Coupling Tissue Type Plasminogen Activator to Carrier Erythrocyte Protects Against Plasma Inhibitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1881-1881
Author(s):  
Kumkum Ganguly ◽  
Douglas B. Cines ◽  
Vladimir R. Muzykantov
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Tissue Type ◽  
Cell Surfaces ◽  
Mouse Blood ◽  
Type Plasminogen Activator ◽  
Α2 Macroglobulin ◽  
Tissue Plasminogen ◽  
Pai 1

Abstract Conjugating tissue plasminogen activator (tPA) to carrier red blood cells (RBC) restricts its permeation into tissues and pre-existing hemostatic clots, minimizing side effects, and prolongs its circulation, which permits it to be incorporated within nascent clot which it lyses from inside. We now report that RBC/tPA dissolves clots formed from mouse blood more effectively than free tPA, while they were equipotent against clots formed in vitro from PAI-1 KO mouse blood. To test whether tPA acquires resistance to plasma inhibitors when conjugated to RBC, we compared the activity of RBC/tPA vs free tPA in the presence of purified PAI-1, α2 macroglobulin (α2M) and α1anti-trypsin (α1AT). At equimolar concentrations, PAI-1 completely inhibited the activity of soluble tPA in vitro, whereas RBC/tPA retained 100% and 75% of its amidolytic and fibrinolytic activity, respectively. RBC/tPA, but not free tPA, was also resistant to equimolar concentrations of α2M and α1AT. However, tPA coupled to RBC pre-incubated with a mixture of hyaluronidase, heparinase and neuraminidase was as susceptible to inactivation by PAI-1, α2M and α1AT as free tPA. Further, tPA coupled to glycocalyx-stripped RBC bound two-fold more 125I-labeled PAI-1 than tPA coupled to naïve RBC. We conclude that the RBC glycocalyx protects tPA from inactivation by PA inhibitors, likely by steric hindrance. This unexpected benefit may enhance the utility of RBC/tPA in thromboprophylaxis and suggests a previous under-appreciated role for the glycocalyx in modulating PA activity on the vasculature and other cell surfaces.

Abstract T P75: Temporal Profiles of Plasminogen Activator Inhibitor-1 Concentration and Activity Changes in Circulation after Focal Stroke and Tissue Type Plasminogen Activator Administration in Rats

Stroke ◽  
2014 ◽  
Vol 45 (suppl_1) ◽  
Author(s):  
Qi Liu ◽  
Xiang Fan ◽  
Helen Brogren ◽  
Ming-Ming Ning ◽  
Eng H Lo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Temporal Profiles ◽  
Activator Inhibitor ◽  
Pai 1

Aims: Plasminogen activator inhibitor-1 (PAI-1) is the main and potent endogenous tissue-type plasminogen activator (tPA) inhibitor, but an important question on whether PAI-1 in blood stream responds and interferes with the exogenously administered tPA remains unexplored. We for the first time investigated temporal profiles of PAI-1 concentration and activity in circulation after stroke and tPA administration in rats. Methods: Permanent MCAO focal stroke of rats were treated with saline or 10mg/kg tPA at 3 hours after stroke (n=10 per group). Plasma (platelet free) PAI-1 antigen and activity levels were measured by ELISA at before stroke, 3, 4.5 (1.5 hours after saline or tPA treatments) and 24 hours after stroke. Since vascular endothelial cells and platelets are two major cellular sources for PAI-1 in circulation, we measured releases of PAI-1 from cultured endothelial cells and isolated platelets after direct tPA (4 μg/ml) exposures for 60 min in vitro by ELISA (n=4 per group). Results: At 3 hours after stroke, both plasma PAI-1 antigen and activity were significantly increased (3.09±0.67, and 3.42±0.57 fold of before stroke baseline, respectively, all data are expressed as mean±SE). At 4.5 hours after stroke, intravenous tPA administration significantly further elevated PAI-1 antigen levels (5.26±1.24), while as expected that tPA neutralized most elevated PAI-1 activity (0.33±0.05). At 24 hours after stroke, PAI-1 antigen levels returned to the before baseline level, however, there was a significantly higher PAI-1 activity (2.51±0.53) in tPA treated rats. In vitro tPA exposures significantly increased PAI-1 releases into culture medium in cultured endothelial cells (1.65±0.08) and platelets (2.02±0.17). Conclution: Our experimental results suggest that tPA administration may further elevate stroke-increased blood PAI-1 concentration, but also increase PAI-1 activity at late 24 hours after stroke. The increased PAI-1 releases after tPA exposures in vitro suggest tPA may directly stimulate PAI-1 secretions from vascular walls and circulation platelets, which partially contributes to the PAI-1 elevation observed in focal stroke rats. The underlying regulation mechanisms and pathological consequence need further investigation.

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 Characterization and fibrinolytic properties of human omental tissue mesothelial cells. Comparison with endothelial cells

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1490-1497
Author(s):  
VW van Hinsbergh ◽  
T Kooistra ◽  
MA Scheffer ◽  
J Hajo van Bockel ◽  
GN van Muijen
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Subcutaneous Fat ◽  
Mesothelial Cells ◽  
Tissue Type ◽  
Microvascular Endothelial Cells ◽  
Type Plasminogen Activator ◽  
Microvascular Endothelial ◽  
Pai 1

It has been reported that omental fat tissue is a good source of human microvascular endothelial cells. By characterization we demonstrate that the epitheloid cells isolated from omental tissue are not endothelial cells, but mesothelial cells. They contain abundant cytokeratins 8 and 18, which are absent in endothelial cells, and vimentin. No staining with the endothelial-specific antibodies EN-4 and PAL-E is observed. A faint and diffuse staining of von Willebrand factor (vWF) is seen in mesothelial cells, whereas microvascular endothelial cells from subcutaneous fat display vWF in distinct granular structures. Human peritoneal mesothelium produces plasminogen activator-dependent fibrinolytic activity, which is essential in the resolution of fibrous exudates and may therefore be important in preventing the formation of fibrous peritoneal adhesions. This fibrinolytic activity is plasminogen activator-dependent, but has not been fully characterized. We report here that human omental tissue mesothelial cells in vitro produce large amounts of tissue-type plasminogen activator (t-PA), together with type 1 and 2 plasminogen activator inhibitor (PAI-1 and PAI-2). PAI-1 is predominantly secreted into the culture medium, whereas the major part of PAI-2 is found in the cells. No urokinase-type plasminogen activator is detected. On stimulation with the inflammatory mediator tumor necrosis factor (TNF), at least a threefold decrease in t-PA antigen is observed, together with an increase in both PAI-1 and PAI-2. TNF also induces a marked change in cell shape. Whereas TNF and bacterial lipopolysaccharide (LPS) have similar effects on the production of PA inhibitor by human endothelial cells, LPS has no or only a relatively small effect on the fibrinolytic properties of mesothelial cells. The decreased fibrinolytic activity induced by the cytokine TNF may impair the natural dissolution of fibrin deposits at the peritoneum in the presence of an inflammatory reaction.

scholarly journals Arsenite Inhibits Tissue-Type Plasminogen Activator Synthesis through NRF2 Activation in Cultured Human Vascular Endothelial EA.hy926 Cells

2021 ◽  
Vol 22 (2) ◽  
pp. 739
Author(s):  
Tsuyoshi Nakano ◽  
Tsutomu Takahashi ◽  
Chika Yamamoto ◽  
Eiko Yoshida ◽  
Toshiyuki Kaji ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Vascular Endothelial Cells ◽  
Tissue Type ◽  
Vascular Endothelial ◽  
Tissue Type Plasminogen Activator ◽  
Nrf2 Pathway ◽  
Type Plasminogen Activator ◽  
Ea.Hy926 Cells ◽  
Pai 1

Chronic arsenic exposure is known to be related to the progression of atherosclerosis. However, the pathogenic mechanisms of arsenic-induced atherosclerosis have not been fully elucidated. Because disruption of the blood coagulation/fibrinolytic system is involved in the development of arteriosclerosis, we investigated the effect of arsenite on fibrinolytic activity in human vascular endothelial EA.hy926 cells in the present study. Fibrinolysis depends on the balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) secreted from vascular endothelial cells. We found that arsenite reduced fibrinolytic t-PA activity by inhibiting its synthesis without affecting PAI-1 production. The inhibitory effect of arsenite on t-PA expression was partially recovered by the reactive oxygen species (ROS) scavenger Trolox. The nuclear factor erythroid 2 related factor 2 (NRF2) pathway is known to be activated by arsenite via ROS production. We confirmed that arsenite activated the NRF2 pathway, and arsenite-induced inhibition of fibrinolytic t-PA activity was abrogated in NRF2-knockdown EA.hy926 cells. These results suggest that arsenite inhibits the fibrinolytic activity of t-PA by selectively suppressing its synthesis via activation of the NRF2 pathway in vascular endothelial cells.

scholarly journals Fibrinolysis in pregnancy: a study of plasminogen activator inhibitors

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 460-466 ◽  
Author(s):  
EK Kruithof ◽  
C Tran-Thang ◽  
A Gudinchet ◽  
J Hauert ◽  
G Nicoloso ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Fibrin Plate ◽  
Pai 1 ◽  
In Pregnancy ◽  
Plasma Activity

During pregnancy the plasma concentration of two different inhibitors of plasminogen activators (PAIs) increases. The only one found in the plasma of nonpregnant women (PAI1) is immunologically related to a PAI of endothelial cells; its plasma activity, as deduced from the inhibition of single-chain tissue-type plasminogen activator (t-PA), increased from 3.4 +/- 2.3 U/mL (mean +/- 95% confidence limits) in the plasma of nonpregnant women to 29 +/- 7 U/mL at term, and its antigen level, measured by a radioimmunoassay, increased from 54 +/- 17 ng/mL to 144 +/- 25 ng/mL. In pregnancy plasma a second PAI (PAI 2) related to a PAI found in placenta extracts was observed. Its level, quantified with a radioimmunoassay, increased from below the detection limit (approximately 10 ng/mL) in normal plasma to 260 ng/mL at term. One hour after delivery, PAI 1 activities and antigen decreased sharply, but the PAI 2 antigen levels remained constant. Three days later, the PAI 1 antigen levels had fallen to normal levels, but the PAI 2 antigen levels were still at least eightfold above the nonpregnant values. During pregnancy, the t-PA and prourokinase (u-PA) antigen concentrations increased 50% and 200%, respectively, whereas the plasminogen and alpha 2-antiplasmin levels remained constant. Despite the large variations in the levels of PAs and PAIs, the overall fibrinolytic activity as measured in diluted plasma by a radioiodinated fibrin plate assay did not change significantly. Just after delivery, a great increase in the t-PA antigen levels was observed. Three to five days after delivery most parameters of the fibrinolytic system were normal again. Our results demonstrate that during pregnancy and in the puerperium profound alterations of the fibrinolytic system occur that are characterized by increases in PAs and their inhibitors, but these alterations do not affect the overall fibrinolytic activity.

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.

Enhanced Tissue Factor Pathway Activity and Fibrin Turnover in the Alveolar Compartment of Patients with Interstitial Lung Disease

2000 ◽  
Vol 83 (06) ◽  
pp. 853-860 ◽  
Author(s):  
A. Günther ◽  
P. Mosavi ◽  
C. Ruppert ◽  
S. Heinemann ◽  
B. Temmesfeld ◽  
...  
Keyword(s):  
Interstitial Lung Disease ◽  
Lung Disease ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Lung Compliance ◽  
Tissue Type ◽  
Type Plasminogen Activator ◽  
Order Of Magnitude ◽  
Pai 1 ◽  
D Dimer

SummaryBronchoalveolar lavage fluids (BALF) from patients with hyper- sensitivity pneumonitis (HP; n = 35), idiopathic pulmonary fibrosis (IPF, n = 41) and sarcoidosis (SARC, n = 48) were investigated for alterations in the alveolar hemostatic balance. Healthy individuals (n = 21) served as Controls. Procoagulant activity (PCA), tissue factor (TF) activity and F VII activity were assessed by means of specific recalcification assays. The overall fibrinolytic activity (FA) was measured using the 125I-labeled fibrin plate assay. Fibrinopeptide A (FP-A), D-Dimer, plasminogen activators (PA) of the urokinase (u-PA) or tissue type (t-PA), PA-Inhibitor I (PAI-1) and α2-antiplasmin (α2-AP) were determined by ELISA technique. As compared to Controls, all groups with interstitial lung disease (ILD) displayed an increase in BALF PCA by approximately one order of magnitude, and this was ascribed to enhanced TF activity by >98%. Accordingly, F VII-activity was increased in all ILD groups, and elevated FP-A levels were noted. There was no significant difference in procoagulant activi- ties between the different ILD entities, but the increase in TF was significantly correlated with deterioration of lung compliance. Overall fibrinolytic activity did not significantly differ between ILD entities and Controls, although some reduction in IPF subjects was observed. Nevertheless, changes in the profile of the different pro- and anti- fibrinolytic compounds were noted. U-PA, but not t-PA levels were significantly reduced in all ILD groups. α2-AP was markedly elevated throughout, whereas PAI-1 levels were lowered. As a balance of enhanced procoagulant and sustained overall fibrinolytic activity, lavage D-dimer levels were elevated by more than one order of magnitude in all ILD patients. We conclude that the predominant alteration in alveolar hemostatic balance in all groups of ILD patients is an enhancement in TF factor pathway activity. Concomitantly, various compounds of the (anti-)fibrinolytic pathways present with altered concentrations, but the overall BALF fibrinolytic activity is largely unchanged. The net enhancement of fibrin turnover is significantly correlated with the decrease in lung compliance. Abbreviations: α2-AP – α2-antiplasmin; ARDS – acute respiratory distress syndrome; BAL – bronchoalveolar lavage; BALF – BAL fluids; BSA – bovine serum albumin; FEV1 – forced expired volume within 1 s; FP-A – fibrinopeptide A; FVC – forced vital capacity; ILD – interstitial lung disease; IPF – idiopathic pulmonary fibrosis; HP – hypersensitivity pneumonitis; PAI-1 – plasminogen-activator-inhibitor-1; PBS – phosphate buffered saline; PCA – procoagulant activity; PL – phospholipid; PPQ – phospholipid-proteinquotient; SARC – sarcoidosis; t-PA – tissue-type plasminogen activator; u-PA – urokinase-type plasminogen activator

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 Characterization and fibrinolytic properties of human omental tissue mesothelial cells. Comparison with endothelial cells

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1490-1497 ◽  
Author(s):  
VW van Hinsbergh ◽  
T Kooistra ◽  
MA Scheffer ◽  
J Hajo van Bockel ◽  
GN van Muijen
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Subcutaneous Fat ◽  
Mesothelial Cells ◽  
Tissue Type ◽  
Microvascular Endothelial Cells ◽  
Type Plasminogen Activator ◽  
Microvascular Endothelial ◽  
Pai 1

Abstract It has been reported that omental fat tissue is a good source of human microvascular endothelial cells. By characterization we demonstrate that the epitheloid cells isolated from omental tissue are not endothelial cells, but mesothelial cells. They contain abundant cytokeratins 8 and 18, which are absent in endothelial cells, and vimentin. No staining with the endothelial-specific antibodies EN-4 and PAL-E is observed. A faint and diffuse staining of von Willebrand factor (vWF) is seen in mesothelial cells, whereas microvascular endothelial cells from subcutaneous fat display vWF in distinct granular structures. Human peritoneal mesothelium produces plasminogen activator-dependent fibrinolytic activity, which is essential in the resolution of fibrous exudates and may therefore be important in preventing the formation of fibrous peritoneal adhesions. This fibrinolytic activity is plasminogen activator-dependent, but has not been fully characterized. We report here that human omental tissue mesothelial cells in vitro produce large amounts of tissue-type plasminogen activator (t-PA), together with type 1 and 2 plasminogen activator inhibitor (PAI-1 and PAI-2). PAI-1 is predominantly secreted into the culture medium, whereas the major part of PAI-2 is found in the cells. No urokinase-type plasminogen activator is detected. On stimulation with the inflammatory mediator tumor necrosis factor (TNF), at least a threefold decrease in t-PA antigen is observed, together with an increase in both PAI-1 and PAI-2. TNF also induces a marked change in cell shape. Whereas TNF and bacterial lipopolysaccharide (LPS) have similar effects on the production of PA inhibitor by human endothelial cells, LPS has no or only a relatively small effect on the fibrinolytic properties of mesothelial cells. The decreased fibrinolytic activity induced by the cytokine TNF may impair the natural dissolution of fibrin deposits at the peritoneum in the presence of an inflammatory reaction.

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