Kinetic analysis of plasminogen activation by purified plasma kallikren

Abstract

Kinetics of plasminogen activation by purified activated plasma kallikein have been studied in a purified system using Glu-plasminogen as a substrate. A synthetic paranitroanilide substrate was used for quantification of the formed plasmin. In that system kallikrein cleaved plasminogen with a K m value of 0.56μM, a K cat of 1.6x10 −4s and a catalytic efficiency k cat/K m of 2.7×10 −4s −1. Addition of CNBr fibrinogen fragments resulted in an increase of K m to 1.18μM, an increase of k cat to 5.1x10 −s −1 ad an increase in the catalytic rate constant k cat/K m to 4.3×10 −4s −4 μM −1. Addition of purified high molecular weight kininogen had no effect on the kinetics of plasminogen activation whether or not stimulating fibrinogen fragments were present. A stimulating effect of fibrinogen fragments could also be shown for the cleavage of the low molecular weight peranitroanilide substrate li-D-Pro-Phe-Arg-pNA by kallikrein; in that system the k cat for substrate cleavage by kallikrein increased from 200s −1 to 280s −1 while the K m value remained unchanged. From these data it can be concluded that based enzyme kinetic studies plasminogen activator activity of purified plasma kallikrein is about 1/1000 of that of high molecular weight urokinase and is only slightly influenced by addition of stimulating fibrinogen fragments. Addition of high molecular weight kininogen does not affect plasminogen activator activity of purified plasma kallikeren.