ABSTRACT
A recombinant peptide fragment of vitronectin (rVN143), that includes the Arg-Gly-Asp (RGD) cell recognition site, was expressed in Escherichia coli using a prokaryotic expression system. The addition of recombinant rVN143 peptide enhances cell adhesion and proliferation similar (approximately 70%) to those of native VN.
Subject(s)
Biomimetic Materials/metabolism , Escherichia coli/metabolism , Fibroblasts/drug effects , Fibroblasts/physiology , Protein Engineering/methods , Vitronectin/biosynthesis , Vitronectin/pharmacology , Binding Sites , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Division/drug effects , Cell Division/physiology , Cloning, Molecular , Dose-Response Relationship, Drug , Escherichia coli/genetics , Fibroblasts/cytology , Gene Expression Regulation, Bacterial/physiology , Gingiva/metabolism , Humans , Peptide Fragments/biosynthesis , Peptide Fragments/chemistry , Protein Binding , Structure-Activity Relationship , Vitronectin/classificationABSTRACT
The biological functions of vitronectin (Vn) are dependent on its conformation. Whereas plasma Vn is present in a conformation that does not bind to heparin, platelet Vn has been recognized to be in a multimeric, conformationally altered form. To further understand the characteristics of platelet Vn, the molecules present in plasma and total and size-fractionated platelet releasates were compared (1) immunologically using three conformationally sensitive epitope-defined monoclonal antibodies, (2) functionally for their ability to interact with heparin, and (3) structurally using denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Our data indicate that Vn is present in platelet releasates in two molecular weight (M(r) forms. The high M(r) fractions contain conformationally and structurally altered Vn capable of interacting with heparin, and this form is distinct from plasma Vn and purified denatured Vn. In contrast, the lower M(r) forms of Vn are similar to plasma Vn. To determine if the presence of multimeric Vn requires platelet activation, platelets were disintegrated by sonication and fractionated by density gradients. Combined sodium dodecyl sulfate-PAGE (SDS-PAGE) and immunoblotting analysis showed a codistribution of multimeric Vn and type 1 plasminogen activator inhibitor in alpha-granule-rich fractions. Thus, platelet Vn is stored in a structurally and functionally distinct form from the molecule in plasma, raising the possibility that platelet-derived heparin-binding competent Vn will accumulate in areas of vascular injury.