Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1991-12-18
1995-05-16
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
435 691, 435 703, 435131, 435215, 530412, C07K 312, C12N 506, C12N 972, C12P 900
Patent
active
054160065
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to forms of plasminogen activators, their preparation, pharmaceutical compositions containing them, and antibodies specifically recognizing them.
Plasminogen activation (PA) is required intravascularly for digesting fibrin clots, and extravascularly for regulating the interactions between cell surfaces and the protein components of the extracellular matrix and of the basement membrane. The product of the PA reaction, plasmin, can degrade fibrin, proteoglycans, fibronectin, laminin and some collagens; in addition, it is also able to activate latent collagenases. The PA system is composed of the two plasminogen activating enzymes, urokinase (u-PA) and tissue PA (t-PA), and by specific PA inhibitors (PAI-1 and PAI-2). In addition, a specific receptor has been identified for u-PA. While the function of the catalytic moiety of these proteases is better understood, that of the regulatory domains is less clear and only now is information being gathered. For example in u-PA, the growth factor domain is responsible for the binding to the u-PA receptor. In t-PA, the finger domain and the second kringle domain mediate the binding of the enzyme to fibrin.
This system allows the formation of plasmin, both in solution and surface-bound. For the latter, the same cells may have receptors for both plasminogen and plasminogen activators as is the case for the human U937 cells and HT1080 cells. Second, in HT1080 cells, receptor-bound u-PA is able to produce receptor-bound plasmin. Surface plasmin-directed proteolysis appears to have several peculiarities: a) receptor-bound pro-u-PA is activated to two-chain u-PA with a rate that is much faster than in solution; b) surface-bound plasmin is resistant to .alpha.-2 antiplasmin; and c) receptor-bound u-PA, on the other hand, is active and accessible to inhibition by PAI-1, at least in U937 cells.
u-PA is secreted as a 431 amino acids inactive protein, pro-urokinase (pro-u-PA), which can be bound to surface receptors (u-PAR) and activated by a single proteolytic cleavage. In the malignant A431 cell line, all u-PAR sites are occupied by pro-u-PA ligand produced by the same cells in an autocrine way. Similar results have been reported for a variety of u-PA-producing human tumor cells.
Pro-u-PA is cleaved between amino acid 157 (Lys) and 158 (Ile) to activate the proenzyme. Cleavage results in the formation of the two-chain active u-PA. Although not known in detail, this cleavage is thought to change the secondary and tertiary structure of protease so as to unmask the active site and make it accessible to the substrate (plasminogen) and the inhibitors (PAI-1, PAI-2, protease nexin, .alpha..sub.2 -macroglobulin, and others). Cleavage of pro-u-PA at the activation site in fact results in a conformational change and in the gain of plasminogen activator activity, both with natural and synthetic substrates.
Plasminogen activators (u-PA, t-PA, pro-u-PA, streptokinase and derivatives thereof) are or can be utilized in thromboembolic therapy. One of the problems connected with this therapy is the very high dosage required which is, at least in part, due to the very rapid clearance of these agents. Possible reasons for the short half-life include binding to specific circulating inhibitors, binding to receptors, internalization and degradation of inhibitor-bound and/or receptor-bound plasminogen activator.
The data presented in Examples 8, 9 and 10 show that phosphorylated pro-u-PA is receptor-bound, that binding to receptor is not required for phosphorylation, and that phosphorylated two-chain u-PA shows a dramatic decrease in its sensitivity to PAI-1. It is therefore proposed: firstly, that phosphorylated plasminogen activators may have a longer half-life than non-phosphorylated plasminogen activators since it would not bind PAI-1. Secondly, that fully phosphorylated plasminogen activators could be employed in thromboembolic therapy since, as their non-phosphorylated homologues, they can be activated, e.g. in the case of pro-u-PA to its two-chain active forms (s
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Blasi Francesco
Correas Isabel
Mastronicola Maria R.
Stoppelli Maria P.
Welinder Karen G.
Golden Matthew J.
Jacobson Dian C.
Wax Robert A.
White John P.
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