Analysis of the mechanism of damage produced by thiazole orange photoinactivation in apheresis platelets

Abstract

Background: Pathogen Reduction Technologies (PRTs) are broad spectrum nucleic acid replication-blocking antimicrobial treatments designed to mitigate risk of infection from blood product transfusions. Thiazole Orange (TO), a photosensitizing nucleic acid dye, was previously shown to photoinactivate several types of bacterial and viral pathogens in RBC suspensions without adverse effects on function. In this report we extended TO treatment to platelet concentrates (PCs) to see whether it is compatible with in vitro platelet functions also, and thus, could serve as a candidate technology for further evaluation.

Material and methods: PCs were treated with TO, and an effective treatment dose for inactivation of Staphylococci was identified. Platelet function and physiology were then evaluated by various assays in vitro.

Results: Phototreatment of PCs yielded significant reduction (≥4-log) in Staphylococci at TO concentrations ≥20 μM. However, treatment with TO reduced aggregation response to collagen over time, and platelets became unresponsive by 24 hours post-treatment (from >80% at 1 h to 0% at 24 h). TO treatment also significantly increased CD62P expression (<1% CD62P+ for untreated and >50% for TO treated at 1 h) and induced apoptosis in platelets (<1% Annexin V+ for untreated and >50% for TO treated at 1 h) and damaged mitochondrial DNA. A mitochondria-targeted antioxidant and reactive oxygen species (ROS) scavenger Mito-Tempo mitigated these adverse effects.

Discussion: The results demonstrate that TO compromises mitochondria and perturbs internal signaling that activates platelets and triggers apoptosis. This study illustrates that protecting platelet mitochondria and its functions should be a fundamental consideration in selecting a PRT for transfusion units containing platelets, such as PCs.

Figure 1. Thiazole orange (TO) photoinactivates staphylococcal species in PCs

CFUs of (A) S. aureus or (B) S. epidermidis were enumerated via pour plating of serially diluted 0.5 mL samples taken before treatment (0 min) after addition of TO (15 min), and after phototreatment (post-inactivation). Graphs are from one representative donor (n=4), with samples plated in quadruplicate. (C) Log reduction of staphylococcal species was dose-dependent, and 20 μM was sufficient to consistently reduce bacteria by ≥4-log CFU. (D) Treatment with less than 10 μM inactivated bacteria by ≤1-log CFU. Data shown is mean +SD.

Figure 2. TO localises to mitochondria and alters platelet metabolism

Platelets were incubated with 20 μM TO followed by photoexcitation and MitoTracker Deep Red to label mitochondria. (A) Confocal microscopy shows colocalisation of signal from TO and MitoTracker, though the colocalisation is not selective (n=3). Blood gas measurements were taken at indicated timepoints to measure glucose (B) and lactose (C) concentration. Data shown is mean+SD for n=4 donors.

Figure 3. Flow cytometry shows that TO treatment increases a population of activated and apoptotic platelets

At indicated times following treatment, platelets were labeled with an APC-conjugated antibody for CD62P (A and B) or Annexin V-APC (C and D). (A) Representative histogram overlays for CD62P expression. (B) Graph shows mean (with SD, n=3 donors) %CD62P+ cells. (C) Representative histogram overlays for Annexin V labeling. (D) Graph shows mean (with SD, n=3 donors) %Annexin V+ cells.

Figure 4. TO significantly decreases aggregation

(A) Platelets were evaluated in a light transmission aggregation (LTA) assay using 10 μg/mL collagen as an agonist. (B) Platelets were treated with 10 μM MitoTempo ± 20 μM TO and evaluated in LTA assay alongside samples in (A). Treatment with MitoTempo delayed the adverse effects of TO treatment on aggregation, indicating that these effects are primarily driven by mitochondrial ROS. For (A) and (B): representative trace at 4 h, with data in graph from duplicate measurements of one representative donor (n=7, **p<0.01, ***p<0.001 by t-test). Data shown is mean +SD.

Figure 5. TO treatment activates PKC isoforms and p38 MAPK

Western blot of platelet lysates collected pre- and post-phototreatment (immediately following light exposure) probed with (A) anti-PKC substrate motif, which binds to any protein with a phosphorylated motif unique to PKC isoforms, or (B) anti-phospho-p38 MAPK antibodies. Blots shown are representative of results from three different donors.

Figure 6. Inhibition assay for mtDNA damage

Mitochondrial DNA was isolated from platelets before and after light exposure in the presence or absence of TO and/or MitoTempo. (A) Threshold cycle values for amplification of short fragments via qPCR of platelet mtDNA. (B) Agarose gel of PCR products from long-range PCR reaction of mtDNA shows inhibition of PCR for TO-treated samples in presence/absence of Mito-Tempo (Lanes 6 and 8). Data shown is mean+SD representative of results from three different donors.