Platelets-Derived Integrin and Tetraspanin-Rich Tethers (PITTs) Drive Pulmonary Inflammation

Integrins are conformationally activatable heterodimeric transmembrane receptors and bidirectional signaling molecules. The dominant platelet integrin, αIIbβ3 (~ 100,000 copies/platelet), associates with the tetraspanin CD9 and is essential for hemostasis and thrombosis, but its role in thrombo-inflammation is less well defined.

Pulmonary inflammation and acute respiratory distress syndrome (ARDS) are hallmarks of severe SARS-CoV-2 infection induced coronavirus disease 2019 (COVID-19). Numerous studies have provided evidence for aberrant platelet activation and/or altered platelet function in affected patients and experimental as well as clinical data point to a role of platelets in fueling pulmonary thrombo-inflammation in this setting.

We analyzed peripheral platelets from patients with severe Covid-19 and detected a paradoxical partial loss of αIIbβ3/CD9 while the cells were apparently not in an activated state. Blood smears from these patients revealed numerous tether-like structures attached to platelets, which we termed Platelet-derived Integrin and Tetraspanin-enriched Tethers (PITTs), due to their enrichment in αIIbβ3/CD9 and the absence of other central platelet receptors. Strikingly, PITT formation was observed in the lungs of mice subjected to pulmonary infection/inflammation models (e.g., via intranasal instillation of Lipopolysaccharide [LPS], S. pneumoniae, or S. aureus), but not in control mice. These PITTs were deposited on endothelial cells and interacted with neutrophils. LPS-induced PITT deposition was abolished in von Willebrand factor-deficient (vWf^-/-) mice, which also showed reduced pulmonary neutrophil recruitment, inflammation and tissue damage.

We reproduced the deposition of PITTs on immobilized vWF under flow using platelets from newly created GFP-tagged integrin αIIb-expressing (Itga2b-GFP^tg/tg) mice, allowing for visualization of both extracellular and intracellular integrin. Interestingly, PITT formation was even more pronounced on surface-adsorbed anti-αIIbβ3 antibodies (MWReg30, JON mAbs), indicating that the immobilization of αIIbβ3 on otherwise resting platelets could trigger PITT formation. Adding anti-αIIbβ3 antibodies in solution resulted in rapid clustering of integrin αIIbβ3, along with CD9 and signaling molecules into distinct membrane microdomains, which we termed ‘disintegration’ complexes (DISCs). Under shear conditions, these DISCs serve as building blocks for PITTs.

In vivo, intravenous injection of anti-αIIbβ3 mAbs led to rapid platelet recruitment to the liver and binding of the antibody-opsonized platelets to endothelial cells. αIIbβ3/CD9 clustered at the platelet ‘rear edge’ and segregated into PITTs, which were deposited on endothelial cells. Remarkably, PITT formation was not solely dependent on shear forces but also required intracellular signaling, as evidenced by the significantly reduced PITT formation in Rac1-deficient platelets.

Collectively, our data provide novel insights into integrin regulation by mechanosensing in platelets and potentially other cells, highlighting a unique mechanism by which integrin αIIbβ3 can be irreversibly removed from cells in a process involving mechanical forces as well as intracellular signaling, driving thrombo-inflammatory pathologies.

No relevant conflicts of interest to declare.