The influence of fibrin(ogen) fragments on the kinetic parameters of the tissue‐type plasminogen‐activator‐mediated activation of different forms of plasminogen

In the present work we have determined K m,app and k cat,app values for tissue‐type plasminogen‐activator‐catalyzed activation of Glu‐plasminogen, Lys‐plasminogen and mini‐plasminogen in the absence and in the presence of fibrinogen‐derived fragments. These were CNBr fragment 2, the Aα chain remnant of CNBr fragment 2 (Aα 148–207) and plasmin‐generated fragment D‐EGTA. The time course of plasmin formation from the various types of plasminogen (plg) was measured spectrophotometrically in a coupled assay system where d ‐valyl‐ l ‐leucyl‐ l ‐lysine p ‐nitroanilide served as a plasmin substrate. The kinetic constants are summarized as follows. (Values in parentheses are concentrations at which the minimum K m,app and maximum k cat,app value is reached.) Type of plg Added fragment None CNBr fragment 2 Aα 148–207 D‐EGTA K m k cat K m k cat K m k cat K m k cat nM min −1 nM min −1 (nM) nM min −1 (nM) nM min −1 (nM) Glu 53 0.12 3 (10) 3.5 (350) 16 (350) 8.6 (17000) 14 (500) 0.58 (750) LYS 30 0.37 10 (10) 13 (700) 26 (170) 16.3 (17000) 13 (750) 1.15 (750) Mini 61 0.21 no significant effects no significant effects 13 (500) 0.36 (375) In conclusion our results show that CNBr fragment 2, Aα 148–207 and to some extent D‐EGTA mimic the accelerating effect of fibrin. The first two of these fragments did not accelerate activation of mini‐plasminogen, lacking the kringle structures I–IV. This suggests that the stimulating effects of these two fragments were dependent on the presence of kringles I–IV of the plasminogen molecule.