Pathogen reduction diminishes the protective effects of platelets on endothelial barrier permeability in vitro

Figures

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  Figure 1.. Preparation of NPR and PR platelets. In each trial (n = 3), platelets from multiple donors (n = 6) were pooled and then split into 2 identical units. Using the INTERCEPT blood system, PR platelets were treated with amotosalen, which, under UVA illumination, resulted in nucleic acid modification to prevent pathogen replication. NPR platelets were then compared with PR platelets from the same pooled donors in subsequent experiments. This procedure was conducted for each of the 3 trials used. Other than treatment with pathogen reduction, the NPR and PR platelets underwent identical preparation, storage, and experimental procedures.

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  Figure 2.. NPR and PR platelet counts decrease with transport between facilities and storage. (A) NPR and PR platelet counts are shown on the day of collection before splitting (day 1, before treatment), after splitting and with either no additional preparation or pathogen reduction treatment (day 1 post-treatment NPR and PR, respectively), and on subsequent experimental days (days 3, 5, 7, and 10). (B) Percentage of initial platelet count in NPR (orange) and PR (blue) platelet groups is shown over time. Individual data points from each trial are shown at each time point. ∗P < .05 comparing the difference between median NPR and PR platelets by 2-way ANOVA with the Tukey honestly significant difference test post hoc at the given time point.

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  Figure 3.. NPR and PR platelets attenuate thrombin-induced disruption of adherens and tight junctions. (A) Treatment with NPR or PR platelets lessened the loss of endothelial junctional proteins in response to thrombin challenge. HUVEC monolayers were stained for VE-cadherin (green), F-actin with phalloidin (red), and DAPI (4′,6-diamidino-2-phenylindole; blue) in images 1, 3, 5, and 7 as well as ZO-1 (white) in images 2, 4, 6, and 8. White arrows highlight gaps between endothelial cells. Platelets from trials 2 and 3 were tested on days 3, 5, 7, and 10, and representative images from trial 2, day 5 are shown. (B) Quantified gap measurement was significantly less in day 3 NPR platelets (orange) compared with PR platelets (blue), but that difference was not maintained as the platelets aged. In addition, there was an increase in gap size between cells with thrombin alone compared with control and a statistically significant decrease with both treatment of NPR and PR platelets. Differences were calculated with 2-way ANOVA. ∗P < .05. ns, nonsignificance.

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  Figure 4.. NPR platelets (orange) and PR platelets (blue) have a differential response to thrombin challenge across an endothelial monolayer as measured by ECIS. (A-B) NPR and PR platelet treatment of HUVECs demonstrated a similar dose-response as measured by the AUC of transendothelial electrical resistance. This dose-response curve was preserved in both day 3 (A) and day 7 (B) platelets and there were no significant differences between NPR and PR response for a given concentration. (C-D) NPR platelets were more protective at lower concentrations against thrombin challenge (0.2 U/mL) as measured by AUC of transendothelial electrical resistance on day 3 (C) but this difference was not sustained to day 7 (D). For all studies, samples were performed with 3 to 8 replicates with means and standard deviations of representative samples shown. Analysis was conducted with 2-way ANOVA and the Tukey test post hoc. ∗P < .05. Thr, thrombin.

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  Figure 5.. NPR and PR platelets demonstrate variable but overall similar aggregation. (A) Under light transmission aggregometry induced by thrombin, aggregation of NPR and PR platelets had similar tracing morphology and no difference in slope or AUC across experimental days. Platelets from trials 1 and 2 were tested and representative curves of day 3 and day 7 platelets (trial 1) are shown. Both slope and AUC for replicates were compared using the t test and P < .05 was considered significant. (B) Under impedance aggregometry induced by collagen or thrombin analog, whereas both NPR (orange) and PR (blue) platelets (trial 3) demonstrated aggregation, NPR platelets exhibited a more robust response. Data are shown as impedance over time normalized to the baseline and as a calculated AUC. The red arrows indicate the time the agonist was administered. CI, confidence interval.