Tyrosine phosphorylation of the adhesion molecule VE-cadherin is assumed to affect endothelial junction integrity. However, it remains unclear whether tyrosine residues of VE-cadherin are required for the induction of vascular permeability and the regulation of leukocyte extravasation in vivo. We found here that knock-in mice expressing a Y685F mutant of VE-cadherin had impaired induction of vascular permeability, but those expressing a Y731F mutant did not. In contrast, mice expressing the Y731F VE-cadherin mutant showed decreased neutrophil-extravasation in cremaster tissue, but those expressing the Y685F mutant did not. Whereas inflammatory mediators induced the phosphorylation of Tyr685 in vivo, Tyr731 showed high baseline phosphorylation. Leukocytes triggered dephosphorylation of Tyr731 via the tyrosine phosphatase SHP-2, which allowed the adaptin AP-2 to bind and initiate endocytosis of VE-cadherin. Thus, Tyr685 and Tyr731 of VE-cadherin distinctly and selectively regulate the induction of vascular permeability or leukocyte extravasation.

SARS-CoV-2 vaccine ChAdOx1 nCov-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models and analysis of VITT patient samples we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the EDTA-containing vaccine. Injected vaccine increased vascular leakage in mice leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release NETs in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drive thrombosis in VITT. The data support a two-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

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Angiogenic blood vessel growth requires several distinct but integrated cellular activities. Endothelial cell sprouting and proliferation lead to the expansion of the vasculature and give rise to a highly branched, immature plexus, which is subsequently reorganized into a mature and stable network. Although it is known that integrin-mediated cell-matrix interactions are indispensable for embryonic angiogenesis, little is known about the function of integrins in different steps of vascular morphogenesis. Here, by investigating the integrin b1-subunit with inducible and endothelial-specific gene targeting in the postnatal mouse retina, we show that b1 integrin promotes endothelial sprouting but is a negative regulator of proliferation. In maturing vessels, integrin b1 is indispensable for proper localization of VE-cadherin and thereby cell-cell junction integrity. The sum of our findings establishes that integrin b1 has critical functions in the growing and maturing vasculature, and is required for the formation of stable, non-leaky blood vessels.