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

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.Vascular injury or inflammation results in the activation of thrombin (1, 2). Thrombin cleaves and activates multiple substrates to mediate hemostasis through fibrin clot formation and platelet aggregation (3-7) and also regulates blood coagulation by activating the anticoagulant protein C pathway (8). The predominant mechanism for the clearance of thrombin is through inhibition by antithrombin III (ATIII), 1 a member of the family of serine protease inhibitors known as serpins (1, 9 -11). The physiological importance of this process is illustrated by the observation that individuals possessing inherited ATIII deficiency or insufficiency are subject to recurrent thrombosis (12).ATIII is a 58-kDa glycoprotein that inhibits thrombin in two kinetically distinct steps: the formation of a weak initial complex followed by rapid conversion to a stable complex (13). Based on the crystal structures of ATIII (14) and other serpins in cleaved (15, 16) and uncleaved (17) forms, inhibition has been proposed to proceed through the recognition of the Arg 393 -Ser 394 bond in an exposed loop of ATIII as a substrate. However, upon cleavage, a conformational change in the inhibitor has been proposed to result in the trapping of the enzyme either as a tetrahedral intermediate or as an acyl-enzyme. Unlike the intermediate typically formed with substrates, the intermediate formed between thrombin and ATIII is stable and resistant to hydrolysis perhaps due to the conformational change which may exclude water from the active site (9).The rate-limiting step in the inhibition of thrombin by ATIII is the formation of the initial complex (ϳ2 ϫ 10 5 M Ϫ1 min Ϫ1 ) which can be accelerated by 3 orders of...

Roles of Low Specificity and Cofactor Interaction Sites on Thrombin during Factor XIII Activation

“…5B), and this is located within exosite I. We compared our results with previously published findings of the residues involved in thrombomodulin-enhanced activation of protein C (19) by thrombin and those involved in heparin-dependent antithrombin inhibition of thrombin (20). The substrates involved in each of the respective reactions (factor XIII, protein C, and antithrombin), which have either procoagulant or anticoagulant activities, are either directly recognized or influenced by the common residues on thrombin, Glu-229, Arg-233, and Trp-50 (Fig.…”

“…A library of 53 thrombin mutants encompassing a total of 78 surface exposed charged and polar residues mutated to alanine was prepared and checked for amidolytic activity as described previously (Table I) (20,23). These mutant thrombins were screened for their ability to activate factor XIII using a modification of a 5-(biotinamido)pentylamine incorporation assay (24).…”

“…For this purpose, we have utilized a library of 53 thrombin mutants encompassing a total of 78 surface exposed, charged, and polar residues mutated to alanine. This library has previously been used to identify residues of thrombin involved in the interaction with many of its substrates including protein C, thrombinactivatable fibrinolysis inhibitor, fibrinogen (19), antithrombin (20), factor V (21), and factor VIII (22). Here, we have used the library to investigate which residues of thrombin are involved in direct factor XIII activation and in the fibrin-enhanced reaction.…”

Molecular Mapping of the Thrombin-Heparin Cofactor II Complex

“…Inhibition rate constants for these thrombin mutants (k 2 values) were reduced from 2-6-fold when compared with wild-type thrombin (k 2 ϭ 2.38 Ϯ 0.26 M Ϫ1 min Ϫ1 ; Table II). These results suggest that a substantial number of thrombin residues are involved in the recognition of HCII in the absence of glycosaminoglycans, in contrast to AT inhibition of thrombin (37).…”

“…HCII-Thrombin Active Site Region Interactions-Residues located around or near the active site, particularly residue Trp 50 (56,57), are critical for the thrombin-HCII interaction (this study), thrombin-AT interaction (37,56,57), activation of factor V (34), protein C activation (33), and TAFI activation and the thrombin-thrombomodulin interaction (33). Logically, these results suggest that mutating residues in the active site region disrupts the interaction of thrombin with macromolec- ular substrates.…”