Abstract 2901: Doppler Ultrasound Enhances the Thrombolytic Activity of Tissue-Plasminogen Activator-Loaded Echogenic Liposomes

Background: Ultrasound has been shown to enhance thrombolysis when used in conjunction with a thrombolytic agent. We have loaded echogenic liposomes (ELIP) with tissue plasminogen activator (tPA) and shown that mechanical non-imaging 1 MHz ultrasound can enhance thrombolysis in an in vitro clot model. This study aimed to evaluate the efficacy of clinical Doppler ultrasound to enhance the thrombolytic effect of our tPA-ELIP. Materials and Methods: A standard reproducible whole blood porcine clot model was used. Clots were blotted dry, weighed, and placed in 10 ml fresh frozen porcine plasma. Clots were treated with tPA-ELIP with and without Doppler ultrasound (pulsed wave ultrasound, 6.6 MHz, 2 minute-treatment time). After 30 minutes, clots were similarly weighed. Data are reported as percent clot mass loss. Samples of the plasma were assayed for D-dimer levels. Results: Exposure of tPA-ELIP to clinical Doppler ultrasound enhanced thrombolysis (p=0.015 vs. tPA alone or Doppler ultrasound alone; Figure ). There was a trend towards higher D-dimer levels in the clots treated with tPA-ELIP + clinical Doppler ultrasound, corresponding to the higher percent clot mass loss seen in this treatment group. Conclusions: This study demonstrates the ability of clinical Doppler ultrasound to enhance the thrombolytic effect of our tPA-ELIP in an in vitro clot model. This demonstrates the potential to translate our novel thrombolytic technique of combining a thrombolytic-loaded contrast agent and ultrasound to patient care.

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Ultrasound-Enhanced Thrombolytic Effect of Tissue Plasminogen Activator–Loaded Echogenic Liposomes in an In Vivo Rabbit Aorta Thrombus Model—Brief Report

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.111.225938 ◽

2011 ◽

Vol 31 (6) ◽

pp. 1357-1359 ◽

Cited By ~ 33

Author(s):

Susan T. Laing ◽

Melanie Moody ◽

Beverly Smulevitz ◽

Hyunggun Kim ◽

Patrick Kee ◽

...

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Rabbit Aorta ◽

Echogenic Liposomes ◽

Tissue Plasminogen ◽

Thrombolytic Effect

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Plasma crosslinked fibrin polymers: quantitation based on tissue plasminogen activator conversion to D-dimer and measurement in normal and patients with acute thrombotic disorders

Blood ◽

10.1182/blood.v80.3.709.709 ◽

1992 ◽

Vol 80 (3) ◽

pp. 709-717 ◽

Cited By ~ 17

Author(s):

A Kornberg ◽

CW Francis ◽

VJ Marder

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Western Blotting ◽

Gel Electrophoresis ◽

Degradation Products ◽

Thrombotic Disease ◽

Tissue Plasminogen ◽

D Dimer

Abstract Plasma crosslinked fibrin polymers (XLFP) are formed as a result of in vivo hemostatic activation and are elevated in thrombotic disease. We have investigated the plasmic degradation of plasma XLFP in vitro to provide information regarding the pattern of crosslinking and the composition of degradation products. Plasma XLFP were identified by sodium dodecyl sulfate (SDS)-agarose electrophoresis and Western blotting and quantitated by gel scanning. D-dimer was measured by enzyme-linked immunosorbent assay and the results were verified by SDS- polyacrylamide gel electrophoresis and Western blotting of the digests. Complete degradation of XLFP occurred only after supplementation of plasma with plasminogen (5 U/mL) and incubation with recombinant tissue plasminogen activator (rt-PA), indicating that the normal plasma plasminogen concentration limits plasmic degradation in vitro. Gel electrophoresis showed that the principal terminal degradation products of XLDP were fragments D, DD, and E, indicating that crosslinking occurred primarily through gamma chain dimers. After adding a low concentration of thrombin to plasma in vitro, XLFP increased progressively before clotting, and the concentration correlated with the increase in the D-dimer concentration after degradation (r = .98). Plasma XLFP and D-dimer concentrations in plasmic digests were significantly elevated in patients with stroke (150 +/- 83 micrograms/mL and 88 +/- 32 micrograms/mL), myocardial infarction (217 +/- 110 micrograms/mL and 84 +/- 30 micrograms/mL), and venous thrombosis (187 +/- 80 micrograms/mL and 86 +/- 19 micrograms/mL) compared with normals (28 +/- 12 micrograms/mL and 25 +/- 7 micrograms/mL). There was a strong correlation between the plasma concentration of XLFP and the D-dimer immunoreactivity of plasma after plasmic degradation (r = .87). The results indicate that XLFP in plasma are crosslinked primarily through gamma chains and degrade to fragment DD with plasminogen activation. Also, the immunoreactivity of in vitro plasmic digests of plasma reflects the concentration of XLFP and may provide a useful indirect measure of in vivo hemostatic activation in patients with thrombotic disease.

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Plasma crosslinked fibrin polymers: quantitation based on tissue plasminogen activator conversion to D-dimer and measurement in normal and patients with acute thrombotic disorders

Blood ◽

10.1182/blood.v80.3.709.bloodjournal803709 ◽

1992 ◽

Vol 80 (3) ◽

pp. 709-717

Author(s):

A Kornberg ◽

CW Francis ◽

VJ Marder

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Western Blotting ◽

Gel Electrophoresis ◽

Degradation Products ◽

Thrombotic Disease ◽

Tissue Plasminogen ◽

D Dimer

Plasma crosslinked fibrin polymers (XLFP) are formed as a result of in vivo hemostatic activation and are elevated in thrombotic disease. We have investigated the plasmic degradation of plasma XLFP in vitro to provide information regarding the pattern of crosslinking and the composition of degradation products. Plasma XLFP were identified by sodium dodecyl sulfate (SDS)-agarose electrophoresis and Western blotting and quantitated by gel scanning. D-dimer was measured by enzyme-linked immunosorbent assay and the results were verified by SDS- polyacrylamide gel electrophoresis and Western blotting of the digests. Complete degradation of XLFP occurred only after supplementation of plasma with plasminogen (5 U/mL) and incubation with recombinant tissue plasminogen activator (rt-PA), indicating that the normal plasma plasminogen concentration limits plasmic degradation in vitro. Gel electrophoresis showed that the principal terminal degradation products of XLDP were fragments D, DD, and E, indicating that crosslinking occurred primarily through gamma chain dimers. After adding a low concentration of thrombin to plasma in vitro, XLFP increased progressively before clotting, and the concentration correlated with the increase in the D-dimer concentration after degradation (r = .98). Plasma XLFP and D-dimer concentrations in plasmic digests were significantly elevated in patients with stroke (150 +/- 83 micrograms/mL and 88 +/- 32 micrograms/mL), myocardial infarction (217 +/- 110 micrograms/mL and 84 +/- 30 micrograms/mL), and venous thrombosis (187 +/- 80 micrograms/mL and 86 +/- 19 micrograms/mL) compared with normals (28 +/- 12 micrograms/mL and 25 +/- 7 micrograms/mL). There was a strong correlation between the plasma concentration of XLFP and the D-dimer immunoreactivity of plasma after plasmic degradation (r = .87). The results indicate that XLFP in plasma are crosslinked primarily through gamma chains and degrade to fragment DD with plasminogen activation. Also, the immunoreactivity of in vitro plasmic digests of plasma reflects the concentration of XLFP and may provide a useful indirect measure of in vivo hemostatic activation in patients with thrombotic disease.

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Tissue plasminogen activator as a novel diagnostic aid in acute pulmonary embolism

VASA ◽

10.1024/0301-1526/a000392 ◽

2014 ◽

Vol 43 (6) ◽

pp. 450-458 ◽

Cited By ~ 1

Author(s):

Julio Flores ◽

Ángel García-Avello ◽

Esther Alonso ◽

Antonio Ruíz ◽

Olga Navarrete ◽

...

Keyword(s):

Pulmonary Embolism ◽

Negative Predictive Value ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Predictive Value ◽

Acute Pulmonary Embolism ◽

Enzyme Linked Immunosorbent Assay ◽

Diagnostic Utility ◽

Tissue Plasminogen ◽

D Dimer

Background: We evaluated the diagnostic efficacy of tissue plasminogen activator (tPA), using an enzyme-linked immunosorbent assay (ELISA) and compared it with an ELISA D-dimer (VIDAS D-dimer) in acute pulmonary embolism (PE). Patients and methods: We studied 127 consecutive outpatients with clinically suspected PE. The diagnosis of PE was based on a clinical probability pretest for PE and a strict protocol of imaging studies. A plasma sample to measure the levels of tPA and D-dimer was obtained at enrollment. Diagnostic accuracy for tPA and D-dimer was determined by the area under the receiver operating characteristic (ROC) curve. Sensitivity, specificity, predictive values, and the diagnostic utility of tPA with a cutoff of 8.5 ng/mL and D-dimer with a cutoff of 500 ng/mL, were calculated for PE diagnosis. Results: PE was confirmed in 41 patients (32 %). Areas under ROC curves were 0.86 for D-dimer and 0.71 for tPA. The sensitivity/negative predictive value for D-dimer using a cutoff of 500 ng/mL, and tPA using a cutoff of 8.5 ng/mL, were 95 % (95 % CI, 88–100 %)/95 % (95 % CI, 88–100 %) and 95 % (95 % CI, 88–100 %)/94 %), respectively. The diagnostic utility to exclude PE was 28.3 % (95 % CI, 21–37 %) for D-dimer and 24.4 % (95 % CI, 17–33 %) for tPA. Conclusions: The tPA with a cutoff of 8.5 ng/mL has a high sensitivity and negative predictive value for exclusion of PE, similar to those observed for the VIDAS D-dimer with a cutoff of 500 ng/mL, although the diagnostic utility was slightly higher for the D-dimer.

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Binding of Tissue Plasminogen Activator to Vascular Grafts

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646541 ◽

1989 ◽

Vol 61 (01) ◽

pp. 131-136 ◽

Cited By ~ 8

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.

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Tissue Plasminogen Activator Plasma Levels as a Potential Diagnostic Aid in Acute Pulmonary Embolism

Archives of Pathology & Laboratory Medicine ◽

10.5858/2003-127-0310-tpapla ◽

2003 ◽

Vol 127 (3) ◽

pp. 310-315

Author(s):

Julio Flores ◽

Angel García-Avello ◽

Victor M. Flores ◽

JoséL. Navarro ◽

Felipe Canseco ◽

...

Keyword(s):

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Lower Limb ◽

Pulmonary Angiography ◽

Enzyme Linked Immunosorbent Assay ◽

Clinical Probability ◽

Tissue Plasminogen ◽

Lung Scan ◽

Pai 1 ◽

D Dimer

Abstract Context.—Pulmonary embolism (PE) is a potentially fatal and frequent complication of deep venous thrombosis, and the most reliable techniques for the diagnosis of PE are not universally available and have other limitations. Objective.—To determine the efficacy of 4 different fibrinolysis system parameters, namely, tissue plasminogen activator (tPA), tissue plasminogen activator inhibitor type 1 (PAI-1), plasmin-antiplasmin complexes (PAP), and D-dimer, in the diagnosis of acute PE. Setting.—A 350-bed university hospital serving an area with 280 000 inhabitants. Patients.—Sixty-six consecutive outpatients with clinically suspected PE. The diagnosis of PE was based on ventilation-perfusion (V/Q) lung scan in combination with clinical assessment, lower limb study, and (when required) pulmonary angiography. Main Outcome Measures.—At the moment of clinical suspicion, a sample of venous blood was obtained to measure levels of tPA, PAI-1, PAP, and D-dimer using an enzyme-linked immunosorbent assay method. Results.—Twenty-seven patients (41%) were classified as PE positive (high clinical probability and V/Q lung scan [n = 12], nondiagnostic V/Q lung scan and high clinical probability [n = 1], inconclusive V/Q lung scan and positive lower limb examination for deep venous thrombosis [n = 11], and positive pulmonary angiography [n = 3]), and 39 patients (59%) were classified PE negative. The sensitivity/negative predictive value for tPA, using a cutoff of 8.5 ng/mL, and PAI-1, using a cutoff of 15 ng/mL, were 100%/100% and 100%/100%, respectively. A tPA level lower than 8.5 ng/mL occurred in 13 (19.7%; all PE negative) of 66 patients with suspected PE, and PAI-1 levels were lower than 15 ng/mL in 9 (13.6%; all PE negative) of 66 patients with suspected PE. The D-dimer, using a cutoff of 500 ng/mL, showed a sensitivity and negative predictive value of 92.6% and 87.5%, respectively. Conclusions.—Our data indicate that tPA and PAI-1 levels are potentially useful in ruling out PE, although tPA seems to be the better parameter. The sensitivity levels and negative predictive values for the rapid enzyme-linked immunosorbent assay for D-dimer used in this investigation were low compared with previous studies using the same test.

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Uptake and Degradation of Tissue Plasminogen Activator in Rat Liver

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647518 ◽

1988 ◽

Vol 59 (03) ◽

pp. 474-479 ◽

Cited By ~ 35

Author(s):

Monica Einarsson ◽

Bård Smedsrød ◽

Håkan Pertoft

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Rat Liver ◽

Liver Cells ◽

Terminal Phase ◽

Tissue Plasminogen ◽

Liver Endothelial Cells ◽

Parenchymal Cells

SummaryThe mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(α), and the terminal phase, t1/2(β), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells.Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells.Competitivb inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types.These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.

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Tissue Plasminogen Activator Expression in Endothelial Cells Exposed to Cyclic Strain in Vitro

Cell Transplantation ◽

10.1177/096368979200100108 ◽

1992 ◽

Vol 1 (1) ◽

pp. 43-50 ◽

Cited By ~ 46

Author(s):

Toshiaki Iba ◽

Bauer E. Sumpio

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Cyclic Strain ◽

Neutral Position ◽

Messenger Ribonucleic Acid ◽

Tissue Plasminogen ◽

Pai 1

The effects of cyclic strain on the production of tissue plasminogen activator (tPA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured endothelial cells (EC) were examined. Human saphenous vein EC were seeded in selective areas of culture plates with flexible membrane bottoms (corresponding to specific strain regions) and grown to confluence. Membranes were deformed by vacuum (-20 kPa) at 60 cycles/min (0.5 s strain alternating with 0.5 s relaxation in the neutral position) for 5 days. EC grown in the periphery were subjected to 7-24% strain, while cells grown in the center experienced less than 7% strain. The results show a significant increase in immunoreactive tPA production on days 1, 3 and 5 compared to day 0 in EC subjected to more than 7% cyclic strain. There was no significant elevation of tPA in the medium of EC subjected to less than 7% strain. tPA activity could only be detected in the medium of EC subjected to more than 7% cyclic strain. PAI-1 levels in the medium were not significantly different in either group. In addition, immunocytochemical detection of intracellular tPA and messenger ribonucleic acid (mRNA) expression of tPA (assessed by the reverse transcriptase polymerase chain reaction utilizing tPA specific sense and antisense primers) was significantly increased in EC subjected to more than 7% cyclic strain. We conclude that a 60 cycles/min regimen of strain that is greater than 7% can selectively stimulate tPA production by EC in vitro and may contribute to the relative nonthrombogenicity of the endothelium in vivo.

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