In vitro and in vivo antiangiogenic activity of a novel deca-peptide derived from human tissue-type plasminogen activator kringle 2

SummaryFunctional assay of extrinsic (tissue-type) plasminogen activator (EPA) in plasma on fibrin plates was evaluated. Using specific quenching antibodies, we demonstrated the method to be specific for EPA under all conditions tested. Contributions of urokinases and intrinsic activators were excluded. The quantity of EPA in blood samples, as compared with purified uterine tissue activator, shows 1 blood activator unit (BAU) to be comparable to 0.93 ng.The median values for EPA activity for healthy volunteers were: baseline, 1.9 BAU/ml (n = 123); diurnal, 5.5 BAU/ml (n = 12); DDAVP administration, 11.7 BAU/ml (n = 39); exhaustive exercise, 25 BAU/ml (n = 24); venous occlusion (15 min), 35 BAU/ml (n = 61). A large inter-individual variation in EPA activity was found, while individual baseline values tended to be constant for periods of weeks.In vitro in blood EPA activity shows a disappearance of 50% in about 90 min at 37° C; EPA activity in euglobulin fractions is stable for ≤2 hr at 37° C.A rapid decrease in EPA activity occurs in vivo, as noted after extracorporal circulation and exercise stimulation (t½ decay, 2-5 min).

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The specific roles of finger and kringle 2 domains of tissue-type plasminogen activator during in vitro fibrinolysis.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)99924-2 ◽

1994 ◽

Vol 269 (17) ◽

pp. 12639-12644

Author(s):

A.J. Horrevoets ◽

A. Smilde ◽

C. de Vries ◽

H. Pannekoek

Keyword(s):

Plasminogen Activator ◽

Tissue Type ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Kringle 2

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Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction

Blood ◽

10.1182/blood.v79.6.1420.bloodjournal7961420 ◽

1992 ◽

Vol 79 (6) ◽

pp. 1420-1427 ◽

Cited By ~ 1

Author(s):

S Kunitada ◽

GA FitzGerald ◽

DJ Fitzgerald

Keyword(s):

Plasminogen Activator ◽

Tissue Type ◽

Clot Lysis ◽

Plasminogen Activator Inhibitor 1 ◽

Clot Retraction ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Pai 1

Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t- PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet- induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.

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Monoclonal Antibodies to Tissue-Type Plasminogen Activator which Prolong its Clearance In Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646571 ◽

1989 ◽

Vol 61 (02) ◽

pp. 259-261

Author(s):

Thomas M Reilly ◽

Robert M Knabb ◽

Andrew T Chiu ◽

David L Bradfute ◽

Pieter B M W M Timmermans

Keyword(s):

Monoclonal Antibodies ◽

Plasminogen Activator ◽

Half Life ◽

Cell Receptor ◽

Tissue Type ◽

Human Hepatoma Cells ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator

SummaryThree murine monoclonal antibodies to tissue-type plasminogen activator (t-PA) were evaluated for their effects on the binding of iodinated t-PA to cultured human hepatoma cells (Hep G2), and on extending the half-life of t-PA injected into rabbits. Two of the antibodies, AE5 and EG2, significantly inhibited t-PA binding in vitro, and extended the in vivo half-life of t-PA four to five-fold. A third antibody, BA10, which had a much smaller inhibitory effect on t-PA binding, had no influence in extending t-PA’s half-life. MOPC-21, a control antibody not directed to t-PA, had no effect on either test. Our results are the first to correlate different compounds’ effects on t-PA binding with their ability to retard t-PA clearance in vivo, and provide additional evidence for the importance of a liver cell receptor in the t-PA clearance process.

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EVIDENCE FOR SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR AND SINGLE CHAIN UROKINASE ON IN VITRO PLASMA CLOT LYSIS

10.1055/s-0038-1642908 ◽

1987 ◽

Author(s):

R S Rappaport ◽

M R Blume ◽

R L Vogel ◽

M H Levner ◽

P P Hung

Keyword(s):

Plasminogen Activator ◽

Tissue Type ◽

Clot Lysis ◽

Single Chain ◽

Plasma Clot ◽

Tissue Type Plasminogen Activator ◽

Molar Ratios ◽

Type Plasminogen Activator

There is mounting evidence from animal models and the clinic that combination thrombolytic therapy with tissue-type plasminogen activator (tPA) and single chain urokinase (scuPA) is synergistic. Yet, efforts to demonstrate synergism between these two plasminogen activators in vitro have met with discordant results. Collen et al (Thromb. Haemostasis, 56:35, 1986) reported an absence of synergism between these two agents on clot lysis in an in vitro plasma milieu when they were evaluated at molar ratios of 1:4 (tPA:scuPA and vice versa). Gurewich and Pannell (Thromb. Res., 44:217, 1986), however, reported a synergistic effect on fibrin-specific clot lysis in vitro when the agents were combined in concentrations exceeding molar ratios of 1:4 (tPA:scuPA). Here, we present evidence that synergism between tPA and scuPA may be demonstrated in vitro provided that the molar ratio of tPA to scuPA exceeds 1:4 and that the concentration of clot bound or unbound tPA is minimized. In order to achieve this experimental condition, the standard in vitro plasma clot lysis assay was modified. Human plasma clots were incubated first for a short time in plasma containing varying amounts of tPA. After incubation, the clots were washed thoroughly and reimmersed in plasma alone or in plasma containing varying amounts of scuPA or tPA. Under these conditions, lysis proceeded at a greater rate and to a greater extent when tPA clots were immersed in plasma containing an appropriate amount of scuPA than when they were immersed in plasma alone or in plasma containing appropriate amounts of tPA. Lysis of untreated clots or clots exposed first to scuPA and then to plasma containing varying amounts of scuPA proceeded far less efficiently with a characteristic lag. The enhanced lysis produced by tPA and scuPA obeyed the classical definition of synergy: the same biological effect can be obtained with two drugs together at algebraic fractional combinations of less than 1 (Berenbaum, M.C., Clin. Exp. Immunol., 28:1-18, 1977). Thus, conditions that more closely mimic the in vivo situation resulting from a bolus injection of tPA followed by infusion with scuPA, may provide a system for duplication of in vivo synergism in. vi tro and investigation of the mechanism thereof.

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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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