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Indre Bielskus, Kevin Carey, Nicholas Maxwell Pfahler, Michael David Miazga, Michael Giovingo, Paul A Knepper; Thrombin generation test of TLR4 antagonists compared to conventional anti-platelet drugs. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4596. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Primary open-angle glaucoma (POAG) is a devastating disease of unknown etiology. Our lab has previously shown that both POAG and Alzheimer’s disease (AD) have a microvascular component. In this study, three inhibitors of the innate immune system receptor toll-4: resveratrol (R), quercetin (Q), and a µ-opioid antagonist (N) were compared to four commonly used anti-platelet drugs—aspirin, ibuprofen, dabigatran, and rivaroxaban. The purpose of this study was to determine the efficacy of RQN as a potential new anti-platelet therapy.
Whole blood was collected from control, POAG, and AD patients after obtaining informed consent and International Review Board approval. Following the method of Jobe et al (Blood;2015), washed platelets were isolated by centrifugation. Platelets (2x108/ml) were activated with convulxin and thrombin, followed by addition of coagulation Factors V, X, and prothrombin, which exposed phosphotidylserine residues and converted prothrombin to thrombin. Chromogenic substrate S-2238 (Chromogenix) was used to determine thrombin generation as measured by a spectrophotometer at 405nm, and normalized to a standard curve. Base levels of thrombin generation were measured in controls, POAG, and AD. Three concentrations of RQN were tested, along with commonly used anti-platelet drugs — aspirin, ibuprofen, rivaroxaban, and dabigatran—in clinically used dosages.
RQN at a 1μM concentration significantly reduced thrombin generation of washed platelets in controls (p=0.007), POAG (p=0.003), and Alzheimer’s disease (p=0.01). There was also significant reduction of thrombin generation at higher RQN concentrations and with use of other anti-platelet drugs, as shown in Table 1. RQN was as effective as conventional anti-platelet drugs in reducing thrombin generation.
RQN was effective in reducing thrombin generation in platelets. RQN blocks activation of the innate immune system and subsequent platelet activation through the three signaling pathways of the TLR4 receptor. In contrast, aspirin and ibuprofen act on the cyclooxygenase 2 pathway, dabigatran serves as a direct thrombin inhibitor, and rivaroxaban acts as a Factor X inhibitor. RQN prevents thrombin generation in a manner similar to conventional anti-platelet drugs; therefore, these results represent a new class of anti-platelet drugs to treat platelet-associated disease, namely 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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