STABLE EXPRESSION OF HUMAN PLATELET-DERIVED GROWTH-FACTOR-B CHAIN BY BOVINE AORTIC ENDOTHELIAL-CELLS - MATRIX ASSOCIATION AND SELECTIVE PROTEOLYTIC CLEAVAGE BY THROMBIN

The localization of platelet-derived growth factor (PDGF) B chain and the regulation of its release by thrombin were investigated in bovine aortic endothelial cells stably transfected with the cDNA for human PD GF B chain (c-sis). Northern blot analysis of c-sis transfected cells revealed increased expression of PDGF B mRNA and constitutive release of 5-10 fold greater amounts of PDGF than in control cells. Incubation with bovine cy-thrombin further induced PDGF release into the conditi oned medium. Metabolic labeling of bovine aortic endothelial cells ove rexpressing c-sis revealed an inefficient rate of constitutive PDGF re lease, with the majority of newly synthesized PDGF remaining extracell ular matrix-associated. Thrombin treatment, however, led to a dramatic increase in the amount of PDGF released into the medium due to select ive proteolytic cleavage of matrix-associated precursors. Incubation w ith a synthetic peptide representing residues 212-230 of precursor PDG F B chain, previously shown to induce the release of PDGF from cell- o r matrix-associated heparan sulfate proteoglycans, led to the release of slightly larger species of PDGF which were susceptible to proteolyt ic cleavage by thrombin in vitro. In addition, PDGF precursors immunop recipitated from cells were also cleaved by thrombin in vitro. We have demonstrated, using normal diploid endothelial cells overexpressing c -sis, that PDGF B chain is stably expressed as a matrix-associated pro tein which is either inefficiently released into the medium or cleaved by paracrine proteases such as thrombin. Modulation of PDGF release b y selective cleavage of preformed, matrix-bound precursors may represe nt a significant mechanism for acute regulation of release of this gro wth factor independent of changes in the rate of synthesis.