ALTERATION OF VITRONECTIN - CHARACTERIZATION OF CHANGES INDUCED BY TREATMENT WITH UREA

Vitronectin circulates in blood as a 70-kDa monomer that interacts wit h complexes generated in the terminal steps of the coagulation and com plement cascades. Vitronectin complexed to thrombin-antithrombin or C5 b-9 is conformationally altered as evidenced by enhanced reactivity wi th monoclonal antibody 8E6 and binding to heparin; these same alterati ons also occur when vitronectin is treated with urea (Tomasini, B. R., and Mosher, D. F. (1988) Blood 72,903-912; Hogasenk, K., Molnes, T. E ., and Harboe, M. (1992) J. BioL Chem. 267, 23076-23082). We have modi fied the purifications of native and urea-treated vitronectin to bette r control conformational state and characterized the alterations induc ed by urea. After treatment with N-ethylmaleimide to prevent formation of disulfide-linked multimers, purification in 8 M urea, and dialysis against physiological saline, vitronectin was largely oligomeric (app roximately 800 kDa) as assessed by gel filtration and polyacrylamide g el electrophoresis in the absence of sodium dodecyl sulfate. Oligomeri c urea-treated vitronectin reacted more strongly with the 8E6 antibody , bound biotinylated heparin more strongly, and neutralized the antico agulant activity of heparin better than monomeric altered vitronectin or native vitronectin. After incubation with urea at 25-degrees-C, nat ive vitronectin, treated during purification with dithionitrobenzoic a cid to force free sulfhydryls to intramolecular disulfides, exhibited increased reactivity with antibody 8E6, increased binding to heparin, and oligomerization. In addition, incubation in urea caused rearrangem ent of disulfides as assessed by loss of the light chain of two-chain vitronectin. The transition for these effects occurred between 2 and 4 M urea. Thus, an irreversible conformational alteration occurs upon t reatment of vitronectin with urea, resulting in oligomers that bind av idly to heparin.