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

Abstract

Thrombin inactivation by heparin cofactor II (HCII) is accelerated by ternary complex formation with heparin. The novel active-site-labeled thrombins, [4′F]FPR-T and [6F]FFR-T, and the exosite I probe, Hir-(54–65)(SO3-), characterized thrombin exosite I and II interactions with HCII and heparin in the complex. HCII binding to exosite I of heparin-bound [4′F]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)(SO3-) 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)(SO3-) 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.