Abstract
Purpose :
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.
Methods :
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.
Results :
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.
Conclusions :
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.