Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex.
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.
Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. ingrid.verhamme@vanderbilt.edu
Ingrid M
IM
Verhamme
D005456:Fluorescent Dyes / Q000737:chemistry*; D006493:Heparin / Q000378:metabolism*; D015844:Heparin Cofactor II / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D013917:Thrombin / Q000378:metabolism*
D005456:Fluorescent Dyes; D015844:Heparin Cofactor II; D006493:Heparin; D013917:Thrombin