Elimination of Native and Carbohydrate-Modified Tissue Plasminogen Activator in Rabbits

SummaryThe elimination of native and carbohydrate-modified tissue type plasminogen activator (t-PA) after an i. v. bolus injection was studied in rabbits. t-PA with a low content of mannose (mant-PA) was obtained by treatment with α-mannosidase, which cleaves terminal mannose from the carbohydrate side chains of the molecule. About 50% of the total mannose was removed from the native t-PA.Clearance of t-PA in rabbits was followed by measurements of both the fibrinolytic activity of the activators and the radioactivity of the iodine-labelled compounds. A biphasic mode of elimination of the fibrinolytic activity as well as of the radioactivity was found with both the native and the carbohydrate modified t-PA. The initial half-life (t½α) was the same, about 1.5 minutes, for both compounds, irrespective of method of analysis. The late half-life (t½β) was also the same, about 15 minutes, for both compounds. However, the β-elimination of native t-PA could only be determined accurately from radioactivity data in the dosages tested, i.e. up to 2 mg of t-PA. No dose dependency in the elimination pattern was observed.In gel filtration experiments, with plasma from the rabbits, it was demonstrated that in addition to fibrinolytically active t-PA and man-t-PA, both fibrinolytically inactive high molecular weight complexes and low molecular weight degradation products of the activators were present in plasma after injection of the compounds.The second phase of elimination .(β) was much more pronounced after mannosidase treatment of the t-PA. The proportion of molecules eliminated with a longer half-life was increased 6-7 times, from about 4% for native t-PA to about 25% for carbohydrate-modified t-PA.Thus, a simple reduction of terminal mannose in the carbohydrate side chains of t-PA does not result in altered half-lives. However, a significantly greater proportion of molecules were eliminated in the late β-phase after the carbohydrate modification. The data also support our recent finding that the mannose receptor on the liver cells is at least in part responsible for the clearance of t-PA.

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Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653442 ◽

1981 ◽

Vol 46 (03) ◽

pp. 658-661 ◽

Cited By ~ 112

Author(s):

C Korninger ◽

J M Stassen ◽

D Collen

Keyword(s):

Plasminogen Activator ◽

Intravenous Injection ◽

Active Site ◽

Half Life ◽

Degradation Products ◽

Gel Filtration ◽

Tissue Type ◽

Organ Distribution ◽

Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

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FIBRINOLYTIC ACTIVITY IN MALIGNANCY

10.1055/s-0038-1643186 ◽

1987 ◽

Author(s):

A Aranda ◽

J A Páramo ◽

B Cuesta ◽

J Fernández ◽

M J Paloma ◽

...

Keyword(s):

Plasminogen Activator ◽

Fibrinolytic Activity ◽

Degradation Products ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Clinical Complications ◽

Significant Prolongation ◽

Type Plasminogen Activator ◽

Lung Malignancies ◽

Euglobulin Lysis Time

Euglobulin lysis time (ELT), euglobulin fibrinolytic activity on fibrin plate (EFA), plasminogen, tissue-type plasminogen activator (t-PA) activity and antigen, ;2-antiplasmin (α2-AP), plasminogen activator inhibitor (PAI) and fibrinogen degradation products (FDP) were determined in a group of 149 patients (mean age 57 ± 13 years, 93 male), with different malignancies (digestive 61, lung 18, urological 24, ginaecological 16, others 30). Findings were compared with those of 44 age-sex matched healthy subjects. There was a significant prolongation of ELT (p <0.003), a decrease of plasminogen (p < 0.004) and an increase of PAI (p < 0.0001) in patients as compared to controls, being similar EFA, t-PA, α2AP and FDP. No differences in any ot these parameters were found in patients with metastatic disease (n= 65) as compared with those with local disease (n= 84). As regards to tumour localization, plasminogen and t-PA decrease were more pronounced in lung malignancies and PAI increase in lung and digestive malignancies. We conclude that there is an impairment in blood fibrinolytic activity in malignancy. Reduced fibrinolysis seems to be related to localization but not to degree of tumoral activity. That might have clinical complications.

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Evidence for carbohydrate-independent endocytosis of tissue-type plasminogen activator by liver cells

Biochemical Journal ◽

10.1042/bj2850799 ◽

1992 ◽

Vol 285 (3) ◽

pp. 799-804 ◽

Cited By ~ 5

Author(s):

E Stang ◽

N Roos ◽

M Schlüter ◽

T Berg ◽

J Krause

Keyword(s):

Plasminogen Activator ◽

Liver Cell ◽

Hepatic Clearance ◽

Mannose Receptor ◽

Cell Types ◽

Tissue Type ◽

Side Chains ◽

Tissue Type Plasminogen Activator ◽

Hepatic Cells ◽

Type Plasminogen Activator

In the liver, tissue-type plasminogen activator (t-PA) is endocytosed by hepatic parenchymal (PC), endothelial (EC) and Kupffer (KC) cells. Although the endocytosis is receptor-mediated, it remains a matter of discussion which receptors are involved in this catabolic process. To evaluate the role of a protein-specific receptor, as well as the possible involvement of the galactose receptor on PC and the mannose receptor on EC, we have employed different glycosylation variants of t-PA in biochemical and immunocytochemical studies. Partial or total removal of carbohydrate side-chains by endoglycosidases did not prevent clearance and hepatic endocytosis of t-PA by either of the liver cell types. Blockade of the galactose and mannose receptors by co-application of a large excess of the glycoprotein ovalbumin remained without effect on the binding and uptake of t-PA by hepatic cells. However, the contribution of different liver cell types to the hepatic clearance of t-PA was to a certain extent dependent on the type of oligosaccharide chains removed. The mannose receptor on EC is partially responsible for the clearance of t-PA by this cell type, whereas the galactose receptor does not seem to be involved in this process. The results obtained in this study further demonstrate that the major portion of the hepatic catabolism of t-PA is independent of its carbohydrate side-chains.

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Sustained Fibrinolysis After Administration of t-PA Despite Its Short Half-Life in the Circulation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651057 ◽

1987 ◽

Vol 57 (01) ◽

pp. 035-040 ◽

Cited By ~ 89

Author(s):

Paul R Eisenberg ◽

Laurence A Sherman ◽

Alan J Tiefenbrunn ◽

Philip A Ludbrook ◽

Burton E Sobel ◽

...

Keyword(s):

Half Life ◽

Fibrinolytic Activity ◽

Degradation Products ◽

Clinical Success ◽

Tissue Type ◽

Tissue Type Plasminogen Activator ◽

Fibrin Degradation Products ◽

Short Half Life ◽

Type Plasminogen Activator ◽

The Difference

SummaryTo characterize the duration of the fibrinolytic response to tissue-type plasminogen activator (t-PA) and streptokinase (SK) in patients with acute myocardial infarction we serially assayed crosslinked fibrin degradation products (XL-FDP) and Bβ15-42 fibrinopeptide. Use of specific monoclonal antibodies permitted quantification and differentiation of fibrin from fibrinogen degradation products. Marked elevations of XL-FDP occurred within 1 hour after administration of t-PA (n = 13) or SK (n = 35) to >1000 ng/ml in 79% of the patients. All patients given t-PA exhibited elevations of XL-FDP >1000 ng/ml, most exhibited values >5000 ng/ml (79% of patients). In contrast 6 of the patients given SK failed to exhibit XL-FDP >1000 ng/ml. XL-FDP >5000 ng/ml occurred in only 14%. The difference in the response to t-PA compared to SK was particularly striking 7 hours or more after administration of activator at which time XL-FDP were markedly elevated in patients given t-PA (5821 ± 1683 ng/ ml) compared with decreasing values in patients given SK (2924 ± 1186 ng/ml) (p <0.01). Levels of Bβ315-42 were significantly higher after t-PA compared with SK beginning 3 hours after treatment, consistent with a greater intensity of fibrinolytic response to t-PA. Marked elevations of this short lived degradation product of fibrin (t1/2 = 10-20 minutes) in the samples drawn late after administration of t-PA (44.3 ±12.8 nM) but not after SK (11.7 ± 4.5 nM) confirmed prolonged fibrinolytic activity of plasmin after t-PA. There was no discernible relationship between the extent of fibrinolysis as assessed by XL-FDP and Bβ 15-42 and the total dose of t-PA administered or the duration of the infusion. Elevations of XL-FDP invariably occurred after SK, and were not significantly different in patients with or without recanalization. Thus “clinical success” of coronary thrombolysis appears to depend on a favorable balance between thrombosis and fibrinolysis rather than the intensity of fibrinolysis alone. The prolonged fibrinolytic activity after t-PA appears to reflect the enhanced binding of this activator to fibrin and is likely to result in more sustained and hence more effective fibrinolysis with t-PA compared to SK despite the short half-life of t-PA (t1/2 = 6 minutes) in the circulation.

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FIBRINOLYSIS AFTER HEART TRANSPLANTATION

10.1055/s-0038-1643114 ◽

1987 ◽

Author(s):

J A Páramo ◽

R Arcas ◽

J Fernández ◽

J Herreros ◽

R Llorens ◽

...

Keyword(s):

Plasminogen Activator ◽

Fibrinolytic Activity ◽

Cardiac Transplantation ◽

Degradation Products ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Inhibitor Activity ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Activator Inhibitor

Some aspects of the function of the fibrinolytic system were investigated in 12 patients undergoing cardiac transplantation. Plasminogen, euglobulin fibrinolytic activity (EFA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor activity (PAI),α2-antiplasmin (α2-Ap) and fibrinogen degradation products (FDP) were determined preo-peratively and on postoperative days 1 and 5. Results showed a significant decrease of plasminogen (p <0.005), EFA (p <0.0001) and t-PA (p ^.0.001) on postoperative day 1 as compared to the baseline value, followed by recovery on day 5. There was a significant increase of PAI (p < 0 . 005) , α2-AP (p <0.0001) and FDP (p < 0.02) on postoperative day 1 as compared to the preoperative value. PAI and FDP reached the baseline value on postoperative day 5, but α2AP also increased on postoperative day 5. Our data show that there is an impairment in blood fibrinolytic activity early after cardiac transplantation, mainly related to a decrease of plasminogen and t-PA and a increase of PAI and α2AP. The clinical relevance of these data needs further evaluation.

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Structural Studies of Fibrinolysis by Electron and Light Microscopy

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615843 ◽

1999 ◽

Vol 82 (08) ◽

pp. 277-282 ◽

Cited By ~ 37

Author(s):

Yuri Veklich ◽

Jean-Philippe Collet ◽

Charles Francis ◽

John W. Weisel

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Plasminogen Activator ◽

Structural Changes ◽

Molecular Mechanisms ◽

Degradation Products ◽

Molecular Model ◽

Three Dimensional ◽

Tissue Type ◽

Type Plasminogen Activator

IntroductionMuch is known about the fibrinolytic system that converts fibrin-bound plasminogen to the active protease, plasmin, using plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plasmin then cleaves fibrin at specific sites and generates soluble fragments, many of which have been characterized, providing the basis for a molecular model of the polypeptide chain degradation.1-3 Soluble degradation products of fibrin have also been characterized by transmission electron microscopy, yielding a model for their structure.4 Moreover, high resolution, three-dimensional structures of certain fibrinogen fragments has provided a wealth of information that may be useful in understanding how various proteins bind to fibrin and the overall process of fibrinolysis (Doolittle, this volume).5,6 Both the rate of fibrinolysis and the structures of soluble derivatives are determined in part by the fibrin network structure itself. Furthermore, the activation of plasminogen by t-PA is accelerated by the conversion of fibrinogen to fibrin, and this reaction is also affected by the structure of the fibrin. For example, clots made of thin fibers have a decreased rate of conversion of plasminogen to plasmin by t-PA, and they generally are lysed more slowly than clots composed of thick fibers.7-9 Under other conditions, however, clots made of thin fibers may be lysed more rapidly.10 In addition, fibrin clots composed of abnormally thin fibers formed from certain dysfibrinogens display decreased plasminogen binding and a lower rate of fibrinolysis.11-13 Therefore, our increasing knowledge of various dysfibrinogenemias will aid our understanding of mechanisms of fibrinolysis (Matsuda, this volume).14,15 To account for these diverse observations and more fully understand the molecular basis of fibrinolysis, more knowledge of the physical changes in the fibrin matrix that precede solubilization is required. In this report, we summarize recent experiments utilizing transmission and scanning electron microscopy and confocal light microscopy to provide information about the structural changes occurring in polymerized fibrin during fibrinolysis. Many of the results of these experiments were unexpected and suggest some aspects of potential molecular mechanisms of fibrinolysis, which will also be described here.

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Binding of 131I-Labeled Tissue-Type Plasminogen Activator on De-Endothelialized Lesions in Rabbits

Nuklearmedizin ◽

10.1055/s-0038-1628893 ◽

1987 ◽

Vol 26 (05) ◽

pp. 224-228 ◽

Cited By ~ 2

Author(s):

Y. Isaka ◽

H. Etani ◽

K. Kimura ◽

S. Yoneda ◽

T. Kamada ◽

...

Keyword(s):

Plasminogen Activator ◽

Abdominal Aorta ◽

Half Life ◽

Balloon Catheter ◽

Biochemical Properties ◽

Tissue Type ◽

Fibrin Deposition ◽

Tissue Type Plasminogen Activator ◽

High Affinity ◽

Type Plasminogen Activator

Tissue-type plasminogen activator (t-PA) which has a high affinity for fibrin in the clot, was labeled with 131I by the iodogen method, and its binding to de-endothelialized lesions in the rabbit was measured to assess the detectability of thrombi. The de-endothelialized lesion was induced in the abdominal aorta with a Fogarty 4F balloon catheter. Two hours after the de-endothelialization, 131I-labeled t-PA (125 ± 46 μCi) was injected intravenously. The initial half-life of the agent in blood (n = 12) was 2.9 ± 0.4 min. The degree of binding of 131I-labeled t-PA to the de-endothelialized lesion was evaluated at 15 min (n = 6) or at 30 min (n = 6) after injection of the agent. In spite of the retention of the biochemical properties of 131I-labeled t-PA and the presence of fibrin deposition at the de-endothelialized lesion, the binding of t-PA to the lesion was not sufficiently strong. Lesion-to-control ratios (cpm/g/cpm/g) were 1.65 ± 0.40 (at 15 min) and 1.39 ± 1.31 (at 30 min), and lesion-to-blood ratios were 1.39 ± 0.32 (at 15 min) and 1.36 ± 0.23 (at 30 min). These results suggest that radiolabeled t-PA may be inappropriate as a radiopharmaceutical for the scintigraphic detection of a pre-existing thrombotic lesion.

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Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646342 ◽

1992 ◽

Vol 68 (06) ◽

pp. 672-677 ◽

Cited By ~ 4

Author(s):

Hitoshi Yahara ◽

Keiji Matsumoto ◽

Hiroyuki Maruyama ◽

Tetsuya Nagaoka ◽

Yasuhiro Ikenaka ◽

...

Keyword(s):

Amino Acid ◽

Plasminogen Activator ◽

Half Life ◽

Tissue Type ◽

Clot Lysis ◽

Amino Acid Substitutions ◽

Wild Type ◽

Plasma Clot ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

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Enhanced Effective Fibrinolysis following the Neutralization of Heparin in Open Heart Surgery Increases the Risk of Post-Surgical Bleeding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645202 ◽

1990 ◽

Vol 63 (02) ◽

pp. 241-245 ◽

Cited By ~ 49

Author(s):

Jørgen Gram ◽

Thomas Janetzko ◽

Jørgen Jespersen ◽

Hans Dietrich Bruhn

Keyword(s):

Blood Loss ◽

Plasminogen Activator ◽

Heart Surgery ◽

Degradation Products ◽

Open Heart Surgery ◽

Tissue Type ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Chest Tube Drain ◽

Median Blood Loss

SummaryThe tissue-type plasminogen activator related fibrinolytic system was studied in 24 patients undergoing cardiopulmonary bypass surgery. The degradation of fibrinogen and fibrin was followed during and after surgery by means of new sensitive and specific assays and the changes were related to the blood loss measured in the chest tube drain during the first 24 postoperative hours. Although tissue-type plasminogen activator was significantly released into the circulation during the period of extracor-poreal circulation (p <0.01), constantly low levels of fibrinogen degradation products indicated that a systemic generation of plasmin could be controlled by the naturally occurring inhibitors. Following extracorporeal circulation heparin was neutralized by protamine chloride, and in relation to the subsequent generation of fibrin, there was a short period with increased concentrations of fibrinogen degradation products (p <0.01) and a prolonged period of degradation of cross-linked fibrin, as detected by increased concentrations of D-Dimer until 24 h after surgery (p <0.01). Patients with a higher than the median blood loss (520 ml) in the chest tube drain had a significantly higher increase of D-Dimer than patients with a lower than the median blood loss (p <0.05).We conclude that the incorporation of tissue-type plasminogen activator into fibrin and the in situ activation of plasminogen enhance local fibrinolysis, thereby increasing the risk of bleeding in patients undergoing open heart surgery

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Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647645 ◽

1988 ◽

Vol 60 (01) ◽

pp. 107-112 ◽

Cited By ~ 4

Author(s):

Roy Harris ◽

Louis Garcia Frade ◽

Lesley J Creighton ◽

Paul S Gascoine ◽

Maher M Alexandroni ◽

...

Keyword(s):

Molecular Weight ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Half Life ◽

Recombinant Tissue Plasminogen Activator ◽

Rat Plasma ◽

High Molecular Weight Complex ◽

Molecular Weights ◽

Major Activity ◽

Tissue Plasminogen

SummaryThe catabolism of recombinant tissue plasminogen activator (rt-PA) was investigated after injection of radiolabelled material into rats. Both Iodogen and Chloramine T iodination procedures yielded similar biological activity loss in the resultant labelled rt-PA and had half lives in the rat circulation of 1 and 3 min respectively. Complex formation of rt-PA was investigated by HPLC gel exclusion (TSK G3000 SW) fractionation of rat plasma samples taken 1-2 min after 125I-rt-PA injection. A series of radiolabelled complexes of varying molecular weights were found. However, 60% of the counts were associated with a single large molecular weight complex (350–500 kDa) which was undetectable by immunologically based assays (ELISA and BIA) and showed only low activity with a functional promoter-type t-PA assay. Two major activity peaks in the HPLC fractions were associated with Tree t-PA and a complex having a molecular weight of ̴ 180 kDa. HPLC fractionation to produce these three peaks at various timed intervals after injection of 125I-rt-PA showed each to have a similar initial rate half life in the rat circulation of 4-5 min. The function of these complexes as yet is unclear but since a high proportion of rt-PA is associated with a high molecular weight complex with a short half life in the rat, we suggest that the formation of this complex may be a mechanism by which t-PA activity is initially regulated and finally cleared from the rat circulation.

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