title: The thrombin-sensitive region of protein S mediates phospholipid-dependent interaction with factor Xa.
abstract: To test the hypothesis that factor Xa (fXa) interacts with protein S, fXa was labeled active-site specifically with a dansyl (D) dye via a Glu-Gly-Arg (EGR) tether to yield DEGR-fXa(i). When protein S was added to phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicle-bound DEGR-fXa(i), the anisotropy of the dansyl moiety was altered from 0.219 +/- 0.002 to 0.245 +/- 0.003. This change in dansyl anisotropy was not observed when DEGR-Xa(i) was titrated with protein S in the absence of PC/PS vesicles, or in the presence of 100% PC vesicles, or when PC/PS vesicle-bound DEGR-fXa(i) was titrated with thrombin-cleaved protein S. The protein S-dependent dansyl fluorescence change was specific for fXa because it was not observed for two homologous and similarly labeled DEGR-fIXa(i) and DEGR-fVIIa(i). Furthermore, protein S specifically and saturably altered the fluorescence anisotropy of PC/PS-bound active site-labeled LWB-FPR-fXa(i) (Kd = 33 nm) and was photocross-linked to PC/PS-bound LWB-FPR-fXa(i) analog, independently confirming the above results. Chemically synthesized microprotein S, comprising residues 1-116 of protein S and including the gamma-carboxyglutamic-rich domain, the thrombin-sensitive region (TSR), and the first epidermal growth factor-like domain (EGF1) of protein S, altered the anisotropy of PC/PS-bound DEGR-fXa(i) from 0.219 to 0.242, similar to the effect of the protein S titration (Kd = 303 nm), suggesting that microprotein S binds to DEGR-fXa(i). To identify individual protein S domain(s) that binds DEGR-fXa(i), the EGF1 and TSR domains were chemically synthesized and studied. The TSR altered the anisotropy of DEGR-fXa(i) by approximately 16% (Kd = 3.9 microm), but the EGF1 domain had no effect on the signal. In controls, the TSR domain did not alter the anisotropy of DEGR-fIXa(i) and DEGR-fVIIa(i), respectively. These data demonstrate that membrane-bound fXa binding to protein S involves the TSR of protein S.
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: Tilman M
authors.initials: TM
authors.lastname: Hackeng
authors.forename: Philip E
authors.initials: PE
authors.lastname: Dawson
authors.forename: John H
authors.initials: JH
authors.lastname: Griffin
mesh_terms: D015951:Factor Xa / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes; D006801:Humans; D008567:Membranes, Artificial; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017293:Protein S / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D017293:Protein S; D013917:Thrombin; D015951:Factor Xa
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