Isolation and Characterization of Poecistasin, an Anti-Thrombotic Antistasin-Type Serine Protease Inhibitor from Leech Poecilobdella manillensis.

Xiaopeng Tang,Ren Lai,Pengpeng Li,Zilei Duan,Mengrou Chen,James Mwangi

doi:10.3390/toxins10110429

Abstract

Antistasin, first identified as a potent inhibitor of the blood coagulation factor Xa, is a novel family of serine protease inhibitors. In this study, we purified a novel antistasin-type inhibitor from leech Poecilobdella manillensis called poecistasin. Amino acid sequencing of this 48-amino-acid protein revealed that poecistasin was an antistasin-type inhibitor known to consist of only one domain. Poecistasin inhibited factor XIIa, kallikrein, trypsin, and elastase, but had no inhibitory effect on factor Xa and thrombin. Poecistasin showed anticoagulant activities. It prolonged the activated partial thromboplastin time and inhibited FeCl3-induced carotid artery thrombus formation, implying its potent function in helping Poecilobdella manillensis to take a blood meal from the host by inhibiting coagulation. Poecistasin also suppressed ischemic stroke symptoms in transient middle cerebral artery occlusion mice model. Our results suggest that poecistasin from the leech Poecilobdella manillensis plays a crucial role in blood-sucking and may be an excellent candidate for the development of clinical anti-thrombosis and anti-ischemic stroke medicines.

Highlights

Protease inhibitors play important roles in the biological purposes of venomous animals, for example, predation and defense [1,2]
P. manillensis secretions were diluted in phosphate buffer (PB buffer) and three fractions were obtained in the chromatographic step using the Sephadex G-50 column (Figure 1A)
The fraction that inhibits the factor XIIa (FXIIa) activity was subjected to a reverse-phase high performance liquid chromatography (RP-HPLC) using a C8 column (Figure 1C), and the peak with inhibitory activity on

Summary

Introduction

Protease inhibitors play important roles in the biological purposes of venomous animals, for example, predation and defense [1,2]. Toxins 2018, 10, 429 the Kazal [6,7,8,9], Kunitz [10,11,12], Bowman-Birk [13,14,15], SSI [16,17,18,19] and Chelonianin [20,21] families. Antistasin is another kind of serine protease inhibitor that contains cysteine-rich 119-amino-acid protein isolated from Mexican leech Haementeria officinalis [22]. Antistasin showed no close sequence similarity to other known protease inhibitors and became the prototype of a novel family [23,24]

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Discussion

Conclusion