Plasminogen activation by recombinant tissue plasminogen activator and potentiation by fibrin: a refined kinetic analysis

Abstract

From previous studies, it is generally accepted that fibrin potentiates the tissue plasminogen activator (t-PA) mediated activation of plasminogen (PLgN) by substantially lowering the Km of the reaction. However, in the majority of these studies, kinetic constants for PLgN activation by t-PA were derived from experiments where dissimilar PLgN concentration ranges were employed in enzyme assays with and without fibrin. From this study, and employing a similar PLgN substrate range (0.07–1.0μM) in reactions with and without fibrin I (des-AA-), we show that by Michaelis-Menten kinetics, there is little alteration in the Km of the above reaction with fibrin addition, while kcat increases up to 10-fold. In the absence of fibrin I, recombinant two chain t-PA (rTCt-PA) yielded the following kinetic parameters: kcat 0.065s−1 and 0.30s−1, for Glu- and Lys-PLgN respectively, Km 0.45 μM for both. In the presence of fibrin I between 0.05 and 0.3μM, and with rTCt-PA as the enzyme, the activation of Glu- and Lys-PLgN obeyed Michaelis-Menten kinetics over the substrate range 0.07 to 0.7 μM. For PLgN activation at infinite fibrin concentration, values for kcat were estimated as 0.60s−1 and 1.25s−1, values for Km as 0.31 and 0.16 μM, and values for KF (the dissociation constant for the fibrin-rTCt-PA complex) as 0.075 and 0.070 μM, for Glu- and Lys-PLgN respectively. To determine whether the apparent Michaelis-Menten parameters for t-PA activation of PLgN are dependent on the employed substrate concentration range, the kinetics of this reaction were reassessed over a broad PLgN range (0.002-30μM). For rTCt-PA activation of Lys-PLgN, reaction kinetics were found to be non-standard, and consistent with cooperativity. The reaction yielded two sets of parameters by Michaelis-Menten kinetics; kcat values were 0.26 and 0.89s−1, and Km values were 0.30 and 2.7 μM. Taken as a whole, data presented here suggest that in the majority of previous studies, the kinetic constants estimated for reactions in the absence and presence of fibrin might be inappropriate for comparison, owing to the possibility of t-PA exhibiting non-standard Michaelis-Menten kinetics.