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RSC Advances PAPER View Article Online View Journal | View Issue Interaction of the synthetic antithrombotic peptide P10 with thrombin: a spectroscopy study Cite this: RSC Adv.,2019,9, 18498 Fangyuan Chen, Han Jiang, Wenwei Chen and Guangrong Huang * Thrombin is a critical serine protease in the coagulation system and is widely used as a target protein for antithrombotics. Spectroscopic analysis is a simple and effective method that is used to study the interaction between small molecules and proteins. In this study, the interactions of a potential antithrombotic peptide AGFAGDDAPR (P10) with thrombin were investigated by fluorescence spectroscopy, UV-vis spectroscopy, circular dichroism, Fourier-transform infrared spectroscopy and Raman spectroscopy, respectively. The results showed that the peptide P10 bonded to thrombin via hydrogen bonding and van der Waals forces, resulting in fluorescence quenching. And, the secondary structure of Received 22nd April 2019 thrombin changed, the b-sheet decreased, and the random coil increased. The peptide P10 bonded to Accepted 29th May 2019 proline and lysine, and changed the space structure of thrombin, resulting in inhibition of thrombin activity. DOI: 10.1039/c9ra02994j The results contributed to exploration of the mechanism of this potential antithrombotic drug interaction Creative Commons Attribution 3.0 Unported Licence. rsc.li/rsc-advances with thrombin in order to provide a preliminary understanding of the pharmacodynamic properties of P10. Introduction isolated and identied from natural materials and have demonstrated many biologically active functions such as The coagulation process is a cascade of proteolytic reactions, bacteriostatic, antioxidant, hypotensive, hypoglycemic, etc.10,11 divided into an intrinsic pathway and an extrinsic pathway. Many naturally occurring antithrombotic biologically active Although the cascade starts with two different mechanisms, its peptides have been found to be specic inhibitors of thrombin. ultimate goal is to convert soluble brinogen to insoluble brin. Typically, a 60-amino acid peptide puried from the extract of This article is licensed under a Both pathways are combined at the activation step of thrombin Ornithodoros moubata,12 a peptide called draculin isolated from in the cascade.1 Therefore, thrombin is considered as a critical the saliva of the Desmodus rotundus13 and AduNAP4 from the serine protease in the blood coagulation process.2 In general, human hookworm14 have already displayed antithrombotic Open Access Article. Published on 11 June 2019. Downloaded 10/2/2021 9:15:30 PM. thrombin consists of a 36-residue light chain and a 259-residue activity via interacting with thrombin. In particular, hirudin, heavy chain that can be mainly divided into the active site, a peptide with 65 or 66 amino acids extracted from leeches, has exosite 1 and exosite 2, which are the regions where thrombin been widely studied as a highly effective direct thrombin exerts its coagulation function.3,4 The thrombin active site inhibitor.6,15,16 Some peptides derived from organism proteins cutting brinogen into brin is located in the gap formed at the by enzymatic hydrolysis also exhibit antithrombotic activity, interface of the two barrels.5 The substrate recognition of such as peptide FQSEEQQQTEDELQDK from casein hydroly- thrombin is regulated by two external sites responsible for sate.17 In particular, a peptide P10 (AGFAGDDAPR) isolated substrate turning and orientation in the protease catalytic site. from Tenebrio molitor with enzymatic hydrolysate exhibited Two exosites regulate the substrate recognition of thrombin, antithrombotic activity in our previous study. which are remote from the active site and responsible for Information about the binding between antithrombotic substrate turning and orientation.6 Exosite 1 binds to the peptides and thrombin is the basis for understanding the extracellular domain of brinogen, thrombomodulin and the pharmacodynamic properties of antithrombotic peptides. At type-1 protease-activated receptor on the platelet. Exosite 2 present, studies on the interaction of antithrombotic peptides binds to b2-glycoprotein-1 and determines the position of and thrombin mainly focus on methods of X-ray diffraction and thrombin on the platelets by interacting with glycoprotein Iba molecular simulation.18,19 But they have limitations in the receptors.7–9 understanding of molecular structure, function, and physical Peptides have attracted great interest in medical or health properties in solution which is required by the latest develop- protection in the past few decades. Various peptides have been ments in protein structure research. Spectral methods that are not limited to the morphology of molecules have attracted great interest and have been used to effectively reveal the structure of Key Lab of Marine Food Quality and Hazard Controlling Technology of Zhejiang proteins in solution.20 In this work, the interaction between an Province, College of Life Sciences, China Jiliang University, Hangzhou, China. antithrombotic peptide P10 and thrombin was studied by E-mail: [email protected]; Tel: +86 571 8687 5628 18498 | RSC Adv.,2019,9, 18498–18505 This journal is © The Royal Society of Chemistry 2019 View Article Online Paper RSC Advances uorescence spectroscopy and UV-vis absorption spectra. subunit binding, substrate binding and denaturation can lead Circular dichroism (CD), Fourier-transform infrared spectros- to changes in the intrinsic uorescence of thrombin.28 The copy (FT-IR) and Raman spectroscopy were used to analyze the effect of P10 on the uorescence intensity of thrombin is shown effect of peptides on the thrombin structure. The mechanism of in Fig. 2A. The excitation wavelength was xed at 280 nm, and interaction of the peptide with the thrombin molecule is ex- thrombin had a strong uorescence emission band at 331 nm. pected to be initially revealed. At the same time, it also aims to The uorescence emission peaks at 331 nm indicated that the provide a comprehensive and detailed spectroscopic analysis tryptophan residues were the major contributing uorophores method for revealing the interaction between small molecule in all three protein species. Obviously, the uorescence inten- ligands and macromolecular receptors. sity of thrombin reduced gradually with the P10 concentration, but the maximum emission wavelength and shape of the peak was not affected. This suggests that P10 might interact with Results and discussion thrombin and quench its intrinsic uorescence. No spectral The antithrombotic activity of the synthesized peptide P10 shi of the emission spectrum was observed in the P10 plus thrombin mixture, indicating that the hydrophobicity and The in vitro antithrombotic activity of P10 was measured polarity of the chromophore did not change.29 quantitatively and the results are shown in Fig. 1A. The peptide The quenching mechanism. The Stern–Volmer plot at 290 K concentration showed a dose-responsive behavior to the and 310 K was plotted to determine the quenching mechanism antithrombotic activity. These data were processed using the À1 (static quenching or dynamic quenching), as shown in Fig. 2B. probit program in SPSS to give the IC50 ¼ 0.16 mg mL (inhi- F and F represented the uorescence intensity of thrombin in bition of 50% thrombus formation) (Fig. 1B).21 Ren et al.22 0 the absence and presence of P10, respectively. And [Q] repre- puried an anticoagulant peptide with the sequence sented the concentration of the quencher. In general, the VEPVTVNPHE from Buthus martensii Karsch, which inhibited À1 stability and quenching constants of the compounds formed thrombin activity with an IC50 of 0.012 mg mL . In addition, Creative Commons Attribution 3.0 Unported Licence. under the static quenching mechanism decrease with three peptides SWAQL, GNHEAGE and CFNEYE were puried increasing temperature. The interaction under dynamic from the Alcalase 2.4L hydrolysate of peanut protein, showing quenching increases the number of effective collisions and complete inhibition of thrombin activity at a concentration of À enhances energy transfer, thus increasing the quenching 0.4 mg mL 1.23 However, the synthesized KNAENELGEVTVR constant of the uorescent material as the temperature illustrated 67.21% antithrombotic activity at a concentration of À increases.26,30 The ratio of F /F was linear with the concentration 1mgmL 1.24 Although P10 had a low antithrombotic activity, it 0 of P10, and the slope of the quenching curve at 290 K was higher may reduce the probability of bleeding or other side effects.25 than that at 310 K (Fig. 2B). This indicated that uorescence quenching between This article is licensed under a Fluorescence quenching of thrombin by P10 thrombin and P10 was static quenching. Fluorescence quenching. Fluorescence spectroscopy To con rm this conclusion, the uorescence quenching data – 31 provides information on the structure and kinetics of the was further analyzed by the Stern Volmer eqn (1). Where Ksv Open Access Article. Published on 11 June 2019. Downloaded 10/2/2021 9:15:30 PM. – protein that is oen used to study protein folding and binding represented the quenching constant of the Stern Volmer. reactions.26 The thrombin molecular structure contains 11 F 0 ¼ 1 þ K ½Q (1) tyrosine residues, 11 phenylalanine residues and 8 tryptophan F sv residues. The uorescence intensity of tryptophan is greatest in these uorescent chromogenic amino acids. When the excita- À By calculation, the values of K were 2.35Â103 M 1 (290 K) sv tion