Lyso-Sulfatide Binds Factor Xa and Inhibits Thrombin Generation by the Prothrombinase Complex
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.