An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly.
A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5-pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl-Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.
Church
W R
WR
Department of Biochemistry, University of Vermont, Burlington 05405.
Messier
T L
TL
Tucker
M M
MM
Mann
K G
KG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000990:Antithrombin III / Q000378:metabolism; D002417:Cattle; D004285:Dogs; D004792:Enzyme Precursors / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D005170:Factor X / Q000037:antagonists & inhibitors / Q000276:immunology*; D015951:Factor Xa; D009842:Oligopeptides / Q000378:metabolism; D011487:Protein Conformation; D011516:Prothrombin / Q000378:metabolism*; D011817:Rabbits; D015842:Serine Proteinase Inhibitors; D013045:Species Specificity; D013552:Swine; D013925:Thromboplastin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D000939:Epitopes; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; C015608:benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide; D000990:Antithrombin III; D011516:Prothrombin; D005170:Factor X; D013925:Thromboplastin; C012007:prethrombins; D015951:Factor Xa