title: Shedding of heparan sulfate proteoglycan by stimulated endothelial cells: evidence for proteolysis of cell-surface molecules.
abstract: Activation of endothelial cells by cytokines and endotoxin causes procoagulant and pro-inflammatory changes over a period of hours. We postulated that the same functional state might be achieved more rapidly by changes in the metabolism of heparan sulfate, which supports many of the normal functions of endothelial cells. We previously found that binding of anti-endothelial cell antibodies and activation of complement on endothelial cells causes the rapid shedding of endothelial cell heparan sulfate. Here we report the biochemical mechanism responsible for the release of the heparan sulfate. Stimulation of endothelial cells by anti-endothelial cell antibodies and complement resulted in the release of 35S-heparan sulfate proteoglycan and partially degraded 35S-heparan sulfate chains. Degradation of the 35S-heparan sulfate chains was not necessary for release since heparin and suramin prevented cleavage of the heparan sulfate but did not inhibit release from stimulated endothelial cells. The 35S-heparan sulfate proteoglycan released from endothelial cells originated from the cell surface and had a core protein similar in size (70.5 kD) to syndecan-1. Release was due to proteolytic cleavage of the protein core by serine and/or cysteine proteinases since the release of heparan sulfate was inhibited 87% by antipain and 53% by leupeptin. Release of heparan sulfate coincided with a decrease of approximately 7 kD in the mass of the protein core and with a loss of hydrophobicity of the proteoglycan, consistent with the loss of the hydrophobic transmembrane domain. The cleavage and release of cell-surface 35S-heparan sulfate proteoglycan might be a novel mechanism by which endothelial cells may rapidly acquire the functional properties of activated endothelial cells.
authors.affiliation: Department of Surgery, Duke University Medical Center Durham, North Carolina 27710, USA.
authors.forename: N S
authors.initials: NS
authors.lastname: Ihrcke
authors.forename: J L
authors.initials: JL
authors.lastname: Platt
mesh_terms: D000818:Animals; D000937:Antigen-Antibody Reactions; D002478:Cells, Cultured; D003167:Complement Activation; D003165:Complement System Proteins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D005966:Glucuronidase; D006026:Glycoside Hydrolases / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012995:Solubility; D013552:Swine; D000099175:Heparanase
chemical_list: D003165:Complement System Proteins; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D005966:Glucuronidase; D006026:Glycoside Hydrolases; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D000099175:Heparanase; C071927:perlecan