Abstract
Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Thrombin is inhibited by the serpins antithrombin III and heparin cofactor II in a reaction that is dramatically accelerated by glycosaminoglycans. The structural basis of the interaction with these inhibitors was investigated by introducing single amino acid substitutions into the anion-binding exosite (R68E, R70E) and unique insertion loops (K52E, K154A) of thrombin. The rate of inhibition of these recombinant thrombins by antithrombin III and heparin cofactor II was determined in the absence and presence of glycosaminoglycan. The second order rate constant (k2) for inhibition by antithrombin III without heparin was 3.7 x 10(5) M-1 min-1 for wild-type thrombin; rates for the mutant thrombins varied less than 2-fold. For inhibition by antithrombin III with heparin, the rate constant was 4.5 x 10(8) M-1 min-1 for wild-type thrombin with no significant differences between any of the recombinant thrombins. In contrast, the rate constant for inhibition by heparin cofactor II without glycosaminoglycan was 4.3 x 10(4) M-1 min-1 for wild-type thrombin; rates were 10-fold slower for thrombin K52E and 2- to 3-fold slower for thrombins R68E and R70E. The rate constants for inhibition of wild-type thrombin by HCII in the presence of heparin or dermatan sulfate were 9.2 x 10(8) M-1 min-1 and 9.0 x 10(8) M-1 min-1, respectively. Compared to wild-type thrombin, the rate of inhibition by HCII with glycosaminoglycan was 5- to 15-fold slower for thrombins K52E and R70E and 50- to over 100-fold slower for thrombin R68E. Thrombin K154A was inhibited by heparin cofactor II with rates similar to wild-type thrombin in all assays. These results suggest that heparin cofactor II interacts with residue Lys-52 in the proposed S1' subsite and with residues Arg-68 and Arg-70 in the anion-binding exosite of thrombin, and that these interactions contribute to the molecular basis of heparin cofactor II specificity for thrombin.
Highlights
From the §Howard HughesMedical Institute, Departments of $Medicine and TBiochemistry & Molecular Biophysics, The Jewish Hospital of St
The structural basisof the inter- substrates, the anion-bindingexosite (2, 4,5 ). This exosite is action with these inhibitors was investigated by intro-distinct from the active site and corresponds to a cluster of ducing single amino acid substitutions into the anion- basic amino acids along theactive site cleft on the carboxylbindingexosite(R68E,R70E) and unique insertion terminal side of the scissile bond [3]
In con- inhibitor, thehydroxyl groupof the active site serine interacts trast, the ratceonstant for inhibitiobny heparin cofac- with the P, residue of the serpin, resulting in the formation tor 11 without glycosaminoglycan was 4.3 X lo4 M" of a protease-inhibitor complex [10, 11].The P1 residue of min" forwild-typethrombin; rateswere 10-fold the reactive loop is a major determinant of target protease slower for thrombin K52E and 2- to %fold slower forspecificity [12]
Summary
Was accelerated over 1000-fold by heparinto amean rate constant of 4.5 X lo M" min" (TableI).Thisrate of. Similar results were reported previously under slightly surface residues of thrombin (K52, R68, and R70) appear to different conditions for plasma-derived thrombin, wild-type, selectively affect thrombin interaction with HCII. The absence of significant differences in K,,, for S-2238 strate competition assaywas used to determine the effect of (Table I) indicates that the differences in observed glycosa- these mutations on the inhibitioonf thrombin by HCII in the minoglycan-dependenitnhibitionrates were nodt ueto presence of heparin (Fig. 2B).
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