BINDING AND PROCESSING OF MULTIMERIC VITRONECTIN BY VASCULAR ENDOTHELIAL-CELLS

The multifunctional adhesive glycoprotein vitronectin (VN) undergoes a unique conformational transition from the plasma form into a multimer ic form that represents the reactive heparin-binding form. In this stu dy we investigated the interaction of multimeric vitronectin (VNmult) or VN-gold conjugates (which are equivalent in biochemical properties) with confluent and subconfluent monolayers of porcine endothelial cel ls. Time-dependent direct binding of radiolabeled VNmult to the lumina l face of endothelial cells at 37 degrees C was observed which was com peted by heparin, whereas plasma VN showed hardly any binding. At 4 de grees C binding of VNmult remained cell-associated, whereas after 6 hr at 37 degrees C a major portion of the ligand was translocated throug h cells and was associated with the subcellular matrix. Cytochemical s tudies with VN-gold conjugates were performed to demonstrate uptake of VNmult. At 4 degrees C only surface decoration of cells with gold lab el was seen, which was totally reversible in the presence of heparin. Subsequent incubation for various time intervals at 37 degrees C revea led disappearance of gold label from the surface and accumulation of c onjugates in a perinuclear distribution inside the cells as judged bot h by electron microscopy and after silver enhancement by light microsc opy. Cross-sections of endothelial cells demonstrated the inclusion of VN-gold conjugates in coated pits, endosomes, and in lysosomal compar tments dose to the nucleus. Within 2-6 hr a portion of VN-gold conjuga tes had accumulated with proteoglycans at the matrix face. These data provide strong evidence for specific routing of a portion of VNmult fr om the circulation into extravascular spaces, where the protein is bel ieved to fulfill major adhesive and regulatory functions particularly as co-factor in plasminogen activation and immune defense.