Activation of prothrombin by two subtilisin-like serine proteases from Acremonium sp. Chunli Liu a , Yasuhiko Matsushita b , Kosuke Shimizu a , Koichi Makimura c , Keiji Hasumi a, * a Department of Applied Biological Science, Tokyo Noko University, 3-5-8 Saiwaicho, Fuchu-shi, Tokyo 183-8509, Japan b Gene Research Center, Tokyo Noko University, 3-5-8 Saiwaicho, Fuchu-shi, Tokyo 183-8509, Japan c Teikyo University Institute of Medical Mycology, 359 Ohtsuka, Hachioji, Tokyo 192-0395, Japan Received 13 April 2007 Available online 30 April 2007 Abstract Two novel subtilisin-like serine proteases (AS-E1 and -E2) that activate prothrombin have been identified in a culture of the fungus Acremonium sp. The enzymes were purified through repeated hydrophobic interaction chromatography. The N-terminal sequences of AS-E1 (34.4 kDa) and AS-E2 (32 kDa) showed high similarity to the internal sequences of two distinct subtilisin-like hypothetical pro- teins from Chaetomium globosum. Both enzymes proteolytically activated prothrombin to meizothrombin(desF1)-like molecules, while the activation cleavage seemed to occur at a site (Tyr 316 -Ile 317 ) that is four residues proximal to the canonical Xa cleavage site (Arg 320 -Ile 321 ). Both enzymes inhibited plasma clotting, possibly due to extensive degradation of fibrinogen and production of meizo- thrombin(desF1)-like molecule. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Subtilase; Subtilisin-like serine protease; Prothrombin; Meizothrombin(desF1); Blood coagulation; Hemostasis The blood coagulation is an important defense system, protecting the body against blood loss from injured vessels. Prothrombin is a vitamin K-dependent zymogen that is converted to thrombin during the penultimate step of the blood coagulation cascade. Under physiological condi- tions, prothrombin is activated to thrombin on cell surfaces by the prothrombinase complex consisting of Xa, Va, and phospholipid membranes [1]. Although Xa alone is capable of the activation, the rate is <10 5 times as compared to the activation by the prothrombinase [2]. Thrombin promotes plugging of damaged vessels by activating platelets and converting fibrinogen to a fibrin clot [3,4]. In addition to the hemostatic role, thrombin is also involved in the inflammation processes [5]. Proteases from foreign sources are thought to be virulence factors in inflammatory events occurring at infected sites. An example is several snake venom enzymes that activate prothrombin [6]. With regard to microbial proteases that activate prothrombin, only a few enzymes have been studied in detail. These include metalloproteinases from Staphylococcus aureus [7] and Bacillus megaterium (bacillolysin MA) [8], and cysteine proteinases from Porphyromonas gingivalis (gingipains) [9]. In our attempts of a screen of microorganisms that mod- ulate coagulation and fibrinolytic systems, we have found that a fungus produced potent enzymes that activate pro- thrombin. In the present study, we describe the identifica- tion, purification, and properties of two novel enzymes belonging to a family of the serine protease subtilase. Experimental procedures Microorganism and purification of AS-E1 and -E2. Strain F11177 was originally isolated from a soil sample and identified as Aremonium sp. based on morphological studies and 28S rDNA D1–D2 domain sequence (Supplementary Methods). The enzymes were produced as described in Supplementary Methods. Culture supernatant (451 ml) was brought to 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.04.133 * Corresponding author. Fax: +81 42 367 5708. E-mail address: hasumi@cc.tuat.ac.jp (K. Hasumi). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 358 (2007) 356–362