Monolith/Hydrogel composites as triamcinolone acetonide carriers for curing corneal neovascularization in mice by inhibiting the fibrinolytic system

SummaryThe role of fibrinolytic system components in thrombus formation and removal in vivo was investigated in groups of six mice deficient in urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), or plasminogen activator inhibitor-1 (PAI-1) (u-PA-/-, t-PA-/- or PAI-1-/-, respectively) or of their wild type controls (u-PA+/+, t-PA+/+ or PAI-1+/+). Thrombus was induced in the murine carotid artery by endothelial injury using the photochemical reaction between rose bengal and green light (540 nm). Blood flow was continuously monitored for 90 min on day 0 and for 20 min on days 1, 2 and 3. The times to occlusion after the initiation of endothelial injury in u-PA+/+, t-PA+/+ or PAI-1+/+ mice were 9.4 ± 1.3, 9.8 ± 1.1 or 9.7 ± 1.6 min, respectively. u-PA-/- and t-PA-/- mice were indistinguishable from controls, whereas that of PAI-1-/- mice were significantly prolonged (18.4 ± 3.7 min). Occlusion persisted for the initial 90 min observation period in 10 of 18 wild type mice and was followed by cyclic reflow and reocclusion in the remaining 8 mice. At day 1, persistent occlusion was observed in 1 wild type mouse, 8 mice had cyclic reflow and reocclusion and 9 mice had persistent reflow. At day 2, all injured arteries had persistent reflow. Persistent occlusion for 90 min on day 0 was observed in 3 u-PA-/-, in all t-PA-/- mice at day 1 and in 2 of the t-PA-/-mice at day 2 (p <0.01 versus wild type mice). Persistent patency was observed in all PAI-1-/- mice at day 1 and in 5 of the 6 u-PA-/- mice at day 2 (both p <0.05 versus wild type mice). In conclusion, t-PA increases the rate of clot lysis after endothelial injury, PAI-1 reduces the time to occlusion and delays clot lysis, whereas u-PA has little effect on thrombus formation and spontaneous lysis.

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Detection of both Type 1 and Type 2 Plasminogen Activator Inhibitors in Human Monocytes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645688 ◽

1990 ◽

Vol 63 (01) ◽

pp. 067-071 ◽

Cited By ~ 10

Author(s):

Joan C Castellote ◽

Enric Grau ◽

Maria A Linde ◽

Nuria Pujol-Moix ◽

Miquel LI Rutllant

Keyword(s):

Molecular Mass ◽

Plasminogen Activator ◽

Human Peripheral Blood ◽

Direct Interaction ◽

Apparent Molecular Weight ◽

Fibrinolytic System ◽

Human Monocytes ◽

Blood Monocytes ◽

Single Chain ◽

Sds Page

SummaryIncreasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125Ifibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.

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Interaction of Plasminogen Activators and Plasminogen with Heparin: Effect of Ionic Strength

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649685 ◽

1993 ◽

Vol 70 (05) ◽

pp. 867-872 ◽

Cited By ~ 21

Author(s):

Dingeman C Rijken ◽

Gerard A W de Munk ◽

Annie F H Jie

Keyword(s):

Molecular Weight ◽

Ionic Strength ◽

Plasminogen Activator ◽

Plasminogen Activators ◽

Fibrinolytic System ◽

Tissue Type ◽

Single Chain ◽

Physiological Conditions ◽

Amino Terminal ◽

Type Plasminogen Activator

SummaryIn order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparinagarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

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Plasminogen Activation In Vivo upon Intravenous Infusion of DDAVP

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648390 ◽

1992 ◽

Vol 67 (01) ◽

pp. 111-116 ◽

Cited By ~ 64

Author(s):

Marcel Levi ◽

Jan Paul de Boer ◽

Dorina Roem ◽

Jan Wouter ten Cate ◽

C Erik Hack

Keyword(s):

Monoclonal Antibodies ◽

Plasminogen Activator ◽

Plasma Levels ◽

Fold Increase ◽

Fibrinolytic System ◽

Plasminogen Activation ◽

Plasminogen Activator Activity ◽

Insight Into

SummaryInfusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established.A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor α2 antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated α2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system.Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex.We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

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On the Fibrinolytic System in Aged Rats, and Its Reactivity to Endotoxin and Cytokines

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648525 ◽

1992 ◽

Vol 67 (06) ◽

pp. 697-701 ◽

Cited By ~ 6

Author(s):

J J Emeis ◽

A Brouwer ◽

R J Barelds ◽

M A Horan ◽

S K Durham ◽

...

Keyword(s):

Plasminogen Activator ◽

Fibrinolytic Activity ◽

Fibrinolytic System ◽

Tissue Type ◽

High Dose ◽

Base Line ◽

Aged Rats ◽

Tissue Type Plasminogen Activator ◽

Young Rats ◽

Type Plasminogen Activator

SummaryAged rats are more susceptible to endotoxin-induced effects, including microthrombosis and platelet aggregation, than are young rats. To investigate whether changes in the fibrinolytic system might be involved, we investigated the fibrinolytic activity in plasma euglobulin fractions and tissues (lung and heart) of young (6-months old) and aged (24-months old) rats under baseline conditions and after challenge with endotoxin. Aged rats had lower plasma levels of tissue-type plasminogen activator (t-PA) and of urokinase-type PA (u-PA) activity. PA inhibitor (PAI) activity was higher in the plasma of aged rats, as was t-PA activity in lung and heart.Rats were treated with either a low dose (1 μg/kg) or a high dose (10 mg/kg) of endotoxin. Both treatments induced a transient phase of increased blood fibrinolytic activity, as evidenced by higher levels of tissue-type plasminogen activator (t-PA) activity and decreased levels of PA inhibitor (PAI) activity. Over time, the fibrinolytic activity decreased, probably due to increased levels of PA inhibitor.Both the early increase in t-PA activity, and the subsequent increase in PAI activity, were more pronounced in the aged rats, as compared with the younger rats, after the high dose of endotoxin. The aged rats also responded to an injection of interleukin-1β or tumor necrosis factor-α with a larger increase of PAI activity than did the younger rats.Together the data suggest that, compared to young rats, aged rats have a decreased base-line plasma fibrinolytic activity, while their fibrinolytic system is more responsive to challenge by endotoxin and cytokines.

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On the Behaviour of a Human Serum Proactivator Reacting with Streptokinase during Artificial Activation of the Fibrinolytic System

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654581 ◽

1961 ◽

Vol 06 (03) ◽

pp. 498-503 ◽

Cited By ~ 1

Author(s):

W Doleschel ◽

W Auerswald

Keyword(s):

Human Serum ◽

Fibrinolytic System ◽

Spontaneous Activation ◽

Significant Change ◽

Artificial Activation ◽

Activator System

SummaryDuring “spontaneous” activation of a human euglobulin preparation in suitably spaced samples — while plasminogen became progressively converted into piasmin — the proactivator content was tested by addition of equal amounts of streptokinase and evaluation of the lytic activities on heated and normal bovine fibrin plates. Indepedently of the decreasing content of plasminogen the proactivator which could be activated by streptokinase showed no significant change of concentration. These observation indicate that plasminogen is not acting as proactivator and that there exists a separate proactivator-activator system of the fibrinolytic mechanism in human serum.

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Effect of Bentonite on Fibrinolytic System in Serum

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654768 ◽

1963 ◽

Vol 10 (01) ◽

pp. 120-132 ◽

Cited By ~ 2

Author(s):

E. S Olesen

Keyword(s):

Guinea Pig ◽

Plasminogen Activator ◽

Fibrinolytic Activity ◽

High Potential ◽

Fibrinolytic System ◽

Fibrinolytic Agents ◽

Isoelectric Precipitation ◽

Active Protease ◽

Pig Serum

SummaryTreatment of serum with bentonite led to a reduced content of inhibitors of trypsin and urokinase in the isoelectrically precipitated euglobulin, and removed fibrinolytic agents and precursors from serum. Bentonite-treated serum added to untreated serum reduced precipitation of the above inhibitors, and presumably also precipitation of inhibitors against a plasminogen activator of serum.Bentonite-treated serum (whether from pig, ox, guinea-pig, or man), added to untreated guinea-pig serum, produced fibrinolytic activity on isoelectric precipitation of the mixture; the activity of the euglobulin was due to an activator of plasminogen as well as an active protease, probably plasmin. The described effects of bentonite-treated serum are similar to those previously reported for anionic polyelectrolytes. Possible mechanisms are discussed.The “non-specific” activation of fibrinolytic activity by means of bentonite emphasizes that guinea-pig serum [which is characterized by a high potential for “nonspecific” activation of its fibrinolytic system Olesen (1962)] contains all the elements required for the formation of an activator of plasminogen, and thus the activation of its plasminogen to plasmin.

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