Proteinase 3 interacts with a 111-kD membrane molecule of human umbilical vein endothelial cells

Proteinase 3 (PR3) is the major autoantigen of antineutrophil cytoplasmic a ntibodies in Wegener's granulomatosis. Previously, it was demonstrated that PR3 induces apoptosis of human endothelial cells and that PR3 contributes to endothelial cell activation by enhancing interleukin-8 production. The p resent study demonstrates that PR3 binds specifically to human umbilical ve in endothelial cells (HUVEC). Digoxigenin (DIG)-labeled PR3 bound readily t o HUVEC cultured on coverslips. By fluorescence-activated cell sorter analy sis, a homogeneous binding of PR3 to HUVEC, using either DIG-labeled or unl abeled PR3, was observed. No detectable membrane expression of PR3 was obse rved after either tumor necrosis factor-alpha stimulation or in nonstimulat ed HUVEC. The binding of PR3-DIG to HUVEC was dose-dependent and was inhibi ted by unlabeled PR3. Scatchard analysis revealed 2000 binding sites per ce ll, with a K-d of 0.1 mu M. Affinity precipitation of biotin-labeled HUVEC membrane proteins with protein G-Sepharose bearing PR3 resulted in specific precipitation of a membrane molecule with a molecular weight of 111 kD und er nonreducing conditions and 52 and 63 kD under reducing conditions. It is hypothesized that PR3, either released systemically or locally at inflamma tory sites following activation of primed polymorphonuclear neutrophils, ma y lead to endothelial cell injury and activation of endothelial cells.