Histidine-rich glycoprotein modulation of the anticoagulant activity of heparin. Evidence for a mechanism involving competition with both antithrombin and thrombin for heparin binding.
Heparin binding to rabbit histidine-rich glycoprotein (HRG) was studied in a purified system, allowing for determination of a heparin dissociation constant of approximately 5.5 X 10(-8) M for the interaction with HRG at pH 7.0. The strong interaction between heparin and HRG was demonstrated to be competitive with the binding of both antithrombin and thrombin to the heparin chain. HRG was further tested as a modulator of the anticoagulant activity of heparin by comparing rates of the heparin-catalyzed reaction between antithrombin and thrombin in the presence and absence of added HRG. The heparin-antithrombin-thrombin reaction was modeled using the formalism of a two-substrate enzyme-catalyzed reaction with heparin as the enzyme and HRG analyzed as an enzyme inhibitor. HRG was shown to compete with both antithrombin and thrombin for binding to heparin by this kinetic analysis. Thus, both the kinetic and heparin-binding data indicate that the mechanism by which HRG modulates heparin anticoagulant activity involves competition for heparin with both the inhibitor and the protease. Inhibition by HRG of the heparin-catalyzed reaction was found to be highly dependent on pH, with a sharp increase in inhibition from about 15% to greater than 90% observed as pH was lowered from 7.4 to 7.0. Since little change in the rate of the heparin-catalyzed inhibition of thrombin by antithrombin occurs in this pH region, the dramatic change in HRG inhibition seen upon pH titration may reflect increasing ionic interaction between heparin and HRG due to the protonation of histidine residues which occurs in this pH region.
D000991:Antithrombins / Q000378:metabolism*; D001667:Binding, Competitive; D006493:Heparin / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D011506:Proteins / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism*; D000099053:Histidine-Rich Glycoprotein
D000991:Antithrombins; D011506:Proteins; D000099053:Histidine-Rich Glycoprotein; D006493:Heparin; D013917:Thrombin