In Vitro Study of the Fibrinolytic Activity via Single Chain Urokinase-Type Plasminogen Activator and Molecular Docking of FGFC1

Abstract

Fungi fibrinolytic compound 1 (FGFC1) is a rare marine-derived compound that can enhance fibrinolysis both in vitro and in vivo. The fibrinolytic activity characterization of FGFC1 mediated by plasminogen (Glu-/Lys-) and a single-chain urokinase-type plasminogen activator (pro-uPA) was further evaluated. The binding sites and mode of binding between FGFC1 and plasminogen were investigated by means of a combination of in vitro experiments and molecular docking. A 2.2-fold enhancement of fibrinolytic activity was achieved at 0.096 mM FGFC1, whereas the inhibition of fibrinolytic activity occurred when the FGFC1 concentration was above 0.24 mM. The inhibition of fibrinolytic activity of FGFC1 by 6-aminohexanoic acid (EACA) and tranexamic acid (TXA) together with the docking results revealed that the lysine-binding sites (LBSs) play a crucial role in the process of FGFC1 binding to plasminogen. The action mechanism of FGFC1 binding to plasminogen was inferred, and FGFC1 was able to induce plasminogen to exhibit an open conformation by binding through the LBSs. The molecular docking results showed that docking of ligands (EACA, FGFC1) with receptors (KR1-KR5) mainly occurred through hydrophilic and hydrophobic interactions. In addition, the binding affinity values of EACA to KR1-KR5 were -5.2, -4.3, -3.7, -4.5, and -4.3 kcal/moL, respectively, and those of FGFC1 to KR1-KR5 were -7.4, -9.0, -6.3, -8.3, and -6.7 kcal/moL, respectively. The findings demonstrate that both EACA and FGFC1 bound to KR1-KR5 with moderately high affinity. This study could provide a theoretical basis for the clinical pharmacology of FGFC1 and establish a foundation for practical applications of FGFC1.

Keywords: FGFC1; fibrinolytic properties; molecular docking; plasminogen; pro-uPA.

Figure 1

The role of the single-chain urokinase-type plasminogen activator (pro-uPA) in the fibrinolytic activity of fungi fibrinolytic compound 1 (FGFC1). Plot of absorbance at 405 nm (A405) versus time (A) and the values of K_n (B) at different pro-uPA concentrations (0–18 nM). The reaction was incubated at 37 °C and measured every 5 min for 120 min. The results are expressed as the mean ± SD performed in triplicate. The concentration of Glu-plasminogen and FGFC1 in the reaction system were 40 nM and 0.048 mM, respectively.

Figure 2

The role of Glu-plasminogen in the fibrinolytic activity of FGFC1. Plot of A₄₀₅ versus time (A) and the values of K_n (B) at different Glu-plasminogen concentrations (0–80 nM). The reaction was incubated at 37 °C and measured every five minutes for 120 min. The results are expressed as the mean ± SD, performed in triplicate. The concentration of pro-uPA and FGFC1 in the reaction system were 9 nM and 0.018 mM, respectively.

Figure 3

The fibrinolytic characterization of FGFC1 mediated by pro-uPA and Glu-plasminogen. Plot of A₄₀₅ versus time (A) and the values of K_n (B) at different FGFC1 concentrations (0–0.36 mM). The reaction was incubated at 37 °C and measured every five min for 120 min. The results are expressed as the mean ± SD, performed in triplicate. The concentration of pro-uPA and Glu-plasminogen in the reaction system were 9 nM and 4 nM, respectively.

Figure 4

The fibrinolytic characterization of FGFC1 mediated by pro-uPA and Lys-plasminogen. Plot of A₄₀₅ versus time (A) and the values of Kn (B) at different FGFC1 concentrations (0–0.36 mM). The reaction was incubated at 37 °C and measured every five minutes for 120 min. The results are expressed as the mean ± SD, performed in triplicate. The concentration of pro-uPA and Lys-plasminogen in the reaction system were 9 and 4 nM, respectively.

Figure 5

The fibrinolytic activity of FGFC1 at different concentrations of EACA (0–216 mM) (A), TXA (0–21.6 mM) (B), and SBTI (0–120 µM) (C). The reaction was incubated at 37 °C and measured at 405 nm using a microplate reader every five minutes for 120 min. Results are expressed as a percentage of the fibrinolytic activity in the absence of EACA, TXA, and SBTI. The results are expressed as the mean ± SD, performed in triplicate. The concentration of FGFC1 for the FGFC1 + EACA, FGFC1 + TXA, and FGFC1 + SBTI assays was 0.12 mM.

Figure 6

Chemical structures of EACA (A) and FGFC1 (B).

Figure 7

The docking results of ligands (EACA and FGFC1) with kringle domains (KR1–KR5) (A–E). In each group, the docking results of EACA (top) and FGFC1 (bottom) with KR1–KR5 are shown, along with the binding site (left) and amino acid residues involved (right). The dotted lines represent hydrogen bonds between the ligand and amino acid residues. KR1–KR5 are shown in surface representation, whereas EACA and FGFC1 are shown as sticks.

Figure 7

The docking results of ligands (EACA and FGFC1) with kringle domains (KR1–KR5) (A–E). In each group, the docking results of EACA (top) and FGFC1 (bottom) with KR1–KR5 are shown, along with the binding site (left) and amino acid residues involved (right). The dotted lines represent hydrogen bonds between the ligand and amino acid residues. KR1–KR5 are shown in surface representation, whereas EACA and FGFC1 are shown as sticks.

Figure 7

The docking results of ligands (EACA and FGFC1) with kringle domains (KR1–KR5) (A–E). In each group, the docking results of EACA (top) and FGFC1 (bottom) with KR1–KR5 are shown, along with the binding site (left) and amino acid residues involved (right). The dotted lines represent hydrogen bonds between the ligand and amino acid residues. KR1–KR5 are shown in surface representation, whereas EACA and FGFC1 are shown as sticks.

Figure 7

The docking results of ligands (EACA and FGFC1) with kringle domains (KR1–KR5) (A–E). In each group, the docking results of EACA (top) and FGFC1 (bottom) with KR1–KR5 are shown, along with the binding site (left) and amino acid residues involved (right). The dotted lines represent hydrogen bonds between the ligand and amino acid residues. KR1–KR5 are shown in surface representation, whereas EACA and FGFC1 are shown as sticks.

Figure 7

The docking results of ligands (EACA and FGFC1) with kringle domains (KR1–KR5) (A–E). In each group, the docking results of EACA (top) and FGFC1 (bottom) with KR1–KR5 are shown, along with the binding site (left) and amino acid residues involved (right). The dotted lines represent hydrogen bonds between the ligand and amino acid residues. KR1–KR5 are shown in surface representation, whereas EACA and FGFC1 are shown as sticks.

Figure 8

Schematic diagram of optimal conformational interaction between FGFC1 and plasminogen. (A) Binding site and (B) the 3D model of FGFC1 docking with protein.

Figure 9

A model of the mechanism of plasminogen activation by FGFC1.