Anticoagulant activity of a natural protein purified from Hypomesus olidus

Abstract A novel anticoagulant protein (E-II-1) was separated and purified from Hypomesus olidus, a unique freshwater fish in northern China. E-II-1 had a molecular mass of approximately 40 kDa with no subunits. The high content of hydrophobic amino acids and negatively charged amino acids in E-II-1 demonstrated that the amino acid compositions might contribute to the anticoagulant activity. E-II-1 contained α-helices 16.75%, β-sheets 42.67%, β-turn 25.58% and random coil 15.00%. In vitro blood coagulation time assay, E-II-1 significantly prolonged the activated partial thrombin time in a dose-dependent manner. Results indicated that E-II-1 acted as anticoagulants through the endogenous pathway with an inhibition of FXa. The specific activity of E-II-1 was 103.50 U/mg at a concentration of 1.00 mg/mL. Therefore, E-II-1 might be one of the promising anticoagulants originated from natural food sources with more safety and less side effects.


Introduction
Human blood coagulation is a complicated process and a series of blood coagulation factors are involved (Guria & Guria 2015), any abnormal factor could lead to the disorders of the blood coagulation. Anticoagulant proteins can slow down the coagulation of blood and prevent the formation of thrombosis to some degree, so the importance of anticoagulant proteins is becoming increasingly recognised. A variety of anticoagulant proteins had been isolated and characterised from natural organisms (Silva et al. 2011;Choi et al. 2013;Thakur et al. 2014). However, few anticoagulant proteins have been isolated from the edible materials so far, especially from the freshwater fish. Hypomesus olidus is a typical freshwater fish in northern China, also known as the Cucumber Fish. This paper focus on separating and purifying the anticoagulant protein from H. olidus, looking forward to provide some support for the prevention of thrombotic diseases or applications of the nutritional foods.

Isolation and purification of anticoagulant protein E-II-1
H. olidus was identified by professors Jingbo Liu (Jilin University) and Gui-Qin Wang (Jilin Agricultural University). A voucher specimen had been kept at the Herbarium of the Institute of Zoology (Chinese Academy of Sciences), and the voucher specimen number was 130915. The identified photo number in Chinese Field Herbarium was 514196.
A novel anticoagulant protein (E-II-1) from H. olidus was separated and purified by a series of steps ( Figure S3). The homogeneity of E-II-1 was confirmed by the elution of one peak with retention time 38.21 min ( Figure S9). E-II-1 showed a single band of approximately 40 kDa, suggesting no subunits contained ( Figure S1 (b)). The molecular mass of E-II-1 was significantly different with other anticoagulant proteins from fish (Jung et al. 2003;Rajapakse et al. 2005), indicated that E-II-1 was a novel anticoagulant protein. E-II-1 had a specific activity of 103.50 U/mg (Table S1), with a purity of 88.13% (calculated by the protein content).

Characteristics of E-II-1
E-II-1 contained hydrophobic amino acid (HAA) 40.29, negatively charged amino acid (NCAA) 23.70 and positively charged amino acid (PCAA) 19.17 (Table S2), which was consistent with that of BmK (Ren et al. 2014). It was reported that NCAA could bind to positively charged thrombin to perform an anticoagulant reaction (Ren et al. 2014). Thus, the high content of HAA and NCAA might be necessary for anticoagulant activities.

Anticoagulant activity in vitro
E-II-1 prolonged APTT significantly, neither PT nor TT were influenced (Figure 1). Moreover, E-II-1 had an inhibitory effect on the factor Xa (but not on IIa) under the existence of AT III ( Figure S12). Results demonstrated that E-II-1 acted only through the endogenous coagulation pathway. Generally, anticoagulant proteins separated from snake venom (Thakur et al. 2014(Thakur et al. , 2015 had higher activities than those of food sources (Silva et al. 2011;Faggio et al. 2016;Sabbione et al. 2015). The specific activity of Rusviprotease from snake venom (Thakur et al. 2015) was 9.30 × 10 4 U/mg, far higher than 103.50 U/mg of E-II-1 in this paper. However, the anticoagulant activity of Codiase from Codium fragile (Choi et al. 2013) was 61.50 U/mg, which was lower than E-II-1. Though the anticoagulant activity of E-II-1 from H. olidus was not high, it might make up the deficiency that high-activity anticoagulants lead to bleeding easily or other side effects.

Conclusion
A fish protein (E-II-1) with anticoagulant activity was separated and purified from H. olidus for the first time. E-II-1 affected the coagulation system through the endogenous pathway with an FXa inhibition. More efforts were necessary to elucidate further anticoagulant activity mechanisms and the structural characteristics of E-II-1. In addition, E-II-1 could be utilised in the anticoagulant therapies or functional foods which would promote good health and well-being.

Supplementary material
Experimental details relating to this paper are available online.

Disclosure statement
No potential conflict of interest was reported by the authors.