Mechanism of potentiation of tissue-type plasminogen activator.

The conversion of plasminogen to plasmin catalyzed by tissue-type plasminogen activator is accelerated in the presence of fibrin, leading to an increased dissolution of fibrin clots. This rate enhancement is mimicked by a dodecapeptide segment of the fibrin molecule containing the sequence A alpha 149-160 (RLEVDIDIKIRS). At low concentrations of the dodecapeptide, the potentiation increases with concentration, but at high concentrations, the stimulation effect diminishes, giving rise to a bell-shaped curve. The maximum rate enhancement of about 10 fold is achieved at a concentration of 85 micrograms/mL. This concentration dependent phenomenon is also observed for two synthetic peptide analogues, GLEVDIDIKIRS and RGGGGGGGKIRS, although the acceleration potential is less. These results indicate that the N-terminal amino acids are not critical for the rate enhancement. The bell-shaped activity-concentration curve suggests that the dodecapeptide may bind to both plasminogen and t-PA. This speculation is further supported by the modification of the potentiator. When the A alpha 149-160 dodecapeptide is pretreated with trypsin or phenyl glyoxal, the potentiation activity is eliminated. We speculate that the acceleration of the plasminogen-to-plasmin reaction catalyzed by t-PA is achieved through the action of the stimulator to bring the enzyme and its substrate together as a bi-dentate cross-linker. This effect increases the apparent concentration of the substrate at the enzyme active site, and is reflected as a decrease in Michaelis-Menten constant.