Enhanced Detection and Functional Analysis of Reticulated Platelets in Myocardial Ischemia/Reperfusion Injury

Introduction: Myocardial ischemia/reperfusion (I/R) injury occurs when an occluded coronary vessel is reopened during myocardial infarction (MI), significantly impacting infarct size and patient outcomes. Platelets are key players in the inflammatory response associated with I/R injury, but the role of reticulated platelets (RP)-the youngest and most reactive subset-remains poorly understood (Ibrahim et al., 2014). Traditional methods for detecting RP such as RNA staining and size-based separation are limited by variability in platelet size and potential labeling of other cell types and nucleic acids (Hille et al., 2020). This study aims to improve RP detection and investigate their role in myocardial damage using innovative detection and mouse models. Methods: The kinetics of RP infiltration into lesioned myocardium were assessed using mice engineered to tag ribosomal protein in platelets - a characteristic feature of RP. Pf4-Cre:RiboTag mice expressing a haemagglutinin (HA.11) tag in ribosomes under the control of the platelet factor 4 promoter were used. Male Pf4-Cre mice (10-14 weeks) underwent 30 minutes of left anterior descending (LAD) artery ligation followed by 1 or 2 days of reperfusion. Blood was collected via terminal cardiac puncture and diluted with Dulbecco's phosphate buffered saline (DPBS+/+). Platelet agonist (400 µM PAR4 peptide) was added or omitted, and samples were stained with platelet surface markers (CD41/61 (JON/A), CD62P, CD42b) and anti-HA.11 antibody (RiboTag). Analysis was conducted using a FACSCanto II cytometer and FlowJo software. Hearts were perfused with cold DPBS, embedded in O.C.T. medium, snap-frozen, and stored at -80°C until analysis. Myocardial sections were stained with fluorophore-conjugated antibodies targeting CD31, CD41, HA.11 (RiboTag), and Hoechst, then imaged using confocal microscopy. Results: I/R injury led to a modest increase in circulating RP levels 1 day post-MI compared to sham-operated mice (12% ± 0.7% vs. 10% ± 0.3%, MI vs. sham respectively, n=7, p= 0.0168). By day 2 post-MI, RP levels in circulation increased 1.7-fold to 18% ± 1.6% vs. 10.4% ± 0.7% (MI vs. sham respectively, n=7, p= 0.0019). Despite this increase, there was no significant difference in platelet count between naïve and day 2 post-MI (432 ± 78 vs. 487 ± 18 x103/µL) . RP exhibited significantly heightened alpha granule release (P-selectin surface expression) and integrin (GPIIb/IIIa) activation upon agonist stimulation compared to non-RP. RP responsiveness to PAR4 agonist was higher in sham-operated mice than in mice 1 or 2 days post-I/R. RP are thus hyper-reactive in comparison to non-RP, but the reactivity of circulating RP decreases 1 day after I/R injury, possibly due to complex formation with leukocytes (Starz et al., 2022), or recruitment to the peri-infarct zone. RP displayed a more pro-inflammatory phenotype (CD41/CD61+, CD62P+) (Spurgeon, Frelinger, 2023) than non-RP 2 days post-MI. By microscopy, platelets were found to translocate into the affected myocardium during I/R injury, preferentially translocating to the infarct core and the peri-infarct zone (including the vessel wall lining), with fewer platelets observed in remote areas such as the left atrium. In contrast, co-staining of platelets carrying the RiboTag (to identify RP) showed that RP predominantly invaded the peri-infarct zone. Conclusions: Our findings reveal that platelets infiltrate infarcted and reperfused hearts, with RP preferentially translocating to the peri-infarct zone. RP exhibit hyper-reactivity and enhanced pro-inflammatory features following I/R injury in both mice and humans (Stratz et al., 2016). These results achieved through advanced RP staining methods and a binary RP detection mouse model (Pf4-Cre), underscore the complex role of RP in myocardial I/R injury.