A14-01 Megakaryocyte/platelet-derived Thrombospondin 1 Protects Alveolar Barrier Integrity by Preserving Basement Membrane Col6a1 From Neutrophil-mediated Lung Injury

Abstract Rationale Matrix remodeling is an early event of lung injury characterized by loss of basement membrane (BM), a structural scaffold critical for maintaining alveolar barrier integrity. The matricellular protein thrombospondin 1 (TSP1) is known to interact with basement membrane proteins such as collagens and laminins, but its direct role in stabilizing the BM structural network after injury remains unclear. Given that megakaryocytes (Mgk) and platelets (Plt) are the major source of TSP1, we hypothesized that Mgk/plt-derived TSP1 stabilizes the alveolar interstitial matrix by regulating the BM network and protects barrier integrity during injury. Methods Mgk/plt-specific TSP1 knockout (PF4Cre, Thbs1flox) and WT littermate mice were subjected to lung injury by intratracheal challenge of Pseudomonas aeruginosa live bacteria or sterile cell-free supernatant. 24 hours post-injury, alveolar permeability and structure were assessed using intravital multiphoton microscopy in reporter (RosamT/mG) mouse lines of Mgk/plt TSP KO and WT, Evans blue extravasation, histologic injury scoring, and lung mechanics by flexiVent. Bronchoalveolar lavage (BAL) was analyzed by label-free LC/MS-MS proteomics. Neutrophil activity and matrix degradation were assessed by BAL neutrophil elastase (NE), MPO, MMP-9, pro-COL1A1, COL4A1, and COL6A1 ELISA quantification. BALF CD63, lung neutrophils and Mgks were measured by flow cytometry. Neutrophil contribution to matrix injury was tested by depleting neutrophils in vivo using anti-Ly6G antibody. Results Deletion of Mgk/plt TSP1 results in significant impairment of lung structure and function, marked by elevated lung tissue injury scoring, extensive airspace dextran accumulation by intravital imaging, increased lung Evans blue extravasation, and reduced lung compliance with increased tissue elastance. BAL proteomic profiling revealed significant enrichment of molecular pathways of neutrophil degranulation, platelet activation, and ECM-organization with specific loss of basement membrane proteins such as COL6A1 after injury. Despite comparable neutrophil levels, Mgk/plt TSP1 KO mice show elevated BAL neutrophil activity markers, such as free NE, MPO, MMP-9, and increased surface-bound CD63 on neutrophils, consistent with BAL proteome analysis. Live intravital and second harmonic generation imaging analysis show a striking increase in Mgk events and fibrillar collagen levels in regions of airspace leak. Neutrophil depletion restored alveolar barrier integrity (BAL total protein and IgM), reduced neutrophil activity (BAL NE), decreased lung Mgk counts, and restored BAL COL6A1 levels, suggesting a neutrophil-dependent mechanism of matrix stabilization during lung injury. Conclusion Mgk/plt-derived TSP1 is a critical regulator of early basement membrane stabilization by shielding COL6A1 from neutrophil-mediated proteolytic tissue damage at the alveolar barrier interface. This abstract is funded by: National Heart, Lung, and Blood Institute, National Institutes of Health, Awards R01HL177904, R01 HL136143, P01 HL114453 (J.S.L.).