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Nicholas Pfahler, Indre Bielskus, Thomas Cronin, James Haney, Jordan Hill, Michael Giovingo, Thomas Patrianakos, Nicholas J Volpe, Paul A Knepper; CD36 Ligand Beta-Amyloid Promotes Coagulation and Inflammation Through Toll-Like Receptor 4. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3721.
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Primary open-angle glaucoma (POAG), Alzheimer’s disease (AD), and age-related macular degeneration (AMD) are common neurodegenerative diseases without known cause. The toxic protein beta-amyloid (Aβ) has been implicated as a common etiological factor in these diseases and may lead to a unifying hypothesis of degeneration. Aβ activation of CD36 and Toll-like receptor 4 (TLR4) is implicated in retinal degeneration and hypercoagulation. Previously, our laboratory reported that POAG and AD patients have increased superactivated platelets (SAPs) – a procoagulant subtype of activated platelets – which are prevented by inhibiting TLR4. The purpose of this study was to examine the interaction of CD36, TLR4, and Aβ in platelet activation and assess levels of receptor expression in POAG.
Whole blood was obtained from control subjects (n=4) and POAG patients (n=4), anticoagulated, centrifuged at 200xg for 15 min to isolate platelets, washed twice, and resuspended at 5x107/mL. CD36 inhibitor (sulfosuccinimidyl oleate (SSO), 100 μM; Sigma), TLR4 inhibitor (TAK-242, 10 μM; Sigma), or buffer was added before challenge with agonists thrombin/convulxin (T/C) or Aβ. Samples were stained with conjugated antibodies and analyzed with a Cyan ADP flow cytometer. CD36 and TLR4 levels were determined using mean fluorescence of anti-CD36 and anti-TLR4 antibodies. Activated platelets were defined as CD41+/PAC1+, while SAPs were defined as CD41+/PAC1-/Fibrinogen+.
Platelet CD36 was significantly upregulated in POAG patients compared with controls (p<0.007), while TLR4 levels trended upward. In resting conditions, TAK-242 increased levels of CD36 (p=0.03). Aβ (500 nM) induced SAP formation (p=0.0001) and increased expression of CD36 and TLR4 (p<0.0001). POAG patients exhibited increased SAPs in response to T/C (p<0.001) and Aβ (p=0.02) compared with controls. SSO inhibited CD36/TLR4 expression and SAP formation when challenged by Aβ but not T/C, whereas TAK was effective in both conditions. SSO had no effect when platelets were challenged with T/C, whereas TAK-242 inhibited SAP formation (p<0.01).
Our results suggest that (1) Aβ binds to platelet CD36 and activates TLR4/6 through receptor cross-talk and (2) CD36-TLR signaling modulates platelet activation and enhances the formation of SAPs. Increased platelet CD36 and TLR4 levels may account for platelet hyperreactivity in POAG and AD patients.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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