Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation.

Thioredoxin (Trx) is an oxidoreductase with important physiological function. Imbalances in the NADPH/thioredoxin reductase/thioredoxin system are associated with a number of pathologies, particularly cancer, and a number of clinical trials for thioredoxin and thioredoxin reductase inhibitors have been carried out or are underway. Due to the emerging role and importance of oxidoreductases for haemostasis and the current interest in developing inhibitors for clinical use, we thought it pertinent to assess whether inhibition of the NADPH/thioredoxin reductase/thioredoxin system affects platelet function and thrombosis. We used small molecule inhibitors of Trx (PMX 464 and PX-12) to determine whether Trx activity influences platelet function, as well as an unbiased proteomics approach to identify potential Trx substrates on the surface of platelets that might contribute to platelet reactivity and function. Using LC-MS/MS we found that PMX 464 and PX-12 affected the oxidation state of thiols in a number of cell surface proteins. Key surface receptors for platelet adhesion and activation were affected, including the collagen receptor GPVI and the von Willebrand factor receptor, GPIb. To experimentally validate these findings we assessed platelet function in the presence of PMX 464, PX-12, and rutin (a selective inhibitor of the related protein disulphide isomerase). In agreement with the proteomics data, small molecule inhibitors of thioredoxin selectively inhibited GPVI-mediated platelet activation, and attenuated ristocetin-induced GPIb-vWF-mediated platelet agglutination, thus validating the findings of the proteomics study. These data reveal a novel role for thioredoxin in regulating platelet reactivity via proteins required for early platelet responses at sites of vessel injury (GPVI and GPIb). This work also highlights a potential opportunity for repurposing of PMX 464 and PX-12 as antiplatelet agents.CM is funded by Medical Research Council Grant No G9826026. AR was funded by a British Heart Foundation Centre of Research Excellence-funded Vacation Studentship. CHC is funded by British Heart Foundation Fellowship FS/11/49/28751.This is the final version of the article. It first appeared from PLOS via https://doi.org/10.1371/journal.pone.016300

GPVI-mediated Ca²⁺ release is sensitive to inhibitors of Trx-R and Trx.

<p>Washed human platelets were loaded with 3 μM fura2-AM and Ca²⁺ release monitored at 340nm and 380nm. Fluorescence was monitored for 5 cycles to obtain a baseline reading, following which drug was added for approx. three minutes for a further 10 cycles, before addition of agonist (1 μg/ml CRP, 0.5 U/ml thrombin, 0.3 ∞M U46619, or 20 μM ADP). PMX 464 and PX-12 inhibited CRP-XL-induced Ca2+ release in a concentration-dependent manner (A), with a modest, but non-significant effect on Ca2+ release induced by ADP (B). PX-12 induced Ca2+ release independently of agonist at or above concentrations of 10 μM (C and D). PMX 464 inhibited Ca2+ release by U46619 at 0.1μM (E, R² 0.6), but not at 0.3 μM or 1 μM. Rutin had no effect on Ca2+ release by any agonist (F, thrombin, black triangles; CRP-XL black circles; U46619, black diamonds; ADP, black squares). To determine whether Trx and PDI inhibitors were inducing receptor shedding, whole blood was incubated with drug (30 μM rutin, 10 μM PX-12, 30 μM PMX 464) or vehicle for 10 minutes (G) or 30 minutes (H), to which was added anti-GPVI antibody conjugated to AlexaFluor 647 to quantify cell surface levels for GPVI by flow cytometry (n = 4, +SEM, one-way ANOVA, Bonferroni post-hoc test).</p

Trx inhibitors alter the abundance of peptides with free thiols on the surface of resting human platelets.

<p>Trx inhibitors alter the abundance of peptides with free thiols on the surface of resting human platelets.</p

PMX 464 and PX-12 inhibit thrombus formation over Type I collagen in whole blood under flow conditions.

<p>Images of the channels are shown in A (30 μM PMX 464) and D (3 μM PX-12) and quantified to show variation between donors for both drugs (B and E, respectively), as well as an overall summary (C and F, respectively), n = 5, +/- SEM, 2-tailed t-test with Bonferroni correction.</p

PMX 464 selectively inhibits CRP-XL-induced aggregation in PRP in a time-dependent manner.

<p>PRP isolated from healthy human volunteers was incubated with 30 μM PMX 464 or vehicle (DMSO) for 30s, 2m or 10m at room temperature before being assess by light transmission aggregometry (A); example traces are shown in B. PMX 464 selectively and significantly inhibited CRP-XL-induced aggregation with a 10 minutes incubation (C, DMSO in white, PMX 464 in black, n = 4, +SEM, 2-way ANOVA). Increasing the incubation time of PMX 464 potentiated the level of inhibition seen with CRP-XL-induced PRP aggregation (D, black bars = 2 minutes, white bars = 10 minutes, horizontal stripes = 30 minutes). As shown in E and F, a 10 min incubation with 30 μM PMX 464 increased the EC50 of CRP-XL ~5-fold (E, 8 pM to 40 pM, p = 0.0316, two-tailed t-test with Bonferroni correction), while a 30 minute incubation with 10 μM PMX 464 increased the EC₅₀ of CRP-XL by 5pM to 30pM (~6-fold, p = 0.0334, two-tailed t-test with Bonferroni correction). PX-12 (30 minutes, 3 μM PX-12) did not inhibit CRP-XL-induced PRP aggregation (G; n = 4, p = 0.1, 2-tailed t-test with Bonferroni correction).</p

PMX 464 attenuates ristocetin-induced platelet agglutination.

<p>PMX 464 (30 μM) reduced platelet agglutination induced by ristocetin in platelet rich plasma (A-D, n = 5, +/- SEM, one-way ANOVA, Bonferroni multiple comparisons test). Samples of washed platelets incubated with drug or vehicle were separated by SDS-PAGE and levels of GPIb assessed by Western blotting (E).</p