Conversion of antithrombin from an inhibitor of thrombin to a substrate with reduced heparin affinity and enhanced conformational stability by binding of a tetradecapeptide corresponding to the P1 to P14 region of the putative reactive bond loop of the inhibitor.

I Björk,K Ylinenjärvi,S T Olson,P E Bock

doi:10.1016/s0021-9258(18)46042-5

I Björk, K Ylinenjärvi + Show 2 more

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https://doi.org/10.1016/s0021-9258(18)46042-5

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Abstract

A synthetic tetradecapeptide having the sequence of the region of the antithrombin chain amino-terminal to the reactive bond, i.e. comprising residues P1 to P14, was shown to form a tight equimolar complex with antithrombin. A similar complex has previously been demonstrated between alpha 1-proteinase inhibitor and the analogous peptide of this inhibitor (Schulze, A. J., Baumann, U., Knof, S., Jaeger, E., Huber, R. and Laurell, C.-B. (1990) Eur. J. Biochem. 194, 51-56). The antithrombin-peptide complex had a conformation similar to that of reactive bond-cleaved antithrombin and, like the cleaved inhibitor, also had a higher conformational stability and lower heparin affinity than intact antithrombin. These properties suggest that the peptide bound to intact antithrombin at the same site that the P1 to P14 segment of the inhibitor occupies in reactive-bond-cleaved antithrombin, i.e. was incorporated as a sixth strand in the middle of the major beta-sheet, the A sheet. The extent of complex formation was reduced in the presence of heparin with high affinity for antithrombin, which is consistent with heparin binding and peptide incorporation being linked. Antithrombin in the complex with the tetradecapeptide had lost its ability to inactivate thrombin, but the reactive bond of the inhibitor was cleaved as in a normal substrate. These observations suggest a model, analogous to that proposed for alpha 1-proteinase inhibitor (Engh, R.A., Wright, H.T., and Huber, R. (1990) Protein Eng. 3, 469-477) for the structure of intact antithrombin, in which the A sheet contains only five strands and the P1 to P14 segment of the chain forms part of an exposed loop of the protein. The results further support a reaction model for serpins in which partial insertion of this loop into the A sheet is required for trapping of a proteinase in a stable complex, and complete insertion is responsible for the conformational change accompanying cleavage of the reactive bond of the inhibitor.

Highlights

From the $Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, S-751 23 Uppsala, Sweden,the llDiuision of Biochemical Research, Henry Ford Hospital, Detroit, Michigan 48202, and the IlDepartment of Pathology, Vanderbilt University School of Medicine, Nashuille, Tennessee 37232-2561
We show that a tetradecapeptide with the sequence of the analogous
The results were expressed as antithrombin activity

Summary

Binding of Reactive Loop Peptide to Antithrombin

EDTA, 0.1% (w/v) polyethylene glycol 6000,pH 7.4, containing 1pg/ The relative molecular mass of the N-acetylated tetradecapeptide was ml of unfractionated heparin. A volume of 100 pl of this dilution was 1345. All experiments were done in 0.02 M sodium phosphate, 0.1 M buffer without heparin, and the mixture was incubated for 1min at NaCl, 100 p~ EDTA, pH 7.4, unless otherwise indicated. 100 pl of the mixture to a cuvette containing the thrombin substrate D-phenylalanyl-L-pipecolyl-L-arginylp-nitroanilide

RESULTS

Amino acid Antithrombin peptide comnlex

Binding of RPeeLapoctotiidpvee

DISCUSSION

Binding Aofn tRPi tetLehoaprocototimipdvebe i n