Release of heparan sulfate proteoglycans from cultured aortic endothelial cells by thrombin.
Anticoagulant heparan sulfate proteoglycans have been shown to be released from cultured endothelial cells. The effect of thrombin on their release was investigated. Thrombin at more than one unit/ml accelerated the release of [35S] sulfate-labeled glycosaminoglycans from cultured porcine aortic endothelial cells. The effect of thrombin reached a maximum after one hour of incubation, and was dependent on the enzyme concentration. 10 unit/ml of thrombin released approximately twice as much amount of 35S-glycosaminoglycans as did Hanks' balanced salt solution alone. When the active site of thrombin was blocked by either diisopropylfluorophosphate or hirudin, the enzyme effect was completely abolished. Released glycosaminoglycans were resistant to chondroitin ABC lyase digestions, but degraded by either heparitinase or nitrous acid treatments. Released 35S-materials were precipitated with trichloroacetic acid and shown to be degraded into smaller molecules after alkali treatment on Sepharose CL-6B gel filtration chromatography. On the other hand, thrombin treatment of 51Cr-labeled cells did not cause the release of radioactivity. These results indicate that thrombin potentiates the release of heparan sulfate proteoglycans from cultured aortic endothelial cells without causing an appreciable damage to the cells. The effect of thrombin is active site-dependent and requires a relatively high enzyme concentration.
Shimada
K
K
Ozawa
T
T
D000818:Animals; D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured; D004727:Endothelium / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism*; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors
D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D013917:Thrombin