Phenomenological analysis of the clotting curve

A model-independent (phenomenological) characterization of the clotting curve is proposed. Three parameters are used to encapsulate the main features of the increase in absorbance observed at 350 nm due to the reaction of thrombin with fibrinogen that leads to clot formation: (1) the maximum increase in absorbance per unit time, delta Am, at the inflection point of the clotting curve; (2) the time needed to reach the maximum increase in absorbance, tm; and (3) the clotting time, tc, obtained from extrapolation of the slope at tm to the zero absorbance baseline. Clotting curves at low fibrinogen concentrations (0.125 divided by 0.250 microM), well below the Km, where thrombin amidase activity is rate-limiting with respect to the subsequent aggregation process, have been measured under a wide variety of experimental conditions, (i.e., as a function of thrombin concentration, pH and temperature) in order to explore the basic response of each parameter to changes in solution conditions. Under all conditions examined in this study we have observed that tm and tc are linked through a linear relationship that appears to be an important invariant property of the clotting curve, regardless of experimental conditions. No such clear relationship exists between delta Am and tc, with tc being associated with several possible values of delta Am and vice versa, depending upon solution conditions. It is proposed that tc is strictly dependent on thrombin amidase activity, while delta Am reflects properties of the aggregation process leading to clot formation. The clotting time shows a pH and temperature dependence that closely resembles that of Km/Vm for synthetic amide substrates. Furthermore, tc changes linearly with either the inverse thrombin concentration and the concentration of competitive inhibitors of fibrinogen binding to thrombin, as expected for the ratio Km/Vm. We show how the analysis of clotting curves obtained at different thrombin and inhibitor concentrations yields a quantitative measure of KI that is in excellent agreement with the value determined independently from steady-state measurements of thrombin amidase activity.