Design and Evaluation of Novel Peptides Targeting Protein Disulfide Isomerase as Antiplatelet Agents

Figures

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  Figure 1. Rational design and characterization of the 34-peptide candidate library. (A) Circular dendrogram illustrating the structural homology relationships among various Protein Disulfide Isomerase (PDI) structures retrieved from thePDB. PDB accession codes are indicated for each structure. (B) WebLogo representation showing sequence conservation of the region surrounding the PDI catalytic active site. The overall height of each stack indicates the sequence conservation at that position, while the height of the letters within the stack reflects the relative frequency of the respective amino acid. Amino acids are colored according to their chemical properties: polar (green), basic (blue), acidic (red), and hydrophobic (black). The absolute conservation of the active CGHC motif (positions 8–11) is highlighted. (C) Summary table listing the amino acid sequences and code designations (p1 to p34) of all 34 peptides included in the initial screening library. (ACM) denotes acetamidomethyl cysteine protection in p33.Standard one-letter amino acid abbreviations used in this figure are: A, Alanine; C, Cysteine; D, Aspartic acid; E, Glutamic acid; F, Phenylalanine; G, Glycine; H, Histidine; I,…

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  Figure 2. Machine learning-based prediction and rational selection of candidate peptides. (A) Receiver Operating Characteristic (ROC) curve and (B) Precision–Recall (PR) curve demonstrating the predictive performance of the ESM-2-based model on the validation (VAL) and independent test (TEST) sets. (C) t-SNE dimensionality reduction in the peptide library’s latent space, revealing five distinct structural families (clusters 0–4). Marker colors indicate the predicted interaction score. (D) Ranking of the 34 peptides based on their prediction score (x-axis) and probability of interaction (color gradient). Bubble size represents the estimated number of contacts. The five peptides strategically selected for in vitro validation (p7, p21, p23, p27, and p33), representing both the highest-affinity group and moderate-affinity candidates for model validation, are highlighted with black circles. The dashed line in (A) denotes a random model where the true positive rate equals the false positive rate.

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  Figure 3. PDI reductase inhibitory activity of the candidate peptides. (A) Representative kinetic curves of PDI-mediated insulin reduction monitored by absorbance at 600 nm over time. (B) Percentage of PDI reductase activity in the presence of the candidate peptides (10 µM) relative to the uninhibited PDI control (100%). BSA (bovine serum albumin) was used as a negative background control. Data are expressed as mean ± SD of three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA (**** p < 0.0001 vs. Control + PDI).

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  Figure 4. Cytotoxicity evaluation of the candidate peptides on human platelets. The cellular safety of the peptides was assessed by measuring LDH release from washed platelets after incubation at (A) 30 µM and (B) 60 µM peptide concentrations. Data are expressed as the percentage of LDH release relative to the maximum lysis control (Triton X-100), representing the mean ± SD of six independent experiments using blood from different healthy donors (n = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (ns: not significant vs. Control; **** p < 0.0001 vs. Triton).

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  Figure 5. Inhibitory effect of p23 on collagen-induced platelet aggregation. The antiplatelet activity of p23 was evaluated in washed platelets (A,B) and platelet-rich plasma (PRP) (C,D) stimulated with 2 µg/mL of collagen. (A,C) Bar graphs representing the percentage of platelet aggregation at increasing concentrations of p23 (0–60 µM). Statistical significance was determined compared to the vehicle control (0 µM) (* p < 0.05, **** p < 0.0001). (B,D) Non-linear regression curves showing the percentage of aggregation inhibition used to calculate the half-maximal inhibitory concentration (IC50). In washed platelets, p23 exhibited an IC50 of 34.17 ± 8.35 µM (B), while in PRP, the IC50 was 53.91 ± 22.17 µM (D). Data are presented as mean ± SD of five independent experiments using blood from different healthy donors (n = 5).

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  Figure 6. Atomistic resolution of the PDI-p23 complex in covalent and standard states. Representative 3D snapshots obtained from the 500 ns MD simulations with PDI shown in grey. (A) The top panels depict the post-reaction covalent state, highlighting the formation of the mixed disulfide bond between PDI-C400 and p23-C11. (B) The bottom panels display the pre-reaction standard state, where the reactive cysteines remain separated. In the zoomed-in views (right), the PDI residues located within a 4.5 Å radius of the peptide are indicated with oval labels. Residues establishing non-covalent interactions with p23 (cyan) are highlighted in dark grey, while the catalytic C400 residue is distinctly highlighted in light grey.

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  Figure 7. Peptide p23 inhibits GPIIb/IIIa integrin activation without affecting platelet granule secretion. Washed platelets were pre-incubated with vehicle or increasing concentrations of p23 (10, 30, and 60 µM) and evaluated in a resting state (unstimulated, white bars) or after stimulation with 2 µg/mL of collagen (light blue bars). Surface expression of specific activation markers was analyzed by flow cytometry. (A) PAC-1 binding, which reports the active conformation of the GPIIb/IIIa complex. (B) Fibrinogen binding to the platelet surface. (C) P-selectin surface expression, a marker for α-granule secretion. (D) CD63 surface expression, a marker for dense granule secretion. All data are expressed as mean values normalized to the resting control (0 µM p23, unstimulated) ± SD of seven independent experiments using blood from different healthy donors (n = 7). Statistical significance was determined using Two-way ANOVA followed by Bonferroni’s posthoc test comparing treatments to their respective vehicle control (* p < 0.05, *** p < 0.001, **** p < 0.0001, ns = not significant).

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  Figure 8. Peptide p23 selectively inhibits thrombus formation without affecting clot retraction and clotting times. (A) Representative fluorescence microscopy images of in vitro thrombus formation in human whole blood perfused over a collagen-coated surface. Platelets were fluorescently labeled and treated with either vehicle (Control) or 30 µM of p23. (B) Quantification of total thrombus area (µm2). (C) Representative macroscopic images of the clot retraction process over 90 min and the final isolated clots. (D) Quantification of clot retraction expressed as the residual weight (grams) of the clots after 90 min. Eptifibatide (EPT) was used as a positive control for retraction inhibition. (E) Prothrombin Time (PT) and (F) Activated Partial Thromboplastin Time (APTT) in human plasma treated with vehicle, p23, or Apixaban (positive control). All data are presented as mean ± SD of independent experiments using blood from different healthy donors (in vitro thrombosis n = 7; clot retraction n = 8; PT and APTT n = 6). Statistical significance was determined using One-way ANOVA compared to the respective control (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant).