Antithrombin III Basel. Identification of a Pro-Leu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity.

J Y Chang,T H Tran

doi:10.1016/s0021-9258(17)36071-4

Abstract

Antithrombin III Basel is a hereditary abnormal antithrombin with normal progressive inhibition activity (normal reactive site) and reduced heparin cofactor activity (impaired heparin binding site). Structures of antithrombin III Basel and normal antithrombin III isolated from the same patient were compared by peptide mapping using the dimethylaminoazobenzene isothiocyanate precolumn derivatization technique. Of the approximately 50 tryptic peptides of normal and abnormal antithrombin III, one peptide comprising residues 40-46 had a different retention time in reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide from antithrombin III Basel had a single substitution of Pro (normal) by Leu (abnormal) at position 41. This substitution is close to an Arg (residue 47) and a Trp (residue 49) which have previously been shown to be critical for heparin binding by antithrombin III. Although additional amino acid substitutions in antithrombin III Basel cannot be ruled out, this Pro-Leu replacement could cause a conformational change by increasing both the helical structure and the hydrophobicity around residue 41. These data suggest that: (i) the heparin binding site of antithrombin III encompasses the region containing residues 41, 47, and 49; and (ii) the impaired heparin cofactor activity of antithrombin III Basel is likely due to a conformational change of the heparin binding site induced by the Pro-Leu substitution at position 41.

Highlights

From the ‘$PharmaceuticalsResearch Laboratories, Ciba-GeigyLimited, CH-4002Basel, Switzerland and
Another interesting case is antithrombin I11 Toyama which has a Cys instead of Arg at position 47 [11].This Arg-Cys substitution causes complete loss of its heparin cofactor activity, but has no effect on its progressive inhibition of thrombin through the reactive site [11].Comparison of sequence homology among antithrombin 111, a-antitrypsin,and ovalbumin [19] further reveals that antithrombin I11 has a unique amino-terminal sequence. These findings indicate that the heparin binding site of antithrombin I11 includes the region surrounding residues 47 and 49
Al- Antithrombin I11 Basel, like antithrombin I11 Toyama, is a thoughadditionalaminoacidsubstitutionsinantigenetically abnormal antithrombin I11 isolated from a patient thrombin 111 Basel cannot be ruled out, this Pro-Leu with recurrent episodes of superficial thrombophlebitis [20]

Summary

RESULTS

Quantitative Amino-terminal Mappingof Tryptic Digests of Normal and Abnormal Antithrombin ZZZ-The experiment was undertaken to investigate thestructural difference of homologous proteinssurrounding the Arg/Lys-X residues. Assuming quantitative trypsin digestion at each Arg/Lys-X bond (Fig. I), any amino acid substitution at Arg/Lys positions or those immediately followArg/Lys should lead to quantitative difference in the recoveries of amino-terminal amino acids. This information is useful here because basic amino acids [3,7] have been shown to be crucial for the heparin binding site of antithrombin 111. The results (Fig. 1) show that the recoveries of amino-terminal amino acids from tryptic digests of normal antithrombin I11 agree closely with those calculated from the amino acid sequence [25], and thereis no significant difference between the recoveries from both normal andabnormalantithrombin 111.

Time m i n

DISCUSSION

AT I l l Barel