Design and Pharmacological Characterization of a Novel Antithrombotic P2Y1 Receptor-Based Vaccine

Figures

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  Figure 1. A cartoon model depicting potential inhibition of ADP-induced activation of the P2Y1-R by EL2Vac. Vaccination with EL2Vac will trigger an immune response and generate antibodies targeted against the EL2 of P2Y1-R, which will prevent ADP from binding, thereby inhibiting ADP-induced platelet activation.

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  Figure 2. Antibody concentration in mice vaccinated with EL2Vac, EL2rVac, or VLP. Mice were immunized with VLP coupled with the EL2 sequence (CGGTGVRKNK-TITCYDTTSDEYLRSYF; T192-F215), random EL2 (LYKDGTICTYVFYNKRRSDTETST), or VLP. EL2Ab, which was measured, including after six months, in the vaccinated mice using ELISA and the EL2 cognate peptide, was detectable at significant levels in the EL2Vac mice, but not the EL2rVac and VLP controls. The titers were assessed in five distinct vaccination groups, each comprising five mice (** p ≤ 0.001); the abbreviation NS denotes insignificance (p ≥ 0.05).

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  Figure 3. Platelet aggregation assessment in the vaccinated mice. Platelets were obtained from mice vaccinated with 20 µg of EL2Vac or EL2rVac and stimulated with three different agonists, (A) 10 µM ADP, (B) 1 µM U46619, (C) 5 μg/mL collagen or (D) 2 μg/mL collagen before their aggregation response was measured in a lumi-aggregometry system. EL2Vac showed selective inhibition of ADP- and low-dose collagen-induced platelet aggregation. Each test was performed in triplicate using three separate groups, with blood pooled from six mice per group each time.

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  Figure 4. P-selectin expression assessment in the vaccinated mice. Platelets were obtained from mice vaccinated with EL2Vac or EL2rVac and stimulated with two different agonists, 10 µM ADP or 1 µM U46619, before their alpha granule secretion/P-selectin expression was measured using flow cytometry. This experiment was conducted in triplicate, and blood was collected from eight mice and pooled per group. The error bars represent the SEM, whereas NS denotes insignificance (** p ≤ 0.001).

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  Figure 5. Occlusion time in the vaccinated mice. Mice vaccinated with EL2Vac or EL2rVac were subjected to the FeCl3-induced carotid artery injury thrombosis model. The time for occlusion was found to be delayed in EL2Vac, unlike EL2rVac, and it persisted six months later. Each point represents a single animal (n = 5 for EL2rVac and EL2Vac, and n = 6 for the EL2Vac 6-month experiment); NS denotes insignificance, ** p ≤ 0.001.

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  Figure 6. Tail bleeding time in the vaccinated mice. Mice vaccinated with EL2Vac or EL2rVac were subjected to the tail bleeding time assay. The bleeding time was not impacted in the EL2Vac or EL2rVac mice. Each point represents a single animal (n = 9 for EL2rVac and EL2Vac). The abbreviation NS denotes insignificance (p ≥ 0.05).

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  Figure 7. The effects of EL2Vac on ADP-triggered reduction in cAMP levels in platelets. cAMP levels were measured in EL2rVac and EL2Vac mice, as discussed in Methods. The experiment was conducted in triplicate, and blood was collected from five to six mice and pooled per group. *** p ≤ 0.0001; the abbreviation NS denotes insignificance (p ≥ 0.05).