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. 2021 Mar 16;12:1693. doi: 10.1038/s41467-021-21754-8

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Determination of best-matched substrates from the positional amino acid preferences using positional substrate preferences (log₂FC enrichment compared to random distribution). The best-matched positions selected for the substrate design are highlighted in bold squares. b Representation of -log₁₀ p-values of AA selected for the octapeptide design to assay protease activity of α-Thrombin and aFX. c The MS1 intensity integrated area for targeted octapeptide substrates and the corresponding cleavage fragments after incubation with activated α-Thrombin and aFX over time (0–240 min). d In silico docking of activated α-Thrombin and aFX with the two model octapeptides obtained by HPEPDOCK software. The docked peptides are shown in stick mode and proteases (α-Thrombin 1ppb; aFX 1g2l) in surface representation. The location of active site (AS) and specific active pocket sites (S1–S4) are indicated in different colors (green, orange, red and purple). e Determination of substrate selectivity measured by the relative reaction velocities (RFU/s) of fluorescent substrate processing for activated α-Thrombin and aFX tested with a panel of closely related coagulation proteases (n = 3 independent replicates). Data are presented as mean values±SD. f The calculated k_cat/K_M values (M⁻¹s⁻¹) for activated α Thrombin and aFX. Source data are provided as a Source Data file.