Single-cell multiomics of human thrombosis reveals immunothrombolytic monocyte-neutrophil interactions and hypoxia-driven thrombus resolution

Abstract Thrombotic diseases remain a leading cause of death and disability worldwide, yet our understanding of the immunological mechanisms driving thrombosis in humans remains incomplete. While extensive research in animal models has elucidated key inflammatory pathways contributing to thrombus formation, the immune cell landscape within human thrombi is poorly understood. Here, we present the first in-depth single-cell and multi-omics analysis of human thrombi, providing unprecedented insights into the immune landscape of thrombosis. By leveraging state-of-the-art single-cell RNA sequencing (scRNA-seq), CITE-seq, and FACS, we dissect the cellular heterogeneity and functional programs of immune cells within human thrombi. This approach reveals thrombus-resolving immune axes, which challenge the conventional paradigm of innate immune cells as purely prothrombotic agents. Our analysis identifies specialized monocyte and neutrophil subsets with distinct transcriptomic and functional properties. Within the thrombus environment, neutrophils undergo a striking shift towards a pro-thrombolytic phenotype, characterized by high expression of urokinase PLAU/PLAUR, thrombomodulin, and vascular endothelial growth factors (VEGFA and VEGFB). This hypoxia-driven thrombus neutrophil subset displays hallmarks of angiogenesis and fibrinolysis. Similarly, we uncover a unique CD16⁺ NR4A1high non-classical monocyte population, which plays a central role in neutrophil recruitment through the secretion of CXCL8, CXCL2, CXCL1, and CXCL16. These monocytes upregulate thrombolytic and anti-coagulant pathways, suggesting a dual role in both sustaining inflammation and promoting thrombus resolution. To further elucidate the functional relevance of these findings, we employed reverse translational approaches using in vitro assays and an in vivo murine thrombosis model. Our experiments confirm that NR4A1high monocytes are essential for the continuous recruitment of neutrophils into thrombi, leading to the accumulation of hypoxia-stimulated, pro-thrombolytic neutrophils. Depletion of these monocytes in vivo significantly reduces neutrophil influx and exacerbates thrombosis, underscoring their functional importance in immune-thrombolysis. Furthermore, pharmacological stabilization of HIF-1α in neutrophils enhances their fibrinolytic potential, suggesting that hypoxia-mediated signaling within the thrombus microenvironment actively promotes thrombus resolution rather than merely reflecting passive ischemic injury. This study provides the first high-resolution immune landscape of human thrombosis. We redefine the role of innate immune cells in thrombosis, demonstrating that human thrombi harbor distinct thrombus-resolving immune programs beyond previously described pro-thrombotic mechanisms. These findings open new avenues for immune-modulatory therapies aimed at enhancing thrombus resolution while mitigating the bleeding risks associated with conventional thrombolytic treatments.