Conformational Activation of Antithrombin by Heparin Involves an Altered Exosite Interaction with Protease

Izaguirre, Gonzalo; Águila, Sonia; Qi, Lixin; Swanson, Richard; Roth, Ryan; Rezaie, Alireza R.; Gettins, Peter G.W.; Olson, Steven T.

doi:10.1074/jbc.m114.611707

Cited by 27 publications

(34 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, AT I207T exhibited a modest increase in inhibition stoichiometry that was more predominant in heparin-catalyzed reactions. Heparin vastly accelerates the rates of thrombin and fXa inactivation by AT; however, it also moderately promotes the substrate pathway of the branched mechanism (8), and the findings of Á guila et al (3) are consistent with this. AT I207A, which was created for comparison, behaved largely as a substrate with both proteinases but with still measurable inhibitory activity.…”

supporting

confidence: 79%

A novel antithrombin domain dictates the journey's end of a proteinase

Verhamme

2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

Edited by Norma AllewellAntithrombin (AT) is an anticoagulant serpin that irreversibly inactivates the clotting proteinases factor Xa and thrombin by forming covalent complexes with them. Mutations in its critical domains, such as those that impair the conformational rearrangement required for proteinase inactivation, increase the risk of venous thrombosis. Á guila et al. characterize for the first time the destabilizing effects of mutations in the region of AT that makes contact with the proteinase in the final acyl-enzyme complex. Their work adds new insight into the unique structural intricacies of the inhibitory mechanism.Inhibitory serpins (serine proteinase inhibitors) belong to a superfamily of proteins with a characteristic fold of three ␤ sheets, eight or nine ␣ helices, and a reactive center loop (RCL) 2 that contains a specific proteinase cleavage site (1). Of the ϳ1,500 identified serpin gene sequences, 36 are present in humans where they perform diverse functions. Antithrombin (AT), one of the 29 known human inhibitory serpins, is a major regulator of hemostasis, as inhibition of its targets thrombin and factor Xa (FXa) leads to a reduction in clotting. Inhibitory serpins are metastable: They undergo one of the largest known conformational changes upon forming covalent complexes with their proteinase targets (2). Whereas the importance of the RCL structure in this conformational change is well documented, much less is known about the structural requirements of residues in the base of the serpin, in particular the region that becomes the "landing place" for the inactivated proteinase in the covalent complex. Á guila et al. (3) now present clinical and biochemical proof that this region in the serpin is critical for inhibitory function and may be considered a new regulatory domain.Serpin action has been likened to that of a mousetrap. In the Michaelis complex, the proteinase is docked to the RCL of the native serpin; upon cleavage of the reactive site in the RCL, an acyl-enzyme intermediate is formed, and the N-terminal RCL fragment inserts as an extra strand into the principal ␤ sheet of AT, rapidly translocating the attached proteinase 180°to the distal end of the serpin (Fig. 1, A and B). In this energetically favorable and stable complex, the proteinase active site is distorted, and the serpin acts as a suicide substrate (1, 2, 4, 5). Proteinase translocation, final docking, and distortion are steps in the inhibitory pathway of the branched serpin mechanism (Fig. 1C). Disturbing the serpin structure favors the alternative side of the branched mechanism, the substrate pathway, in which the acyl-enzyme intermediate completes hydrolysis to form a cleaved serpin and regenerated proteinase.The elaborate structure of inhibitory serpins makes them particularly prone to dysfunction caused by mutations. Moderate and even mild AT deficiency, as observed in patients with heterozygous mutations, significantly increases the risk of deep vein thrombosis and pulmonary embolism. In type II AT deficiency, normal a...

show abstract

supporting

confidence: 79%

A novel antithrombin domain dictates the journey's end of a proteinase

Verhamme

2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The dissociation constant of these variants was lower than control antithrombin (Table 2). This suggested that the mutations caused a slight activation of the inhibitor in the absence of heparin, a finding observed with other antithrombin variants (6).…”

Section: Heparin Affinity Of I207t and I207a Variantsmentioning

confidence: 68%

“…Heparin activates antithrombin by two mechanisms. In one mechanism, heparin serves as a polysaccharide bridge to promote the encounter between protease and antithrombin, whereas in the second mechanism, the serpin is activated through conformational changes, which enhance reactivity with protease and involve several exosite interactions (3)(4)(5)(6). The first mechanism is dominant with the protease, thrombin, and the second selectively enhances antithrombin reactivity with factor Xa and factor IXa.…”

mentioning

confidence: 99%

Disease-causing mutations in the serpin antithrombin reveal a key domain critical for inhibiting protease activities

Águila

Izaguirre

Martínez‐Martínez

et al. 2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Edited by Norma AllewellAntithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin-protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin-protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.Serine protease inhibitors (serpins) are a superfamily of proteins that control proteases involved in inflammation, complement, coagulation, and fibrinolytic pathways (1). Inhibitory serpins exert control over their target proteases by an unusual branched pathway suicide substrate mechanism. Antithrombin inhibits its target proteases by this branched pathway in a manner that preferentially promotes the formation of a stable inhibitory covalent complex with the target protease instead of allowing the proteolytic cleavage of the inhibitor (2). This serpin requires activation by heparin to achieve a physiologically significant rate of inhibition of its target proteases. Heparin activates antithrombin by two mechanisms. In one mechanism, heparin serves as a polysaccharide bridge to promote the encounter between protease and antithrombin, whereas in the second mechanism, the serpin is activated through conformational changes, which enhance reactivity with protease and involve several exosite interactions (3-6). The first mechanism is dominant with the protease, thrombin, and the second selectively enhances antithrombin reactivity with factor Xa and factor IXa. In the serpin inhibitory mechanism, target proteases initially recognize and bind a substrate amino acid sequence in the reactive center loop (RCL) 3 of the...

show abstract

“…5). FXa interacts directly with AT at a specific exosite exposed on binding to heparin (Izaguirre et al, 2014). In addition, in the presence of calcium, a single heparin chain can bind to both AT and FXa directly, enhancing the AT-FXa interaction further (Rezaie and Olson, 2000;Lin et al, 2001), but it is not an absolute requirement.…”

Section: A Antithrombin-mediated Inhibition Of Factor Iia (Thrombin)mentioning

confidence: 99%