Process Biochemistry 48 (2013) 340–350 Contents lists available at SciVerse ScienceDirect Process Biochemistry jo u rn al hom epa ge: www .elsevier.com/locate/procbio Direct acting anti-thrombotic serine protease from brown seaweed Costaria costata Dae-Won Kim a , Kumar Sapkota a,d , Jun-Hui Choi a , Yoon-Sik Kim b , Seung Kim c,∗∗ , Sung-Jun Kim a,∗ a Department of Biotechnology, Chosun University, Gwangju 501-759, Republic of Korea b Department of Clinical Laboratory Science, Dongkang College, Gwangju 500-714, Republic of Korea c Department of Alternative Medicine, Gwangju University, Gwangju 503-703, Republic of Korea d Central Department of Zoology, Tribhuvan University, Kirtipur, Kathmandu, Nepal a r t i c l e i n f o Article history: Received 30 October 2012 Received in revised form 4 December 2012 Accepted 14 December 2012 Available online 25 December 2012 Keywords: Algal protease Costaria costata Fibrino(geno)lysis Thrombosis a b s t r a c t A direct acting antithrombotic serine protease (CCP) was purified from brown seaweed Costaria costata. CCP was a monomeric protease with molecular mass of 60,547.598 daltons as determined by mass spec- trometry. The N-terminal sequence of CCP was SCNSCLDKVDADGLN. Proteolytic activity was inhibited by PMSF and APMSF. CCP exhibited high amidolytic activity toward substrate S-2251 with apparent K m and V max values were 14.5 M and 183.5 U/ml respectively. Fibrin plate and fibrin zymography results revealed that CCP was able to degrade fibrin clots directly. It specifically hydrolyzed A and  and B and  chains followed by  and – chains of human fibrinogen and fibrin respectively. Cleavage of fibrin clot and fibrinogen was emphasized by observing the alteration of secondary structure using FTIR spectroscopy. Morphological alteration of fibrin clot was also evidenced by fluorescent microscopic obser- vation. CCP reduced thrombus effectively in vitro. In vivo observation showed that it prevented/decreased thrombus formation in carrageenan-induced mice tail model. CCP prolonged activated partial thrombo- plastin time (APTT) and had little effect on prothrombin time (PT). Platelet function analyzer (PFA-100) tests showed that CCP prolonged closure time (CT). These data suggest that CCP could have therapeutic potential for the treatment of thrombosis. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction The circulatory disorders of both arterial and venous systems such as myocardial infarction, stroke, deep-vein thrombosis, and pulmonary embolism are substantial and rapidly growing prob- lem around the world. The abnormal blood clot formation inside the blood vessels usually leads to these devastating diseases. Fibrin and fibrinogen play critically important roles in this event. Fibrin is a fibrous protein and plays a principle role in blood clot- ting. Fibrinogen is a plasma glycoprotein that consists of a pair of three polypeptide A, B and  chains [1]. Thrombin cataly- ses the conversion of soluble fibrinogen into insoluble fibrin, the major structural component of blood clots. Fibrinolysis is a crucial mechanism, in which the insoluble fibrin fiber is hydrolyzed into fibrin degradation products by an enzyme system present in the blood [2]. The fibrinolytic process is mediated by a serine protease ∗ Corresponding author at: Department of Biotechnology, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea. Tel.: +82 62 230 6664; fax: +82 62 230 6664. ∗∗ Corresponding author. Tel.: +82 62 670 2718. E-mail addresses: seungk@gwangju.ac.kr (S. Kim), sjbkim@chosun.ac.kr (S.-J. Kim). plasmin, which is derived from plasminogen by plasminogen activators (PAs) such as tissue plasminogen activator (t-PA), and urokinase (u-PA), and is regulated by relevant inhibitors- 2-antiplasmin, and plasminogen activator inhibitor-1 (PAI-1). Fibrinolytic agents also referred to as PAs, have the unique abil- ity to dissolve the blood clots through the activation of intrinsic components of the fibrinolytic system [3]. All currently clinically available fibrinolytic (thrombolytic) agents including anticoagu- lants, antiplatelet and direct thrombolytics, are most effective in treating thrombosis. However, all these agents have significant drawbacks, including bleeding complications [4]. Therefore, the search for safe and effective antithrombotic agent with novel mech- anisms of action that can dissolve a thrombus reliably is needed. Recently, antithrombotic agents from plant sources have received considerable attention for their safe and potential benefi- cial properties. Marine algae have been used as food and medicine for centuries in many countries [5]. They are recognized as rich sources of diverse bioactive substances with important biomed- ical potential such as anticoagulant, antiviral, anti-inflammatory, anti-oxidant, and anti-cancer [6]. Anticoagulant activities, partic- ularly from polysaccharides of marine algae have been reported several decades ago and more than 60 species have been identified to possess such properties [7,8]. Brown seaweeds as staple item 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.12.012