BINDING-SITES FOR BLOOD-COAGULATION FACTOR XA AND PROTEIN-S INVOLVINGRESIDUES-493-506 IN FACTOR VA

Inactivation due to cleavage of Factor Va (FVa) at Arg 506 by activate d protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic ove rlapping pentadecapeptides comprising FVa heavy chain residues 481-525 were tested for their ability to inhibit prothrombin activation by pr othrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The m ost potent inhibition was observed for peptide VP493 (residues 493-506 ), with 50% inhibition at 2.5 mu M VP493 also inhibited FXa in plasma in FXa-1-stage clotting assays by 50% at 3 mu M. When the C-terminal c arboxamide group of VP493 was replaced by a carboxyl group, most proth rombinase inhibitory activity was lost. VP493 preincubated with FXa in hibited prothrombinase with a pattern of mixed inhibition. Homologous peptides from Factor VIII sequences did not inhibit prothrombinase. Af finity-purified antibodies to VP493 inhibited prothrombinase activity and prolonged FXa-1-stage clotting times. VP493 also blocked the abili ty of protein S to inhibit prothrombinase independently of APC. Immobi lized VP493 bound specifically with similar affinity to both FXa and p rotein S (K-d similar to 40 nM), but did not measurably bind prothromb in or APC. These studies suggest that FVa residues 493-506 contribute to binding sites for both FXa and protein S, providing a rationale for the ability of protein S to negate the protective effect of FXa towar d APC cleavage of FVa. Possible loss of this FVa binding site for FXa due to cleavage at Arg 506 by APC may help explain why this cleavage c auses 40% decrease in FVa activity and facilitates inactivation of FVa .