Partial activation of antithrombin without heparin through deletion of a unique sequence on the reactive site loop of the serpin.
Native antithrombin (AT) has an inactive reactive site loop conformation unless it is activated by a unique pentasaccharide fragment of heparin (H(5)). Structural data suggests that this may be due to preinsertion of two N-terminal residues of the reactive site loop of the serpin into the A-beta-sheet of the molecule. Relative to alpha(1)-antitrypsin, the reactive site loop of AT has three additional residues, Arg(399), Val(400), and Thr(401), at the C-terminal P' end of the loop. To determine whether a longer reactive site loop of AT is responsible for loop preinsertion in the native conformation, mutants of the serpin were expressed in which these residues were individually or in combination deleted. Kinetic analysis suggested that deletion of two residues, Val(400) and Thr(401), changed the solution equilibrium of the serpin in favor of the active conformation, thereby enhancing the inhibition of factor Xa by an order of magnitude independent of H(5). Interestingly, the reactivity of this mutant with thrombin was impaired by the same order of magnitude in the absence, but not in the presence of H(5). These results suggest that a longer reactive site loop in AT is responsible for its inactive native conformation toward factor Xa, while at same time AT requires this feature to regulate the activity of thrombin.
Rezaie
Alireza R
AR
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA. rezaiear@slu.edu
D000991:Antithrombins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism*; D010949:Plasma / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000991:Antithrombins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006493:Heparin