Identification of a factor IX/IXa binding protein on the endothelial cell surface.
Endothelium provides a specific binding site for Factor IX/IXa which can propagate activation of coagulation by promoting Factor IXa-VIII-mediated activation of Factor X. In this report the endothelial cell Factor IX/IXa binding site has been identified and the coagulant function of the receptor blocked. Studies using [3H]Factor IX derivatized with the photoaffinity labeling agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) and cultured bovine endothelial cells demonstrated cross-linking to a trypsin-sensitive cell surface protein of Mr approximately equal to 140,000. Immunoprecipitation of metabolically labeled endothelium with Factor IX derivatized with the cleavable cross-linking agent N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate and antibody to Factor IX demonstrated the endothelial cell origin of the Mr 140,000 cell surface protein. Blockade of the Factor IX/IXa binding protein by covalently linking SANPAH-5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa or SANPAH-Factor IX prevented both specific Factor IXa binding and effective Factor IXa-VIII-mediated activation of Factor X on endothelium. Following extraction of endothelium with detergents, Factor IX/IXa binding activity was solubilized and could be assayed using a polyvinyl chloride plate binding assay. Western blots of cell extracts demonstrated binding of 125I-Factor IX at Mr approximately equal to 140,000 which was blocked by excess Factor IX, but not antisera to Factor VIII, von Willebrand factor, alpha 2-macroglobulin, or epidermal growth factor receptor. These data indicate that endothelium provides a distinct binding site for Factor IX/IXa consisting, at least in part, of a membrane protein which can modulate the coagulant activity of Factor IXa on the cell surface.
D000818:Animals; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004727:Endothelium / Q000378:metabolism*; D005164:Factor IX / Q000378:metabolism*; D015949:Factor IXa; D005169:Factor VIII / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D001386:Azides; D003432:Cross-Linking Reagents; D003902:Detergents; D011956:Receptors, Cell Surface; C052276:factor IX-IXa receptor; C033169:3-((4-azidophenyl)dithio)propionic N-hydroxysuccinimide; D005169:Factor VIII; D005164:Factor IX; D005170:Factor X; D004492:Edetic Acid; D010738:Type C Phospholipases; D015949:Factor IXa; D014357:Trypsin