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Ferritin binds to light chain of human H-kininogen and inhibits kallikrein-mediated bradykinin release.

ABSTRACT: Ferritin is an iron-storage protein that exists in both intracellular and extracellular compartments. We have previously identified H-kininogen (high-molecular-weight kininogen) as a ferritin-binding protein [Torti and Torti (1998) J. Biol. Chem. 273, 13630-13635]. H-Kininogen is a precursor of the potent pro-inflammatory peptide bradykinin, which is released from H-kininogen following cleavage of H-kininogen by the serine protease kallikrein. In this report, we demonstrate that binding of ferritin to H-kininogen occurs via the modified light chain of H-kininogen, and that ferritin binds preferentially to activated H-kininogen. We further demonstrate that binding of ferritin to H-kininogen retards the proteolytic cleavage of H-kininogen by kallikrein and its subsequent release of bradykinin from H-kininogen. Ferritin does not interfere with the ability of kallikrein to digest a synthetic substrate, suggesting that ferritin specifically impedes the ability of kallikrein to digest H-kininogen, perhaps by steric hindrance. Based on these results, we propose a model of sequential H-kininogen cleavage and ferritin binding. These results are consistent with the hypothesis that the binding of ferritin to H-kininogen may serve to modulate bradykinin release.

SUBMITTER: Parthasarathy N

PROVIDER: S-EPMC1222672 | biostudies-other | 2002 Jul

REPOSITORIES: biostudies-other

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Project description:Essentials Kallikrein amplifies contact activation and is a potential target for preventing thrombosis. We developed and characterized a kallikrein aptamer using convergent evolution and kinetic assays. Kall1-T4 prolongs intrinsic clotting time by inhibiting factor XIIa-mediated prekallikrein activation. Kall1-T4 decreases high-molecular-weight kininogen cleavage and bradykinin release. SUMMARY:Background Plasma kallikrein is a serine protease that plays an integral role in many biological processes, including coagulation, inflammation, and fibrinolysis. The main function of kallikrein in coagulation is the amplification of activated factor XII (FXIIa) production, which ultimately leads to thrombin generation and fibrin clot formation. Kallikrein is generated by FXIIa-mediated cleavage of the zymogen prekallikrein, which is usually complexed with the non-enzymatic cofactor high molecular weight kininogen (HK). HK also serves as a substrate for kallikrein to generate the proinflammatory peptide bradykinin (BK). Interestingly, prekallikrein-deficient mice are protected from thrombotic events while retaining normal hemostatic capacity. Therefore, therapeutic targeting of kallikrein may provide a safer alternative to traditional anticoagulants with anti-inflammatory benefits. Objectives To isolate and characterize an RNA aptamer that binds to and inhibits plasma kallikrein, and to elucidate its mechanism of action. Methods and Results Using convergent Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated an RNA aptamer that targets kallikrein. This aptamer, Kall1-T4, specifically binds to both prekallikrein and kallikrein with similar subnanomolar binding affinities, and dose-dependently prolongs fibrin clot formation in an activated partial thromboplastin time (APTT) coagulation assay. In a purified in vitro system, Kall1-T4 inhibits the reciprocal activation of prekallikrein and FXII primarily by reducing the rate of FXIIa-mediated prekallikrein activation. Additionally, Kall1-T4 significantly reduces kallikrein-mediated HK cleavage and subsequent BK release. Conclusions We have isolated a specific and potent inhibitor of prekallikrein/kallikrein activity that serves as a powerful tool for further elucidating the role of kallikrein in thrombosis and inflammation.

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Ferritin binds to light chain of human H-kininogen and inhibits kallikrein-mediated bradykinin release.

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