September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
A new look at platelet inhibition and neuroprotection
Author Affiliations & Notes
  • Kevin Carey
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Indre Bielskus
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Mit Patel
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Kelsey Green
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Andrius Lelis
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Christopher Wanderling
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Paulius Kuprys
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
  • Paul A Knepper
    Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States
    Ophthalmology, Northwestern University, Chicago, Illinois, United States
  • Footnotes
    Commercial Relationships   Kevin Carey, None; Indre Bielskus, None; Mit Patel, None; Kelsey Green, None; Andrius Lelis, None; Christopher Wanderling, None; Paulius Kuprys, None; Paul Knepper, Testog Inc (P)
  • Footnotes
    Support  BrightFocus Foundation Grant G2011-047, National Eye Institute Grant P30EY01792
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 2561. doi:
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      Kevin Carey, Indre Bielskus, Mit Patel, Kelsey Green, Andrius Lelis, Christopher Wanderling, Paulius Kuprys, Paul A Knepper; A new look at platelet inhibition and neuroprotection. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2561.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Microvascular disease is an important etiological factor in primary open angle glaucoma (POAG). Our recent studies indicate that POAG patients have microvascular changes present in non-ocular sites, increased number of superactivated platelets (SAPs) which are prothrombogenic, and the importance of the innate immune system, specifically Toll-4 (TLR-4) antagonists in preventing cell death. The purpose is to evaluate antagonists of the three legs of the TLR-4 pathway—the TLR-4 receptor (naltrexone), the down-stream myD88-dependent pathway (quercetin), and the down-stream myD88-independent pathway (resveratrol)—to reduce SAPs.

Methods : Whole blood was collected via venipuncture from control subjects (n=7), anticoagulated, centrifuged to isolate the platelet rich plasma, and treated with peptide gly-pro-arg-pro-NH2 (Sigma) to prevent coagulation and succinylated biotinylated-fibrinogen (gift from Dr. George Dale) to identify SAPs. Platelets were pre-incubated with varying μM concentrations of resveratrol, quercetin and naltrexone alone or in combinations. The platelets were activated with thrombin and convulxin, and stained with fluorophore conjugated antibodies to identify platelet status by flow cytometry: CD41-phycoerythrin (Invitrogen) for all platelets; PAC-1-FITC (BD Biosciences) for activated platelets; streptavidin-APC (BD Biosciences) for SAPs. The Chou-Talalay theorem was used to determine synergistic drug effects and is based on the median-effect equation to provide the common link between single entity and multiple entities.

Results : The Chou-Talalay theorem identified the optimum drug combination and concentration to be 1 μM resveratrol, 1 μM quercetin, and 1 μM naltrexone which yielded a 63% reduction in SAPs (p<0.02). The Chou-Talalay index, in which a value >1 indicates antagonism, 1 indicates additivity, and <1 indicates synergism, gives a value of 0.015, suggesting a strong synergistic drug effect. Other combinations of the three drugs revealed the importance of blocking each leg of the TLR-4 signaling pathway.

Conclusions : Resveratrol, quercetin, and naltrexone act synergistically to reduce SAP levels, with the most synergistic dose being 1 μM of each drug. Significantly, the synergistic dose of the three-drug combination is obtainable by oral administration. While further trials are required to determine in vivo drug effects, our initial results provide a new look at reducing platelet activity in microvascular diseases.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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