmin more specifically than Bi-VSP. These findings demonstrate a novel mechanism by which bumblebee venom affects the hemostatic system through the antifibrinolytic activity of Bi-KTI and through Bi-VSP-mediated fibrin(ogen)olytic activities, raising interest in Bi-KTI and Bi-VSP as potential clinical agents. Citation: Choo YM, Lee KS, Yoon HJ, Qiu Y, Wan H, et al. (2012) Antifibrinolytic Role of a Bee Venom Serine Protease Inhibitor That Acts as a Plasmin Inhibitor. PLoS ONE 7(2): e32269. doi:10.1371/journal.pone.0032269 Editor: Matthew Bogyo, Stanford University, United States of America Received October 11, 2011; Accepted January 24, 2012; Published February 16, 2012 Copyright: 2012 Choo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the Agenda Project (200901OFT102966290) of the Rural Development Administration, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: brjin@dau.ac.kr . These authors contributed equally to this work. Introduction Serine proteases and serine protease inhibitors, which are found in diverse organisms, are of broad interest because they have diverse physiological functions and affect processes, such as the immune response, hemostasis, fibrinolysis, and the elimination of inflammation [1–3]. Serine proteases and serine protease inhib- itors have been found in snake venom in which many serine proteases exhibit fibrin(ogen)olytic activity [4–6] and serine protease inhibitors demonstrate antifibrinolytic activity [7–10]. Bumblebee (Bombus spp.) venom contains three major compo- nents: bombolitin, phospholipase A2, and serine proteases [11–14]. Our previous studies provided the first evidence of the fibrin (ogen)olytic activity of bumblebee venom serine proteases, which act as prothrombin activators, thrombin-like proteases, and plasmin-like proteases [13,14]. Although several Kunitz-type serine protease inhibitors have been reported to be present in snake venom [7,15–17], the role of serine protease inhibitors in bee venom remains unknown. Although bee venom has attracted considerable interest as a rich source of pharmacological substances [18] and has been used traditionally for the treatment of various diseases [19], the mechanism by which bee venom affects the hemostatic system remains poorly understood. In this study, we showed that the bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) is a plasmin inhibitor that exhibits antifibrino- lytic activity. We also determined how Bi-KTI and B. ignitus venom serine protease (Bi-VSP) are involved in fibrinolysis. The present study demonstrates that Bi-KTI acts as an antifibrinolytic agent, providing support for the use of Bi-KTI as a potential clinical agent. Results and Discussion Bi-KTI is a bee venom Kunitz-type serine protease inhibitor To explore the role of serine protease inhibitors in bee venom, we identified an expressed sequence tag (EST) for a gene encoding a venom serine protease inhibitor (Bi-KTI) in a B. ignitus cDNA library. Bt-KTI consists of 82 amino acids (aa), including a predicted 24-aa signal peptide and a 58-aa mature peptide (GenBank accession number JN381496). Database searches showed that the mature Bt-KTI peptide contains features consistent with snake venom Kunitz-type inhibitors [7,15–17], including six conserved cysteine residues and a P1 site (Figure 1A). Recombinant Bi-KTI was expressed as a 6.5-kDa peptide in baculovirus-infected insect cells (Figure 1B). Using recombinant Bi-KTI, we investigated the inhibitory effects of the enzyme and found that Bi-KTI is a Kunitz-type trypsin-like inhibitor (Figure 1C). Collectively, these data indicate that Bi-KTI is a member of the Kunitz-type inhibitor family [7,15–17]. Bi-KTI acts as a plasmin inhibitor Given that Bi-KTI is a Kunitz-type inhibitor [7–9], we first assessed whether Bi-KTI inhibits plasmin by determining the time course of human fibrin degradation. We found that Bi-KTI significantly inhibited the degradation of fibrin into fibrin degradation products (FDPs) (Figure 2A). To obtain direct PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32269