Structural basis of the cofactor function of denatured albumin in plasminogen activation by tissue-type plasminogen activator

In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.

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Enhancement of tissue-type plasminogen activator (t-PA)-mediated plasminogen activation on the surface of endothelial cells by t-PA receptor

Blood Coagulation & Fibrinolysis ◽

10.1097/00001721-200107000-00013 ◽

2001 ◽

Vol 12 (5) ◽

pp. 413-414

Author(s):

H. Fukao ◽

S. Ueshima ◽

K. Okada ◽

O. Matsuo

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Tissue Type ◽

Plasminogen Activation ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator

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The Effect of Human Recombinant Tissue-Type Plasminogen Activator on Clinical and Laboratory Parameters of Hemostasis and Systemic Plasminogen Activation in the Dog and the Rat

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647044 ◽

1988 ◽

Vol 60 (02) ◽

pp. 271-279 ◽

Cited By ~ 3

Author(s):

John C Bloom ◽

Teresa S Sellers ◽

Gary C Gries ◽

Eric B Wheeldon ◽

Susan R O'Brien ◽

...

Keyword(s):

Plasminogen Activator ◽

Degradation Products ◽

Tissue Type ◽

Fibrinogen Concentration ◽

Plasminogen Activation ◽

Post Mortem ◽

Major Hemorrhage ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Dose Dependent

SummaryThe effect of human recombinant tissue-type plasminogen activator (rt-PA) on parameters of hemostasis and systemic plasminogen activation was studied in the dog and rat. Effects on screening coagulation times, fibrinogen concentration, fibrin/fibrinogen degradation products, and plasminogen and α2-anti- plasmin (α2-AP) activities in plasma were examined following single bolus injections of 0.5-5.0 mg/kg, single and repeated 3 hr infusions of 0.75-7.5 mg/kg and 24 hr infusions of 6.0 and 30.0 mg/kg administered intravenously to dogs. Rats receiving single or 14 daily injections of 5.0-30.0 mg/kg i.v. were similarly monitored. Systemic fibrinogenolysis (>50% decrease in fibrinogen, plasminogen or α2-AP values) was observed in dogs receiving ≥1.0 mg/kg as a bolus, ≥3.75 mg/kg (20.8 μg or 1.19 × 104 IU kg−1min−1) as a 3 hr infusion and >6 mg/kg (4.2 μg or 2.42 × 103IU kg−1min−1) as a 24 hr infusion; and in rats treated with bolus injections of 30 mg/kg rt-PA. Clinical and laboratory indications of impaired hemostasis and bleeding (anemia, prolonged coagulation times and post-mortem evidence of hemorrhage) were associated with these effects, which together were dose-dependent and influenced by the rate of infusion. The incidence of major hemorrhage was variable and limited to animals receiving prolonged (24 hr) or repeated infusions.

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A Simple, Sensitive Spectrophotometric Assay for Extrinsic (Tissue-Type) Plasminogen Activator Applicable to Measurements in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657277 ◽

1982 ◽

Vol 48 (03) ◽

pp. 266-269 ◽

Cited By ~ 345

Author(s):

J H Verheijen ◽

E Mullaart ◽

G T G Chang ◽

C Kluft ◽

G Wijngaards

Keyword(s):

Plasminogen Activator ◽

Cyanogen Bromide ◽

Spectrophotometric Assay ◽

Tissue Type ◽

Plasminogen Activation ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Specific Stimulation ◽

Assay Conditions ◽

Stimulation Of

SummaryAn indirect spectrophotometric assay for extrinsic plasminogen activator has been devised, which is based on the parabolic assay of Drapier et al. (5). The system contains activator, plasminogen, the synthetic plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251, Kabi) and a mixture of soluble fibrinogen fragments prepared by treatment of fibrinogen with cyanogen bromide. The addition of these fibrinogen fragments considerably enhances the sensitivity and specificity of the method owing to specific stimulation of the plasminogen activation by extrinsic plasminogen activator.The assay conditions were optimized and the application for extrinsic plasminogen activator measurements in plasma euglobulin fractions is demonstrated.

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Dysfibrinogenemia (Fibrinogen Dusard) Associated with Impaired Fibrin-Enhanced Plasminogen Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661033 ◽

1984 ◽

Vol 51 (01) ◽

pp. 108-109 ◽

Cited By ~ 57

Author(s):

H R Lijnen ◽

J Soria ◽

C Soria ◽

D Collen ◽

J P Caen

Keyword(s):

Pulmonary Embolism ◽

Plasminogen Activator ◽

Tissue Type ◽

Plasminogen Activation ◽

Recurrent Thrombosis ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

History Of

SummaryThe fibrin-mediated enhancement of the activation of plasminogen by tissue-type plasminogen activator observed with normal fibrin, is strongly decreased with fibrin Dusard, although the binding of tissue-type plasminogen activator to this fibrin is normal. This impaired fibrin-mediated plasminogen activation is most likely related to the history of recurrent thrombosis and pulmonary embolism observed in this family.

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A monoclonal antibody preventing binding of tissue-type plasminogen activator to fibrin: useful to monitor fibrinogen breakdown during t-PA infusion

Blood ◽

10.1182/blood.v67.5.1482.1482 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1482-1487 ◽

Cited By ~ 16

Author(s):

P Holvoet ◽

HR Lijnen ◽

D Collen

Keyword(s):

Plasminogen Activator ◽

Tissue Type ◽

Clot Lysis ◽

Plasminogen Activation ◽

Plasma Clot ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Additional Support

Abstract One (MA-1C8) of 36 monoclonal antibodies obtained by fusion of P3X63- Ag8–6.5.3 myeloma cells with spleen cells of mice immunized with purified human tissue-type plasminogen activator (t-PA) blocked the activity of t-PA on fibrin plates but not on chromogenic substrates. MA- 1C8 at a concentration of 200 micrograms/mL inhibited plasma clot lysis and binding of t-PA to the clot. MA-1C8 had no influence on the activation of plasminogen by t-PA, which obeys Michaelis-Menten kinetics with Km = 105 mumol/L and kcat = 0.05 s-1; however, it abolished the influence of CNBr-digested fibrinogen on Km. These findings confirm that the stimulatory effect of fibrin on the activation of plasminogen by t-PA is mediated by binding of t-PA to fibrin and provide additional support for the kinetic model. Addition of t-PA to pooled fresh human plasma to a concentration of 5 micrograms/mL resulted in extensive fibrinogen breakdown after incubation for one hour at 37 degrees C or during storage at -20 degrees C for one day. In both instances, fibrinogen degradation was completely prevented by addition of MA-1C8 to a concentration of 200 micrograms/mL of plasma. MA-1C8 also effectively prevented in vitro fibrinogen degradation and in vitro plasminogen activation in plasma samples obtained during infusion of recombinant t-PA in patients with thromboembolic disease. Thus, MA-1C8 is a useful tool for discriminating between in vivo and in vitro fibrinolysis during thrombolytic therapy with t-PA.

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A Refined Kinetic Analysis of Plasminogen Activation by Recombinant Bovine Tissue-Type Plasminogen Activator Indicates Two Interconvertible Activator Forms†

Biochemistry ◽

10.1021/bi9806697 ◽

1998 ◽

Vol 37 (36) ◽

pp. 12631-12639 ◽

Cited By ~ 6

Author(s):

Laust B. Johnsen ◽

Peter Ravn ◽

Lars Berglund ◽

Torben E. Petersen ◽

Lone K. Rasmussen ◽

...

Keyword(s):

Kinetic Analysis ◽

Plasminogen Activator ◽

Tissue Type ◽

Plasminogen Activation ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Bovine Tissue

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Like Fibrin, (DD)E, the Major Degradation Product of Crosslinked Fibrin, Protects Plasmin from Inhibition by α2-antiplasmin

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615612 ◽

2001 ◽

Vol 85 (03) ◽

pp. 502-508 ◽

Cited By ~ 11

Author(s):

Agnes Lee ◽

James Fredenburgh ◽

Ronald Stewart ◽

Janice Rischke ◽

Jeffrey Weitz

Keyword(s):

Protective Effect ◽

Plasminogen Activator ◽

Degradation Product ◽

Tissue Type ◽

Plasminogen Activation ◽

Tissue Type Plasminogen Activator ◽

Potent Stimulator ◽

Type Plasminogen Activator ◽

Kd Values ◽

Major Degradation Product

SummaryPlasmin generation is localized to the fibrin surface because tissue-type plasminogen activator (t-PA) and plasminogen bind to fibrin, an interaction that stimulates plasminogen activation over a hundred-fold. To ensure efficient fibrinolysis, plasmin bound to fibrin is protected from inhibition by α2-antiplasmin. (DD)E, a major soluble degradation product of cross-linked fibrin that is a potent stimulator of t-PA, compromises the fibrin-specificity of t-PA by promoting systemic activation of plasminogen. In this study we investigated whether (DD)E also protects plasmin from inhibition by α2-antiplasmin, facilitating degradation of this soluble t-PA effector. (DD)E and fibrin reduce the rate of plasmin inhibition by α2-antiplasmin by 5- and 10-fold, respectively. Kringle-dependent binding of plasmin to (DD)E and fibrin, with Kd values of 52 and 410 nM, respectively, contributes to the protective effect. When (DD)E is extensively degraded by plasmin, yielding uncomplexed fragment E and (DD), protection of plasmin from inhibition by α2-antiplasmin is attenuated. These studies indicate that (DD)E-bound plasmin, whose generation reflects the ability of (DD)E to stimulate plasminogen activation by t-PA, has the capacity to degrade (DD)E by virtue of its resistance to inhibition. This provides a mechanism to limit the concentration of (DD)E and maintain the fibrin-specificity of t-PA.

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