Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with a lower apparent molecular mass

Nielsen, Lene; Andreasen, Peter A.; Grøndahl-Hansen, J.; Skriver, Lars; Danø, Keld

doi:10.1016/0014-5793(86)80261-7

Cited by 63 publications

(29 citation statements)

References 33 publications

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“…The same result was obtained for the PAI-1 of fibrosarcoma cells [35]. However the N-terminal valine predicted from the cDNA sequence of the endothelial PAI-1 [32,33] has been confirmed by amino acid sequencing [32].…”

Section: Preparationsupporting

confidence: 63%

Plasminogen activator inhibitor from human endothelial cells. Purification and partial characterization

et al. 1987

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An inhibitor of plasminogen activator was purified to apparent homogeneity from human umbilical vein endothelial cell conditioned medium. The purification was achieved by a speedy and simple two-step procedure, without the use of denaturants. The purified protein was a single-chain glycoprotein with apparent molecular mass of 48 kDa.The purified inhibitor had a specific activity of 8500 U/mg protein and the activity could be stimulated about fourteenfold by treatment with denaturants.An antiserum to the purified inhibitor was raised in rabbits. It recognised plasminogen activator inhibitor from platelets and plasma as well as from cultured endothelial cells. The immunoglobulin fraction of the antiserum neutralised the functional activity of the inhibitor from all these sources.The activity of the fibrinolytic system depends on the cleavage of the zymogen plasminogen to form plasmin by plasminogen activators. Two major immunologically distinct types of plasminogen activator, tissue-type (tPA) and urokinase (u-PA) have been identified (reviewed in [l]). t-PA is present in normal resting plasma at a molecular mass of about 110 kDa [2 -41, in contrast to the 65-kDa form isolated from tissues [5]. The high-molecular-mass t-PA in plasma is now known to be an equimolar complex of t-PA with a plasminogen activator inhibitor (PAI) [3, 41. Inhibitors active against both t-PA and u-PA have been discovered in bovine [6] and human [7 -91 endothelial cells, in platelets [lo, 111 and in plasma [3, 12 -141. Immunological cross-reactivity between the bovine endothelial and human platelet inhibitor has been established [15] and this type of PA1 is now designated PAI-1 [16].The bovine endothelial cell PA1 is in an inactive or latent form that is converted to an active form in the presence of denaturants such as SDS and guanidinium hydrochloride [17]. This inhibitor has been purified using SDS/polyacrylamide gel electrophoresis (SDS-PAGE) as the final preparative step; thus the purified product was active [18].In the study reported here, we aimed to purify PA1 from human endothelial cells in the absence of denaturants. T h s Correspondence to

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Section: Preparationsupporting

confidence: 63%

Plasminogen activator inhibitor from human endothelial cells. Purification and partial characterization

et al. 1987

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“…N-terminal sequence analysis was performed after the incubations. At both temperatures, we found the two original N-terminal sequences of PAI-1, SAVHHPP-and VHHPPSY-, differing by the two N-terminal amino acids (Andreasen et al, 1986a). One new N-terminal sequence, MAPEEII-, appeared after incubation at O"C, but not at 37°C.…”

Section: Inhibitory Activity Of Pai-1 At Different Temperaturesmentioning

confidence: 76%

“…To produce human PAI-1 in a baculo-virus expression system, full-length human PAI-1 cDNA (a 2.2-kb fragment; Andreasen et al, 1986a) was cloned into the BurnHT site of the baculo-virus transfer vector pVLl392. Spodoptoru frugiperda cells (SF9 cells, from American Type Culture Collection, Rockville MD) were grown in BaculoGoldTM protein-free insect medium (Pharmingen, San Diego CA) supplemented with antibiotics (100 IU/ml penicillin and 50 pg/ml streptomycin; (Gibco).…”

Section: Methodsmentioning

confidence: 99%

“…In addition, PAI-1 is involved in clearance of uPA and tPA from the extracellular space, by the specific and avid binding of activator/PAI-I complexes to a,-macroglobulin receptor/low-density-lipoprotein receptor-related protein, glycoprotein 330 (see review by Andreasen et al, 1994), and very-low-density-lipoprotein receptor (Heegaard et al, 1995). PAI-1 exists in a 379-residue and a 381-residue variant, differing by two N-terminal amino acids (Andreasen et al, 1986a). The inhibitory activity of PAI-1 is stabilized by vitronectin (see review by Preissner and Jenne, 1991).…”

mentioning

confidence: 99%

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Conformational Changes of the Reactive‐Centre Loop and β‐Strand 5A Accompany Temperature‐Dependent Inhibitor‐Substrate Transition of Plasminogen‐Activator Inhibitor 1

Kjøller

Martensen

Sottrup‐Jensen

et al. 1996

European Journal of Biochemistry

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We have studied conformational changes of type-I plasminogen-activator inhibitor (PAI-1) during a temperature-dependent inhibitor -substrate transition by measuring susceptibility of the molecule to nontarget proteinases. When incubated at 0°C instead of the normally used 37"C, a tenfold decrease in the specific inhibitory activity of active PAI-1 was observed. Accordingly, PAI-I was recovered in a reactivecentre-cleaved form from incubations with urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) at 0"C, but not at 37°C. It thus behaved as a substrate for the target proteinases at the lower temperature. Active PAI-1 was exposed to a variety of non-target proteinases, including elastase, papain, thermolysin, trypsin, and V8 proteinase. It was found that specific peptide bonds in the reactive centre loop (RCL) and strand 5 in @-sheet A (sSA) had a temperature-dependent proteolytic susceptibility, while the P,,-P,, (E332-S333) bond, forming the hinge between s5A and the RCL, showed indistinguishable susceptibility to proteolysis by V8 proteinase at 0" and 37°C. In latent and reactivecentre-cleaved PAI-1, all the bonds were resistant to proteolysis at the higher as well as the lower temperature. An anti-PAI-I monoclonal antibody maintained the inhibitory activity of PAIL1 and prevented reactive centre cleavage at 0 "C, and thus prevented substrate behaviour. Concomitantly, it caused specific changes in proteolytic susceptibility of sSA and the RCL, but it did not affect cleavage of the P,,-PI, bond by V8 proteinase. Our observations suggest that temperature-dependent conformational changes of 8-sheet A and the RCL determine whether the serpin act as an inhibitor or a substrate. Furthermore they suggest that the RCL of PAI-I is fully extracted from 8-sheet A in the inhibitory as well as in the substrate form, favoring a so-called induced conformational state model to explain why inhibitory activity requires partial insertion of the RCL into 8-sheet A.Keywords: conformation ; plasminogen-activator inhibitor; proteolysis ; reactive-centre loop; serpin.The serpin superfamily includes of a large number serine proteinase inhibitors and a variety of proteins with other or unknown functions (see reviews by Huber and Carrell, 1989;Potempa et al., 1994). The molecular mechanism of the inhibitory function of all the serine proteinase inhibitor members of the superfamily is thought to be similar, on the basis of a high percentage of sequence identity and the same over-all fold, as shown by X-ray crystal analysis. A key feature is an approximately 20-amino-acid peptide segment, the reactive-centre loop (RCL) which protrudes from the main body of the molecule (see reviews by Bode and Huber, 1992; Stein and Carrell, 1995).

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“…PAI-1 is able to inhibit u-PA activity and tumour cell invasion in an in vitro system (Nielsen et al, 1986;Cajot et al, 1990;Cubellis et al, 1990), but the practical action of PAI-I in vivo, especially in cancer cell invasion and metastasis, has remained obscure. Bouchet et al (1994) confirmed the poor prognosis of breast cancer patients whose tumours contained a large amount of PAl-1.…”

Section: Discussionmentioning

confidence: 99%

Significance of plasminogen activator inhibitor 2 as a prognostic marker in primary lung cancer: association of decreased plasminogen activator inhibitor 2 with lymph node metastasis

Yoshino

Endo²,

Watanabe³

et al. 1998

Br J Cancer

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Summary The expression of urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and plasminogen activator inhibitor (PAI) 1 and 2 was examined in 105 cases of primary lung cancer tissue using immunohistochemical staining and reverse transcriptase polymerase chain reaction (RT-PCR) techniques. The expression of u-PA, u-PAR and PAI-1 was detected in approximately 80% of primary lung cancers, whereas detectable PAI-2 expression was observed only in half of the overall cases. We assessed the relationships between the expression pattern and clinicopathological findings and found that a diminished expression level of PAI-2 was significantly correlated with lymph node metastasis and a poor prognosis. These results indicate that PAI-2 may play a critical role in the regulation of extracellular matrix degradation during tumour cell invasion and metastasis, and the expression of PAI-2 may be useful as a marker for evaluating the prognosis of lung cancer.

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Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with a lower apparent molecular mass

Cited by 63 publications

References 33 publications

Plasminogen activator inhibitor from human endothelial cells. Purification and partial characterization

Plasminogen activator inhibitor from human endothelial cells. Purification and partial characterization

Conformational Changes of the Reactive‐Centre Loop and β‐Strand 5A Accompany Temperature‐Dependent Inhibitor‐Substrate Transition of Plasminogen‐Activator Inhibitor 1

Significance of plasminogen activator inhibitor 2 as a prognostic marker in primary lung cancer: association of decreased plasminogen activator inhibitor 2 with lymph node metastasis

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