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-fXai. 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-Xai 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-fXai 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-fVIIai. Furthermore, protein S specifically and saturably altered the fluorescence anisotropy of PC/PS-bound active site-labeled LWB-FPR-fXai (Kd = 33 nM) and was photocross-linked to PC/PS-bound LWB-FPR-fXai analog, independently confirming the above results. Chemically synthesized microprotein S, comprising residues 1-116 of protein S and including the γ-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-fXai 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-fXai. To identify individual protein S domain(s) that binds DEGR-fXai, the EGF1 and TSR domains were chemically synthesized and studied. The TSR altered the anisotropy of DEGR-fXai by ∼16% (Kd = 3.9 μM), but the EGF1 domain had no effect on the signal. In controls, the TSR domain did not alter the anisotropy of DEGR-fIXai and DEGR-fVIIai, respectively. These data demonstrate that membrane-bound fXa binding to protein S involves the TSR of protein S. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.