Strategies for the Improvement of Thrombolytic Agents

Lijnen, H.R.; Collen, D.

doi:10.1055/s-0038-1646377

Cited by 143 publications

(54 citation statements)

References 222 publications

(238 reference statements)

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“…The plasmin thus activated can proteolytically degrade the fibrin network associated with blood clots. The role of t-PA as an initiator in thrombolysis has been exploited as a thrombolytic agent (3)(4)(5). However, the half-life of t-PA in plasma is very short, ranging from 1-4 min in rodents (6-11) to 5-10 min in humans (12,13).…”

Section: Mh1cj Cells Comigrated With the Large Subunit Of Lrp/a2mrmentioning

confidence: 99%

Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator.

Williams

Strickland

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

300

191

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Tissue-type plasminogen activator (t-PA), a serine protease that catalyzes the initial and rate-limiting step in the fibrinolytic cascade, is cleared rapidly in vivo by the liver. Using chemical crosslinking, we have recently identified a plasminogen-activator inhibitor type 1 (PAI-1)-independent t-PA clearance receptor on rat hepatoma MH1C1 cells with a relative molecular mass of %-500 kDa. Another recently identified membrane receptor, low density lipoprotein receptorrelated protein/a2-macroglobulin receptor (LRP/a2MR), was also detected on MH1Cj hepatoma cells by using immunoprecipitation with anti-LRP/a2MR antibody. When analyzed by SDS/PAGE, we found the t-PA receptor identified on MH1Cj cells comigrated with the large subunit of LRP/a2MR.The t-PA receptor was immunoprecipitated by an anti-LRP/a2MR antibody after chemical crosslinking of specifically bound 12'I-labeled t-PA to its receptor. Through chemical crosslinking studies, we found that t-PA and methylamineactivated a2-macroglobulin could bind to LRP/a2MR simultaneously without competing with one another for binding, suggesting that the two ligands bound to two independent sites on the LRP/a2MR molecule. Furthermore, a 39-kDa protein, which modulates ligand binding to LRP/a2MR, was also found to inhibit t-PA binding to its receptor. These data thus show that the t-PA clearance receptor identified on MH1Cj hepatoma cells is LRP/a2MR.Tissue-type plasminogen activator (t-PA) plays an essential role in the fibrinolytic process by catalyzing the conversion of the zymogen plasminogen to plasmin (1,2). The plasmin thus activated can proteolytically degrade the fibrin network associated with blood clots. The role of t-PA as an initiator in thrombolysis has been exploited as a thrombolytic agent (3-5). However, the half-life of t-PA in plasma is very short, ranging from 1-4 min in rodents (6-11) to 5-10 min in humans (12, 13).In vivo studies have shown that the liver is the major organ responsible for t-PA clearance (6-11). Kinetic studies have suggested that specific receptors exhibiting characteristics of receptor-mediated endocytosis are involved in the clearance process. At least two classes of clearance mechanisms exist: a noncarbohydrate-mediated pathway via hepatocytes and a carbohydrate-mediated pathway via endothelial cells (14). In hepatocytes, detailed studies of the clearance mechanism have revealed two types of clearance receptor: plasminogenactivator inhibitor type 1 (PAI-1)-dependent (15)(16)(17)(18)(19)(20) and -independent (11,21) receptors. Recent studies from our laboratory have identified a PAI-1-independent t-PA receptor on rat hepatoma MH1C1 cells (22). Binding of t-PA to this receptor occurs predominantly via the free form and requires neither the protease active site nor the presence of bioactive PAI-1. This situation contrasts with the PAI-1-dependent t-PA receptor we have previously characterized on human hepatoma HepG2 cells, for which the predominant specificbinding species is the t-PA-PAI-1 complex (16)(17)(18)(19)22). Simi...

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Section: Mh1cj Cells Comigrated With the Large Subunit Of Lrp/a2mrmentioning

confidence: 99%

Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator.

Williams

Strickland

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

300

191

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“…PAM-acts as a suicide substrate for both t-PA and urokinase, a plasminogen activator with many structural similarities to t-PA (1,2). These plasminogen activator-inhibitor complexes are cleared from the circulation by the liver (1,6,7). In fact, in a perfused liver system t-PA-PAI-1 complex is cleared considerably faster than free t-PA (8).…”

mentioning

confidence: 99%

“…PAl-i reacts rapidly with a series of positively charged residues located near the catalytic site of t-PA (3), forming a heterodimeric complex that is stable to treatment with SDS (4,5). PAM-acts as a suicide substrate for both t-PA and urokinase, a plasminogen activator with many structural similarities to t-PA (1,2). These plasminogen activator-inhibitor complexes are cleared from the circulation by the liver (1,6,7).…”

mentioning

confidence: 99%

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Complexes of tissue-type plasminogen activator and its serpin inhibitor plasminogen-activator inhibitor type 1 are internalized by means of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

Orth

Madison

Gething

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

233

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Tissue-type plasmingen activator and urokinase are serine proteases secreted by many cell types that participate in biological processes, such as tissue restructuring, cell migration, and tumor metalst . Clinically, these proteases are used to dissolve coronary fibrin clots that are the proximal causes of acute myocardial infarction. In vivo, the activity of these enzymes is controlled by plasminn-activator inhibitors, members of the serpin family of protease inhibitors. This study shows that tissue-type plsminogen activator-inhibitor complexes bind in solution to low density lipoprotein receptorrelated protein (LRP), a large heterodimeric ubiquitous membrane receptor. In cultured cells, endocytosis and degradation of these complexes is reduced by polyclonal antibodies directed against LRP and inhibited by a Mr 39,000 protein that binds to LRP and inhibits its interaction with previously known ligands, including apolipoprotein E and a2-macroglobulin. We propose a role for LRP in the clearance of plasminogen activatorinhibitor complexes that is analogous to its function in the endocytosis of a2-macroglobulin-protease complexes.Tissue-type plasminogen activator (t-PA) is a blood-borne serine protease of Mr -66,000 that converts the zymogen plasminogen to the highly active fibrinolytic protease plasmin. This activity is exploited clinically to dissolve thrombi that form in coronary arteries and cause acute myocardial infarction. t-PA is secreted as an enzymatically active protein of 527 amino acids. The N-terminal portion of the molecule is organized into four highly conserved structural motifs, whereas the C-terminal portion is a typical serine protease and contains a characteristic catalytic triad (1).Under normal physiological conditions, the concentration of t-PA in plasma is low (:0.1 nM), and much of the endogenous protein circulates as an enzymatically inactive complex composed of t-PA and a powerful inhibitor, the serpin plasminogen-activator inhibitor type 1 (PAI-1) (1, 2). PAl-i reacts rapidly with a series of positively charged residues located near the catalytic site of t-PA (3), forming a heterodimeric complex that is stable to treatment with SDS (4, 5). PAM-acts as a suicide substrate for both t-PA and urokinase, a plasminogen activator with many structural similarities to t-PA (1, 2). These plasminogen activator-inhibitor complexes are cleared from the circulation by the liver (1, 6, 7). In fact, in a perfused liver system t-PA-PAI-1 complex is cleared considerably faster than free t-PA (8). The hepatic receptor(s) responsible for this efficient removal of t-PA-PAI-1 complexes have not been identified, although studies with the human hepatic cell line HepG2 have shown that interaction between t-PA and PAT-i associated with the extracellular matrix is required for recognition and endocytosis (9, 10).The requirement for complex formation of a protease with its inhibitor for efficient receptor-mediated endocytosis is not unprecedented. The protease inhibitor a2-macroglobulin forms a complex with...

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“…However, when administered in therapeutic doses, t-PA, due to its limited fibrin selectivity, causes plasminemia that may contribute to bleeding complications (Rao et al, 1988;Arnold et al, 1989). Therefore, considerable efforts have been devoted to the design of new variants of t-PA exhibiting improved fibrin selectivity (Higgins and Bennett, 1990;Lijnen and Collen, 1991). Recently, a novel mutein of t-PA called TNK, which is more fibrin selective than t-PA, has been characterized Collen et al, 1994).…”

mentioning

confidence: 99%

Structural Features Mediating Fibrin Selectivity of Vampire Bat Plasminogen Activators

Bringmann¹,

Gruber²,

Liese³

et al. 1995

Journal of Biological Chemistry

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The distinguishing characteristic of vampire bat (Desmodus rotundus) salivary plasminogen activators (DSPAs) is their strict requirement for fibrin as a cofactor. DSPAs consist of structural modules known from urokinase (u-PA) and tissue-type plasminogen activator (t-PA) such as finger (F), epidermal growth factor (E), kringle (K), and protease (P), combining to four genetically and biochemically distinct isoenzymes, exhibiting the formulas FEKP (DSPA␣1 and ␣2) and EKP and KP (DSPA␤ and DSPA␥). Only DSPA␣1 and ␣2 bind to fibrin. All DSPAs are single-chain molecules, displaying substantial amidolytic activity. In a plasminogen activation assay, all four DSPAs are almost inactive in the absence of fibrin but strongly stimulated by fibrin addition. The catalytic efficiency (k cat /K m ) of DSPA␣1 increases 10 5 -fold, whereas the corresponding value of t-PA is only 550. The ratio of the bimolecular rate constants of plasminogen activation in the presence of fibrin versus fibrinogen (fibrin selectivity) of DSPA␣1, ␣2, ␤, ␥, and t-PA was found to be 13,000, 6500, 250, 90, and 72, respectively. Whereas all DSPAs are therefore more fibrin dependent and fibrin selective than t-PA, the extent depends on the respective presence of the various domains. The introduction of a plasmin-sensitive cleavage site in a position akin to the one in t-PA partially obliterates fibrin cofactor requirement. Fibrin dependence and fibrin selectivity of DSPAs are accordingly mediated by fibrin binding, which involves the F domain, as yet undefined determinants within the K and P domains, and by the absence of a plasmin-sensitive activation site. These findings transcend the current understanding of fibrin-mediated stimulation of plasminogen activation: in addition to fibrin binding, specific protein-protein interactions come into play, which stabilize the enzyme in its active conformation.

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Strategies for the Improvement of Thrombolytic Agents

Cited by 143 publications

References 222 publications

Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator.

Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator.

Complexes of tissue-type plasminogen activator and its serpin inhibitor plasminogen-activator inhibitor type 1 are internalized by means of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

Structural Features Mediating Fibrin Selectivity of Vampire Bat Plasminogen Activators

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