Immunothrombolytic monocyte-neutrophil axes dominate the single-cell landscape of human thrombosis

Abstract Thrombotic diseases remain the major cause of death and disability worldwide with insufficient preventive and therapeutic strategies available. In the last decades a prominent inflammatory component has been identified as a key driver in the initiation and propagation of thrombosis – named thromboinflammation. However, a comprehensive investigation of the human immune system in thromboinflammation, beyond histological quantification, is lacking, which is essential for the development of novel therapeutic approaches. We therefore mapped the trajectories, functional states, and intercommunication of immune cells in stroke thrombi, retrieved by thrombectomy, at single-cell resolution. We reveal distinct leukocyte subpopulations with prothrombotic and, surprisingly, prominent fibrinolytic properties characterized by aberrant activation of intracellular host defense as well as hypoxia induced pathways. A prominent thrombolytic PLAU high , PLAUR high , THBD high thrombus neutrophil subset, also expressing high levels of pro-recanalizing VEGFA and VEGFB, dominated the thrombus neutrophil environment. On the other hand CD16 high NR4A1 high non-classical monocytes with strong CXCL8, CXCL2, CXCL1 and CXCL16 mediated neutrophil- attracting and PLAU, PLAUR, THBD and TFPI mediated thrombolytic properties defined the thrombus monocyte environment. These thrombus monocyte subsets were characterized by high expression of TIMP1 and TREM1. These novel innate immune- cell subsets provide insights into the thrombogenic and pro-resolving properties of innate immune-cells. To provide mechanistic insight into these multi-omic findings, we utilized reverse translation approaches. In vitro as well as murine in vivo thrombosis models underlined the causal relevance of these immune-cell axes for thrombolysis: NR4A1 high thrombus monocytes acquired a neutrophil-chemoattractive transcriptomic phenotype, neutrophils continuously infiltrated established murine thrombi in vivo and acquired a HIF1α-mediated thrombolytic phenotype in vitro . A depletion of NR4A1 high thrombus monocytes reduced thrombus neutrophil influx and exacerbated thrombosis in vivo . Together, this unravels cross-communicating monocyte and neutrophil subsets with thrombus-resolving properties and provide a publicly accessible immune-landscape of thrombosis. This provides a valuable resource for future research on thrombo- inflammation and might pave the way for novel immune-modulatory approaches for prevention or resolution of thrombosis.