Abstract
Heparin regulates the inhibitory activity of antithrombin. It has been proposed that residues P15 and P14 are expelled from beta-sheet A of antithrombin by heparin binding, permitting better interaction of the reactive center loop with factor Xa. We have made a P14 antithrombin variant (S380E) to create an activated inhibitory form of antithrombin in which P14 is already expelled from beta-sheet A. S380E antithrombin fluorescence is enhanced 35 +/- 5% compared with control antithrombin. There is minimal further increase in antithrombin fluorescence upon heparin binding. The variant has a 5 degrees C lower T(m) than control antithrombin. The variant is an inhibitor of proteinases and has a nearly 200-fold increased basal rate of inhibition of factor Xa, after correction for an increased stoichiometry of inhibition. This is comparable to that of antithrombin activated by high affinity heparin pentasaccharide. Full-length high affinity heparin causes only a 7-fold additional increase in rate and a large increase in stoichiometry of inhibition. In contrast, the basal rate of inhibition of thrombin is similar to that of control antithrombin but is increased 300-fold by heparin. These findings suggest that the native state of the S380E variant exists in a loop-expelled conformation that is consequently highly reactive toward factor Xa.
Highlights
Both biochemical [5, 6] and x-ray diffraction studies [7,8,9,10,11] support a mechanism for the heparin-induced conformational change in the reactive center loop in which the P15 and P14 residues at the N-terminal end of the reactive center loop, which are inserted into -sheet A as strand 4 in the low reactivity state of native antithrombin, are expelled from the -sheet upon heparin binding (Fig. 1) to give a reactive center loop that is very much more reactive toward factor Xa
Fluorescence Emission Spectra of S380E Antithrombin in the Absence and Presence of Heparin—An approximately 35– 40% enhancement of the fluorescence emission spectrum of plasma and recombinant wild-type antithrombin, together with a 2-nm blue shift in the position of the emission maximum occur as a result of the conformational changes that occur upon heparin binding [26]
In the absence of heparin, we found that the S380E variant had a normalized fluorescence intensity that was already 35 Ϯ 5% higher than that of control antithrombin, suggesting that the conformation of the variant was similar to that of heparinactivated antithrombin, and that the environments of tryptophans 225 and 307 had been changed in the manner expected from expulsion of the reactive center loop from -sheet A
Summary
Production and Isolation of Antithrombin—S380E antithrombin was created using human antithrombin cDNA on an N135Q background. Except for the reactions of factor Xa and thrombin with control antithrombin and of thrombin with S380E variant antithrombin, for which the second order rate constants were slow, all rate constant determinations were made from continuous progress curve assays, using the change in absorbance at 405 nm resulting from hydrolysis of chromogenic substrate. For these measurements the antithrombin:proteinase ratio was at least five times the SI to maintain pseudo first order reaction conditions. Full-length high affinity heparin, Mr 15,000, was prepared by fractionation of heparin first by size-exclusion chromatography and by antithrombin affinity chromatography, as described [25]
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