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Paul A Knepper, Nicholas Maxwell Pfahler, Indre Bielskus, Michael Giovingo, Nicholas J Volpe; Platelet TLR4 expression in degenerative disease. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5166. doi: https://doi.org/.
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High-tension glaucoma (HTG), normal-tension glaucoma (NTG), and Alzheimer’s disease (AD) are common ocular and neurodegenerative diseases that feature a proinflammatory phenotype. Previously, our laboratory found that HTG and AD, but not NTG, subjects exhibited increased levels of procoagulant platelets that were reduced by inhibitors of innate immune receptor toll-like receptor 4 (TLR4). One explanation could be that platelets are primed by increased TLR4 signaling as a result of increased levels of tissue injury, as observed in HTG and AD. This study tested the hypothesis that platelet TLR4 expression is higher in HTG and AD, but not NTG, subjects at baseline and in response to platelet activation.
Whole blood was obtained from control (n=5), NTG (n=5), HTG (n=5), and AD (n=4) subjects after informed consent. Platelets were isolated by centrifugation, stained by monoclonal phycoerythrin (PE)-labeled anti-human TLR4 or IgGk1 antibodies in baseline conditions or after challenge with thrombin for 5 minutes, and analyzed by flow cytometry. Quantitation was performed using a flow cytometric PE fluorescence quantitation kit (BD Biosciences). IgGk1 fluorescence was subtracted to control for background. Two-way ANOVA and two-tailed Student’s t-test were used for statistical analysis.
The number of TLR4 receptors/platelet in baseline conditions was significantly increased in AD subjects (463.4±63.6) compared to controls (365.5±36.0, p=0.02), but only slightly increased in HTG subjects (395.2±40.5) and unchanged in NTG subjects (357.6±58.3). After stimulation with thrombin/convulxin, the number of TLR4 receptors/platelet increased to 1716.5±184.5 in control subjects (p<0.001), 1952.9±148.9 in NTG subjects (p<0.001), 2155.7±198.1 in HTG subjects (p<0.001), and 2293.2±298.9 in AD subjects (p<0.001). The increase in TLR4 induced by thrombin/convulxin was significantly higher in AD (p=0.009) and HTG (p=0.007) subjects compared to controls.
Resting platelet TLR4 expression was significantly increased in AD but not HTG or NTG subjects. Platelet activation induced significant increases in TLR4 in all subjects and these increases were significantly higher in both AD and HTG compared to control or NTG subjects. The 5-fold increase in TLR4 is likely due to release of the α-granule store and insertion into the plasma membrane. The results suggest that AD and HTG subjects may be at risk for increased inflammation and thrombosis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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