Monoclonal Antibody-based Immunoassays for the Specific Quantitation of Rat PAI-1 Antigen and Activity in Biological Samples

Ngo, Thu Hoa; Verheyen, Stefan; Knockaert, I; Declerck, Paul

doi:10.1055/s-0037-1615069

Cited by 21 publications

(19 citation statements)

References 36 publications

(41 reference statements)

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“…Human t-PA (predominantly in the single-chain form) was a kind gift from Boehringer Ingelheim (Brussels, Belgium), and human u-PA (consisting of a mixture of high and low molecular weight u-PA in a 75:25 ratio) was a kind gift from Bournonville Pharma (Braine l'Alleud, Belgium). Monoclonal antibodies against rat and human PAI-1 were produced as described previously (14,15 (Arg 260 to Ala) using the appropriate oligonucleotides containing the desired mutations as primer. Polymerase chain reaction was performed essentially as described previously (16), with slight adaptations of the temperatures used for annealing.…”

Section: Methodsmentioning

confidence: 99%

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Identification of a Target Site in Plasminogen Activator Inhibitor-1 That Allows Neutralization of Its Inhibitory Properties Concomitant with an Allosteric Up-regulation of Its Antiadhesive Properties

Ngo¹,

Hoylaerts²,

Knockaert³

et al. 2001

Journal of Biological Chemistry

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The serpin plasminogen activator inhibitor-1 (PAI-1) has a dual function: 1) it plays an important role as a direct inhibitor of the plasminogen activation system, and 2) its interaction with the adhesive glycoprotein vitronectin suggests a role in tissue remodeling and metastasis, independent from its proteinase inhibitory properties. Unique to this serpin is the close association between its conformational and functional properties. Indeed, PAI-1 can occur in an active and a latent conformation, but both functions are exclusively present in the active conformation. We report here the epitope localization and functional effects of a monoclonal antibody (

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Section: Methodsmentioning

confidence: 99%

“…PAI-1 activity was measured as described previously (15). Antibodyinduced inactivation of PAI-1 was evaluated through quantitation of residual PAI-1 activity after incubation of PAI-1 with antibody.…”

Section: Affinity Of Antibodies With Pai-1 Pai-1 Mutants and Vitronmentioning

confidence: 99%

Identification of a Target Site in Plasminogen Activator Inhibitor-1 That Allows Neutralization of Its Inhibitory Properties Concomitant with an Allosteric Up-regulation of Its Antiadhesive Properties

Ngo¹,

Hoylaerts²,

Knockaert³

et al. 2001

Journal of Biological Chemistry

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“…Samples were incubated at 37°C, and aliquots were removed at various times and assayed for their inhibitory activity against t-PA using an immunofunctional assay, adapted to a method described before (27) with minor modifications. Briefly, the samples were incubated with a 2-fold molar excess of t-PA at 37°C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Importance of the Hinge Region between α-Helix F and the Main Part of Serpins, Based upon Identification of the Epitope of Plasminogen Activator Inhibitor Type 1 Neutralizing Antibodies

Bijnens¹,

Gils²,

Knockaert³

et al. 2000

Journal of Biological Chemistry

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The serpin plasminogen activator inhibitor type 1 (PAI-1) is an important protein in the regulation of fibrinolysis and inhibits its target proteinases through formation of a covalent complex. In the present study, we have identified the epitope of two PAI-1 neutralizing monoclonal antibodies (MA-33H1F7 and MA-55F4C12). Based upon differential cross-reactivity data of these monoclonals with PAI-1 from different species and on a sequence alignment between these PAI-1s, combined with the three-dimensional structure, we predicted that the residues Glu Plasminogen activator inhibitor type 1 (PAI-1), 1 a member of the serine proteinase inhibitor (serpin) superfamily (1-4) is an important protein in the regulation of fibrinolysis. PAI-1 is the most important physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma (5).PAI-1 is unique among the serpins because of its functional and conformational flexibility. The active conformation of PAI-1 inhibits its target proteinases by the formation of a stable, inactive complex. After the formation of an initial, reversible Michaelis-like complex, the proteinase cleaves the active site of PAI-1 and forms a stable, covalent complex resulting in the inactivation of the proteinase (6, 7). The structure of a covalent complex between PAI-1 and t-PA in particular, or between a serpin and its target proteinase in general, is presently unknown. However, two different models have been proposed. According to one model, the proteinase moves after the initial attack to the opposite pole of the serpin, thereby resulting in a complete insertion of the N-terminal side of the reactive-site loop (7-9). Alternatively, it was hypothesized that movement of the proteinase following the initial proteinase/ serpin interaction is less extended, yielding a complex in which the N-terminal side of the reactive-site loop is only partially inserted (10, 11), accompanied by a distortion of the catalytic triad of the proteinase (6) and stabilized by multiple interactions between serpin and proteinase.Although PAI-1 is synthesized as an active molecule, it converts spontaneously to an inactive, latent form that can be partially reactivated by denaturing agents (12). In this latent conformation, the active site is inaccessible for the target proteinases as a result of the insertion of the N-terminal side of the reactive-site loop in ␤-sheet A of the PAI-1 molecule (13). In addition, a third conformation with substrate properties has been identified (14 -16). This form of PAI-1 reacts with t-PA or u-PA, resulting in a cleavage of the active site of PAI-1 but without the formation of a covalent complex (17).Previously, we have characterized a panel of monoclonal antibodies that neutralize PAI-1 activity by converting the active pathway into the non-inhibitory substrate pathway (18). For two of these antibodies, MA-55F4C12 and MA-33H1F7, the binding region was found to be located remote of the reactivesite loop, within a region comprising residues at positions 128 -156 (19). Within the three-...

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“…10 MAPK activation was assessed by Western blotting, using cells lysates (20 g) in LLB and antibodies specific for total and phosphorylated p42, p44, or p38 MAPK (Cell Signaling, 1:1000 in TTBS containing 75 mmol/L of NaCl and 0.5% milk powder for 4 hours at 20°C). Iodinated secondary antibodies (Amersham, 1:5000) were detected using a phosphor imager system (Biorad).…”

Section: Biochemical Measurementsmentioning

confidence: 99%

Prorenin Induces Intracellular Signaling in Cardiomyocytes Independently of Angiotensin II

et al. 2006

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Abstract-Tissue accumulation of circulating prorenin results in angiotensin generation, but could also, through binding to the recently cloned (pro)renin receptor, lead to angiotensin-independent effects, like p42/p44 mitogen-activated protein kinase (MAPK) activation and plasminogen-activator inhibitor (PAI)-1 release. Here we investigated whether prorenin exerts angiotensin-independent effects in neonatal rat cardiomyocytes. Polyclonal antibodies detected the (pro)renin receptor in these cells. Prorenin affected neither p42/p44 MAPK nor PAI-1. PAI-1 release did occur during coincubation with angiotensinogen, suggesting that this effect is angiotensin mediated. Prorenin concentrationdependently activated p38 MAPK and simultaneously phosphorylated HSP27. The latter phosphorylation was blocked by the p38 MAPK inhibitor SB203580. Rat microarray gene (nϭ4800) transcription profiling of myocytes stimulated with prorenin detected 260 regulated genes (PϽ0.001 versus control), among which genes downstream of p38 MAPK and HSP27 involved in actin filament dynamics and (cis-)regulated genes confined in blood pressure and diabetes QTL regions, like Syntaxin-7, were overrepresented. Quantitative real-time RT-PCR of 7 selected genes (Opg, Timp1, Best5, Hsp27, Col3a1, and Hk2) revealed temporal regulation, with peak levels occurring after 4 hours of prorenin exposure. This regulation was not altered in the presence of the renin inhibitor aliskiren or the angiotensin II type 1 receptor antagonist eprosartan. Finally, pilot 2D proteomic differential display experiments revealed actin cytoskeleton changes in cardiomyocytes after 48 hours of prorenin stimulation. In conclusion, prorenin exerts angiotensinindependent effects in cardiomyocytes. Prorenin-induced stimulation of the p38 MAPK/HSP27 pathway, resulting in alterations in actin filament dynamics, may underlie the severe cardiac hypertrophy that has been described previously in rats with hepatic prorenin overexpression. (Hypertension. 2006;48:564-571.)

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Monoclonal Antibody-based Immunoassays for the Specific Quantitation of Rat PAI-1 Antigen and Activity in Biological Samples

Cited by 21 publications

References 36 publications

Identification of a Target Site in Plasminogen Activator Inhibitor-1 That Allows Neutralization of Its Inhibitory Properties Concomitant with an Allosteric Up-regulation of Its Antiadhesive Properties

Identification of a Target Site in Plasminogen Activator Inhibitor-1 That Allows Neutralization of Its Inhibitory Properties Concomitant with an Allosteric Up-regulation of Its Antiadhesive Properties

Importance of the Hinge Region between α-Helix F and the Main Part of Serpins, Based upon Identification of the Epitope of Plasminogen Activator Inhibitor Type 1 Neutralizing Antibodies

Prorenin Induces Intracellular Signaling in Cardiomyocytes Independently of Angiotensin II

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