Breath Biopsy Reveals Systemic Immunothrombosis and Its Resolution through Bioorthogonal Dendritic Nanoprobes

Abstract Immunothrombosis, an inflammation‐dependent activation of the coagulation cascade, leads to microthrombi formations in small vessels. It is a dreaded complication of COVID‐19 and a major cause of respiratory failure. Due to their size and disseminated nature, microthrombi are currently undetectable. Here, noninvasive detection of a volatile reporter in the exhaled air is introduced for assessment of systemic immunothrombosis. A dendritic nanoprobe, containing high loading of a thrombin‐sensitive substrate, is selectively cleaved by thrombin, resulting in release of a synthetic bioorthogonal volatile organic compound (VOC). The VOC is quantitated in the exhaled air biopsies via gas chromatography–mass spectrometry (GC–MS), allowing near real‐time assessment of systemic immunothrombosis. The VOC detection can be further improved with more rapid and sensitive MS‐based technologies. The amount of the VOC in the exhaled air decreases with resolution of the microvascular inflammation and intravascular fibrin depositions. Through conjugation of the thrombin‐sensitive peptide with a rhodol derivative, a novel thrombin‐sensitive fluorescent nanoprobe is developed for intravital visualization of thrombin activity in actively growing thrombi. These results establish unprecedented detection of thrombin activity in vivo, addressing this unmet medical need. This novel approach facilitates diagnosis of immunothrombosis in diseases such as diabetic complications, disseminated intravascular coagulation, and COVID‐19.