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.

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

Abstract 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.

Inhibitor-Resistant Tissue-Type Plasminogen Activator: An Improved Thrombolytic Agent In Vitro

1994 ◽  
Vol 71 (01) ◽  
pp. 124-128 ◽  
Author(s):  
R V Shohet ◽  
S Spitzer ◽  
E L Madison ◽  
R Bassel-Duby ◽  
M-J Gething ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryPlatelet-rich clots are inefficiently lysed by current fibrinolytic agents. Platelets contain a great deal of plasminogen activator inhibitor 1 (PAI-1), the principal endogenous inhibitor of tissue-type plasminogen activator (t-PA). We have tested whether PAI-1 resistant t-PAs would be more effective thrombolytic agents in an in vitro model of platelet rich clots. Clots were formed with recalcified human plasma without or with the addition of platelets. The lysis of these clots was followed by the release of incorporated 125I-fibrinogen. Mutant and wild-type t-PA were almost equally effective against clots lacking platelets but the mutant was twice as effective at lysing platelet-rich clots. A mechanism for this effect is suggested by the demonstration that a complex between wild-type t-PA and extruded platelet contents resembles that between purified t-PA and PAI-1 and that the PAI-1 resistant t-PA does not interfere with formation of this adduct. Because of its enhanced ability to lyse platelet-rich clots in vitro, further in vivo work may find that PAI-1 resistant t-PA is a more efficacious therapeutic agent than wild-type t-PA in situations where platelets contribute to the failure of thrombolysis.

scholarly journals Adenovirus-Mediated Transfer of Tissue-Type Plasminogen Activator Augments Thrombolysis in Tissue-Type Plasminogen Activator–Deficient and Plasminogen Activator Inhibitor-1–Overexpressing Mice

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1527-1534
Author(s):  
Peter Carmeliet ◽  
Jean-Marie Stassen ◽  
Ilse Van Vlaenderen ◽  
Robert S. Meidell ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Plasminogen Activator ◽  
Recombinant Adenovirus ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Pai 1

Impaired fibrinolysis, resulting from increased plasminogen activator inhibitor-1 (PAI-1) or reduced tissue-type plasminogen activator (t-PA) plasma levels, may predispose the individual to subacute thrombosis in sepsis and inflammation. The objective of these studies was to show that adenovirus-mediated gene transfer could increase systemic plasma t-PA levels and thrombolytic capacity in animal model systems. Recombinant adenovirus vectors were constructed that express either human wild type or PAI-1–resistant t-PA from the cytomegalovirus (CMV) promoter. Both t-PA-deficient (t-PA−/−) and PAI-1–overexpressing transgenic mice were infected by intravenous injection of these viruses. Intravenous injection of recombinant adenovirus resulted in liver gene transfer, t-PA synthesis, and secretion into the plasma. Virus dose, human t-PA antigen, and activity concentrations in plasma and extent of lysis of a 125I-fibrin–labeled pulmonary embolism were all closely correlated. Plasma t-PA antigen and activity were increased approximately 1,000-fold above normal levels. Clot lysis was significantly increased in mice injected with a t-PA–expressing virus, but not in mice injected with saline or an irrelevant adenovirus. Comparable levels of enzyme activity and clot lysis were obtained with wild type and inhibitor-resistant t-PA viruses. Adenovirus-mediated t-PA gene transfer was found to augment clot lysis as early as 4 hours after infection, but expression levels subsided within 7 days. Adenovirus-mediated transfer of a t-PA gene can effectively increase plasma fibrinolytic activity and either restore (in t-PA–deficient mice) or augment (in PAI-1–overexpressing mice) the thrombolytic capacity in simple animal models of defective fibrinolysis.

The Role of the Finger Domain of Tissue-type Plasminogen Activator (t-PA) and Plasminogen Activator Inhibitor 1 (PAI-1) in Initiation and Progression of Thrombolysis

1994 ◽  
Vol 72 (06) ◽  
pp. 900-905 ◽  
Author(s):  
Harold A R Stringer ◽  
Peter van Swieten ◽  
Anton J G Horrevoets ◽  
Annelies Smilde ◽  
Hans Pannekoek
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Activator Inhibitor ◽  
Pai 1

SummaryWe further investigated the role of the finger (F) and the kringle-2 (K2) domains of tissue-type plasminogen activator (t-PA) in fibrin-stimulated plasminogen activation. To that end, the action of purified (wt) t-PA or of variants lacking F (del.F) or K2 (del.K2) was assessed either in a static, human whole blood clot-lysis system or in whole blood thrombi generated in the “Chandler loop”. In both clot-lysis systems, significant differences were observed for the initiation of thrombolysis with equimolar concentrations of the t-PA variants. A relatively minor “lag phase” occurred in thrombolysis mediated by wt t-PA, whereas a 6.4-fold and 1.6-fold extension is found for del.F and del.K2, respectively. We observed identical lag-times, characteristic for each t-PA variant, in platelet-rich heads and in platelet-poor tails of thrombi. Since plasminogen activator inhibitor 1 (PAI-1) is preferentially retained in the platelet-rich heads, we conclude that the inhibitor does not interfere with the initial stage of thrombolysis but exerts its action in later stages, resulting in a reduction of the rate of clot lysis. A complementation clot-lysis assay was devised to study a potential interplay of del.F and del.K2. Accordingly, clot lysis was determined with combinations of del.F and del.K2 that were inversely varied in relation to equipotent dosage to distinguish between additive, antagonistic or synergistic effects of these variants. The isobole for combinations of del.F and del.K2 shows an independent, additive action of del.F and del.K2 in clot lysis. Under the conditions employed, namely a relatively high concentration of fibrin and Glu-plasminogen and a low concentration of t-PA variant, our data show: i) the crucial role of the F domain and the lack of effect of PAI-1 in initiation of thrombolysis, ii) the lack of importance of the fibrimbinding domains of t-PA and the regulatory role of PAI-1 in advanced stages of thrombolysis.

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.

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.

scholarly journals Lack of interference by heparin with thrombolysis or binding of tissue- type plasminogen activator to thrombi

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1347-1352 ◽  
Author(s):  
ET Fry ◽  
BE Sobel
Keyword(s):  
Plasminogen Activator ◽  
Plasma Concentrations ◽  
Concomitant Administration ◽  
Tissue Type ◽  
Clot Lysis ◽  
Chloromethyl Ketone ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

Abstract Coronary thrombolysis with t-PA is generally implemented with concomitant administration of heparin. However, results of studies in vitro suggest that heparin competes with fibrin for binding of tissue- type plasminogen activator (t-PA), augments activation of free plasminogen, decreases fibrin specificity, and impairs thrombolysis. To define the biological implications of these observations, we characterized effects of therapeutic concentrations of heparin on the binding of t-PA to thrombi formed in whole blood, effects of heparin on activation of plasminogen by t-PA in plasma, and effects of heparin on thrombolysis induced by t-PA in a clot lysis system designed to simulate conditions in vivo. The amount of t-PA bound to thrombi was not affected by heparin (0, 0.5, 1.0, and 5.0 U/mL). When t-PA activity was selectively and irreversibly inhibited by D-Phe-Pro-Arg- chloromethyl ketone (PPACK) the amount of t-PA-PPACK bound was similarly unaffected by heparin. Thrombolysis measured by 125I- fibrin(ogen) release and by reduction of mass of thrombi were not altered by heparin. Heparin did not affect plasminogen consumption induced by t-PA. Plasma concentrations of alpha-2-antiplasmin after exposure of blood to t-PA were less depressed with increasing concentrations of heparin. Thus, heparin in therapeutic concentrations does not interfere with binding of t-PA to thrombi, augment activation of free plasminogen, or inhibit thrombolysis. Accordingly, it appears likely that concomitant administration of heparin will not impair thrombolysis with t-PA implemented clinically.

Generation and in vitro characterisation of inhibitory nanobodies towards plasminogen activator inhibitor 1

2016 ◽  
Vol 116 (12) ◽  
pp. 1032-1040 ◽  
Author(s):  
Xiaohua Zhou ◽  
Maarten L. V. Hendrickx ◽  
Gholamreza Hassanzadeh-Ghassabeh ◽  
Serge Muyldermans ◽  
Paul J. Declerck
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Hinge Region ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Activator Inhibitor ◽  
Pai 1

SummaryPlasminogen activator inhibitor 1 (PAI-1) is the principal physiological inhibitor of tissue-type plasminogen activator (t-PA) and has been identified as a risk factor in cardiovascular diseases. In order to generate nanobodies against PAI-1 to interfere with its functional properties, we constructed three nanobody libraries upon immunisation of three alpacas with three different PAI-1 variants. Three panels of nanobodies were selected against these PAI-1 variants. Evaluation of the amino acid sequence identity of the complementarity determining region-3 (CDR3) reveals 34 clusters in total. Five nanobodies (VHH-s-a98, VHH-2w-64, VHH-s-a27, VHH-s-a93 and VHH-2g-42) representing five clusters exhibit inhibition towards PAI-1 activity. VHH-s-a98 and VHH-2w-64 inhibit both glycosylated and non-glycosylated PAI-1 variants through a substrate-inducing mechanism, and bind to two different regions close to αhC and the hinge region of αhF; the profibrinolytic effect of both nanobodies was confirmed using an in vitro clot lysis assay. VHH-s-a93 may inhibit PAI-1 activity by preventing the formation of the initial PAI-1•t-PA complex formation and binds to the hinge region of the reactive centre loop. Epitopes of VHH-s-a27 and VHH-2g-42 could not be deduced yet. These five nanobodies interfere with PAI-1 activity through different mechanisms and merit further evaluation for the development of future profibrinolytic therapeutics.

scholarly journals Adenovirus-Mediated Transfer of Tissue-Type Plasminogen Activator Augments Thrombolysis in Tissue-Type Plasminogen Activator–Deficient and Plasminogen Activator Inhibitor-1–Overexpressing Mice

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1527-1534 ◽  
Author(s):  
Peter Carmeliet ◽  
Jean-Marie Stassen ◽  
Ilse Van Vlaenderen ◽  
Robert S. Meidell ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Plasminogen Activator ◽  
Recombinant Adenovirus ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Pai 1

AbstractImpaired fibrinolysis, resulting from increased plasminogen activator inhibitor-1 (PAI-1) or reduced tissue-type plasminogen activator (t-PA) plasma levels, may predispose the individual to subacute thrombosis in sepsis and inflammation. The objective of these studies was to show that adenovirus-mediated gene transfer could increase systemic plasma t-PA levels and thrombolytic capacity in animal model systems. Recombinant adenovirus vectors were constructed that express either human wild type or PAI-1–resistant t-PA from the cytomegalovirus (CMV) promoter. Both t-PA-deficient (t-PA−/−) and PAI-1–overexpressing transgenic mice were infected by intravenous injection of these viruses. Intravenous injection of recombinant adenovirus resulted in liver gene transfer, t-PA synthesis, and secretion into the plasma. Virus dose, human t-PA antigen, and activity concentrations in plasma and extent of lysis of a 125I-fibrin–labeled pulmonary embolism were all closely correlated. Plasma t-PA antigen and activity were increased approximately 1,000-fold above normal levels. Clot lysis was significantly increased in mice injected with a t-PA–expressing virus, but not in mice injected with saline or an irrelevant adenovirus. Comparable levels of enzyme activity and clot lysis were obtained with wild type and inhibitor-resistant t-PA viruses. Adenovirus-mediated t-PA gene transfer was found to augment clot lysis as early as 4 hours after infection, but expression levels subsided within 7 days. Adenovirus-mediated transfer of a t-PA gene can effectively increase plasma fibrinolytic activity and either restore (in t-PA–deficient mice) or augment (in PAI-1–overexpressing mice) the thrombolytic capacity in simple animal models of defective fibrinolysis.

scholarly journals Lack of interference by heparin with thrombolysis or binding of tissue- type plasminogen activator to thrombi

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1347-1352
Author(s):  
ET Fry ◽  
BE Sobel
Keyword(s):  
Plasminogen Activator ◽  
Plasma Concentrations ◽  
Concomitant Administration ◽  
Tissue Type ◽  
Clot Lysis ◽  
Chloromethyl Ketone ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

Coronary thrombolysis with t-PA is generally implemented with concomitant administration of heparin. However, results of studies in vitro suggest that heparin competes with fibrin for binding of tissue- type plasminogen activator (t-PA), augments activation of free plasminogen, decreases fibrin specificity, and impairs thrombolysis. To define the biological implications of these observations, we characterized effects of therapeutic concentrations of heparin on the binding of t-PA to thrombi formed in whole blood, effects of heparin on activation of plasminogen by t-PA in plasma, and effects of heparin on thrombolysis induced by t-PA in a clot lysis system designed to simulate conditions in vivo. The amount of t-PA bound to thrombi was not affected by heparin (0, 0.5, 1.0, and 5.0 U/mL). When t-PA activity was selectively and irreversibly inhibited by D-Phe-Pro-Arg- chloromethyl ketone (PPACK) the amount of t-PA-PPACK bound was similarly unaffected by heparin. Thrombolysis measured by 125I- fibrin(ogen) release and by reduction of mass of thrombi were not altered by heparin. Heparin did not affect plasminogen consumption induced by t-PA. Plasma concentrations of alpha-2-antiplasmin after exposure of blood to t-PA were less depressed with increasing concentrations of heparin. Thus, heparin in therapeutic concentrations does not interfere with binding of t-PA to thrombi, augment activation of free plasminogen, or inhibit thrombolysis. Accordingly, it appears likely that concomitant administration of heparin will not impair thrombolysis with t-PA implemented clinically.

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