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


OBJECTIVE
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.
Thelwell
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National Institute for Biological Standards and Control, South Mimms, Herts, UK.
Longstaff
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D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D005337:Fibrin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D005340:Fibrinogen / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D005342:Fibrinolysis; D005786:Gene Expression Regulation; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008956:Models, Chemical; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000378:metabolism*; D015849:Plasminogen Inactivators / Q000378:metabolism; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D017395:Plasminogen Activator Inhibitor 1; D015849:Plasminogen Inactivators; D005337:Fibrin; D005340:Fibrinogen; D010959:Tissue Plasminogen Activator