Tissue Plasminogen Activator: The Biochemistry and Pharmacology of Variants Produced by Mutagenesis

Higgins, Deborah L.; Bennett, William F.

doi:10.1146/annurev.pa.30.040190.000515

Cited by 54 publications

(19 citation statements)

References 45 publications

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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).…”

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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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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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“…To date no further significant progress has been seen with other tPAbased thrombolytics such as monteplase, desmoteplase or pamiteplase for example, or chimeras such as amediplase, or other molecules such as saruplase (single chain urokinase) or SAK [105]. Despite all the detailed investigations of protein biochemistry on these molecules [83,115] the only advantage of the newer thrombolytics is a bolus administration rather than the longer infusion of first generation treatments. Thus, so far the only improvements in thrombolytic therapy relate to the pharmacokinetics of the drugs, not other properties such as fibrin binding or specificity, or inhibitor susceptibility [116].…”

Section: What Can a Model Of Fibrinolysis Achieve?mentioning

confidence: 99%

Fibrinolysis at the Fluid-Solid Interface of Thrombi

Kolev

Longstaff

Machovich

2005

CMCCHA

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Thrombolysis is conventionally regarded as dissolution of the fibrin matrix of thrombi by plasmin, a protease generated by plasminogen activators from its inactive precursor, plasminogen. Typically plasminogen activation occurs on the surface of the clot, where fibrin behaves as a cofactor in this process, and plasmin also initiates its proteolytic action at the fluid-solid interface. Although the basic reactions of the plasminogen/plasmin system in fluid phase are well characterized in terms of classical enzymology, they cannot explain completely the interfacial fibrinolytic events. Recently new methods have been introduced for quantitative evaluation of plasminogen activation on gel-phase fibrin and heterogenous-phase proteolysis, an overview of the new methodology is presented. Following formation of an interfacial lytic zone, fibrin dissolution proceeds through propagation of this zone to the core of the clot, which depends on diffusion and permeation phenomena affected by the composition of thrombi. Phospholipids (originating from platelets) form a diffusion barrier to the thrombolytic agents and also bind some of them; structural cellular proteins (namely myosin) interact with the fibrin fibers masking their cofactor and plasmin-cleavage sites. The contribution of these recent findings to our understanding of the limitations of current thrombolytic therapy is discussed. Finally, attention is focused on the termination of thrombus-associated proteolytic action in an environment abundant in proteinase inhibitors. Thus, combining together the interfacial events in the initiation, progress and termination of thrombolysis, a concept for modeling the thrombus as a temporary fibrinolytic compartment is presented.

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“…Tissue-type plasminogen activator (ref. 5; t-PA), which is normally secreted, is used as a model protein. PCR mutagenesis (6) is used to generate random mutations within the kringle 1 (K1) domain of t-PA. Fluorescence-activated cell sorting (FACS) is used to screen for t-PA mutants possessing loss of an epitope to a specific mAb, whose nonlinear binding domains overlap with the t-PA clearance receptor contact regions.…”

Section: Introductionmentioning

confidence: 99%

Random PCR mutagenesis screening of secreted proteins by direct expression in mammalian cells.

Rice¹,

Goeddel²,

Cachianes³

et al. 1992

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

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We have developed a general method for screening randomly mutagenized expression libraries in mammalian cells by using fluorescence-activated cell sorting (FACS). The cDNA sequence of a secreted protein is randomly mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA is inserted into an expression vector encoding the membrane glycophospholipid anchor sequence of decay-accelerating factor (DAF) fused to the C terminus of the secreted protein. This results in expression of the protein on the cell surface in transiently transfected mammalian cells, which can then be screened by FACS. This method was used to isolate mutants in the kringle 1 (K1) domain of tissue plasminogen activator (t-PA) that would no longer be recognized by a specific monoclonal antibody (mAb387) that inhibits binding of t-PA to its clearance receptor. DNA sequence analysis of the mutants and localization of the mutated residues on a three-dimensional model of the K1 domain identified three key discontinuous amino acid residues that are essential for mAb387 binding. Mutants with changes in any of these three residues were found to have reduced binding to the t-PA receptor on human hepatoma HepG2 cells but to retain full clot lysis activity.Random mutagenesis of a DNA region of interest coupled with a screening system is a powerful method for the study of a gene or its regulatory functions (1, 2). A saturation mutagenesis technique has been developed for expression of mutated surface antigens on the surface of transfected mammalian cells (3,4). The population ofcells expressing mutated antigens that would no longer bind to specific monoclonal antibodies (mAbs) was selected by specific complementmediated lysis of cells that retained the epitope of interest. This technique works only for screening mutated proteins that are normally expressed on the cell surface. It would therefore be useful to develop a screening method for selection of mutagenized proteins that are normally secreted by cells, as many proteins of therapeutic interest are secretory proteins. This paper reports the development of a cloning vector for expression of secretory proteins as fusion proteins on the cell surface of transfected mammalian cells. The secreted protein is displayed on the cell surface by fusion with the glycophospholipid membrane anchor of decayaccelerating factor (DAF). Tissue-type plasminogen activator (ref. 5; t-PA), which is normally secreted, is used as a model protein. PCR mutagenesis (6) is used to generate random mutations within the kringle 1 (K1) domain of t-PA. Fluorescence-activated cell sorting (FACS) is used to screen for t-PA mutants possessing loss of an epitope to a specific mAb, whose nonlinear binding domains overlap with the t-PA clearance receptor contact regions. MATERIALS AND METHODSPlasmid Construction. The t-PA-DAF fusion protein expression vector construct was made by inserting synthetic DNA oligonucleotides encoding the last 37 amino acids of DAF (7) behind the last codon of t-PA. The resulting...

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Tissue Plasminogen Activator: The Biochemistry and Pharmacology of Variants Produced by Mutagenesis

Cited by 54 publications

References 45 publications

Structural Features Mediating Fibrin Selectivity of Vampire Bat Plasminogen Activators

Structural Features Mediating Fibrin Selectivity of Vampire Bat Plasminogen Activators

Fibrinolysis at the Fluid-Solid Interface of Thrombi

Random PCR mutagenesis screening of secreted proteins by direct expression in mammalian cells.

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