The binding of activated protein C to factors V and Va.
Activated protein C has been derivatized with the active site-directed fluorophore 2-(dimethylamino)-6-naphthalenesulfonylglutamylglycylarginyl chloromethyl ketone (2,6-DEGR-APC). Covalently modified activated protein C has been used to investigate the binding interactions of the protein to factors V and Va in the presence of phospholipid vesicles. The fluorescence polarization of the 6-dimethylaminonaphthalene-2-sulfonyl moiety increased saturably with increasing phospholipid concentrations in the presence or absence of factor V or Va. Differences in the limiting polarization values indicated distinguishable differences in the interactions between 2,6-DEGR-APC and phospholipid in the presence of factor V or Va. The dissociation constant calculated for the 2,6-DEGR-APC/phospholipid interaction (7.3 X 10(-8) M) was not significantly altered by factor V but was decreased to 7 X 10(-9) M in the presence of factor Va. The interaction between 2,6-DEGR-APC and factor V or Va was characterized by a 1:1 stoichiometry. The binding of 2,6-DEGR-APC to factor V or Va in the presence of phospholipid could be reduced in a competitive manner by diisopropylphosphofluoridate-treated activated protein C. An analysis of the displacement curves indicated that the binding of 2,6-DEGR-APC was indistinguishable from the binding of diisopropylphosphofluoridate-treated activated protein C. The interaction between 2,6-DEGR-APC and phospholipid-bound factor Va was further examined using the isolated subunits of factor Va. Fluorescence polarization changes observed with component E of Va (light chain) closely corresponded with the changes observed with factor Va, whereas isolated component D (heavy chain) had little influence on the binding of 2,6-DEGR-APC to phospholipid vesicles. The data presented are consistent with the interpretation that component E of factor Va contains a binding site for activated protein C.
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D003619:Dansyl Compounds; D005165:Factor V / Q000378:metabolism*; D015943:Factor Va; D005454:Fluorescence Polarization; D006023:Glycoproteins / Q000378:metabolism*; D007531:Isoflurophate / Q000494:pharmacology; D008433:Mathematics; D010743:Phospholipids / Q000378:metabolism; D011486:Protein C
D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D006023:Glycoproteins; D010743:Phospholipids; D011486:Protein C; D007531:Isoflurophate; D015943:Factor Va; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; D005165:Factor V