Binding of fibrin monomer and heparin to thrombin in a ternary complex alters the environment of the thrombin catalytic site, reduces affinity for hirudin, and inhibits cleavage of fibrinogen.
Interaction of the blood clotting proteinase, thrombin, with fibrin monomer and heparin to form a thrombin.fibrin monomer.heparin ternary complex is accompanied by a change in thrombin catalytic specificity. Equilibrium binding interactions in the assembly of the ternary complex were characterized quantitatively using thrombin labeled at the active site with a fluorescent probe and related to changes in thrombin specificity toward exosite I-dependent binding of hirudin and cleavage of fibrinogen. Changes in the active site environment accompanying binding of heparin or fibrin to thrombin in binary complexes were reported by fluorescence enhancements which contributed additively to the perturbation accompanying formation of the ternary complex. Quantitative analysis of the interactions supports a preferentially ordered path of ternary complex assembly, in which initial binding of heparin to thrombin facilitates binding of fibrin monomer with an approximately 40-fold increased affinity. Binding of fibrin monomer in the ternary complex decreased the affinity of native thrombin for hirudin by >100-fold and inhibited cleavage of fibrinogen, but this inhibition was overcome when fibrin(ogen)-fibrin interactions occurred. These results support a ternary complex model in which heparin binding through exosite II of thrombin facilitates fibrin monomer binding via exosite I, with accompanying changes in thrombin catalytic specificity resulting from perturbations in the active site and reduced accessibility of exosite I to hirudin and fibrinogen.
Hogg
P J
PJ
Centre for Thrombosis and Vascular Research, School of Pathology, The University of New South Wales, Sydney 2052, Australia.
Jackson
C M
CM
Labanowski
J K
JK
Bock
P E
PE
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006629:Hirudins / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005338:Fibrin Fibrinogen Degradation Products; D006629:Hirudins; C011468:fibrinmonomer; C047136:2-anilinonaphthalene-6-sulfonic acid; D005340:Fibrinogen; D006493:Heparin; D013917:Thrombin