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.

Björk, Ingemar; Ylinenjärvi, K; Olson, Steven T.; Bock, Paul E.

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

Cited by 112 publications

(16 citation statements)

References 44 publications

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“…In latent PAI-1, 13 residues of the reactive center loop (P16−P4) are inserted into β-sheet A as strand 4A (). This “insertion” site, between strands 3A and 5A, is an obvious epitope for a serpin inhibitor, as has also been proven for antithrombin ( , ), α 1 -antitrypsin (), and PAI-1 ( , ) using different segments of the loop structure. However, in contrast to the effect of these peptides, AR-H029953XX did not induce cleavage of PAI-1 by tPA.…”

Section: Discussionmentioning

confidence: 67%

Identification of the Binding Site for a Low-Molecular-Weight Inhibitor of Plasminogen Activator Inhibitor Type 1 by Site-Directed Mutagenesis

et al. 1998

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A novel low-molecular-weight inhibitor, AR-H029953XX, was developed from a known fibrinolytic compound, flufenamic acid, which prevented complex formation of human plasminogen activator inhibitor type 1 (PAI-1) with tissue plasminogen activator (tPA) by inhibition of PAI-1. To explore the binding site for AR-H029953XX, mutants of human PAI-1 were constructed by site-directed mutagenesis and were then expressed in CHO cells, purified, activated, and characterized. (1) PAI-1 with mutations in the reactive center loop: L1-PAI-1 (P10, Ser337Glu) had stability and activity similar to those of wild-type PAI-1 (wt-PAI-1), and L2-PAI-1 (P12, Ala335Glu) was highly stable but was a substrate for tPA. (2) PAI-1 with mutations near the binding epitope for the strongly inhibiting monoclonal antibody CLB-2C8: C1-PAI-1 (Phe114Glu), C2-PAI-1 (Val121Phe), C3-PAI-1 (Arg76Glu/Arg115Glu/Arg118Glu), and C4-PAI-1 (Arg115Glu) were all comparable in activity and stability to wt-PAI-1. AR-H029953XX (Ki = 25 microM) prevented complex formation between tPA and active wt-PAI-1 as well as that with mutants L1-, L2-, C1-, C2-, and C4-PAI-1. AR-H029953XX also inhibited binding of these PAI-1 variants to the antibody CLB-2C8, as measured by surface plasmon resonance. In contrast, AR-H029953XX had almost no inhibitory effect on the complex formation of tPA with C3-PAI-1. Moreover, AR-H029953XX had no effect on the binding rate of CLB-2C8 to C3-PAI-1, or on the binding to latent PAI-1 or to cleaved L2-PAI-1. The binding site of AR-H029953XX thus appears to be located in the neighborhood of the postulated epitope for CLB-2C8, near residues Arg76 and/or Arg118. This specific domain of the PAI-1 molecule might thus also be important for the mechanism of inhibitory activity toward tPA. Moreover, the structure of this region in active PAI-1 has to be different from the corresponding regions in latent and cleaved PAI-1.

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Section: Discussionmentioning

confidence: 67%

Identification of the Binding Site for a Low-Molecular-Weight Inhibitor of Plasminogen Activator Inhibitor Type 1 by Site-Directed Mutagenesis

et al. 1998

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Section: Methodsmentioning

confidence: 99%

“…An N-acetylated tetradecapeptide having the sequence of residues 380-393, i.e., the Pi-Pu segment, of human anti-thrombin (Ser-Glu-Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-Ile-Ala-Gly-Arg) was synthesized by the solid-phase procedure (Bjork et al, 1992a). Complexes between bovine or human antithrombin and this peptide were prepared by incubating antithrombin (2 mg/mL) with the peptide (1 mg/mL) for 24 h at 37 °C in 1 M Tris-HCl and 0.5 M formate, pH 7.4, and isolating the complexes by heparin affinity chromatography (Bjork et al, 1992a). The bovine antithrombin-peptide complex had properties identical to those of the complex of human antithrombin with the peptide, indicating that the tetradecapeptide was bound to bovine antithrombin as a middle strand of the main /3-sheet of the inhibitor in a manner similar to that of human antithrombin (Bjork et al, 1992a).…”

Section: Methodsmentioning

confidence: 99%

Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A .beta.-sheet of the inhibitor during trapping of target proteinases

Björk

Nordling²,

St³

1993

Biochemistry

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Identical or highly similar antigenic determinants, not present in the intact inhibitor, were induced in antithrombin on cleavage of the reactive bond, on formation of a complex between antithrombin and a synthetic reactive-loop tetradecapeptide, and on partial denaturation of antithrombin at low concentrations of guanidinium chloride. Previous studies indicate that the common structural feature of these three modified forms of antithrombin is that the region of the reactive-bond loop on the amino-terminal side of the reactive bond, or the corresponding synthetic peptide, is inserted as a middle strand in the main beta-sheet of the inhibitor, the A sheet. The new epitopes in the three modified antithrombin forms therefore most likely are exposed as a result of this insertion. Identical or highly similar epitopes were exposed also in complexes between antithrombin and thrombin or factor Xa, strongly suggesting that a substantial segment of the reactive-bond loop is inserted into the A sheet also in these complexes. In contrast, the new epitopes were not exposed in antithrombin on binding of heparin, implying that the conformational change induced by heparin does not involve such loop insertion. These results provide the first experimental verification of recent hypotheses that insertion of the reactive-bond loop of serpins into the A beta-sheet is involved in the binding of target proteinases.

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“…The interspace between the upper part of β strand 3A and 5A therefore constitutes an attractive binding site for inhibitors of serpin activity. Peptides containing fragments of the reactive-centre loop have indeed been proved to inhibit α 1 -antitrypsin and antithrombin [22,23]. Moreover, the peptides corresponding to P14-P1 (TVASSSTA-VIVSAR) and P14-P7 (TVASSSTA) in PAI-1 have been shown to inhibit PAI-1 activity [24,25].…”

Section: Introductionmentioning

confidence: 98%

Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

et al. 1998

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This is the first reported crystal structure of a complex formed between a serpin and a serpin inhibitor. The localisation of the inhibitory peptide in the complex strongly supports the theory that molecules binding in the space between beta strands 3A and 5A of a serpin are able to prevent insertion of the reactive-centre loop into beta sheet A, thereby abolishing the ability of the serpin to irreversibly inactivate its target enzyme. The characterisation of the two binding sites for the peptide inhibitor provides a solid foundation for computer-aided design of novel, low molecular weight PAI-1 inhibitors.

show abstract

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.

Cited by 112 publications

References 44 publications

Identification of the Binding Site for a Low-Molecular-Weight Inhibitor of Plasminogen Activator Inhibitor Type 1 by Site-Directed Mutagenesis

Identification of the Binding Site for a Low-Molecular-Weight Inhibitor of Plasminogen Activator Inhibitor Type 1 by Site-Directed Mutagenesis

Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A .beta.-sheet of the inhibitor during trapping of target proteinases

Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

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