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

doi:10.1083/jcb.103.3.1129

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

JM Harlan, PJ Thompson, RR Ross and DF Bowen-Pope

Cultured endothelial cells secrete a platelet-derived growth factor- like molecule (PDGFc). We examined the effects of purified human alpha- 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 alpha-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 alpha- thrombin (10 U/ml) with a 4.9 +/- 1.1 fold increase at 24 h (mean +/- SEM of nine experiments, P less than 0.01). alpha-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 alpha-thrombin was inhibited by monospecific antibody to purified human PDGF indicating that the molecule(s) is closely related to PDGF. alpha-Thrombin inactivated with diisopropylfluorophosphate was without stimulatory effect. Lysis of HUVE by repeated cycles of freeze/thaw released minimal PDGFc (less than 0.3 ng per 10(6) cells) compared to levels of PDGFc released into supernatant medium in response to alpha- thrombin (greater than 5.0 ng per 10(6) cells after a 24-h incubation with 10 U/ml alpha-thrombin). Moreover, incubation of freeze/thaw lysates of HUVE with alpha-thrombin failed to release PDGFc. Over a 3-h time course, however, alpha-thrombin-induced secretion of PDGFc was not prevented by cycloheximide. We conclude that alpha-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.
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