Enzymatic properties of t-PA/ α2-macroglobulin complexes

Abstract

Complexes between single chain tissue plasminogen activator (sc-t-PA) or two chain tissue plasminogen activator (tc-t-PA) with α 2-macroglobulin have been made from the purified constituents with α 2-macroglobulin in excess. After separation of the free tissue plasminogen activator (t-PA), some of the properties of the complexes have been evaluated. The specific activities, measured as quotient between t-PA activity and t-PA antigen, are similar for the complexes as compared to the free t-PA derivatives. Indeed, the specific activity for sc-t-PA/ α 2-macroglobulin complex is even slightly higher than for free sc-t-PA, most likely because of complexation to α 2-macroglobulin interferes with the sc-t-PA antigen analysis. The enzymatic properties of the complexes in activating plasminogen were investigated using different plasminogen concentrations, either in the presence of soluble fibrin or in its absence. All reactions obeyed Michaelis-Mentens kinetics. The k cat values were similar in all reactions (0.025–0.044 s −1). However, K m for sc-t-PA/ α 2-macroglobulin was 16 μmol/l in the absence of fibrin and 16 nmol/l in its presence. Thus, a 1000-fold decrease in K m was observed by addition of fibrin. With tc-t-PA/ α 2-macroglobulin complex the K m in the absence of fibrin was 2.1 μmol/l as compared to 18 nmol/l in its presence. This corresponds to a decrease in K m of approximately 120-fold. Inactivation of sc-t-PA/ α 2-macroglobulin complex by α 2-antiplasmin was found to be somewhat slower than the reaction with free t-PA. However, the reaction rate of sc-t-PA/ α 2-macroglobulin by PAI-1 was about 1000-fold slower as compared to the reaction with free t-PA. Furthermore, the time course of the reaction suggests a clearly reversible reaction. The physiologic implications or existence of t-PA/ α 2-macroglobulin complex in vivo are still unknown.