Fibrinolysis is amplified by converting α2-antiplasmin from a plasmin inhibitor to a substrate

I. Y. Sazonova, B. M. Thomas, Inna Gladysheva, A. K. Houng, Guy Reed

Research output: Contribution to journalArticle

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Abstract

α2-antiplasmin (α2-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against α2-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of α2-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to α2-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured α2-AP. Binding studies with chimeric α2-APs revealed that none of the mAbs bound to sites in α2-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of α2-AP, or the reactive center loop in the serpin domain of α2-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of α2-AP (L 13 GNQEPGGQTALKSPPGVCS 32) near the site at which α2-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of α2-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by α2-AP (from 1.1 ± 0.1 to 51 ± 4 and 67 ± 7) indicating that they convert α2-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of α2-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known α2-AP-plasmin contacts to enhance fibrinolysis by converting α2-AP from an inhibitor to a plasmin substrate.

Original languageEnglish (US)
Pages (from-to)2087-2094
Number of pages8
JournalJournal of Thrombosis and Haemostasis
Volume5
Issue number10
DOIs
StatePublished - Oct 1 2007

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Antifibrinolytic Agents
Fibrinolysis
Fibrinolysin
Monoclonal Antibodies
Epitopes
Serpins

All Science Journal Classification (ASJC) codes

  • Hematology

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Fibrinolysis is amplified by converting α2-antiplasmin from a plasmin inhibitor to a substrate. / Sazonova, I. Y.; Thomas, B. M.; Gladysheva, Inna; Houng, A. K.; Reed, Guy.

In: Journal of Thrombosis and Haemostasis, Vol. 5, No. 10, 01.10.2007, p. 2087-2094.

Research output: Contribution to journalArticle

Sazonova, I. Y. ; Thomas, B. M. ; Gladysheva, Inna ; Houng, A. K. ; Reed, Guy. / Fibrinolysis is amplified by converting α2-antiplasmin from a plasmin inhibitor to a substrate. In: Journal of Thrombosis and Haemostasis. 2007 ; Vol. 5, No. 10. pp. 2087-2094.
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abstract = "α2-antiplasmin (α2-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against α2-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of α2-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to α2-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured α2-AP. Binding studies with chimeric α2-APs revealed that none of the mAbs bound to sites in α2-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of α2-AP, or the reactive center loop in the serpin domain of α2-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of α2-AP (L 13 GNQEPGGQTALKSPPGVCS 32) near the site at which α2-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of α2-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by α2-AP (from 1.1 ± 0.1 to 51 ± 4 and 67 ± 7) indicating that they convert α2-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of α2-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known α2-AP-plasmin contacts to enhance fibrinolysis by converting α2-AP from an inhibitor to a plasmin substrate.",
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AU - Reed, Guy

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AB - α2-antiplasmin (α2-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against α2-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of α2-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to α2-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured α2-AP. Binding studies with chimeric α2-APs revealed that none of the mAbs bound to sites in α2-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of α2-AP, or the reactive center loop in the serpin domain of α2-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of α2-AP (L 13 GNQEPGGQTALKSPPGVCS 32) near the site at which α2-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of α2-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by α2-AP (from 1.1 ± 0.1 to 51 ± 4 and 67 ± 7) indicating that they convert α2-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of α2-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known α2-AP-plasmin contacts to enhance fibrinolysis by converting α2-AP from an inhibitor to a plasmin substrate.

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