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.

FIGURE 1.

Interaction of protein S with DEGR-fXa_i in the presence of phospholipid vesicles. Samples containing DEGR-fXa_i (initially 200 nm) in 50 mm Hepes (pH 7. 4), 150 mm NaCl, 2 mm CaCl₂ (buffer A) were titrated with phospholipids (open circles) with either PC/PS (4:1 mole ratio) (A) or 100% PC (B) vesicles prior to the addition of protein S (filled triangles). At the end of the titration, 5 mm EDTA (filled circles) was added to reverse the fluorescence changes. The anisotropy of the dansyl moiety in DEGR-fXa_i was monitored as described under “Experimental Procedures.” C, factor Xa dependence of dansyl anisotropy change. Three parallel titrations were performed. Equimolar concentrations of DEGR-fXa_i (filled circles), DEGR-fIXa_i (open circles), and DEGR-fVIIa_i (open triangles) (initially 200 nm) were each titrated with PC/PS vesicles as described (31, 32) prior to the addition of protein S. Anisotropy (r) was measured as described above.

FIGURE 2.

Interaction of PC/PS-bound Light-with-a-Bite-FPR-fXa_i with protein S. Three parallel titrations were performed. Samples containing equimolar concentrations of LWB-FPR-fXa_i (initially 200 nm) containing PC/PS vesicles were titrated with protein S (open inverted triangles), fVa (open squares), or protein C (open triangles), and the anisotropy (r) of the fluorescein moiety measured as described in the text.

FIGURE 3.

A, the protein S ·LWB-FPR-fXa_i complex was detected using photocross-linking. LWB-FPR-fXa_i (2 μm) in buffer containing buffer A was incubated with PC/PS (25 μm) and protein S (6 μm) prior to exposure to 30 s of 254 nm irradiation. Subsequently, samples were analyzed using SDS-PAGE and visualized using anti-fXa monoclonal as primary antibody in immunoblotting as described under “Experimental Procedures.” Lane 2 contains the reaction mixture prior to UV irradiation and is designated as zero time. Lane 3 contains the reaction mixture after 30 s of exposure to UV light. B, LWB-FPR-fVIIa_i (2 μm) in buffer A was incubated with PC/PS (25 μm) and protein S (6 μm) prior to exposure to UV light as described above. Samples were analyzed by SDS-PAGE and visualized using anti-fVIIa polyclonal (Enzyme Research Laboratories) as primary antibody in immunoblotting. C, LWB-FPR-fXa_i (2 μm) in buffer containing buffer A was incubated with PC/PS (25 μm) and protein S (50 nm) prior to exposure to 30 s of 254 nm irradiation. Subsequently, samples were analyzed using SDS-PAGE and visualized using anti-protein S polyclonal (Dako) as primary antibody in immunoblotting as described under “Experimental Procedures.” Lane 1 contains protein S ·PC/PS complex irradiated with UV light, lane 2 contains LWB-FPR-fXa_I ·PC/PS complex irradiated with UV light, and lane 3 contains the reaction mix irradiated with UV light. Photocross-link between LWB-FPR-fXa_i and protein S is indicated by the arrow.

FIGURE 4.

Interaction of microprotein S with PC/PS-bound DEGR-fXa_i. Samples containing DEGR-fXa_i (initially 200 nm) in buffer A were titrated with PC/PS vesicles (not shown) up to a concentration of 50 μm before the addition of microprotein S (open squares) or thrombin-cleaved protein S (open triangles). r_O is the anisotropy of PC/PS-bound DEGR-fXa_i, whereas r is the anisotropy of the dansyl moiety in DEGR-fXa_i at any point in the subsequent protein titration.

FIGURE 5.

Interaction of PC/PS-bound DEGR-fXa_i with domains of protein S. A, DEGR-fXa_i (initially 200 nm) was titrated with chemically synthesized domains of TSR (filled inverted triangles) or EGF1 (open circles) of protein S. r_O and r are as in the legend to Fig. 3. B, the factor Xa dependence of TSR-dependent change in dansyl anisotropy. Three parallel titrations were performed. Equimolar concentrations of DEGR-fXa_i (closed circles), DEGR-fIXa_i (open triangles), and DEGR-fVIIa_i (open circles) (initially 200 nm) were each titrated with TSR of protein S, and the dansyl anisotropy monitored as described under “Experimental Procedures.”