Fibrinolysis mediated by tissue plasminogen activator. Disclosure of a kinetic transition

Abstract

The rate of 'G1u'-plasminogen activation by tissue plasminogen activator was repeatedly determined during a fibrinolytic process. The process was found to proceed via two distinct phases. The kinetics of each phase obeyed Michaelis-Menten equation: First phase; k,,, about 0.17 s-' and K , about 1 pM, second phase; k,,, about 0.13 s-' and K, about 0.06 pM. Practically identical results were obtained with one-chain as with two-chain tissue plasminogen activator. Transition from first to second phase occurred when the system had been exposed to a certain degree of plasmin digestion. Electrophoretic analysis demonstrated time correlation between the appearance of minimally degraded fibrin (X-fragments) and the transition. No such correlation was found between transition and conversion of 'G1u'-plasminogen to 'Lys'-plasminogen. The effect can result in an acceleration (up to 13-fold) of the fibrinolytic process once a slight degradation of the fibrin has taken place. In vivo, the effect described may constitute a mechanism that protects a fibrin clot from premature lysis.Fibrin deposits in the circulatory tract are degraded to soluble components by the serine protease plasmin. This enzyme is formed from the plasma protein plasminogen, by the action of plasminogen activators. The known plasminogen activators are serine proteases, which cleave one Arg-Val peptide bond in the plasminogen molecule to form the active two-chain enzyme, plasmin.Plasmin shows some substrate specificity and preferentially digests peptide bonds in the fibrin matrix and in the fibrin precursor, fibrinogen. This specificity is far from absolute and plasmin-sensitive bonds are found in most proteins (many of the numerous trypsin-sensitive bonds are also cleaved by plasmin!). Therefore, plasmin generated during fibrinolysis could damage plasma proteins and cell surface proteins. To avoid this, the organism has developed mechanisms that localize the generation of plasmin and confine its action. Two such mechanisms are generally recognized : (a) Tissue plasminogen activator (t-PA), an important plasminogen activator of the blood, is efficient only in the presence of fibrin [l]. In the absence of fibrin, the rate of t-PA catalysed plasminogen activation is low, but in the presence of fibrin the reaction rate increases up to 1000-fold [2]. Plasmin generation is thus localized to fibrin, which then is degraded. Once the fibrin is degraded, the rate of plasmin generation returns to low values. (b) Generated plasmin not engaged inCorrespondence to M. R h b y , Enheten for tillampad Cell-och Molekylarbiologi, Ume5 Universitet, S-90187 Urne5, SwedenAhhreviulions. A405 -492, difference between absorbance at 405 nm and absorbance at 492 nm; SDS, sodium dodecyl sulphate; t-PA, tissue plasminogen activator; u-PA, urinary plasminogen activator (urokinase); pNA, g-nitroanilide.Enzymes. Plasmin (EC 3.4.21 3; urokinase (EC 3.4.21.31 Normally, fibrin clots serve a useful purpose and should not lyse prematurely. Nature has therefore devised mechanisms to avoid thi...

On the Mechanism of the Antifibrinolytic Activity of Plasma Carboxypeptidase B

“…Upon binding to these residues, Pg acquires a highly activable conformation, which plays an essential role in fibrin-specific activation of Pg by t-PA (22), single chain urokinase-type PA (23,24), and staphylokinase (25). Generated Pl is largely protected from inhibition by ␣ 2 -antiplasmin when bound to these residues (26).…”

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. tion upon binding (21)(22)(23)(24)(25). Also, binding of Pl to these sites would protect it from inhibition by ␣ 2 -antiplasmin (26).…”

Interactions between Staphylokinase, Plasmin(ogen), and Fibrin

“…The portion of Pg bound to C-terminal lysines is believed to play an important role in the acceleration of fibrinolysis induced by other plasminogen activators. It is activated by two chain urokinase faster than Glu-Pg in solution (33); it is responsible for the acceleration of Pl generation during the second phase of tissue-type plasminogen activator-induced fibrinolysis (34). Liu and Gurewich (35) have postulated the existence of a unique conformation of Glu-Pg bound to C-terminal lysines on fibrin fragment E-2, which plays an essential role in the mechanism of fibrin-specific plasminogen activation by single-chain urokinase-type plasminogen activator (35,36).…”

Thrombin-thrombomodulin connects coagulation and fibrinolysis: more than an in vitro phenomenon

“…1 Generation of C-terminal basic amino acids provides positive feedback enhancing plasminogen activation. [1][2][3][4] Fibrin is a key component of the fibrinolytic cascade, not only as a substrate but as a cofactor and modulator, and is also the end product of the coagulation cascade. Therefore, fibrinolysis is regulated by all of the regulatory elements attributed to control of coagulation.…”