Plasmin catalyzed fibrinolysis as quantified by a new kinetic method

Kinetics fibrinolysis by plasmin (Pro) was studied by a new method which allowed to describe quantitatively the clot lysis reaction. Cylindrical fibrin (Fn) clots (0.9 ml) were formed in glass tubes of different diameters. After addition of Pm (5 nM1 μM) in buffer over the clots, the lysis kinetics at 37°C was traced by decrease of clot column height with catetometer. Linear rate of lysis (V^I) was measured as tangent of the clot boundary movement in time (mm/min). Because of fibrin surface square (S) did not change during the lysis, the mass rates of lysis (V^M, M/min) were calculated from equation: V^M=v^I·S·[Fn]/v, where [Fn] was Fn concentration in clot, v was liquid phase volume. Dependences of V^M on the Pm and Fn concentrations, as well as on S and shaking rate were obtained. It was found that the dependence of V^M on [Fn] obeyed the Michaelis-Menten law. From the analysis of this plot in Lineweaver-Burk coordinates, the kinetic parameters of fibrinolysis by Pm were found to be k_kat=0.04 s^-1 and K_M=1 μM. The increase of S from 0.05 cm² to 1.4 cm² at the constant [Fn] resulted in the exponential raise of V^M because of the sites available for cleavage by Pm increased with S. The raise of V^M with shaking rate showed that clot lysis is limited to diffusion of Pm through buffer-clot boundary. The supposed method is quantitative, continuous, more simple and available than I¹²⁵-fibrin-labelled method. Its sensibility for Pm is 5 nM and for fibrin lysis is 50 μm. The method permits to vary widely the composition of the solid and liquid phases.