Major cell types in the coronary thrombosis of acute myocardial infarction patients revealed by scRNA-seq

Dear Editor,

Intracoronary thrombosis, resulting from the rupture or erosion of atherosclerotic plaques, is the main cause of acute myocardial infarction (AMI). The role of immune cells in thrombosis has garnered attention as a central contributor to this catastrophic event.¹ To further our understanding, we conducted single-cell RNA sequencing to picture a cellular atlas of aspired intracoronary thrombus.

Our dataset comprised a total of 10 456 cells, and a median of 2786 genes were detected per cell (Figure 1). Unsupervised clustering revealed that the largest part of cells in the intracoronary thrombus were monocytes (29.3%). We further subclustered monocytes and identified eight subclusters (Figure 2A). Cluster 6 was identified as conventional dendritic cells, for the high expression levels of HLA-DRB1 and HLA-DRA. The remaining clusters were categorized based on the relative expression levels of CD14 and FCGR3A, leading to three classifications: classical (CD14⁺⁺/FCGR3A⁻; clusters 0, 2, 4), intermediate (CD14⁺⁺/FCGR3A⁺; clusters 1, 3) and non-classical monocytes (CD14⁺/FCGR3A⁺⁺; clusters 5, 7) (Figure 2B,C). Marker genes and functional heterogeneity for each cluster are presented in Figure 2D,E. Besides, among the monocytes, 14 modules of co-regulated genes were identified by weighted gene co-expression network analysis (WGCNA) (Figure 2F).

Classical monocytes comprised the largest proportion of the monocyte population (60.1%) and exhibited diverse functions. Cluster 0 (30.1%) demonstrated significant activation in response to non-infectious stimuli during AMI, characterized by elevated expression levels of NLRP3, IL-1B and NFKBIA. Module brown (TNF and NF-kappa signalling pathway) displayed a strong correlation with cluster 0 (Figure S1A). Cluster 2 (21.8%) displayed heightened expression of inflammatory mediators including S100A9, S100A8, CTSS and phagocytosis receptor CD36 (Figure 2G). Figure 2H,I proved its presentation. This cluster had a strong correlation with module tan, which is linked to phagosome and leukocyte trans-endothelial migration (Figure S2B). Cluster 4 (6.6%) exhibited elevated expression of T cell activation genes (CD247, NKG7, GNLY) and was associated with module black (nature killer [NK]-mediated cytotoxicity) (Figure S1C).

Intermediate monocytes represented 35.7% of the total monocyte population and comprised two distinct clusters. Cluster 1 (26.9%) showed a pro-fibrotic and anti-inflammatory profile, characterized by high expression levels of genes associated with fibrosis (FN1, SPP1), cardiac protection (CTSL, ANGPTL4), T cell activation (ALCAM), oxidized low-density lipoprotein-induced cell injury (ANGPTL4) and phagocytosis (MERTK). Four modules were predominantly expressed in cluster 1. Notably, module pink facilitated substrate adhesion-dependent cell spreading and endocytosis (Figure S1D), while module green-yellow was associated with focal adhesion, extracellular matrix−receptor interaction and calmodulins (Figure S1E). Intercellular communication network analysis among monocytes indicated a robust connection between clusters 1 and 2 (Figure 2J). Close receptor-ligand pairs included SPP1-CD44 and MIF-CD74 (Figure 2H), both associated with immunosuppression and the M1/M2 macrophage transition.^{2, 3} Cluster 3 (8.8%) exhibited an activated cellular response to heat stress, as evidenced by the high expression of heat shock proteins and the activation of MAPK signalling pathway, indicating its role as a stress-reacted protective monocyte subtype. Module magenta (regulation of cellular response to heat, response to unfolded protein, DNA repairment and IF-8 production) showed preferential expression in cluster 3 (Figure S1F). Additionally, module blue (HIF-1 signalling pathway and glycolysis/gluconeogenesis) reflected metabolic reprogramming in inflammatory macrophages responding to tissue injury in a hypoxic environment⁴ (Figure S1G), significantly correlating with both intermediate monocyte clusters.

Non-classical monocytes, comprising clusters 5 and 7, constituted 4.2% of the total monocyte population. Cluster 5 (2.8%) expressed genes associated with patrolling (VAV2, ITGAL, PKN1 and ARHGEF18, Figure 3A), and showed a predominant role in haemostasis. Upregulated functions included platelet activation, signalling and aggregation, regulation of cell shape, cell surface interactions at the vascular wall and glycoprotein VI−mediated activation cascade (Figure 3B). Furthermore, it facilitated T cell proliferation, neutrophil degranulation, Fc epsilon RI-mediated MAPK activation and cytokine signalling, highlighting its potential involvement in both innate and adaptive inflammatory reactions to plaque eruption/erosion during AMI. CDKN1C, a potent cell-cycle inhibitor, was highly expressed in this subset (Figure 3C), indicative of its proapoptotic and highly differentiated nature. Cluster 7 (1.4%), the smallest monocyte cluster, exhibited pronounced expression of homeostasis-related terms including VEGF signalling, and complement and coagulation cascades. Recruited monocytes are known to amplify thrombosis.⁵ Our study proved that non-classical monocytes, previously thought to be associated with post-AMI healing,⁶ in fact contributed to homeostasis and thrombosis.

To infer cellular state progression, RNA velocities were estimated (Figure 3D). Cluster 0 marked the starting point of pseudotime, representing the most immature monocyte cluster (Figure 3E), while cluster 1 denoted the most advanced stage of differentiation. Intermediate monocytes and other classical clusters differentiated from cluster 0, whereas non-classical monocytes exhibited a more distinct trajectory. These findings suggest that intermediate monocytes arise locally from resident classical monocytes, whereas non-classical monocytes may originate from circulating monocytes. The visualized velocity and the top six pseudotime-dependent genes − including NFKBIA, TIMP1, IL1B, PLIN2, TNFAIP3 and CCL3 − were shown in Figure 3F,G, all of which underscore their pivotal role in the AMI process.^7-9 Both NFKBIA and TNFAIP3 serve as negative inflammatory modulators. Moreover, TIMP1, PLIN2 and CCL3 have all been recognized as predictors of future cardiovascular events in AMI patients.

In addition to monocytes, neutrophils in the coronary thrombus were classified into three types (Figure S2). Cluster 1 (44.9%) represented N1 neutrophils in a heightened inflammatory state. Cluster 2 (2.6%) was a small subpopulation of exhausted neutrophils expressing GRK5.¹⁰ NK cells were subdivided into five clusters (Figure S3), with clusters 0, 1, 2 representing type II NKT cells, while clusters 3 and 4 comprised CD56^dim NK cells. T cells were categorized into five subclusters, with one identified as CD8⁺ T cells (cluster 2) and four as CD4⁺ T cells (clusters 0, 1, 3, 4) (Figure S4). Macrophages constituted a minor population in coronary thrombus and could be subclustered into two types (Figure S5): cluster 0 represented an anti-inflammatory type, whereas cluster 1 exhibited an accelerating phenotype.

In conclusion, we found that SPP1⁺ intermediate monocytes constitute a large proportion of intra-thrombus monocytes and play a significant role in fibrosis, anti-thrombosis and metabolic reprogramming. Contrary to expectations, intra-thrombus non-classical monocytes act as pro-thrombotic patrols, promoting atherothrombosis rather than exhibiting anti-inflammatory effects. Key cell subclusters identified in this study provide potential therapeutic targets for cardiovascular ischemic events.

Z.W. designed the project, analysed the data, interpreted the results and wrote the manuscript. C.C., Z.L., J.L. and Y.S. collected patient samples. J.W. performed nucleus isolation and snRNA-seq. J.G. and L.S. supervised the project.

The authors declare no conflict of interest.

This work was supported by the Beijing Municipal Science and Technology Commission [grant number: Z191100006619106] and the National High Level Hospital Clinical Research Funding [grant number: 2023-GSP-QN-3].

The institutional review board central committee at Fuwai Hospital approved the study (2022-1734), and all eligible patients provided informed consent.