Modulation of thrombin-fibrinogen interaction by specific ion effects.
Steady-state measurements of synthetic substrate hydrolysis by human alpha-thrombin in the presence of human fibrinogen, under experimental conditions where light scattering due to the formation of fibrin aggregates is negligible, have allowed for a quantitative evaluation of Km for fibrinogen. Measurements of Km for fibrinogen carried out at pH 7.5 and 37 degrees C as a function of NaCl, NaBr, KCl, and KBr concentration, from 50 to 500 mM, show that the derivative d ln Km/d ln a +/-, where a +/- is the mean ion activity, is constant over the entire range of salt concentrations and is strictly dependent on the particular salt present in solution. The values of d ln Km/d ln a +/- are found to be equal to 0.75 +/- 0.03 (NaCl), 0.90 +/- 0.01 (NaBr), 0.62 +/- 0.07 (KCl), and 0.60 +/- 0.03 (KBr). Measurements of Km for two synthetic amide substrates, under identical solution conditions, reveal practically no change in Km with salt concentration, while they show a significant decrease in kcat when Na+ salts are replaced by K+ salts. The drastic difference in the salt dependence of Km between fibrinogen and the synthetic amide substrate points out that a significant role may be played by the fibrinogen recognition site in the energetics of thrombin-fibrinogen interaction. The sensitivity of Km for fibrinogen to different salts unequivocally demonstrates that specific ion effects, rather than nonspecific ionic strength effects, modulate thrombin-fibrinogen interaction under experimental conditions of physiological relevance. Analysis of ion effects on clotting curves obtained at pH 7.5 and 37 degrees C also shows a drastic differential effect of cations and anions.(ABSTRACT TRUNCATED AT 250 WORDS)
De Cristofaro
R
R
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Di Cera
E
E
D001667:Binding, Competitive; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D009994:Osmolar Concentration; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D012996:Solutions; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism*; D014867:Water / Q000378:metabolism
D012996:Solutions; D014867:Water; D005340:Fibrinogen; D012964:Sodium; D013917:Thrombin; D011188:Potassium