BINDING OF HUMAN ALPHA-THROMHIN TO PLATELET GPLB - ENERGETICS AND FUNCTIONAL-EFFECTS

Thrombin interaction with platelet glycocalicin (GC), the 140 kDa extr acytoplasmic fragment of the membrane glycoprotein Ib, was investigate d by using a solid-phase assay. Thrombin bound to GC-coated polystyren e wells was detected by measuring the hydrolysis of a chromogenic subs trate. The monoclonal antibody LJ-Ib10, which specifically binds to th e thrombin-binding site of GC, could displace thrombin from immobilize d GC, whereas the monoclonal antibody LJ-Ib1, which interacts with the von Willebrand factor-binding domain of GC, did not affect thrombin b inding to GC. Competitive inhibition of thrombin binding to immobilize d GC was also observed using GC in solution or ligands that bind to th e thrombin heparin-binding site, such as heparin and prothrombin fragm ent 2. Furthermore functional experiments demonstrated that GC binding to thrombin competes with heparin for thrombin inactivation by the an tithrombin III-heparin complex as well. Thrombin-GC interaction was al so studied as a function of temperature over the range 4-37 degrees C. A large negative heat capacity change (Delta C-p,), of -4.14+/-0.8 kJ mol(-1).K-1, was demonstrated to dominate the thermodynamics of throm bin-GC complex-formation. Finally it was demonstrated that GC binding to thrombin can allosterically decrease the enzyme affinity for hirudi n via a simultaneous decrease in association rate and increase in the dissociation velocity of the enzyme-inhibitor adduct. Together these o bservations indicate the GC binding to the heparin-binding domain of t hrombin is largely driven by a hydrophobic effect and that such intera ction can protect the enzyme from inhibition by the heparin-anti-throm bin III complex.