Platelet Toll-like Receptors in Healthy and Acute Myocardial Infarction Subjects

Platelet activation is pathological in acute myocardial infarction (AMI). Despite treatment with dual anti-platelet therapy (DAPT), platelet activation can continue to occur post-AMI and has been linked to an increased risk of recurrent cardiovascular events. Toll-like receptors (TLRs) are important innate immune receptors, and platelets are known to express a subset of TLRs. The functional significance of these platelet-TLR pathways in AMI has not been fully examined but may contribute to persistent post-AMI platelet activation. Platelet-TLR expression, TLR-mediated platelet activation and the platelet effect on leukocyte responses to TLR stimulation were examined in this thesis.   Platelet-TLR expression and TLR-mediated platelet activation was examined for a subset of these receptors (TLR1, 2, 4, 6 and 9) in healthy subjects and in AMI subjects on DAPT. We observed an increase in platelet expression of TLR1, 4 and 9 in AMI platelets compared to healthy subjects. Further investigation into platelet-TLR9 expression showed an increase in expression upon platelet activation in healthy, but not AMI, subjects. We observed direct, dose-dependent platelet activation in response to Pam3CSK4 (TLR2/1 agonist) and ODN2009 (TLR9 agonist) in healthy subjects and in AMI on DAPT. For both cohorts, platelets were also directly activated by a high dose of LPS (TLR4 agonist) but were not directly activated by FSL-1 (TLR6 agonist). These results demonstrate that some (TLR1, 2, 4 and 9), but not other (TLR6), platelet-TLR pathways can cause platelet activation in AMI despite treatment with potent anti-platelet therapy.   For the results described above, we were unable to assess TLR-mediated platelet activation in the absence of anti-platelet therapy in AMI subjects as these drugs are administered before or immediately upon presentation to hospital. It was therefore not possible to exclude the possibility that DAPT was providing a degree of inhibition of platelet activation in AMI patients. To address this, we determined the extent to which aspirin monotherapy or DAPT could inhibit platelet activation in response to TLR2/1, TLR4 and TLR9 stimulation in a cross-over study in healthy subjects. We demonstrated that DAPT only modestly inhibited, and aspirin monotherapy did not inhibit, platelet activation in response to all TLR agonists tested and platelets still became potently activated despite treatment with anti-platelet agents. These platelet-TLRs represent intact on-treatment platelet activation pathways.   Lastly, we determined the extent to which platelets modulate leukocyte responses to TLR2/1, TLR2/6 and TLR4 stimulation. Platelets were able to reduce neutrophil responses to TLR stimulation, and modulated PBMC cytokine and chemokine production in a complex manner following stimulation with LPS and FSL-1. The presence of platelets did not change cytokine/chemokine production in response to Pam3CSK4, demonstrating a TLR agonist-specific manner of platelet modulation. We further investigated the effect of platelets on neutrophil responses to TLR stimulation. With platelets, neutrophil activation was attenuated, and phagocytic activity was increased in unstimulated cultures and in response to various doses of Pam3CSK4 and FSL-1. Neutrophil elastase secretion was attenuated in unstimulated cultures and in response to low-dose stimulation with all three TLR agonists. We show that platelets can both augment and attenuate various markers of neutrophil function.  Together, this work indicates that platelets express functional TLR pathways that can differentially regulate a number of thrombotic and inflammatory responses in healthy subjects and in subjects with AMI.