Persistence of Fibrinolytic Activity in Fragments of Human Veins Cultured in Vitro

1970 ◽  
Vol 24 (01/02) ◽  
pp. 043-047 ◽  
Author(s):  
M Pandolfi
Keyword(s):  
Blood Vessels ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Vasa Vasorum ◽  
Adult Human ◽  
Slide Technique

SummaryExplants from 5 adult human veins were cultured in a fibrinolytically inactive medium for 3 weeks and assayed for the presence of plasminogen activator by the fibrin slide technique. The explants from 3 veins showed fibrinolytic activity confined to their vasa vasorum for the whole duration of the culture; no decrease of activity was seen. The finding suggests that small blood vessels are able to synthesize plasminogen activator.

Endothelial and Smooth Muscle Cells as Antagonists in Vascular Fibrinoysis

1975 ◽  
Author(s):  
V. Noordhoek Hegt
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Blood Vessels ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Vascular System ◽  
Muscle Cells ◽  
Vascular Wall ◽  
Vasa Vasorum ◽  
Slide Technique

Endothelial plasminogen activator activity in different types of human blood vessels obtained from fifty necropsies and thirty-five biopsies was detected and localized by means of plasminogen-rich fibrin slides. Great differences in endothelial activator activity were found along and across (vasa vasorum) the wall of the human vascular system.The same blood vessels were simultaneously investigated by a modified fibrin slide technique using plasminogen-free fibrin slides covered by plasmin to detect and localize inhibition of fibrinolysis in the vascular wall. The great variation in plasmin inhibition in different vessels revealed by this “fibrin slide sandwich technique” appeared to be closely associated with the localization and number of smooth muscle cells present in the walls of the vascular system. Strong plasmin inhibition was generally found at sites which showed no activator activity with the regular fibrin slide technique, while areas with a high endothelial fibrinolytic activity mostly revealed no inhibitory capacity.These results indicate that much of the variation in endothelial fibrinolytic activity on fibrin slides is due to inhibitory effects from the surrounding smooth muscle cells rather than to variability in the plasminogen activator content of the endothelium itself.

Binding of Tissue Plasminogen Activator to Vascular Grafts

1989 ◽  
Vol 61 (01) ◽  
pp. 131-136 ◽  
Author(s):  
Richard A Harvey ◽  
Hugh C Kim ◽  
Jonathan Pincus ◽  
Stanley Z Trooskin ◽  
Josiah N Wilcox ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Polymer Surface ◽  
Enzymic Activity ◽  
Vascular Prostheses ◽  
Chemically Modified ◽  
Insoluble Surfactant ◽  
Tissue Plasminogen

SummaryTissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDM AC). Surfactant- treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I- tPA (5-30 μg 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t½ of appoximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.

scholarly journals Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1354-1362 ◽  
Author(s):  
TC Wun ◽  
A Capuano
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Fibrinolytic Activity ◽  
High Rate ◽  
Bovine Aortic Endothelial Cell ◽  
Hep G2 ◽  
Triton X ◽  
Sepharose 4B

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA- inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT- 1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI- 2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.

scholarly journals Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4204-4213 ◽  
Author(s):  
S Handt ◽  
WG Jerome ◽  
L Tietze ◽  
RR Hantgan
Keyword(s):  
Endothelial Cells ◽  
Blood Vessels ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Time Dependent ◽  
Plasminogen Activator Inhibitor 1 ◽  
Necrosis Factor Alpha ◽  
Activator Inhibitor ◽  
Pai 1

Time-dependent thrombolytic resistance is a critical problem in thrombolytic therapy for acute myocardial infarction. Platelets have been regarded as the main source of plasminogen activator inhibitor-1 (PAI-1) found in occlusive platelet-rich clots. However, endothelial cells are also known to influence the fibrinolytic capacity of blood vessels, but their ability to actively mediate time-dependent thrombolytic resistance has not been fully established. We will show that, in vitro, tumor necrosis factor-alpha-stimulated endothelial cells secrete large amounts of PAI-1 over a period of hours, which then binds to fibrin and protects the clot from tissue plasminogen activator- induced fibrinolysis. In vivo, endothelial cells covering atherosclerotic plaques are influenced by cytokines synthesized by plaque cells. Therefore, we propose that continuous activation of endothelial cells in atherosclerotic blood vessels, followed by elevated PAI-1 secretion and storage of active PAI-1 in the fibrin matrix, leads to clot stabilization. This scenario makes endothelial cells a major factor in time-dependent thrombolytic resistance.

Interaction of a Plasmin A-Chain/t-PA B-Chain Hybrid Enzyme with Plasma Inhibitors In Vivo and In Vitro

1990 ◽  
Vol 63 (03) ◽  
pp. 459-463 ◽  
Author(s):  
S Wilson ◽  
P Chamberlain ◽  
I Dodd ◽  
A Esmail ◽  
J H Robinson
Keyword(s):  
Guinea Pig ◽  
Plasminogen Activator ◽  
Active Site ◽  
Guinea Pigs ◽  
Fibrinolytic Activity ◽  
Pharmacokinetic Profile ◽  
Inhibitory Mechanisms ◽  
A Chain

SummaryA hybrid plasminogen activator consisting of the “A” chain of plasmin linked to the “B” chain of rt-PA was inhibited in vitro in human and guinea pig plasmas 4 to 5-fold more rapidly than its parent activator, two-chain t-PA. Using zymographic and autoradiographic techniques together with the use of immunodepleted plasma the major inhibitor was identified as aIpha-2-antiplasmin. The pharmacokinetic profile of the hybrid in guinea pigs was determined by two different methods: disappearance of fibrinolytic activity and removal of radiolabelled hybrid from the circulation. Fibrinolytic activity was cleared rapidly via inhibitory mechanisms, whilst radiolabelled material was cleared considerably more slowly due to the formation of hybrid-inhibitor complexes. When the active site of the hybrid was reversibly acylated inhibitory mechanisms were evaded and a prolonged pharmacokinetic profile of activity was observed.

Defibrotide Counteracts the Modifications of Anti-Thrombotic Phenotype of Endothelial Cells Induced by Thalidomide.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2502-2502 ◽  
Author(s):  
Cinara Echart ◽  
Barbara Graziadio ◽  
Cinzia Repice ◽  
Mario Boccadoro ◽  
Antonio Palumbo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fibrin Clot ◽  
Rt Pcr ◽  
Plasminogen Activator Inhibitor 1 ◽  
Adhesive Properties ◽  
Pai 1

Abstract Introduction: Patients with Multiple myeloma are at relatively high risk of developing thromboembolic events, usually deep vein thromboses (DVT). There are numerous contributing factors, including therapy, such as thalidomide, where DVT has been identified as a major toxicity, especially when thalidomide is used in combination with other treatments such as dexamethasone. The mechanisms by which thalidomide predisposes to thrombosis are not well understood. Defibrotide (DF) is an orally biovailable polydisperse oligonucleotide with anti-thrombotic, pro-fibrinolytic and anti-adhesive properties. Previously, DF has been shown to dose-dependently counteracted the increase in Plasminogen Activator Inhibitor-1 (PAI-1) expression and decrease on tissue plasminogen activator (t-PA) activity after lipopolysaccharide (LPS) stimulation of endothelial cells in vitro. Methods and Results: We have conducted in vitro studies using human microvascular endothelial cells (HMEC) in order to investigate the effect of different doses of thalidomide on various fibrinolytic factors. In addition, we evaluated whether DF modulates changes of fibrinolysis induced by thalidomide. HMEC were treated with 50 and 100μg/ml of thalidomide for 24 hours in presence and absence of DF (at a dose of 150μg/ml). t-PA and PAI-1 gene expression were evaluated through real time polymerase chain reaction (RT-PCR) of cDNA prepared from HMEC. Release of t-PA and PAI-1 were evaluated by imunoenzymatic assay (ELISA). Furthermore, we evaluated the fibrinolytic activity of cell surpernatant using a fibrin clot plate assay. In this method the fibrin clot was formed by mixing fibrinogen, plasminogen and thrombin. The plasmin generated by the cell surpernatant was able to digest fibrin and also hydrolyzed the chromogenic substrate S-2251. The RT-PCR results showed that thalidomide reduces t-PA (2.2 fold) and increases PAI-1 gene expression (4.0 fold) in HMEC cells, whereas DF was able to counteract this effect by up-regulating the t-PA and down-regulating PAI-1 gene expression induced by thalidomide (8.8 and 2.0 fold, respectivielly). Similar results was observed analyzing t-PA release by HMEC cells treated with different concentrations of thalidomide with and without DF. Thalidomide significantly reduces the t-PA released in both concentrations (p<0.001) and DF significantly increase the release of t-PA reduced by thalidomide (p<0.01). The changes of fibrinolytic activity in HMEC by thalidomide and the capacity of DF to restore the fibrinolysis was confirmed by analyzing the lyses of fibrin clots with endothelial cell surpernatant (p<0.01). Conclusions: These results show that DF is able to counteract the alterations of fibrinolytic factors in HMEC treated with thalidomide. Whilst further studies in preclinical MM models are underway, these data suggest a potential role for DF in the prevention of DVT induced by thalidomide and support ongoing clinical trials of DF in combination with thalidomide-based treatment.

scholarly journals Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

2009 ◽  
Vol 297 (4) ◽  
pp. L568-L577 ◽  
Author(s):  
Andrey A. Komissarov ◽  
Andrew P. Mazar ◽  
Kathy Koenig ◽  
Anna K. Kurdowska ◽  
Steven Idell
Keyword(s):  
Enzymatic Activity ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Receptor Complexes ◽  
Pleural Injury ◽  
Pai 1 ◽  
Formation Of Complexes

The proenzyme single-chain urokinase plasminogen activator (scuPA) more effectively resolved intrapleural loculations in rabbits with tetracycline (TCN)-induced loculation than a range of clinical doses of two-chain uPA (Abbokinase) and demonstrated a trend toward greater efficacy than single-chain tPA (Activase) (Idell S et al., Exp Lung Res 33: 419, 2007.). scuPA more slowly generates durable intrapleural fibrinolytic activity than Abbokinase or Activase, but the interactions of these agents with inhibitors in pleural fluids (PFs) have been poorly understood. PFs from rabbits with TCN-induced pleural injury treated with intrapleural scuPA, its inactive Ser195Ala mutant, Abbokinase, Activase, or vehicle, were analyzed to define the mechanism by which scuPA induces durable fibrinolysis. uPA activity was elevated in PFs of animals treated with scuPA, correlated with the ability to clear pleural loculations, and resisted (70–80%) inhibition by PAI-1. α-macroglobulin (αM) but not urokinase receptor complexes immunoprecipitated from PFs of scuPA-treated rabbits retained uPA activity that resists PAI-1 and activates plasminogen. Conversely, little plasminogen activating or enzymatic activity resistant to PAI-1 was detectable in PFs of rabbits treated with Abbokinase or Activase. Consistent with these findings, PAI-1 interacts with scuPA much slower than with Activase or Abbokinase in vitro. An equilibrium between active and inactive scuPA (kon= 4.3 h−1) limits the rate of its inactivation by PAI-1, favoring formation of complexes with αM. These observations define a newly recognized mechanism that promotes durable intrapleural fibrinolysis via formation of αM/uPA complexes. These complexes promote uPA-mediated plasminogen activation in scuPA-treated rabbits with TCN-induced pleural injury.

scholarly journals In Vitro Study of the Fibrinolytic Activity via Single Chain Urokinase-Type Plasminogen Activator and Molecular Docking of FGFC1

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 1816
Author(s):  
Chunli Gao ◽  
Quan Shen ◽  
Pengjie Tang ◽  
Yuling Cao ◽  
Houwen Lin ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Fibrinolytic Activity ◽  
Hydrophobic Interactions ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Fungi fibrinolytic compound 1 (FGFC1) is a rare marine-derived compound that can enhance fibrinolysis both in vitro and in vivo. The fibrinolytic activity characterization of FGFC1 mediated by plasminogen (Glu-/Lys-) and a single-chain urokinase-type plasminogen activator (pro-uPA) was further evaluated. The binding sites and mode of binding between FGFC1 and plasminogen were investigated by means of a combination of in vitro experiments and molecular docking. A 2.2-fold enhancement of fibrinolytic activity was achieved at 0.096 mM FGFC1, whereas the inhibition of fibrinolytic activity occurred when the FGFC1 concentration was above 0.24 mM. The inhibition of fibrinolytic activity of FGFC1 by 6-aminohexanoic acid (EACA) and tranexamic acid (TXA) together with the docking results revealed that the lysine-binding sites (LBSs) play a crucial role in the process of FGFC1 binding to plasminogen. The action mechanism of FGFC1 binding to plasminogen was inferred, and FGFC1 was able to induce plasminogen to exhibit an open conformation by binding through the LBSs. The molecular docking results showed that docking of ligands (EACA, FGFC1) with receptors (KR1–KR5) mainly occurred through hydrophilic and hydrophobic interactions. In addition, the binding affinity values of EACA to KR1–KR5 were −5.2, −4.3, −3.7, −4.5, and −4.3 kcal/moL, respectively, and those of FGFC1 to KR1–KR5 were −7.4, −9.0, −6.3, −8.3, and −6.7 kcal/moL, respectively. The findings demonstrate that both EACA and FGFC1 bound to KR1–KR5 with moderately high affinity. This study could provide a theoretical basis for the clinical pharmacology of FGFC1 and establish a foundation for practical applications of FGFC1.

scholarly journals Effects of Bacterial Endotoxin on the Fibrinolytic Activity of Normal Human Leukocytes

Blood ◽  
1971 ◽  
Vol 37 (4) ◽  
pp. 447-453 ◽  
Author(s):  
IRA M. GOLDSTEIN ◽  
BARBARA WÜNSCHMANN ◽  
TAGE ASTRUP ◽  
EDWARD S. HENDERSON
Keyword(s):  
Plasminogen Activator ◽  
Bacterial Endotoxin ◽  
Maximum Effect ◽  
Human Leukocytes ◽  
Plasminogen Activator Activity ◽  
Normal Individuals ◽  
Normal Human ◽  
Slide Technique ◽  
Stimulation Of

Abstract Two modifications of Todd’s histochemical fibrin slide technique were used to study the effect of bacterial endotoxin on the plasminogen activator activity of intact normal human leukocytes in vitro. Leukocyte suspensions were obtained from heparinized peripheral blood by dextran sedimentation. Plasminogen activator activity was measured as the percentage of individual cells producing zones of fibrinolysis in plasminogen-rich fibrin. Of the cells from normal individuals, 27 ± 2 per cent were fibrinolytically active. The addition of lipopolysaccharide B from Salmonella abortus equi resulted in a marked increase in the percentage of active cells without impairment of cell viability. The stimulation of plasminogen activator release appeared to be dose-related, since at higher endotoxin concentrations the number of active cells gradually approached the granulocyte level. Maximum effect on leukocytes was observed 2 hours after addition of endotoxin, followed by a steady decline in activity. Plasminogen activator activity in the suspending medium became detectable after 2 hours’ incubation. Simultaneous assays of beta-glucuronidase revealed increasing amounts of this lysosomal enzyme in the suspending medium during incubation. Addition of endotoxin enhanced the rate of this increase. The parallel appearance of plasminogen activator and beta-glucuronidase activities without concomitant cell destruction from unstimulated and endotoxin-treated leukocytes is consistent with the release of both compounds from lysosomes following noncytocidal stimulation.

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