Lorin K. Johnson

The isolation of vascular endothelial cell lines with altered cell surface and platelet-binding properties

Abstract The vascular endothelium consists of a single layer of flattened, contact-inhibited cells that line the inner surface of blood vessels. One important characteristic of these cells is that circulating blood cells should not adhere or become activated by contact with the endothelial cell surface that is exposed to the bloodstream. This non-thrombogenic surface is a characteristic not only of the endothelium in vivo, but also of cultured endothelial cells such as the clonal line of adult bovine aortic endothelium used in this study. Using the mutagen 2-chloroacetaldehyde, we have been able to isolate cell lines of endothelial origin which have stable alterations in morphology and in the ability to bind platelets to the upper cell surface. Other cell surface properties that are altered in these cells include the distribution of a major cell surface glycoprotein (LETS, fibronectin), the response to incubation with a multivalent lectin (concanavalin A) and the distribution of iodinatable cell surface proteins. The binding of platelets to the endothelial cell variants can be quantified by radioisotope labeling of the platelets and measurement of the amount of label on cell-associated platelets. When platelets were incubated with 14 C-serotonin prior to incubation with the variant endothelial cells, it was found that the binding of platelets to these cells was not accompanied by release of serotonin from platelet granules. This is in contrast to the release reported when platelets are allowed to adhere to subendothelial vessel components, and demonstrates that adhesion to endothelial surfaces is dissociable from the platelet release reaction. The use of such endothelial cell variants should allow elucidation of the biochemical properties of the normal endothelial cell surface which render it nonthrombogenic.

A strategy for obtaining active mammalian enzyme from a fusion protein expressed in bacteria using phospholipase A2 as a model

An active preparation of human phospholipase A2 (PLA2) was made after expression as an insoluble fusion protein in Escherichia coli. The new key elements required for PLA2 isolation were the maintenance of the fusion protein in solution after the initial solubilization and the use of a tryptophan cleavage procedure for regeneration of native PLA2 from the fusion protein. The fusion protein was composed of a beta-galactosidase leader peptide incorporating six consecutive threonine residues to aid in insoluble inclusion body formation, followed by a tryptophan adjacent to the N-terminus of PLA2. The fusion protein was purified from cell lysates, and the leader peptide was cleaved on the C-terminal side of the tryptophan residue with N-chlorosuccinimide. The released PLA2 was refolded and renatured to produce an enzyme with activity comparable to that of other phospholipases A2.