Absence of Synergism Between Tissue-Type Plasminogen Activator (t-PA), Single-Chain UrokinaseType Plasminogen Activator (scu-PA) and Urokinase on Clot Lysis in a Plasma Milieu In Vitro

1986 ◽  
Vol 56 (01) ◽  
pp. 035-039 ◽  
Author(s):  
D Collen ◽  
F De Cock ◽  
E Demarsin ◽  
H R Lijnen ◽  
D C Stump
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Dose Response ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryA potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scuPA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in titrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and α2-antiplasmin.t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant α2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal.

ANTITHROMBOTIC EFFECTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR AT PHYSIOLOGICAL BLOOD LEVELS

1987 ◽  
Author(s):  
W Witt ◽  
B Baldus ◽  
P Donner
Keyword(s):  
Plasminogen Activator ◽  
Plasma Levels ◽  
Dose Range ◽  
Tissue Type ◽  
Clot Lysis ◽  
Blood Levels ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

Effective thrombolysis in human patients and experimental animals by tissue-type plasminogen activator (t-PA) usually requires t-PA plasma levels in the microgram range. Compared to that physiological plasma levels of t-PA are about 100 - 1000 times lower. To investigate the effects of t-PA at physiological blood levels rat studies were performed in vitro and in vivo employing highly purified recombinant single-chain t-PA (sct-PA: 500,000 IU/mg).t-PA activity in rat whole blood as assessed by dilute blood clot-lysis time (DBC-LT) was increased by addition of sct-PA as low as 3 ng/ml (20 % decrease in DBC-LT). Injection of brady-kinin 10, 100 and 1000 μg/kg i.v. shortened DBC-LT to 54, 23, and 10 % of controls corresponding to the effect of about 10, 30, and 100 ng/ml sct-PA added in vitro. Infusion of sct-PA 15 - 450 μg/kg/h i.v. shortened DBC-LT ex vivo dose-dependently by 20 - 90 % at steady state levels (n = 5). In the same dose range sct-PA inhibited thrombus formation along a silk thread introduced into an arteriovenous shunt in anaesthetized rats. The reduction in thrombus dry weight was dose-dependent amounting to 33 - 67 % of preapplication values (n = 5 - 8) at 15 - 450 μg/kg/h i.v. sct-PA. Already 50 μg/kg/h sct-PA corresponding to a sct-PA activity of about 15 ng/ml displayed a significant (a = 0.05) effect in this model.The results of this study suggest that t-PA present at physiological resting or activation (bradykinin) levels during acute clot formation may have potent antithrombotic efficacy. This study provides further evidence for the importance of a balance coagulation-fibrinolysis which can be influenced on both sides towards thrombophilia as well as to achieve antithrombotic therapy, e.g. by elevating plasma fibrinolytic activity with low-dose t-PA treatment or with drugs which stimulate the endogenous fibrinolytic potential.

Biochemical and Biological Properties of a Recombinant Tissue-Type Plasminogen Activator Derived from the Rat JMI-229 Cell Line

1992 ◽  
Vol 67 (02) ◽  
pp. 239-247 ◽  
Author(s):  
H R Lijnen ◽  
P D Webb ◽  
B Van Hoef ◽  
F De Cock ◽  
J M Stassen ◽  
...  
Keyword(s):  
Cell Line ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Chain Molecule ◽  
Single Chain ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryRecombinant tissue-type plasminogen activator (rt-PA), produced by expression of the genomic t-PA DNA from the JMI-229 cell line, which is of rat origin, in the host cell line, was purified to homogeneity. JMI-229 rt-PA was obtained essentially as a single chain molecule which was quantitatively converted to a two-chain moiety by treatment with plasmin. The plasminogen activating potential of single chain JMI-229 rt-PA was 5-fold lower than that of commercially available human rt-PA (Actilyse®) in the absence of fibrin, but comparable in the presence of fibrin; it showed a concentration-dependent binding to fibrin, with a significantly more pronounced binding than Actilyse® at low fibrin concentration (85 ± 8% versus 20 ± 7% at 0.025 mg/ml fibrin; p = 0.004). In human plasma in the absence of fibrin, the concentrations of both single chain and two-chain JMI-229 rt-PA required to induce 50% fibrinogen degradation in 2 h, were about 15-fold higher than those of Actilyse®. Both single chain and two-chain forms of JMI-229 rt-PA and of Actilyse® induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma, in the absence of significant systemic fibrinolytic activation. Equally effective concentrations (causing 50% clot lysis in 2 h) were 0.11 or 0.10 pg/ml for single chain or two-chain JMI-229 rt-PA, as compared to 0.11 or 0.15 pg/ml for single chain or two-chain Actilyse®. Continuous infusion over 60 min of single chain JMI-229 rt-PA or Actilyse® in hamsters with a 125I-fibrin-labeled pulmonary embolus, revealed a very similar thrombolytic potency (clot lysis versus dose) and specific thrombolytic activity (clot lysis versus steady state plasma antigen level of t-PA). The initial plasma half-life following intravenous bolus injection of 0.10 mg/kg in hamsters was equally short for JMI-229 rt-PA or Actilyse® (1.2 or 1.4 min respectively).It is concluded that JMI-229 rt-PA has a higher fibrin-affinity and a higher fibrin-specificity in human plasma in the absence of fibrin than Actilyse®, but a comparable thrombolytic potency in a hamster pulmonary embolism model.

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

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.

Influence of Intrinsic and Extrinsic Plasminogen upon the Lysis of Thrombi In Vitro

1991 ◽  
Vol 66 (06) ◽  
pp. 672-677 ◽  
Author(s):  
N Nishino ◽  
V V Kakkar ◽  
M F Scully
Keyword(s):  
Plasminogen Activator ◽  
Dose Response ◽  
Tissue Type ◽  
Single Chain ◽  
Response Curves ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Dose Response Curves

SummaryWhen the rate of lysis of artificial thrombi (prepared from plasma or whole blood) was expressed according to the concentration of tissue type plasminogen activator (t-PA) or single chain urokinase type plasminogen activator (sc-uPA) then bell-shaped dose response curves were obtained, low rates being observed at concentrations of activator greater than 500 units/ml. Bell-shaped dose response curves were not observed for rate of lysis of artificial thrombi over the concentrations of streptokinase tested (SK) or for the lysis of plasma gel clots by any of the activators tested. Further investigation indicated that the preponderant mechanism for dissolution of thrombi at 500 units/ml of t-PA was by activation of the plasminogen within the thrombus (intrinsic) since the plasminogen present in the plasma perfusing the thrombus (extrinsic) rapidly became depleted. On the other hand, at 50 units/ml t-PA the lysis was observed to be due preponderantly to the action of plasmin arising from extrinsic rather than intrinsic plasminogen. If "plasminogen enriched" thrombi were prepared in the presence of Lys plasminogen (Lys-Plg) faster rates of lysis occurred and bell-shaped biometric curves were not observed.

Characterization of Domain Deletion and/or Duplication Mutants of a Recombinant Chimera of Tissue-Type Plasminogen Activator and Urokinase-Type Plasminogen Activator (rt-PA/u-PAI)

1990 ◽  
Vol 64 (01) ◽  
pp. 053-060 ◽  
Author(s):  
L Nelles ◽  
H R Lijnen ◽  
A Van Nuffelen ◽  
E Demarsin ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Catalytic Domain ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Specific Activities

SummaryChimeric molecules comprising the A-chain of tissue-type plasminogen activator (t-PA) and the catalytic domain of urokinase-type plasminogen activator (u-PA) have intact enzymatic characteristics of u-PA, but only partial fibrin-binding properties of t-PA (Nelles et al., J Biol Chem 1987; 262: 10855–62). The following domain deletion and/or duplication mutants of such a t-PA/u-PA chimera were constructed, purified and charactertzed: rt-PA-ΔFE∇/u-PA, with deletion of the finger-like (F) and epidermal growth factor-like (E) domains, rt-PA-ΔK1∇K2/u-PA, with kringle 1 (K1) replaced by a second copy of kringle 2 (K2), and rt-PA-ΔFEK1∇K2/u-PA, with F and E domain deletions in rt-PAΔK1∇K2/u-PA.The specific activities on fibrin plates of the single-chain (sc) chimeras ranged between 68,000 IU/mg for rt-PA-ΔK1∇K2/scu-PA and 200,000 IU/mg for rt-PA-ΔFEK1∇K2/scu-PA, as compared to L20,000 IU/mg for rscu-PA. The specific activities of their plasmin-generated two-chain (tc) derivatives ranged between 120,000 IU/mg for rt-PA-ΔK1∇K2/tcu-PA and 240,000 IU/mg for rt-PA-ΔFEK1∇K2/tcu-PA, as compared to 100,000 IU/mg for rtcu-PA. All two-chain chimeras activated plasminogen following Michaelis-Menten kinetics, with catalytic efficiencies between 0.072 μM−1s−1 for rt-PA-ΔK1∇K2/tcu-pA and 0.081 pM−1 s−1 for rt-PA-ΔFEK1∇K2/tcu-PA, as compared to 0.088 μM−1 s−1 for rtcu-PA. CNBr-digested fibrinogen enhanced the initial rate of plasminogen activation by a factor 2.2 to 6.2, as compared to 4.9 for rtcu-PA. The fibrin-affinity of the chimeras decreased in the order rt-PA > rt-PA-ΔK1∇K2/u-PA > u-PA and that for lysine in the order rt-PA-ΔFEK1∇K2/u-PA > > t-PA/u-PA ⩽ st-PA > rt-PA-ΔFE/u-PA > u-PA. All single-chain plasminogen activators caused a time and concentration-dependent clot lysis in an in vitro plasma clot lysis system, with equi-effective doses (causin g 50% clot lysis in 2 h) ranging between 0.53 and 0.90 μg/ml, as compared to 1 .7 μg/ml for rscu-PA, and were associated with comparable residual fibrinogen levels of approximately 80%.Thus, substitution of K1 by a second copy of K2 in the chimeric protein t-PA/u-PA enhances the affinity for both fibrin and lysine significantly and improves the fibrinolytic potency in an in vitro clot lysis system marginally.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

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

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1420-1427 ◽  
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.

Studies on the Specific Fibrinolytic Effect of Human Extrinsic (Tissue-Type) Plasminogen Activator in Human Blood and in Various Animal Species in Vitro

1981 ◽  
Vol 46 (02) ◽  
pp. 561-565 ◽  
Author(s):  
C Korninger ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Culture Fluid ◽  
The Other ◽  
Tissue Type ◽  
Chain Molecule ◽  
Autologous Plasma ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryHuman extrinsic (tissue-type) plasminogen activator (EPA) was highly purified from the culture fluid of a human melanoma cell line, both as a one-chain or as a two-chain molecule. Its specific fibrinolytic effect on human whole blood clots or plasma clots with different degrees of fibrin crosslinking was evaluated in an in vitro system, composed of a 125I-fibrin labeled clot, hanging in circulating human plasma. After infusion of EPA (30 IU per ml over 3 hrs), non-crosslinked clots lysed more extensively (75-100 percent in 5 hrs) than totally-crosslinked clots (50-65 percent), and no difference was found between one-chain or two-chain EPA. The extent of lysis of totally-crosslinked human or animal plasma clots hanging in autologous plasma induced by EPA varied markedly from one species to the other. When 90 IU of EPA were infused over 3 hrs, crosslinked human plasma clots dissolved for over 95 percent within 5 hrs. Under comparable conditions, the degree of lysis was 80 percent in primate plasma (cynomolgus fascicularis), 60 percent in cat and rabbit plasma, 30 percent in dog plasma and only 10 percent in rat plasma. Systemic activation of the fibrinolytic system in the circulating plasmas was minor and dose-dependent in all species, but complete fibrinogen breakdown was not observed in any species following infusion of up to 90 IU EPA per ml plasma.It is concluded that the human system is more susceptible to EPA induced fibrinolysis than the other animal systems which were investigated, and that even totally-crosslinked clots can be lysed after infusion of EPA.

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

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1482-1487 ◽  
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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