A study of the activation of fibrin-bound plasminogen by tissue-type plasminogen activator, single chain urokinase and sequential combinations of the activators

Abstract

The efficacy of tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) to activate fibrin-bound plasminogen was determined. With the use of a solid-phase model of intact and plasmin-degraded fibrin, the initial rate, Vi (fmol of plasmin/min), of plasmin generation by t-PA, scu-PA and two chain u-PA (tcu-PA) and the ratio, R (mol of plasmin generated/mol of activator) were determined as indicators of the efficiency of plasminogen activation. [Glu)t-plasminogen bound to intact fibrin was most efficiently activated by t-PA (Vi=0.843 and R=4.08), while scu-PA and tcu-PA produced markedly lower activation coefficients (Vi=0.011 and 0.023, and R=0.13 and 0.50 respectively). In contrast, when [Glu] 1-plasminogen was bound to the degraded surface, it became activatable with equivalent efficiencies by both t-PA and scu-PA (R=4.95 and 4.72, respectively). No evidence of Lys-plasminogen formation was observed. Rather, the selective activation of plasminogen bound to degraded fibrin by scu-PA is consistent with a particular interaction of Glu-plasminogen with carboxy-terminal lysine residues on the degraded surface. The consequences of this interaction of scu-PA with plasminogen bound to degraded fibrin on sequential activations of plasminogen by both t-PA and scu-PA were further explored. While the activation by scu-PA and then t-PA produced only an additive effect on plasmin generation, the opposite combination induced a greater than additive affect; the concentration of activators needed to produce 50% of plasmin generation was 70pM for the scu-PA/t-PA combination; in contrast, the amount required to produce a similar degree of activation with the opposite combination (t-PA/scu-PA) was only 17 pM. These results indicate that the potentiation of the scu-PA activity requires both the previous generation of plasmin by t-PA, and plasminogen bound to carboxy-terminal lysine residues of the degraded fibrin surface as a substrate.