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Nicholas Maxwell Pfahler, Indre Bielskus, Michael David Miazga, Kevin Carey, Elani Kaufman, Madalyn Mandich, Michael Giovingo, Paul A Knepper; A novel method to reduce superactivated platelets in POAG and Alzheimer’s disease. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4598.
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Superactivated platelets (SAPs) are increased in primary open-angle glaucoma (POAG) and Alzheimer’s disease (AD). SAPs are a prothrombogenic subpopulation of platelets involved in microvascular disease. Previously, we reported that a synergism of resveratrol (R), quercetin (Q), and a u-opiate antagonist (N) in vitro decreases SAPs in controls by inhibiting the three legs of toll-like receptor 4 (TLR4) signaling (ARVO, 2015). The purpose of this study was to evaluate pre-clinical TLR4 inhibitors in vitro as an anti-platelet therapy for POAG and AD using flow cytometry and to evaluate the in vivo efficacy of TLR4 inhibitors in mice using flow cytometry and capillary microscopy.
Whole blood was drawn by venipuncture after Institutional Review Board and informed consent from POAG (n=8), AD (n=6), and control (n=12) subjects. PRP was isolated, pre-treated with μM doses of RQN, and challenged with thrombin and convulxin. Fluorophore-conjugated antibodies were used to identify SAPs by flow cytometry: anti-CD41-PE identifies all platelets, anti-PAC-1-FITC identifies activated platelets, and streptavidin-APC identifies SAPs. For in vivo studies, mice were pre-treated with 1 μM RQN 3h and 1h before intraperitoneal injection of 15 U/ml thrombin. In one trial (n=10), whole blood was drawn from inferior vena cava in order to analyze SAPs by flow cytometry. In a second trial (n=10), footpad hemorrhages were counted by capillary microscopy. Paired t-tests were used for statistical analysis and the Chou-Talalay method was used to determine synergistic drug effects.
SAPs were significantly increased in POAG (p≤0.001) and AD (p=0.0069) compared with controls. A 1 µM dose of RQN in vitro reduced SAPs from 28.9±6.4% to 13.4±4.5% (a decrease of 53.1%, p≤0.001) in controls, from 52.9±5.3% to 25.4±6.2% (-52.1%, p≤0.001) in POAG, and from 50.6±6.8% to 34.6±8.9% (-31.6%, p=0.0036) in AD. See Figure 1. In mice, 1 µM RQN in vivo reduced SAPs from 74.8±1.6% to 48.8±5.9% (-34.79±5.9%, p=0.0018) and footpad hemorrhages from 31.9 to 12.3 /100 capillaries (-61.6%, p=0.012) compared with controls. RQN at 1 μM gave a Chou-Talalay combination index value of 0.015, suggesting a strong synergistic effect.
RQN decreases SAPs in POAG and AD subjects and decreases SAPs and footpad hemorrhages in thrombin-challenged mice. The RQN combination may represent a potential therapy for microvascular disease in POAG and AD.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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