Expression and Characterization of Recombinant Porcine Plasminogen Activator Inhibitor-1

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 (

Section: Discussionmentioning

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

“…Urokinase-type plasminogen activator (Urokinase Choay ® , Sanofi Winthrop) was a kind gift from Bournonville Pharma (Braine l'Alleud, Belgium). Recombinant human PAI-1, porcine PAI-1, murine PAI-1, and rat PAI-1 were expressed in E. coli and were produced basically as described previously (22)(23)(24). Rabbit PAI-1 (recombinant, expressed in Saccharomyces cerevisiae) was a kind gift from Dr. Hofman (Merck Research Laboratories, West Point, PA) (25).…”

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

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-...

“…2. The sequences of human PAI-1 (huPAI-1) and porcine PAI-1 (poPAI-1) are 91% homologous and have no "obvious functional differences" (46). The rPAI-1 23 region of poPAI-1 contains 16 amino acid variations from the huPAI-1 gene.…”

Section: Isolation Of Porcine Pai-1 Gene Fragments-mentioning

confidence: 99%

“…We have closely examined one deleted PAI-1 gene product (rPAI-1 23 ) that encompasses the porcine PAI-1 gene sequences (46) containing nucleotides 444 -999 corresponding to nucleotides 238 -793 in the human PAI-1 gene (amino acids 80 -265 of mature protein) (47). The recombinant rPAI-1 23 fragment: 1) excluded those sequences coding for the reactive center loop at the carboxyl terminus; 2) eliminated a vitronectin binding site, more than one half of a heparin binding region, and the latency residue at Lys-323 (42); 3) retained vitronectin and a second tPA binding region at amino acids 128 -145, part of a heparin binding site at amino acids 80 -88, a thrombin interactive region at residues 115-148, residues 127-158, which contain the helix F and the loop that connects helix F with helix s3A (48,49).…”

mentioning

confidence: 99%

A Truncated Plasminogen Activator Inhibitor-1 Protein Induces and Inhibits Angiostatin (Kringles 1–3), a Plasminogen Cleavage Product

Mulligan-Kehoe

Wagner

Wieland

et al. 2001

Plasminogen activator inhibitor-1 (PAI-1) is a serpin protease inhibitor that binds plasminogen activators (uPA and tPA) at a reactive center loop located at the carboxyl-terminal amino acid residues 320 -351. The loop is stretched across the top of the active PAI-1 protein maintaining the molecule in a rigid conformation. In the latent PAI-1 conformation, the reactive center loop is inserted into one of the beta sheets, thus making the reactive center loop unavailable for interaction with the plasminogen activators. We truncated porcine PAI-1 at the amino and carboxyl termini to eliminate the reactive center loop, part of a heparin binding site, and a vitronectin binding site. The region we maintained corresponds to amino acids 80 -265 of mature human PAI-1 containing binding sites for vitronectin, heparin (partial), uPA, tPA, fibrin, thrombin, and the helix F region. The interaction of "inactive" PAI-1, rPAI-1 23 , with plasminogen and uPA induces the formation of a proteolytic protein with angiostatin properties. Increasing amounts of rPAI-1 23 inhibit the proteolytic angiostatin fragment. Endothelial cells exposed to exogenous rPAI-1 23 exhibit reduced proliferation, reduced tube formation, and 47% apoptotic cells within 48 h. Transfected endothelial cells secreting rPAI-1 23 have a 30% reduction in proliferation, vastly reduced tube formation, and a 50% reduction in cell migration in the presence of VEGF. These two studies show that rPAI-1 23 interactions with uPA and plasminogen can inhibit plasmin by two mechanisms. In one mechanism, rPAI-1 23 cleaves plasmin to form a proteolytic angiostatin-like protein.In a second mechanism, rPAI-1 23 can bind uPA and/or plasminogen to reduce the number of uPA and plasminogen interactions, hence reducing the amount of plasmin that is produced.