Binding of human alpha-thrombin to platelet GpIb: energetics and functional effects.
Thrombin interaction with platelet glycocalicin (GC), the 140 kDa extracytoplasmic fragment of the membrane glycoprotein Ib, was investigated by using a solid-phase assay. Thrombin bound to GC-coated polystyrene wells was detected by measuring the hydrolysis of a chromogenic substrate. The monoclonal antibody LJ-Ib10, which specifically binds to the thrombin-binding site of GC, could displace thrombin from immobilized GC, whereas the monoclonal antibody LJ-Ib1, which interacts with the von Willebrand factor-binding domain of GC, did not affect thrombin binding to GC. Competitive inhibition of thrombin binding to immobilized GC was also observed using GC in solution or ligands that bind to the thrombin heparin-binding site, such as heparin and prothrombin fragment 2. Furthermore functional experiments demonstrated that GC binding to thrombin competes with heparin for thrombin inactivation by the antithrombin III-heparin complex as well. Thrombin-GC interaction was also studied as a function of temperature over the range 4-37 degreesC. A large negative heat capacity change (DeltaCp), of -4.14+/-0.8 kJ.mol-1.K-1, was demonstrated to dominate the thermodynamics of thrombin-GC complex-formation. Finally it was demonstrated that GC binding to thrombin can allosterically decrease the enzyme affinity for hirudin via a simultaneous decrease in association rate and increase in the dissociation velocity of the enzyme-inhibitor adduct. Together these observations indicate the GC binding to the heparin-binding domain of thrombin is largely driven by a hydrophobic effect and that such interaction can protect the enzyme from inhibition by the heparin-anti-thrombin III complex.
de Cristofaro
R
R
Haemostasis Research Centre, Department of Internal Medicine, Catholic University of Rome, 00168 Rome, Italy.
de Candia
E
E
Croce
G
G
Morosetti
R
R
Landolfi
R
R
D000990:Antithrombin III / Q000378:metabolism; D001667:Binding, Competitive; D006493:Heparin / Q000378:metabolism; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism*; D014909:Wheat Germ Agglutinins
D006629:Hirudins; D019038:Platelet Glycoprotein GPIb-IX Complex; D014909:Wheat Germ Agglutinins; C023592:glycocalicin; D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin