Heparin binding domain of antithrombin III: characterization using a synthetic peptide directed polyclonal antibody.
Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that apparently forms covalent complexes with thrombin. The interaction between ATIII and thrombin is enhanced several thousandfold by the glycosaminoglycan, heparin. We have previously proposed that the heparin binding site of ATIII resides within a region extending from amino acid residues 114-156 [Smith, J. W., & Knauer, D. J. (1987) J. Biol. Chem. 262, 11964-11972]. Computer-assisted analysis of this region revealed the presence of a 22 amino acid domain (residues 124-145), part of which shows a strong potential for the formation of an amphipathic helix: hydrophobic on one face and highly positively charged on the other. In the presence studies, polyclonal antisera were generated against a synthetic peptide corresponding to residues 124-145 in native human ATIII. Affinity-purified IgG from these antisera, as well as monovalent Fab's derived from them, specifically blocked the binding of heparin to ATIII. Additionally, occupancy of the heparin binding site by these same monovalent and bivalent IgG's at least partially substituted for heparin, accelerating linkage formation between ATIII and thrombin. These results provide the first immunological evidence that region 124-145 is directly involved in the binding of heparin to ATIII and that an antibody-induced conformational change within this region can mediate ATIII activation.
Smith
J W
JW
Department of Developmental and Cell Biology, University of California, Irvine 92717.
Dey
N
N
Knauer
D J
DJ
D000906:Antibodies; D000990:Antithrombin III / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G / Q000302:isolation & purification; D011487:Protein Conformation; D012995:Solubility
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D000990:Antithrombin III; D006493:Heparin