Proteinase 3 Interacts with a 111-kD Membrane Molecule of Human Umbilical Vein Endothelial Cells

Proteinase 3 (PR3) is the major autoantigen of anti- neutrophil cytoplasmic antibodies in Wegener's granulomato- sis. Previously, it was demonstrated that PR3 induces apoptosis of human endothelial cells and that PR3 contributes to endo- thelial cell activation by enhancing interleukin-8 production. The present study demonstrates that PR3 binds specifically to human umbilical vein endothelial cells (HUVEC). Digoxigenin (DIG)-labeled PR3 bound readily to HUVEC cultured on cov- erslips. By fluorescence-activated cell sorter analysis, a homo- geneous binding of PR3 to HUVEC, using either DIG-labeled or unlabeled PR3, was observed. No detectable membrane expression of PR3 was observed after either tumor necrosis factor-a stimulation or in nonstimulated HUVEC. The binding of PR3-DIG to HUVEC was dose-dependent and was inhibited by unlabeled PR3. Scatchard analysis revealed 2000 binding sites per cell, with a Kd of 0.1 mM. 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 under nonreducing conditions and 52 and 63 kD under reducing conditions. It is hypothesized that PR3, either released system- ically or locally at inflammatory sites following activation of primed polymorphonuclear neutrophils, may lead to endothe- lial cell injury and activation of endothelial cells.