Heparin Promotes the Binding of Thrombin to Fibrin Polymer

The binding of human cr-thrombin (IIa) to fibrin polymer (FnIIp) was studied in the presence and absence of a high affinity 20,300 M, heparin (H) at pH 7.4,10.15, and 23 “C. In the absence of heparin, thrombin interacts with a high affinity class of binding sites on fibrin polymer with a dissociation constant of 301 f 36 nM in a manner which is independent of the enzyme active site. Studies of thrombin binding as a function of heparin and fibrin polymer concentrations imply that a ternary thrombin-fibrin polymer-heparin complex (IIa.FnIIp*H) is formed. Assembly of the ternary complex occurs randomly through the interactions of all three possible intermediate binary complexes; IIaaH, IIa*FnIIp, and FnIIp*H. Using an independently determined value of 280 f 35 nM for the FnIIp*H dissociation constant, global fits of the binding data yield a dissociation constant of 15 f: 6 nM for the IIa*H interaction and 47 + 9 nM for the IIa*H intermediate binary complex interaction with FnIIp. These studies indicate that heparin enhances the binding of thrombin to fibrin polymer 6.4-fold with an overall dissociation constant for ternary complex formation of 705 nM2. The effect of heparin molecular weight on ternary complex formation has also been investigated. Heparins of molecular weights 11,200-20,300 behave similarly with respect to their influence on ternary complex formation, whereas heparins of lower molecular weight are less effective in promoting thrombin binding to fibrin polymer. This effect of heparin is also independent of whether it has high or low affinity for antithrombin III. The demonstration of the formation of a ternary IIa.FnIIp*H complex complements kinetic evidence indicating the formation of an analogous ternary complex with fibrin II monomer (Hogg, P. J., and Jackson, C. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,36193623). The possible implications of these findings for the in viva distribution and actions of thrombin and the clinical efficacy of heparin are also discussed.