Abstract
Platelet activation is pathological in acute myocardial infarction (AMI) and sustained platelet activation, despite treatment with dual anti-platelet therapy (DAPT), has been linked to an increased risk of recurrent cardiovascular events. In addition to platelets, neutrophils have acquired a distinct role in the pathophysiology of cardiovascular diseases such as AMI. Neutrophils are effector cells of the innate immune system with phagocytotic and antimicrobial activity and also have the ability to produce neutrophil extracellular traps (NETs), structures made up of chromatin and histones which are decorated with proteases and granular proteins. During sterile inflammation such as AMI, neutrophils are activated to produce NETs, which can provide a scaffold for platelets to participate in coronary thrombus formation. Further, thrombogenic platelets have been implicated in mediating further NET formation. This positive feedback loop is beneficial during infections; capturing and preventing the spread of microbes around the body reduces the risk of damage to the host. Yet, the positive feedback loop between these two effector cells may have detrimental effects during AMI; NETs driving platelet activation and platelet activation driving NETs can create an unwanted cycle of increased inflammation and thrombosis. Sustained inflammation and thrombosis can cause damage to the myocardium during AMI. However, the evidence in support of the positive feedback loop between platelets and NETs is controversial. Moreover, it remains to be investigated how the standard treatment following AMI, dual anti-platelet therapy (DAPT), affects the neutrophil-NET-platelet axis.
Here, we investigated the existence of a propagating feedback loop between NETs and platelets and found that cell-free NETs are not sufficient in driving platelet aggregation. Rather activated neutrophils, independent of NET formation, are required to aggregate platelets. Moreover, we found that activated platelets reduce NET formation, contrary to the literature.
We further assessed whether DAPT had any effects on neutrophil function, NET formation, and the dampening effects of platelets on NETosis. Here, we demonstrated that in vitro DAPT directly reduced the release of soluble mediators as well as NET formation. We were unable however, to inhibit platelets in vitro using DAPT, and as such could not investigate the effects of in vitro DAPT-treated platelets on NET formation. To counter this, we conducted a prospective interventional study to examine the effects of in vivo DAPT-treated platelets on NET formation and found that platelets maintained their ability to dampen NET formation despite DAPT. In vivo DAPT also affected the release of soluble mediators from neutrophils, however in a more discriminatory fashion compared to in vitro DAPT. Moreover, NET formation was increased post in vivo DAPT under specific conditions. To address whether these functional changes could be attributed to genomic changes caused by DAPT, we investigated transcriptional changes in 8 healthy participants on DAPT. The inter-individual variability was greater than any differences caused by DAPT and as such, we did not identify differentially expressed genes at the single gene level. When gene clusters were evaluated, we identified changes in gene clusters associated with calcium signalling, indicating that DAPT may affect calcium signalling and we hypothesize that this effect of DAPT primes neutrophils for NET formation, which can be driven by calcium signalling.
Together, this work suggests an alternative model of the neutrophil-NET-platelet axis where NETs are not sufficient to induce platelet aggregation and platelets reduce NET formation. Furthermore, while we report opposing effects of DAPT in vitro and in vivo, DAPT may prime neutrophils for NET formation, while platelets retain their ability to dampen NET formation despite DAPT in vivo.