MODULATION OF THROMBIN FIBRINOGEN INTERACTION BY SPECIFIC ION EFFECTS

Steady-state measurements of synthetic substrate hydrolysis by human a-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 K(m) for fibrinogen. Measurements of K(m) 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 K(m)/d In 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 In K(m)/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 K(m) for two synthetic amide substrates, under identical solution conditions, reveal practically no change in K(m) with salt concentration, while they show a significant decrease in k(cat) when Na+ salts are replaced by K+ salts. The drastic difference in the salt dependence of K(m) 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 K(m) 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. The clotting time is minimum in the presence of NaCl and increases significantly in the presence of NaBr, KCl, or KBr.