Mutagenesis studies toward understanding the mechanism of differential reactivity of factor Xa with the native and heparin-activated antithrombin.
A unique pentasaccharide fragment of high-affinity heparin activates antithrombin (AT) to enhance its rate of complex formation with factor Xa (FXa) by 200-300-fold. Recent results have indicated that the activation of AT is associated with the exposure of a cryptic exosite on the serpin that is an interactive site for FXa in the complex. Previously, we identified Arg(150) on the autolysis loop of FXa as a candidate residue that may specifically interact with the heparin-activated AT. Three other surface loops on FXa including 39, 60, and the sodium-binding 220 loops have been implicated to be critical for the protease interaction with the activated AT. To determine the extent of the contribution of these loops to the specificity of the FXa interaction with activated AT, several loop mutants of the protease were prepared and their reactivity with AT was studied in both the absence and presence of pentasaccharide. Analysis of the inhibition kinetic data suggests that the residues of both 39 and 60 loop make a minor contribution to the recognition of AT in both the native and activated conformation of the serpin. On the other hand, the reactivity of AT with the sodium loop mutants of FXa in the absence of the cofactor was severely impaired. However, the extent of the rate-accelerating effect of pentasaccharide in the AT inhibition of the mutants was not affected. These results suggest that all three loops play a role in the specificity of the FXa-AT interaction; however, neither loop specifically interacts with the activated conformation of the serpin.
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA. rezaiear@slu.edu
Alireza R
AR
Rezaie
Likui
L
Yang
Chandrashekhara
C
Manithody
D019943:Amino Acid Substitution / Q000235:genetics; D000991:Antithrombins / Q000737:chemistry*; D001780:Blood Coagulation Tests; D002460:Cell Line; D002863:Chromogenic Compounds / Q000737:chemistry; D005170:Factor X / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D065427:Factor Xa Inhibitors; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity / Q000235:genetics
D000991:Antithrombins; D002863:Chromogenic Compounds; D065427:Factor Xa Inhibitors; D009844:Oligosaccharides; D011994:Recombinant Proteins; D005170:Factor X; D006493:Heparin; D015951:Factor Xa