Toll-like receptor 9 expression and activation in acute coronary syndrome patients on dual anti-platelet therapy
Research For Life
Reports of research work funded by grants prior to 2016
Clinical Research Laboratory
Thrombotic response to atherosclerotic plaque rupture or erosion is the major underlying cause of acute coronary syndromes (ACS). Platelet activation and aggregation plays a pivotal role in the formation of arterial thrombi in ACS. Dual antiplatelet therapy (DAPT), a combination of aspirin with a P2Y12 receptor antagonist, has been shown to substantially reduce the risk of adverse cardiovascular events and is the current standard of care for ACS. However, a proportion of patients will continue to experience recurrent ischaemic events despite treatment with DAPT. Because platelet activation can occur via multiple signalling pathways, alternative activation pathways that are not currently therapeutically targeted may contribute to this ongoing ischaemic risk in some patients. Platelet toll-like receptors (TLRs) may represent such an alternative pathway.
TLRs are known to be expressed on and within platelets and various immune-driven, anti-microbial effects have been attributed to platelet-TLR agonism. Most papers have reported TLR expression and activation in small cohorts of healthy volunteers but little information is available on how these parameters are modulated in ACS.
The grant given by WMRF in June last year allowed us to pursue the following aims, adapted from the original application submitted in March:
TLR9 is the least well-characterised platelet-TLR but is known to be present in platelets TLR9-mediated platelet activation in response to free radical-altered self-ligands has recently been characterised. TLRs 1, 2 and 6 are expressed on the platelet surface and are known to be pro-atherogenic. TLR2 dimerises with either TLRs 1 or 6 to recognize different bacterial lipopeptides and TLR2/1 agonism induces platelet activation. TLR4 is the most well-characterised platelet-TLR. Significant platelet cell-surface TLR4 expression was reported and LPS administration induces platelet activation and aggregation.
Fig 1. TLR9 expression increases in ACS platelets and upon platelet activation, compared to resting healthy platelets.
Platelet-TLR9 expression, as measured by MFI, was analysed in response to 1 µM and 10 µM TRAP stimulation. Mean ± SD for 5 subjects in each cohort are shown. One way ANOVA was found to be significant (p<0.05) for each parameter and allowed for Student’s t-tests to be performed. * p<0.05, ** p<0.01.
Western blot analysis for TLRs 1, 4, and 6 in unstimulated whole platelet lysates for healthy subjects (H1, H2) and ACS subjects (A1, A2).
We have demonstrated that TLR9 expression was significantly elevated in ACS, compared to healthy platelets. This may indicate increased sensitivity to TLR9 agonists and increased platelet reactivity in these patients. Platelet activation caused increased TLR9 expression in healthy platelets. This is a potential mechanism to explain increased platelet-TLR9 expression in ACS patients who undergo extensive platelet activation during their event. ODN2006-mediated platelet activation in PRP demonstrates the functionality of platelet-TLR9. We are the first to demonstrate ODN2006-mediated platelet activation in ACS patients on DAPT, indicating that the TLR9 pathway is inadequately inhibited by current anti-platelet drugs. Platelet-TLR9 appears to be a novel connection between oxidative stress, infection and platelet activation. Activation of this pathway potentially increases the risk of thrombosis and the risk of adverse cardiovascular events.