The regulation by fibrinogen and fibrin of tissue plasminogen activator kinetics and inhibition by plasminogen activator inhibitor 1

Abstract

Summary. Background: Tissue plasminogen activator (tPA) is unusual in the coagulation and fibrinolysis cascades in that it is produced as an active single-chain enzyme (sctPA) rather than a zymogen. Two chain tPA (tctPA) is produced by plasmin but there are conflicting reports in the literature on the behaviour of sc-and tctPA and little work on inhibition by the specific inhibitor plasminogen activator inhibitor-1 (PAI-1) under physiological conditions. Objectives: To perform a systematic study on the kinetics of sctPA and tctPA as plasminogen activators and targets for PAI-1. Methods: Detailed kinetic studies were performed in solution and in the presence of template stimulators, fibrinogen and fibrin, including native fibrin and partially digested fibrin. Numerical simulation techniques were utilized to cope with the challenges of investigating kinetics of activation and inhibition in the presence of fibrin(ogen). Results: Enzyme efficiency (k cat /K m ) was higher for tctPA than sctPA in solution with chromogenic substrate (3-fold) and plasminogen (7-fold) but in the presence of templates, such as fibrinogen and native or cleaved fibrin, the difference disappeared. sctPA was more susceptible to PAI-1 in buffer solution and in the presence of fibrinogen; however, in the presence of fibrin, PAI-1 inhibited more slowly and there was no difference between sc and tctPA. Conclusions: Fibrinogen and fibrin modulate the activity of tPA differently in regard to their activation of plasminogen and inhibition by PAI-1. Fibrinogen and fibrin stimulate tPA activity against plasminogen but fibrin protects tPA from PAI-1 to promote fibrinolysis.

Unveiling an exceptional zymogen: the single-chain form of tPA is a selective activator of NMDA receptor-dependent signaling and neurotoxicity

“…Each preparation was characterized by immunoblotting ( Figure 2a) and its ability to initiate fibrinolysis. In agreement with previous reports, 2,4 rtPA, dtPA, sc-tPA and tc-tPA displayed similar fibrinolytic activity as revealed by in vitro clot lysis (Figures 2b and c). However, as cleavage of the NMDA receptor GluN1 subunit by tPA does not require fibrin, the different sources of tPA were tested by reference to their fibrin-independent amidolytic activity against a fluorogenic substrate (Spectrofluor 444FL), and confirmed by using plasminogen-casein zymography (Figures 3a-d), unless otherwise stated.…”

“…This observation is independent of their respective proteolytic activity, as sc-and tc-tPA displayed similar dose-dependent fibrinolytic activity (Supplementary Figures 1a and b), and as previously reported. 3,4 In addition, cultures of cortical neurons were subjected to NMDA paradigm as described above in the presence of 60 nM tPA (final total molar concentration) at different sc-tPA to tc-tPA ratios (40 : 60; 60 : 40; 80 : 20; 100 : 0) (Figure 5c, n ¼ 4). Altogether, these data show that the NMDA neurotoxicity of tPA is sc-tPA dose-dependent regardless of the presence of tc-tPA.…”

“…Interestingly, in the presence of fibrin both sctPA and tc-tPA display the same fibrinolytic activity. 4 Fibrin is therefore an allosteric regulator of tPA fibrinolytic activity.…”

“…In the vasculature, it is well established that fibrin stimulates the enzymatic activity of sctPA and tc-tPA to similar levels. 2,4 tPA has also critical functions in the brain parenchyma either as an enzyme, a cytokine-like molecule or a neuromodulator with roles in cell migration, neuronal plasticity and neurodegeneration. [5][6][7] Neurons 8 and glial cells 9,10 secrete tPA in the brain parenchyma where it can controls some plasminogendependent effects.…”

The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies

“…To achieve this, derivatives of pFastBac-tPA 24 were generated by site-directed mutagenesis via QuikChange (Stratagene) according to the manufacturer's protocol. All constructs were checked by sequence analysis.…”