Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Mechanism of the action of SMTP-7, a novel small-molecule modulator of plasminogen activation Keiji Koyanagi, Ritsuko Narasaki, Shingo Yamamichi, Eriko Suzuki and Keiji Hasumi SMTP-7 is a small molecule that promotes the proteolytic activation of plasminogen by relaxing its conformation. SMTP-7 has excellent therapeutic activities against thrombotic stroke in several rodent models. The objective of this study was to elucidate detailed mechanism of the action of SMTP-7 in vitro. We report here that the action of SMTP-7 requires a cofactor with a long-chain alkyl or alkenyl group, and that the fifth kringle domain (kringle 5) of plasminogen is involved in the SMTP-7 action. In this study, we found that the SMTP-7 action to enhance plasminogen activation depended on the presence of a certain type of surfactant, and we screened biologically relevant molecules for their cofactor activity for the SMTP action. As a result, phospholipids, sphingolipids, and oleic acid were found to be active in assisting the SMTP-7 action. On the contrary, stearic acid and bile acids were inactive. Thus, a certain structural element, not only the surface-activating potential, is required for a compound to act as a cofactor for the SMTP-7 action. The plasminogen molecule consists of a PAN domain, five kringle domains, and a serine protease domain. The cofactor-dependent effects of SMTP-7 was observed with plasminogen species including kringle 5 such as intact plasminogen (Glu-plasminogen), des-PAN plasminogen (Lys-plasminogen), and des-[PAN S (kringles 1–4)] plasminogen (mini-plasminogen). However, SMTP-7 effect was not observed with the smallest plasminogen species des-[PAN S (kringles 1–4) and a half of kringle 5)] plasminogen (micro-plasminogen). Thus, kringle 5 is crucial for the action of SMTP-7. Blood Coagul Fibrinolysis 25:316– 321 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Blood Coagulation and Fibrinolysis 2014, 25:316–321 Keywords: conformation, fibrinolysis, plasminogen, SMTP-7 Department of Applied Biological Science, Tokyo Noko University, Tokyo, Japan Correspondence to Keiji Hasumi, Department of Applied Biological Science, Tokyo Noko University, 3-5-8 Saiwaicho, Fuchu-shi, Tokyo 183-8509, Japan Tel: +81 42 367 5710; fax: +81 42 367 5708; e-mail: hasumi@cc.tuat.ac.jp Received 11 April 2013 Revised 24 September 2013 Accepted 13 November 2013 Introduction The plasminogen/plasmin system plays a central role in blood clot lysis [1,2]. The circulating form of human plasminogen (Glu-plasminogen) is a single-chain zymo- gen consisting of an N-terminal PAN domain, five hom- ologous kringle domains, and a trypsin-like serine protease domain. Plasminogen is proteolytically con- verted to the active enzyme plasmin by plasminogen activators such as urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator through specific cleavage at the Arg561-Val562 bond [3]. Plasmi- nogen exhibits a tight conformation due to multiple interdomain interactions [4,5]. The interaction between Lys50 in the PAN domain and the lysine-binding site in the fifth kringle domain (K5) plays a crucial role in maintaining closed conformation [6,7]. The tight confor- mation of plasminogen attenuates the activation by plas- minogen activators [3,8]. Although binding of lysine analogs such as tranexamic acid and 6-aminohexanoic acid to lysine binding sites in some kringle domains leads to relaxation of plasminogen conformation to be suscept- ible to activation by plasminogen activators, these lysine analogs attenuate plasminogen binding to fibrin, which is mediated by lysine binding sites, and thereby inhibits fibrinolysis [9]. We have discovered a family of small molecules desig- nated SMTP from the fungus Stachybotrys microspora [10]. SMTP promotes plasminogen activator-catalyzed plasmi- nogen activation by relaxing plasminogen conformation [10,11]. Unlike lysine analogs, SMTP enhances fibrin- plasminogen binding, suggesting that SMTP relaxes plas- minogen conformation through a mechanism in which the lysine binding sites are not involved, and thus, enabling fibrin binding of plasminogen through lysine binding site(s) [10]. SMTP-7 [11,12], one of the potent SMTP congeners, ameliorates embolic stroke in rodent models [13,14]. These excellent activities may be attributable to the fact that the action of SMTP-7 depends on endogenous plasminogen activators, serving as a physiological on- demand system coping with thrombotic events [10]. In this study, we attempted to elucidate detailed mech- anism of the action of SMTP-7. As a result, we found that the action of SMTP-7 was dependent on a cofactor such as phospholipids, sphingolipids, and oleic acid. The cofactor-dependent SMTP-7 action was observed native plasminogen and truncated form of plasminogen includ- ing intact K5 but not that lacked intact K5. Thus, K5, which harbors a lipid binding site [15], plays a crucial role in phospholipid-dependent SMTP-7 action. 316 Original article 0957-5235 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI:10.1097/MBC.0000000000000032