title: Glucosylceramide, a neutral glycosphingolipid anticoagulant cofactor, enhances the interaction of human- and bovine-activated protein C with negatively charged phospholipid vesicles.
abstract: The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with a K(d) (app) = 16 micro g/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with a K(d) (app) = 3 micro g/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of approximately 63 and approximately 64 A were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming kappa(2) = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent K(d) of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6-7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal gamma-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: Subramanian
authors.initials: S
authors.lastname: Yegneswaran
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Deguchi
authors.forename: John H
authors.initials: JH
authors.lastname: Griffin
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D031541:Fluorescence Resonance Energy Transfer; D005963:Glucosylceramides / Q000494:pharmacology*; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011486:Protein C / Q000378:metabolism*; D011517:Prothrombin Time
chemical_list: D000925:Anticoagulants; D005963:Glucosylceramides; D008081:Liposomes; D010718:Phosphatidylserines; D010743:Phospholipids; D011486:Protein C