Plasminogen activator inhibitor type‐1 : reactive center and amino‐terminal heterogeneity determined by protein and cDNA sequencing

Andreasen, Peter A.; Riccio, Andrea; Welinder, Karen G.; Douglas, Roseileen M.; Sartorio, R.; Nielsen, Lene; Oppenheimer, C L; Blasi, Francesco; Danø, Keld

doi:10.1016/0014-5793(86)81113-9

Cited by 214 publications

(106 citation statements)

References 48 publications

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“…More than 95% of the PAI-1 produced in the CHO cells was secreted into the culture medium demonstrating that the constructed signal sequence functioned efficiently in these cells (data not shown). In agreement with earlier reports for HT 1080 produced PAT-1 [57], the purified recombinant eucaryotic PAI-1 contained two overlapping amino-terminal sequences. Since both of these amino termini fall within the predicted processing window for the signal peptidase [68], we cannot determine whether they represent alternative processing products of the signal peptidase or irthe protein with thc shorter N-terminus results from proteolytic cleavage after the removal of the signal sequence.…”

Section: Discussionsupporting

confidence: 92%

Purification of active human plasminogen activator inhibitor 1 from Escherichia coli

Lawrence

Strandberg

Grundström

et al. 1989

European Journal of Biochemistry

110

105

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Plasminogen activator inhibitor 1 (PAI-1) inhibits both tissue-type plasminogen activator (tPA) and urokinasetype plasminogen activator (uPA) and, therefore, is an important regulator of plasminogen activation. We have developed eucaryotic and procaryotic expression systems for PAI-1 and characterized the recombinant glycosylated and non-glycosylated products, together with a non-recombinant natural control, produced in the histosarcoma cell line HT 1080. For eucaryotic expression, the PAI-1 cDNA was stably transfected into Chinese hamster ovary cells (CHO cells), while procaryotic expression in Escherichiu coli was examined after inserting the DNA sequence encoding the mature PAI-1 protein into an inducible expression vector. Recombinant PAI-1 from CHO cells was purified approximately 50-fold in two steps and was indistinguishable from natural PAI-1. Between 3% and 4% of total cellular protein in the procaryotic expression system consisted of PAI-1, from which it was purified approximately 30-fold, with yields of between 15% and 20%. This PAI-1 formed 1 :I complexes with uPA and also with the single-and two-chain forms of tPA. Kinetic analysis demonstrated that the procaryoteproduced PAI-1 had an inhibitory activity towards all three forms of PA that resembled that of natural PAI-1 with association rate constants of approximately lo7 M-' s-' . In contrast to PAL-1 from eucaryotic cells, the PAI-1 from E. eoli had an inherent activity equal to that of guanidine/HCl-activated natural PAI-1. The activity could not be increased by treatment with denaturants suggesting that the latent form of PAI-1 was absent. However, at 37°C the procaryote-produced PAI-1 lost activity at the same rate as natural PAI-1, with approximately 50% of the activity remaining after 3 h. This activity could be partially restored by treatment with 4 M guanidine/HCl. E. coli-derived PAI-1, added to human plasma and fractionated by Sephacryl S-200 chromatography, eluted in two peaks that were similar to those obtained with guanidine-activated PAT-1 from eucaryotic cells, suggesting that it bound to the PAI-1-binding protein (vitronectin).The generation of controlled proteolytic activity is an important feature of many physiological processes [l]. The plasminogen activator (PA) system is a general proteolytic system that has been shown to be involved in such diverse processes as fibrinolysis, ovulation, cellular migration, angiogenesis and tumor metastasis [2 -61. There are two forms of plasminogen activators in mammals, tissue-type PA (tPA) and urokinase-type PA (uPA). Both PAS convert the inactive zymogen, plasminogen, into the broad-specificity protease plasmin [2]. Regulation of the PA system occurs primarily at the level of PA synthesis and activity [3]. Factors, such as hormones and growth factors have been shown to regulate the PA system by directly modulating gene expression 12, 4, 61. although there are also factors that both potentiate and inhibit the activity of the PA system following release of PAS from cells [2, 31. There are at ...

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Section: Discussionsupporting

confidence: 92%

Purification of active human plasminogen activator inhibitor 1 from Escherichia coli

Lawrence

Strandberg

Grundström

et al. 1989

European Journal of Biochemistry

110

105

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“…The tPA antisense and sense probes were prepared from pBSKShTA (15). The PAI-1 antisense and sense probes were prepared from pBSKShPAI-1 (16). The PAI-2 antisense probe was prepared from pDB4707 (17 (18).…”

Section: Methodsmentioning

confidence: 99%

Plasminogen activation in melanocytic neoplasia

et al. 1993

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A large body of experimental evidence suggests that plasminogen acti vators provide tumoral cells with efficient means to degrade extracellular matrix constituents and thereby facilitate their dissemination to distant sites. Melanocytic neoplasia encompass a spectrum of lesions exhibiting diverse clinical behavior that remain difficult to predict with current histopathological evaluations. Little information concerning the contribu tion of plasminogen activation in diagnostic specimens of human melanocytic tumors is presently available. We thus analyzed biopsy specimens of pigmented skin lesions by histolÃ³gica! techniques that identify the cellular sites of synthesis of plasminogen activators and of their inhibitors and that localize the sites of plasminogen activators-catalyzed enzymatic activities. We found that urokinase-type plasminogen activators (uPA) and plasmin ogen activator inhibitor type 1 mRNAs accumulate in atypical nevocytes and in melanoma cells, but not in benign nevocytes. However, uPAcatalyzed proteolytic activity was detected exclusively in melanomas.These observations suggest that up-regulation of the uPA gene is an early feature of melanocyte transformation and that unbalanced enzyme/ inhibitor activity is associated with the malignant phenotype. By support ing a role for uPA in melanoma invasiveness, they provide a novel tool for the evaluation of atypia in nevi.

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“…Elevated levels of PAI-1 in plasma are associated with an increased risk ofthromboembolism suggesting a critical role of PAI-I in the in vivo regulation of fibrinolysis [2]. PAI-1 is a single-chain glycoprotein and is a member of the serine proteinase inhibitor (serpin) family [3][4][5][6]. PAI-1 inhibits both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) with second-order rate constants of 107 M -~'s -~ [7].…”

Section: Introductionmentioning

confidence: 99%

Characterization of common neoantigenic epitopes generated in plasminogen activator inhibitor‐1 after cleavage of the reactive center loop or after complex formation with various serine proteinases

Debrock

Declerck

1995

FEBS Letters

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Plasminogen activator inhibitor-I (PAl-l), an important risk factor for thrombotic diseases, is a member of the superfamily of serine proteinase inhibitors. To define structural rearrangements occurring during interaction between PAI-I and its target proteinases we have raised monoclonal antibodies against the PAI-IIt-PA complex. Thirteen out of 401 monoclonal antibodies reacted preferentially with the PAI-IIt-PA complex as compared to free PAI-I or free t-PA. Detailed characterization revealed the presence of two non-overlapping neoantigenic epitopes in the PAI-IIt-PA complex. Both neoantigenic epitopes were also exposed after complex formation between PAI-I and either urokinase-type plasminogen activator, plasmin or thrombin as well as after cleavage of the reactive site loop of non-inhibitory substrate type PAI-I variants. Thus, we have identified two neoantigenic epitopes, localized entirely in PAl-l, and commonly exposed after complex formation of active PAI-I with various proteinases or after cleavage of substrate PAI-I. These monoclonal antibodies should facilitate further studies on the mechanism of interaction between various PAI-I forms and its target proteinases.

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Plasminogen activator inhibitor type‐1 : reactive center and amino‐terminal heterogeneity determined by protein and cDNA sequencing

Cited by 214 publications

References 48 publications

Purification of active human plasminogen activator inhibitor 1 from Escherichia coli

Purification of active human plasminogen activator inhibitor 1 from Escherichia coli

Plasminogen activation in melanocytic neoplasia

Characterization of common neoantigenic epitopes generated in plasminogen activator inhibitor‐1 after cleavage of the reactive center loop or after complex formation with various serine proteinases

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