Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA

H R Lijnen; B Van Hoet; F De Cock; D Collen

doi:10.1055/s-0038-1647254

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1990; 64(01): 061-068
DOI: 10.1055/s-0038-1647254

Original Article

Schattauer GmbH Stuttgart

Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA^[*]

Authors

H R Lijnen

The Centerfor Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium
B Van Hoet

The Centerfor Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium
F De Cock

The Centerfor Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium
D Collen

The Centerfor Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium

Abstract

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Summary

The activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala⁷⁴⁰, a recombinant plasminogen with the active site Ser⁷⁴⁰ mutagenaed to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA -Glul²⁷⁵, a recombinant single chain t-PA in which the Arg of the plasmin sensitiv e Arg²⁷⁵- Ile²⁷⁶ peptide bond was substituted with Glu. Conversion of ¹²⁵I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v₀ in pM s⁻¹) per 1 μM enzyme. In the absence of fibrin stimulation, the vs for the activation of nPlg and rPlg-Ala⁷⁴⁰ with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s⁻¹) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s⁻¹). In the presence of 1 μM soluble fibrin monomer (desAAfibrin), the v₀ for nPlg and rPlg-Ala⁷⁴⁰ by single chain rt-PA was also comparable (24 and, 33 pM s-1 respectively), whereas with 1 pM CNBr-digested fibrinogen, the vs for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala⁷⁴⁰ (135 and 7.5 pM s⁻¹ respectively). In contrast, the vs for nPlg and rPlg-Ala⁷⁴⁰ by single chain rt-PA- G1u²⁷⁵, two-chain rt-PA-G1u²⁷⁵ or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.

These findings confirm and establish: 1) that single chain t-PA is an active enzyme both in the presence and absence of fibrin stimulator; 2) that, in a system devoid of plasmin activity (rPlg- Ala⁷⁴⁰), the two-chain form of t-PA is about L5 times more active than the single chain form in the absence of fibrin but equipotent in the presence of desAAfibrin; and 3) that the mechanism of stimulation of plasminogen activation with single chain t-PA by CNBr-digested fibrinogen is different from that by soluble fibrin.

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References

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Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA[*]

Authors

Effect of Fibrin-Like Stimulators on the Activation of Plasminogen by Tissue-Type Plasminogen Activator (t-PA) - Studies with Active Site Mutagenized Plasminogen and Plasmin Resistant t-PA^[*]