α-Thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells

Abstract

Cultured endothelial cells secrete a platelet-derived growth factor-like molecule (PDGFc). We examined the effects of purified human α-thrombin on the production of PDGFc in cultures of human umbilical vein endothelial cells (HUVE) using a specific radioreceptor assay for PDGF. Addition of physiologically relevant concentrations of α-thrombin (0.1 to 10 U/ml) induced a time- and dose-dependent increase in the release of PDGFc into the culture medium. Significant stimulation of PDGFc release was observed as early as 1.5 h after addition of α-thrombin (10 U/ml) with a 4.9 ± 1.1 fold increase at 24 h (mean ± SEM of nine experiments, P < 0.01). α-Thrombin treatment of HUVE did not affect cell viability as assessed by trypan blue dye exclusion. The receptor binding of PDGFc secreted by HUVE in response to α-thrombin was inhibited by monospecific antibody to purified human PDGF indicating that the molecule(s) is closely related to PDGF. α-Thrombin inactivated with diisopropylfluorophosphate was without stimulatory effect. Lysis of HUVE by repeated cycles of freeze/thaw released minimal PDGFc (<0.3 ng per 106 cells) compared to levels of PDGFc released into supernatant medium in response to α-thrombin (>5.0 ng per 106 cells after a 24-h incubation with 10 U/ml α-thrombin). Moreover, incubation of freeze/thaw lysates of HUVE with α-thrombin failed to release PDGFc. Over a 3-h time course, however, α-thrombin-induced secretion of PDGFc was not prevented by cycloheximide. We conclude that α-thrombin induces secretion of PDGFc from HUVE by a nonlytic mechanism requiring the serine esterase activity of the enzyme. Although this effect does not initially require de novo protein synthesis, it does require cell-mediated conversion of PDGFc from an inactive to an active form.