Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex

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• 作者：Ingrid M. Verhamme ; ^{ingrid.verhamme@vanderbilt.edu}
• 关键词：Serpins ; Heparin cofactor II ; Thrombin inactivation ; Heparin ; Fluorescence equilibrium binding ; Stopped-flow fluorescence
• 刊名：Analytical Biochemistry
• 出版年：2012
• 出版时间：15 February 2012
• 年：2012
• 卷：421
• 期：2
• 页码：489-498
• 全文大小：551 K

文摘

Thrombin inactivation by heparin cofactor II (HCII) is accelerated by ternary complex formation with heparin. The novel active-site-labeled thrombins, [4¡äF]FPR-T and [6F]FFR-T, and the exosite I probe, Hir-(54-65)(), characterized thrombin exosite I and II interactions with HCII and heparin in the complex. HCII binding to exosite I of heparin-bound [4¡äF]FPR-T caused a saturable fluorescence increase, absent with antithrombin. Heparin binding to exosite II and a second weaker site caused fluorescence quenching of [6F]-FFR-T, attenuated by simultaneous Hir-(54-65)() binding. Stopped-flow analysis demonstrated ordered assembly of HCII and the [6F]FFR-T¡¤heparin complex, in agreement with tighter heparin binding to thrombin than to HCII. Saturating HCII dependences and bell-shaped heparin dependences of the fluorescence change reported ternary complex formation, consistent with a template mechanism in which the thrombin¡¤heparin complex binds HCII and allowing for interaction of thrombin¡¤(heparin)₂ complexes with HCII. Hir-(54-65)() displacement in reactions with FPR-blocked and active thrombin indicated a concerted action of the active site and exosite I during ternary complex formation. These studies demonstrate that binding of HCII to the thrombin¡¤heparin complex is dramatically enhanced compared with heparin binding alone and that exosite I is still available for ligand or HCII binding when both heparin binding sites on thrombin are saturated.