Functional Requirements for Inhibition of Thrombin by Antithrombin III in the Presence and Absence of Heparin

Manuel Tsiang,Anant K Jain,Craig S Gibbs

doi:10.1074/jbc.272.18.12024

Manuel Tsiang, Anant K Jain + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.18.12024

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Abstract

Mutation of 79 highly exposed amino acids that comprise approximately 62% of the solvent accessible surface of thrombin identified residues that modulate the inhibition of thrombin by antithrombin III, the principal physiological inhibitor of thrombin. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence and absence of heparin (W50A, E229A, and R233A) also decreased hydrolysis of a small tripeptidyl substrate. These residues were clustered around the active site cleft of thrombin and were predicted to interact directly with the "substrate loop" of antithrombin III. Despite the large size of antithrombin III (58 kDa), no residues outside of the active cleft were identified that interact directly with antithrombin III. Mutations that decreased the susceptibility of thrombin to inhibition by antithrombin III in the presence but not in the absence of heparin (R89A/R93A/E94A, R98A, R245A, K248A, K252A/D255A/Q256A) in general did not also affect hydrolysis of the tripeptidyl substrate. These residues were clustered among a patch of basic residues on a surface of thrombin perpendicular to the face containing the active site cleft and were predicted to interact directly with heparin. Three mutations (E25A, R178A/R180A/D183A, and E202A) caused a slight enhancement of inhibition by antithrombin III.

Highlights

Vascular injury or inflammation results in the activation of thrombin [1, 2]
The rate-limiting step in the inhibition of thrombin by antithrombin III (ATIII) is the formation of the initial complex (ϳ2 ϫ 105 MϪ1 minϪ1) which can be accelerated by 3 orders of magnitude (ϳ2 ϫ 108 MϪ1 minϪ1) in the presence of the glycosaminoglycan, heparin
Screening of Thrombin Mutants for Inhibition by Antithrombin III in the Presence and Absence of Heparin—All 56 mutants were screened for inhibition by ATIII both in the presence and absence of heparin

Summary

Chymotrypsinogen numberingb kcat

93 Ϯ 6 95 Ϯ 5 64 Ϯ 3 161 Ϯ 9 191 Ϯ 8 101 Ϯ 5 107 Ϯ 5 121 Ϯ 5 125 Ϯ 6 193 Ϯ 8 112 Ϯ 7 114 Ϯ 5 138 Ϯ 15 250 Ϯ 14 102 Ϯ 7 78 Ϯ 9 130 Ϯ 7 151 Ϯ 5 133 Ϯ 7 139 Ϯ 7 127 Ϯ 7 103 Ϯ 4 151 Ϯ 8 110 Ϯ 4 140 Ϯ 6 90 Ϯ 4 116 Ϯ 5 116 Ϯ 5 149 Ϯ 16 67 Ϯ 4 113 Ϯ 5 114 Ϯ 9 83 Ϯ 5 130 Ϯ 4 96 Ϯ 5 160 Ϯ 7 100 Ϯ 7 79 Ϯ 3 107 Ϯ 4 68 Ϯ 3 105 Ϯ 4 129 Ϯ 7 114 Ϯ 7 97 Ϯ 5 131 Ϯ 7 215 Ϯ 7 144 Ϯ 8 77 Ϯ 4 108 Ϯ 6 103 Ϯ 4 104 Ϯ 4 159 Ϯ 7 160 Ϯ 6. 7Ϯ3 28 Ϯ 6 42 Ϯ 6 29 Ϯ 6 35 Ϯ 7 21 Ϯ 4 24 Ϯ 4 42 Ϯ 9 43 Ϯ 7 37 Ϯ 7 21 Ϯ 4 37 Ϯ 6 36 Ϯ 6 46 Ϯ 13 15 Ϯ 4 49 Ϯ 12 45 Ϯ 17 28 Ϯ 8 36 Ϯ 4 30 Ϯ 7 46 Ϯ 8 32 Ϯ 10 23 Ϯ 3 34 Ϯ 6 20 Ϯ 4 33 Ϯ 6 30 Ϯ 6 10 Ϯ 1 25 Ϯ 5 15 Ϯ 3 40 Ϯ 5 2.7 Ϯ 0.4 14 Ϯ 3 27 Ϯ 6 25 Ϯ 3 30 Ϯ 6 39 Ϯ 7 79 Ϯ 16 a Residues numbered consecutively from the start of the thrombin B chain. This library of mutants was screened for its sensitivity to inhibition by ATIII in the presence and absence of heparin and the functional requirements for ATIII inhibition were compared with those required for the hydrolysis of a small peptidyl substrate that interacts only with the active site cleft

EXPERIMENTAL PROCEDURES

TABLE II Thrombin mutants resistant to inhibition by antithrombin III

RESULTS

DISCUSSION