Circulating S100A8/A9 drives faster platelet COX-1 recovery via MRP4, impairing the duration of aspirin response

Low-dose aspirin is a cornerstone of cardiovascular prevention in high-risk patients, inhibiting platelet cyclooxygenase (COX-1) and reducing thromboxane A2 (TXA2) production for 24 hours. However, the duration of this effect varies among individuals, and the role of inflammation and platelet microRNAs in this variability remains unclear. This study identifies a thromboinflammatory mechanism linked to shorter duration of TXB2 suppression during aspirin therapy. This mechanism involves (1) elevated circulating S100A8/A9, indicating systemic inflammation; (2) reduced platelet microRNA-21-5p (miR-21-5p) and increased circulating miR-21-5p (c-miR-21-5p), suggesting altered microRNA regulation; (3) S100A8/A9-driven upregulation of platelet multidrug resistance protein 4 (MRP4); and (4) enhanced COX-1 expression, resulting in increased TX production. Patients with accelerated COX-1 recovery had increased circulating S100A8/A9 and platelet MRP4, directly related with serum TXB2 and platelet COX-1 mRNA/protein levels. In vitro, recombinant S100A8/A9 (rS100A8/A9) upregulated MRP4 and COX-1 in platelets and DAMI, a human megakaryoblastic cell line, and MRP4 inhibition prevented this effect. Additionally, these patients exhibited lower platelet miR-21-5p levels and higher c-miR-21-5p, inversely and directly related, respectively, to S100A8/A9, MRP4, and serum TXB2 slope. In DAMI cells, rS100A8/A9 reduced platelet miR-21-5p and increased c-miR-21-5p, effects prevented by MRP4 inhibition. During immune inflammation, reflected by high circulating S100A8/A9, once-daily aspirin may incompletely inhibit COX-1 due to miR-21-5p downregulation, higher megakaryocyte/platelet MRP4 expression, COX-1 upregulation driving platelet activation, and c-miR-21-5p release. Circulating S100A8/A9 and miR-21-5p can serve as biomarkers for patients with shorter duration of aspirin effect over 24 hours and potential targets to optimize antiplatelet therapy for high-risk patients.