Kinetics Of Plasminogen Activation By Tissue Plasminogen Activator. Role Of Fibrin

The activation of human plasminogen (P) by two-chain tissue plasminogen activator (A) was studied in the presence of fibrin films (F) of increasing size and surface density. Initial rates of plasminogen activation (v) were determined as a function both of the plasminogen and fibrin concentration. The activation rate was strongly dependent on the presence of fibrin and plots of 1/v versus 1/ [p] or 1 /[F] yielded straight lines. The kinetic data were in agreement with the following reaction scheme.According to this model tissue plasminogen activator would bind to fibrin with a dissociation constant (KF of 0.2 µM and this complex fixes plasminogen with a Michaelis constant (Kp’) of 0.15 µM (Glu-plasminogen) or 0.02 µM (Lys-plasminogen) to form a ternary complex, converted to plasmin with a catalytic rate constant kcat = 0.05 s-1. This mechanism implies that both plasminogen and tissue plasminogen activator are concentrated on the fibrin surface through formation of a fibrin bridge. Activation of plasminogen in the absence of fibrin occurs with Km = 65 µM (Glu-plasminogen) or Km= 19 µM (Lys-plasminogen) and kcat = 0.05 s-1. Our data suggest that fibrin enhances the activation rate of plasminogen by tissue plasminogen activator by increasing the affinity of plasminogen for fibrin-bound tissue plasminogen activator and not by influencing the catalytic efficiency of the enzµMe. These data also support the hypothesis that fibrinolysis is both triggered by and directed towards fibrin.Generated plasmin was quantitated by measuring the rate of solubilization of 125I-labeled fibrin.

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The kinetics of plasminogen activation by thrombin-cleaved pro- urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator

Blood ◽

10.1182/blood.v81.4.980.bloodjournal814980 ◽

1993 ◽

Vol 81 (4) ◽

pp. 980-987

Author(s):

JN Liu ◽

V Gurewich

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Catalytic Efficiency ◽

Clot Lysis ◽

Plasminogen Activation ◽

Single Chain ◽

Carboxypeptidase B ◽

Thrombin Cleavage ◽

Tissue Plasminogen ◽

A Chain

Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A- chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90- fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro- UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb- UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.

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The kinetics of plasminogen activation by thrombin-cleaved pro- urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator

Blood ◽

10.1182/blood.v81.4.980.980 ◽

1993 ◽

Vol 81 (4) ◽

pp. 980-987 ◽

Cited By ~ 29

Author(s):

JN Liu ◽

V Gurewich

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Catalytic Efficiency ◽

Clot Lysis ◽

Plasminogen Activation ◽

Single Chain ◽

Carboxypeptidase B ◽

Thrombin Cleavage ◽

Tissue Plasminogen ◽

A Chain

Abstract Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A- chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90- fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro- UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb- UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.

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Tissue plasminogen activator is a ligand of cation-independent mannose 6-phosphate receptor and consists of glycoforms that contain mannose 6-phosphate

Scientific Reports ◽

10.1038/s41598-021-87579-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

James J. Miller ◽

Richard N. Bohnsack ◽

Linda J. Olson ◽

Mayumi Ishihara ◽

Kazuhiro Aoki ◽

...

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Dependent Manner ◽

Plasminogen Activation ◽

Urokinase Plasminogen Activator Receptor ◽

Plasminogen Activation System ◽

Extracellular Region ◽

Glycosylation Sites ◽

Tissue Plasminogen ◽

Mannose 6 Phosphate

AbstractPlasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand.

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Modulation of tissue plasminogen activator-catalyzed plasminogen activation by synthetic peptides derived from the amino-terminal heparin binding domain of fibronectin.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)46715-9 ◽

1993 ◽

Vol 268 (25) ◽

pp. 18924-18928

Author(s):

M.S. Stack ◽

S.V. Pizzo

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Synthetic Peptides ◽

Binding Domain ◽

Plasminogen Activation ◽

Heparin Binding ◽

Amino Terminal ◽

Tissue Plasminogen ◽

Heparin Binding Domain

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The potentiating effect of platelet on plasminogen activation by tissue plasminogen activator

Thrombosis Research ◽

10.1016/0049-3848(85)90321-4 ◽

1985 ◽

Vol 40 (6) ◽

pp. 853-861 ◽

Cited By ~ 29

Author(s):

K. Deguchi ◽

S. Murashima ◽

S. Shirakawa ◽

C. Soria ◽

J. Soria ◽

...

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Plasminogen Activation ◽

Tissue Plasminogen

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Mechanism of the stimulatory effect of 6-aminohexanoic acid on plasminogen activation by streptokinase or tissue plasminogen activator: The role of chloride

European Journal of Drug Metabolism and Pharmacokinetics ◽

10.1007/bf03220185 ◽

2003 ◽

Vol 28 (4) ◽

pp. 315-320 ◽

Cited By ~ 4

Author(s):

L. Guinn ◽

V. M. Doctor

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Plasminogen Activation ◽

Tissue Plasminogen

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Interaction Of Fibrin And Tissue Plasminogen Activator

10.1055/s-0038-1652740 ◽

1981 ◽

Author(s):

P Wallén ◽

M Rånby

Keyword(s):

Dissociation Constant ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Conformational Changes ◽

Specific Interaction ◽

Kinetic Studies ◽

Test System ◽

Activation Rate ◽

Tissue Plasminogen ◽

Analytical System

Fibrin itself has a marked influence on fibrinolysis induced by tissue plasminogen activator (TA) indicating a specific interaction. The interaction between fibrin and TA is manifested in two ways, 1) a marked stimulating effect of fibrin on the activation of plasminogen; 2) physical ad- sorbtion of TA on fibrin. By measurement in a sensitive analytical system in which the generation of plasmin is followed by a chromogenic substrate it has been shown that TA is a rather poor activator of plasminogen. In the presence of fibrin the kinetics of the activation is dramatically changed. A stimulation up to 1000-fold is obtained at low plasminogen concentrations. As for activation of native plasminogen (Glu-plasminogen) there is a decrease of km (about 15-fold) as well as an increase of kc (about 80-fold). Fibrinogen has comparatively little effect on the activation rate (at the most 10-fold). The amidolytic activity of TA, using an activator sensitive substrate, is not influenced by fibrin indicating that the effect is not due to conformational changes in the active site region of TA.By varying the concentration of fibrin in the test system it has been demonstrated that the stimulation effect increases suddenly at a fibrin concentration of about 0.01μM. It was suggested that this value represents the dissociation constant of the TA-fibrin complex. However, the amount of fibrin necessary for the adsorbtion of TA in purification experiments indicates a significantly higher dissociation constant (about 0.4 μM). An important difference between the purification experiments and the studies on fibrin stimulation is that plasminogen (plasmin) is absent in the former studies. The formation of a triple complex between fibrin, plasminogen and TA may be the explanation for a more efficient binding of TA in the kinetic studies.

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