Transcriptome Analysis Identified SPP1-Positive Monocytes as a Key in Extracellular Matrix Formation in Thrombi.

Abstract

Background: Thrombi follow various natural courses. They are known to become harder over time and may persist long term; some of them can also undergo early spontaneous dissolution and disappearance. Hindering thrombus stability may contribute to the treatment of thrombosis and the prevention of embolisms. However, the detailed mechanisms underlying thrombus maturation remain unclear.

Methods: We compared the RNA expression in 3 thrombi retrieved from cerebral vessels via thrombectomy with that in simultaneously sampled blood. The results were validated using immunohistochemistry on 66 thrombi retrieved from patients with cerebral embolism. Single-cell RNA sequencing data of thrombi from pulmonary arteries were used to uncover the molecular mechanisms.

Results: A total of 1121 genes were upregulated in thrombi. Pathway enrichment and protein-protein interaction analyses revealed upregulation of extracellular matrix formation and identified SPP1 as a hub gene. Immunohistochemistry revealed that expression level of osteopontin (the transcript of SPP1) positively correlated to that of collagen type VI in thrombi. Osteopontin was expressed primarily by monocytes/macrophages in the thrombi, particularly in older ones. Among these 66 patients, the presence of collagen type VI in the thrombus was marginally associated with longer puncture-to-reperfusion time and more passes before reperfusion, indicating that those thrombi were resistant to thrombectomy. Single-cell RNA sequencing of thrombi identified a subpopulation of monocytes/macrophages that highly express SPP1. These SPP1-high monocytes/macrophages highly expressed genes related to the extracellular matrix and communicated closely with fibroblasts.

Conclusions: Collectively, our findings indicate that SPP1-high monocytes/macrophages play a crucial role in thrombus maturation.