Abstract
Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme Factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIIIa. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at positions 467, 511, 652, or 1683 displayed attenuated FXa binding, whereas the prothrombin affinity was unaffected. The affinity toward FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicates that the enzyme:cofactor assembly of the prothrombinase and the tenase complexes are homologous and provide a useful platform for further investigation of specific structural elements involved in the FVa.FXa complex assembly.
Highlights
The formation of thrombin from prothrombin is a key event in the coagulation process
FVIIIa is a cofactor to Factor IXa (FIXa) in the activation of Factor X (FX), a reaction that in many respects is very similar to the prothrombin activation [2]
The results of this study show that regions surrounding residues 467, 511, 652, and 1683 of FVa are important for binding of Factor Xa (FXa) and for the assembly of the prothrombinase complex
Summary
Materials—Bln was from Roche Molecular Biochemicals (Germany), and Bsu36I and BspEI were from New England BioLabs (Beverly, MA). Prothrombinase Assay—Recombinant FV proteins were incubated with 0.5 unit/ml of ␣-thrombin (Hematologic Technologies Inc.) for 10 min at 37 °C prior to analysis in the prothrombinase assay. The formation of membrane-bound FXa1⁄7FVa complexes was measured by determining the rates of prothrombin activation in the presence of phospholipid vesicles at increasing concentrations of FXa and a fixed concentration of FVa. FVa (50 pM) was preincubated for 4 min with FXa (0.1–50 000 pM) and phospholipid vesicles (50 M of 10/90, PS/PC). Binding of FXa to Membrane-bound FVa Using Magnetic Beads—A magnetic bead-based assay for FXa binding to membrane-bound FVa was performed as described by Rudolph, after some modifications [39] In this assay, biotinylated phospholipid vesicles were immobilized on the surface of streptavidin-coated magnetic beads. To measure the binding of FXa to membrane-bound FVa, FVa (0.5 nM) was incubated for 10 min with the phospholipid-coated beads. Statistical Analysis—All results are presented as mean values Ϯ S.E. of three independent experiments performed in duplicate
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