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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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Tissue plasminogen activator (tPA)
The natural thrombolytic process is illustrated in Figure 9.18. Plasmin is a protease which catalyses the proteolytic degradation of fibrin present in clots, thus effectively dissolving the clot. Plasmin is derived from plasminogen, its circulating zymogen. Plasminogen is synthesized in, and released from, the kidneys. It is a single-chain 90 kDa glycoprotein, which is stabilized by several disulphide linkages.
Tissue plasminogen activator (tPA, also known as fibrinokinase) represents the most important physiological activator of plasminogen. tPA is a 527 amino acid serine protease. It is synthesized predominantly in vascular endothelial cells (cells lining the inside of blood vessels) and displays five structural domains, each of which has a specific function (Table 9.11). tPA displays four potential glycosylation sites, three of which are normally glycosylated (residues 117, 184 and 448). The carbohydrate moieties play an important role in mediating hepatic uptake of tPA and hence its clearance from plasma. It is normally found in the blood in two forms; a single-chain polypeptide (type I tPA) and a two-chain structure (type II) proteolytically derived from the single chain structure. The two-chain form is the one predominantly associated with clots undergoing lysis, but both forms display fibrinolytic activity.
Fibrin contains binding sites for both plasminogen and tPA, thus bringing these into close proximity. This facilitates direct activation of the plasminogen at the clot surface (Figure 9.18). This activation process is potentiated by the fact that binding of tPA to fibrin (a) enhances the subsequent binding of plasminogen and (b) increases tPA’s activity towards plasminogen by up to 600-fold.
Figure 9.18. (a) The fibrinolytic system, in which tissue plasminogen activator (tPA) proteolytically
converts the zymogen plasminogen into active plasmin, which in turn degrades the fibrin strands, thus dissolving the clot. tPA and plasminogen both bind to the surface of fibrin strands (b), thus ensuring rapid and efficient activation of the thrombolytic process
Overall, therefore, activation of the thrombolytic cascade occurs exactly where it is needed — on the surface of the clot. This is important as the substrate specificity of plasmin is poor, and circulating plasmin displays the catalytic potential to proteolyse fibrinogen, factor V and factor VIII. Although soluble serum tPA displays a much reduced activity towards plasminogen, some free circulating plasmin is produced by this reaction. If uncontrolled, this could increase the risk of subsequent haemorrhage. This scenario is usually averted, as circulating plasmin is rapidly
BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 383 Table 9.11. The five domains that constitute human tPA and the biological function of each domain
tPA domain
Finger domain (F domain) Protease domain (P domain) Epidermal growth factor domain (EGF domain)
Kringle-1 domain (K domain) Kringle-2 domain (K2 domain)
Promotes tPA binding to fibrin with high affinity Displays plasminogen-specific proteolytic activity Binds hepatic receptors thereby mediating hepatic clearance of tPA from blood Associated with binding to the hepatic receptor Facilitates stimulation of tPA’s proteolytic activity by fibrin
neutralized by another plasma protein, a2-antiplasmin (a2-antiplasmin, a 70 kDa, single-chain glycoprotein, binds plasmin very tightly in a 1:1 complex). In contrast to free plasmin, plasmin present on a clot surface is very slowly inactivated by a1-antiplasmin. The thrombolytic system has thus evolved in a self-regulating fashion, which facilitates efficient clot degradation with minimal potential disruption to other elements of the haemostatic mechanism.
First-generation tPA. Although tPA was first studied in the late 1940s, its extensive characterization was hampered by the low levels at which it is normally synthesized. Detailed studies were facilitated in the 1980s after the discovery that the Bowes melanoma cell line produces and secretes large quantities of this protein. This also facilitated its initial clinical appraisal. The tPA gene was cloned from the melanoma cell line in 1983, and this facilitated subsequent large-scale production in CHO cell lines by recombinant DNA technology. The tPA cDNA contains 2530 nucleotides and encodes a mature protein of 527 amino acids. The glycosylation pattern was similar, although not identical, to the native human molecule. A marketing licence for the product was first issued in the USA to Genentech in 1987 (under the trade name Activase). The therapeutic indication was for the treatment of acute myocardial infarction. The production process entails an initial (10 000 litre) fermentation step, during which the cultured CHO cells produce and secrete tPA into the fermentation medium. After removal of the cells by sub-micron filtration and initial concentration, the product is purified by a combination of several chromatographic steps. The final product has been shown to be greater than 99% pure by several analytical techniques, including HPLC, SDS-PAGE, tryptic mapping and N-terminal sequencing.
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