Fig 1.
Structures of Lyso-SF and Its Analogs.
Fig 2.
Inhibitory activity of lyso-SF.
(A) Effects of Lyso-SF in plasma clotting assays: The effects of lyso-SF on fXa-initiated clotting assays (closed circles) or on IIa-initiated clotting assays (open circles). (B) Effect of Lyso-SF in II-ase assays: The effects of lyso-SF on IIa generation by the II-ase complex were tested in a purified system (see Experimental Procedures). (C) Phospholipid dependence of Lyso-SF inhibition of II-ase: The effects of lyso-SF on IIa generation by the II-ase complex were tested at three different PC/PS vesicle concentrations, no PC/PS vesicles (open circles), 6 μM PC/PS (closed inverted triangles), 15 μM PC/PS (open triangles) and 30 μM PC/PS (closed diamonds).
Fig 3.
Effect of different analogs of Lyso-SF on II activation by gd-fXa/fVa/PL.
The effects of lyso-SF (closed diamonds), psychosine (open squares), glucopsychosine (open triangles), lyso-sphingomyelin (open circles), and N-acetyl sulfatide (closed squares) on the activation of II by gd-fXa/fVa/PL are shown.
Fig 4.
Binding of Lyso-SF to fXa and Gd-fXa using SPR.
Surface Plasmon Resonance (SPR) was used to monitor binding of lyso-SF to BEGR-fXai and BEGR-gd-fXai. (A) Sensorgram depicting the dose-dependent binding of lyso-SF (from top to bottom; 185, 138, 92, 55, 37μM) to BEGR-fXai. Lyso-SF (62.5 μM) did not exhibit any binding to N-acetylsulfatide (dotted line). (B) Sensorgram depicting the dose-dependent binding of lyso-SF (from top to bottom; 92, 55, 37, 18 μM) to BEGR-gd-fXai. (C) PC/PS vesicles exhibited binding to BEGR-fXai but not to BEGR-gd-fXai.
Fig 5.
Fluorescence spectroscopy binding studies.
(A) Binding of Lyso-SF to fXa in the presence or absence of PL Vesicles. The binding of fXa to lyso-SF was monitored by fluorescence spectroscopy. Samples containing DEGR-fXai (initially 200 nM) in 50 mM Hepes (pH 7.4), 150 mM NaCl, 5 mM CaCl2 either in the presence (closed triangles) or absence (closed circles) of PC/PS vesicles were titrated with lyso-SF and the net fluorescence intensity recorded. At the end of the titration, EDTA (10 mM) was added to reverse the fluorescence change. F was the fluorescence intensity at any given point in the titration and Fo was the initial fluorescence intensity, before the addition of lyso-SF. (B) Binding of FVa to DEGR-fXa/PL in the presence or absence of Lyso-SF. DEGR-fXai (initially 200 nM) in 50 mM Hepes (pH 7.4), 150 mM NaCl, 5 mM CaCl2 was titrated with 25 μM PC/PS vesicles prior to the addition of 100 μM lyso-SF (closed circles) or buffer (closed squares). Subsequently the complex was titrated with fVa and the net fluorescence intensity of DEGR-fXai recorded at λex = 340 nm and λem = 530 nm, respectively. F was the fluorescence intensity at any given point in the titration and Fo was the initial fluorescence intensity before the addition of lyso-SF.