"Clotspeed," a Mathematical Simulation of the Functional Properties of Prothrombinase

Prothrombinase is a Ca2+-dependent, 1:1, enzymatic complex of Factor Xa and Factor Va that assembles on the surface of negatively charged phospholipid vesicles or platelets. It catalyzes the proteolytic conversion of prothrombin to the blood-clotting enzyme thrombin. Experimentally determined kinetic parameters, plus Kd and n values for the interaction of substrate, cofactor (Factor Va), and serine protease (Factor Xa) for both phospholipid and each other, were used to develop a model that simulates the functional properties of the enzymatic complex. Through the use of a desk-top computer and a program designated "Clotspeed," the distribution of enzymatic components and substrate between the bulk fluid and phospholipid is determined for a given set of initial concentrations of reaction components. Simulated reaction rates are then calculated from the calculated distributions, fractional binding, and local and bulk concentration of reactants. Predicted behavior includes formal Michaelis-Mentenlike properties for the reaction, increasing apparent Km with increased levels of phospholipid, and apparent inhibition by excess substrate, enzyme, and phospholipid. Inhibition by excess enzyme and phospholipid was demonstrated experimentally in quantitative agreement with predicted results. The model is useful in that it rationalizes well the seemingly unusual properties of prothrombinase in straightforward physical terms, provides a means of rationally choosing experimental conditions to both further test and refine the model, and explores the properties not only of prothrombinase but also other blood-clotting or surface-bound enzymatic complexes.