Plasminogen activator inhibitor 1 (PAI-1): Immunogold localization within platelet-fibrin thrombi

1992 ◽  
Vol 50 (1) ◽  
pp. 810-811
Author(s):  
J.C. Lewis ◽  
R.R. Hantgan ◽  
W.G. Jerome ◽  
K.G. Grant ◽  
A. Dekker ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Vitro Assay ◽  
Fibrin Network ◽  
Pai 1

Thrombosis, the major clinical sequelae to atherosclerosis, is complex and encompasses a multiplicity of interactions among plasma proteins, platelets and other blood cells, and vascular endothelial cells. Thrombolysis, in a fashion paralleling thrombus progression, is also influenced by a multiplicity of interactions, and recent evidence has suggested that both platelets and endothelial cells play a role in prolonging the lytic process. This prolongation is detrimental to prognosis following vascular occlusion. We have previously reported that thrombin-stimulated platelets will prolong clot lysis when included in an in-vitro assay comprised of tissue-type plasminogen activator, plasminogen, and fibrin(ogen). This observation has been expanded in the present study to included TNF stimulated human umbilical vein endothelial cells, and our data document the association of platelet and EC derived PAI-1 with the fibrin network. HUVEC grown on carbon-stabilized, formvar-coated gold grids for whole mount IVEM were stimulated with tumor necrosis factor, prior to clot initiation and subsequent lysis, by addition to the cultures of fibrinogen, t-PA, plasminogen and thrombin-stimulated platelets. At selected times of lysis following polymerization, based upon laser light scattering kinetic studies, the samples were fixed and processed for PAI-1 localization using the immunogold technique. When observed by SEM, the partially lysed thrombi consisted of an anastomosing fibrin network that extended from endothelial cell surfaces (Figure 1). Within the thrombus, the delicate, branching fibrin strands often were focused at points containing the activated platelets. The interaction of fibrin with endothelial cells was evidenced by IVEM as a delicate extracellular array extending between and among adjacent cells (Figure 2 a,b). Immunogold probes, documenting PAI-1, were distributed in clusters along the fibrin (Figures lb,c). PA1-1, although cellular in origin, was not associated with the surfaces of either platelets or endothelial cells. The specificity of PAI-1 localization was verified through inclusion of a non-related immunogold probe which bound in substantially lower concentration and without site selectivity (Figure 2c). We conclude that HUVEC and platelets modulate thrombolysis through the release of PAI-1 which binds to fibrin and retards plasminogen activation.

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.

Fluvastatin Inhibits Basal and Stimulated Plasminogen Activator Inhibitor 1, but Induces Tissue Type Plasminogen Activator in Cultured Human Endothelial Cells

2000 ◽  
Vol 84 (07) ◽  
pp. 59-64 ◽  
Author(s):  
Luciana Mussoni ◽  
Cristina Banfi ◽  
Luigi Sironi ◽  
Magda Arpaia ◽  
Elena Tremoli
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
De Novo ◽  
Umbilical Vein ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Mrna Levels ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe effects of fluvastatin, a synthetic hydroxymethylglutaryl coenzyme A (HMG-CoA) inhibitor, on the biosynthesis of tissue plasminogen activator (t-PA) and of its major physiological inhibitor (plasminogen activator inhibitor type 1, PAI-1) were investigated in cultured human umbilical vein endothelial cells (HUVEC). Fluvastatin (0.1 to 2.5 µM), concentration-dependently reduced the release of PAI-1 antigen by unstimulated HUVEC, subsequent to a reduction in PAI-1 steady-state mRNA levels and de novo protein synthesis. In contrast, it increased t-PA secretion.The drug also reduced PAI-1 antigen secreted in response to 10 µg/ml bacterial lipopolysaccharide (LPS), 100 U/ml tumour necrosis factor α (TNFα) or 0.1 µM phorbol myristate acetate (PMA).Mevalonate (100 µM), a precursor of isoprenoids, added to cells simultaneously with fluvastatin, suppressed the effect of the drug on PAI-1 both in unstimulated and stimulated cells as well as on t-PA antigen. Among intermediates of the isoprenoid pathway, all-trans-geranylgeraniol (5 µM) but not farnesol (10 µM) prevented the effect of 2.5 µM fluvastatin on PAI-1 antigen, which suggests that the former intermediate of the isoprenoid synthesis is responsible for the observed effects.

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 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.

Functional Alterations of Endothelial Cells Cultured In Vitro in Contact with Biomaterials

1992 ◽  
Vol 20 (1) ◽  
pp. 61-65
Author(s):  
Elisabetta Cenni ◽  
Gabriela Ciapetti ◽  
Susanna Stea ◽  
Alessandro Di Leo ◽  
Daniela Cavedagna ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Umbilical Vein ◽  
Control Cell ◽  
Control Level ◽  
Neutral Red ◽  
Cell Counting ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

In order to evaluate in vitro the suitability of various prosthetic materials for endothelial seeding, human endothelial cells derived from the umbilical vein were placed in direct contact with a variety of polymers. As a control, endothelial cells were cultured in the absence of material. After 24, 48, 72 and 96 hours, the cells were counted and a viability test with neutral red was performed. Assays of 6-keto prostaglandinFla (6-keto-PGF1a), tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) were carried out on the supernatants. The cell counting technique demonstrated growth inhibition in the cell populations in contact with Woven Dacron® and Double Velour Dacron® in comparison with control cell cultures. Vital staining with neutral red, always sharply positive in the controls, was weak in the cells placed in contact with the materials. The 6-keto-PGF1a concentration in the supernatant was similar to the control level in the populations in contact with Woven Dacron® and Double Velour Dacron®. The tPA synthesis was higher in the cells exposed to the assayed materials compared to control values, while the PAI-1 concentration in the supernatant was lower in the cultures in contact with all materials.

scholarly journals Platelets inhibit fibrinolysis in vitro by both plasminogen activator inhibitor-1-dependent and -independent mechanisms

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 351-356 ◽  
Author(s):  
WP Fay ◽  
DT Eitzman ◽  
AD Shapiro ◽  
EL Madison ◽  
D Ginsburg
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Clot Lysis ◽  
Homozygous Mutation ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Platelet-rich thrombi are resistant to lysis by tissue-type plasminogen activator (t-PA). Although platelet alpha-granules contain plasminogen activator inhibitor-1 (PAI-1), a fast-acting inhibitor of t-PA, the contribution of PAI-1 to the antifibrinolytic effect of platelets has remained a subject of controversy. We recently reported a patient with a homozygous mutation within the PAI-1 gene that results in complete loss of PAI-1 expression. Platelets from this individual constitute a unique reagent with which to probe the role of platelet PAI-1 in the regulation of fibrinolysis. The effects of PAI-1-deficient platelets were compared with those of normal platelets in an in vitro clot lysis assay. Although the incorporation of PAI-1-deficient platelets into clots resulted in a moderate inhibition of t-PA-mediated fibrinolysis, normal platelets markedly inhibited clot lysis under the same conditions. However, no difference between PAI-1-deficient platelets and platelets with normal PAI-1 content was observed when streptokinase or a PAI-1-resistant t-PA mutant were used to initiate fibrinolysis. In addition, PAI-1-resistant t-PA was significantly more efficient in lysing clots containing normal platelets than wild-type t-PA. We conclude that platelets inhibit t-PA-mediated fibrinolysis by both PAI- 1-dependent and PAI-1-independent mechanisms. These results have important implications for the role of PAI-1 in the resistance of platelet-rich thrombi to lysis in vivo.

scholarly journals Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 533-541 ◽  
Author(s):  
DA Dichek ◽  
O Nussbaum ◽  
SJ Degen ◽  
WF Anderson
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fold Increase ◽  
Retroviral Vector ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

Abstract In an attempt to enhance the fibrinolytic activity of endothelial cells (EC), a retroviral vector containing the human tissue-type plasminogen activator (t-PA) cDNA was constructed. Sheep EC were stably transduced with the vector, resulting in a 30-fold increase in t-PA activity over that detected in EC transduced with a control vector. Southern and Northern analyses confirmed the presence of both the vector sequence and the appropriate mRNA transcripts. Secretion of high levels of recombinant human t-PA continued in vitro for the duration of the experiments, up to 11 weeks after transduction, although the rate of t- PA secretion decreased in some of the EC lines. Zymographic analysis of conditioned medium from t-PA-transduced EC showed the presence of two new molecular species with plasminogen activator activity that could be specifically immunoprecipitated with a monoclonal antihuman t-PA antibody. The relative molecular masses of these species (60 to 80 and 110 Kd) suggest that they represent recombinant human t-PA both free and bound to sheep plasminogen activator inhibitor 1 (PAI-1). Consistent with this interpretation, the 110-Kd species could be specifically immunoprecipitated with antiserum to PAI-1. These studies demonstrate that retroviral vector-mediated gene transfer may be used to increase total EC fibrinolytic activity.

scholarly journals Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 533-541
Author(s):  
DA Dichek ◽  
O Nussbaum ◽  
SJ Degen ◽  
WF Anderson
Keyword(s):  
Endothelial Cells ◽  
Gene Transfer ◽  
Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Fold Increase ◽  
Retroviral Vector ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Pai 1

In an attempt to enhance the fibrinolytic activity of endothelial cells (EC), a retroviral vector containing the human tissue-type plasminogen activator (t-PA) cDNA was constructed. Sheep EC were stably transduced with the vector, resulting in a 30-fold increase in t-PA activity over that detected in EC transduced with a control vector. Southern and Northern analyses confirmed the presence of both the vector sequence and the appropriate mRNA transcripts. Secretion of high levels of recombinant human t-PA continued in vitro for the duration of the experiments, up to 11 weeks after transduction, although the rate of t- PA secretion decreased in some of the EC lines. Zymographic analysis of conditioned medium from t-PA-transduced EC showed the presence of two new molecular species with plasminogen activator activity that could be specifically immunoprecipitated with a monoclonal antihuman t-PA antibody. The relative molecular masses of these species (60 to 80 and 110 Kd) suggest that they represent recombinant human t-PA both free and bound to sheep plasminogen activator inhibitor 1 (PAI-1). Consistent with this interpretation, the 110-Kd species could be specifically immunoprecipitated with antiserum to PAI-1. These studies demonstrate that retroviral vector-mediated gene transfer may be used to increase total EC fibrinolytic activity.

scholarly journals Platelets inhibit fibrinolysis in vitro by both plasminogen activator inhibitor-1-dependent and -independent mechanisms

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 351-356 ◽  
Author(s):  
WP Fay ◽  
DT Eitzman ◽  
AD Shapiro ◽  
EL Madison ◽  
D Ginsburg
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Clot Lysis ◽  
Homozygous Mutation ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Platelet-rich thrombi are resistant to lysis by tissue-type plasminogen activator (t-PA). Although platelet alpha-granules contain plasminogen activator inhibitor-1 (PAI-1), a fast-acting inhibitor of t-PA, the contribution of PAI-1 to the antifibrinolytic effect of platelets has remained a subject of controversy. We recently reported a patient with a homozygous mutation within the PAI-1 gene that results in complete loss of PAI-1 expression. Platelets from this individual constitute a unique reagent with which to probe the role of platelet PAI-1 in the regulation of fibrinolysis. The effects of PAI-1-deficient platelets were compared with those of normal platelets in an in vitro clot lysis assay. Although the incorporation of PAI-1-deficient platelets into clots resulted in a moderate inhibition of t-PA-mediated fibrinolysis, normal platelets markedly inhibited clot lysis under the same conditions. However, no difference between PAI-1-deficient platelets and platelets with normal PAI-1 content was observed when streptokinase or a PAI-1-resistant t-PA mutant were used to initiate fibrinolysis. In addition, PAI-1-resistant t-PA was significantly more efficient in lysing clots containing normal platelets than wild-type t-PA. We conclude that platelets inhibit t-PA-mediated fibrinolysis by both PAI- 1-dependent and PAI-1-independent mechanisms. These results have important implications for the role of PAI-1 in the resistance of platelet-rich thrombi to lysis in vivo.

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