Development of the Antithrombotic Peptide LEKNSTY Targeting the Collagen Surface: II. Improvement of Plasma Stability

Abstract

The development of antithrombotic peptides targeting collagen was proven effective, and an effective antithrombotic peptide LEKNSTY was obtained in part I. However, the plasma stability of LEKNSTY was found to be not good enough. In this part, the LEKNSTY was further optimized for improvement in plasma stability by substitution using d-amino acid residues. Two novel antithrombotic peptides LekNStY and lEKnsTy were designed, where lowercase letters represent d-amino acid residues. Improvements in plasma stability of both LekNStY and lEKnsTy were experimentally confirmed. Moreover, good binding of these antithrombotic peptides on the collagen surface was confirmed by molecular dynamics simulation and experimental validation. For example, a K d of only 0.75 ± 0.10 μM was observed for lEKnsTy. Moreover, LekNStY and lEKnsTy were found to inhibit platelet adhesion on the collagen surface more effectively than LEKNSTY, and the IC 50 of lEKnsTy was only 2/5 of that of LEKNSTY. These results confirmed the successful design of LekNStY and lEKnsTy that had good plasma stability and could effectively inhibit arterial thrombosis, which would be helpful for the research into interfaces involved in thrombus formation and the development of antithrombotic nanomedicine.

Design and characterization of antithrombotic ClEKnsTy-Au nanoparticles as diagnostic and therapeutic reagents

“…The binding free energy was calculated by the protocol that used in the literature 37,38 and our previous work. 16,18,19,39–41 The equations were described briefly as follows.Δ G bind = Δ G polar + Δ G nonpolar Δ G polar = Δ G elec + Δ G PB Δ G nonpolar = Δ G vdW + Δ G SASA …”

“…31 The D-amino acid structure was obtained in our previous work. 18,19 The protonation state of residues was set according to a pH of 7. The proteins were dissolved in physiological saline, where the water molecule was treated using a TIP3P model.…”

“…The binding free energy was calculated by the protocol that used in the literature 37,38 and our previous work. 16,18,19,[39][40][41] The equations were described briefly as follows.…”

“…[12][13][14][15] In our previous work, the antithrombotic peptides LWWNSYY and lEKnsTy (lowercase letters represent D-amino acid residues) were designed targeting collagen, which were confirmed as being capable of binding on the collagen surface and thus inhibiting thrombus formation at the early stage of thrombus formation using both in vitro and in vivo experiments. [16][17][18][19] However, these results focused on the inhibition of thrombus formation, but did not have the function related to diagnosis.…”

Development of antithrombotic peptides based on the molecular interactions between von Willebrand factor and GPIbα

“…This strategy was established and successfully used to examine the protein interface in various systems including a collagen-GPIba complex. [41][42][43][44][45][46] Inhibitors targeting the vWF A1 domain were then designed based on the molecular interactions, followed by the screening and experimental validations.…”