Design, synthesis and In vitro evaluation of potent, novel, small molecule inhibitors of plasminogen activator inhibitor-1

SummaryThe relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.

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Localization of a Vitronectin Binding Region of Plasminogen Activator Inhibitor-1

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653876 ◽

1995 ◽

Vol 73 (05) ◽

pp. 829-834 ◽

Cited By ~ 11

Author(s):

Jaya Padmanabhan ◽

David C Sane

Keyword(s):

Binding Site ◽

Plasminogen Activator ◽

Inhibitory Activity ◽

Plasminogen Activator Inhibitor ◽

Structural Homology ◽

Binding Region ◽

Plasminogen Activator Inhibitor 1 ◽

Activator Inhibitor ◽

Pai 1

SummaryThe PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70,1-105,1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.

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Faculty Opinions recommendation of Tiplaxtinin, a novel, orally efficacious inhibitor of plasminogen activator inhibitor-1: design, synthesis, and preclinical characterization.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1019931.226492 ◽

2004 ◽

Author(s):

Thomas W von Geldern

Keyword(s):

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Plasminogen Activator Inhibitor 1 ◽

Design Synthesis ◽

Activator Inhibitor ◽

Inhibitor Of Plasminogen Activator

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Synthesis and secretion of plasminogen activator inhibitor 1 by human endothelial cells in vitro. Effect of active site mutagenized tissue-type plasminogen activator.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)35242-0 ◽

1991 ◽

Vol 266 (2) ◽

pp. 792-797

Author(s):

K Bartha ◽

P J Declerck ◽

H Moreau ◽

L Nelles ◽

D Collen

Keyword(s):

Plasminogen Activator ◽

Active Site ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Human Endothelial Cells ◽

Plasminogen Activator Inhibitor 1 ◽

Type Plasminogen Activator ◽

Vitro Effect ◽

Activator Inhibitor

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Abstract T P75: Temporal Profiles of Plasminogen Activator Inhibitor-1 Concentration and Activity Changes in Circulation after Focal Stroke and Tissue Type Plasminogen Activator Administration in Rats

Stroke ◽

10.1161/str.45.suppl_1.tp75 ◽

2014 ◽

Vol 45 (suppl_1) ◽

Author(s):

Qi Liu ◽

Xiang Fan ◽

Helen Brogren ◽

Ming-Ming Ning ◽

Eng H Lo ◽

...

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Plasminogen Activator Inhibitor 1 ◽

Type Plasminogen Activator ◽

Temporal Profiles ◽

Activator Inhibitor ◽

Pai 1

Aims: Plasminogen activator inhibitor-1 (PAI-1) is the main and potent endogenous tissue-type plasminogen activator (tPA) inhibitor, but an important question on whether PAI-1 in blood stream responds and interferes with the exogenously administered tPA remains unexplored. We for the first time investigated temporal profiles of PAI-1 concentration and activity in circulation after stroke and tPA administration in rats. Methods: Permanent MCAO focal stroke of rats were treated with saline or 10mg/kg tPA at 3 hours after stroke (n=10 per group). Plasma (platelet free) PAI-1 antigen and activity levels were measured by ELISA at before stroke, 3, 4.5 (1.5 hours after saline or tPA treatments) and 24 hours after stroke. Since vascular endothelial cells and platelets are two major cellular sources for PAI-1 in circulation, we measured releases of PAI-1 from cultured endothelial cells and isolated platelets after direct tPA (4 μg/ml) exposures for 60 min in vitro by ELISA (n=4 per group). Results: At 3 hours after stroke, both plasma PAI-1 antigen and activity were significantly increased (3.09±0.67, and 3.42±0.57 fold of before stroke baseline, respectively, all data are expressed as mean±SE). At 4.5 hours after stroke, intravenous tPA administration significantly further elevated PAI-1 antigen levels (5.26±1.24), while as expected that tPA neutralized most elevated PAI-1 activity (0.33±0.05). At 24 hours after stroke, PAI-1 antigen levels returned to the before baseline level, however, there was a significantly higher PAI-1 activity (2.51±0.53) in tPA treated rats. In vitro tPA exposures significantly increased PAI-1 releases into culture medium in cultured endothelial cells (1.65±0.08) and platelets (2.02±0.17). Conclution: Our experimental results suggest that tPA administration may further elevate stroke-increased blood PAI-1 concentration, but also increase PAI-1 activity at late 24 hours after stroke. The increased PAI-1 releases after tPA exposures in vitro suggest tPA may directly stimulate PAI-1 secretions from vascular walls and circulation platelets, which partially contributes to the PAI-1 elevation observed in focal stroke rats. The underlying regulation mechanisms and pathological consequence need further investigation.

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Fibrate-modulated Expression of Fibrinogen, Plasminogen Activator Inhibitor-1 and Apolipoprotein A-I in Cultured Cynomolgus Monkey Hepatocytes

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615393 ◽

1998 ◽

Vol 80 (12) ◽

pp. 942-948 ◽

Cited By ~ 29

Author(s):

M. Kockx ◽

H. M. G. Princen ◽

T. Kooistra

Keyword(s):

Plasminogen Activator ◽

Cynomolgus Monkey ◽

Plasma Concentrations ◽

Plasminogen Activator Inhibitor ◽

Peroxisome Proliferator ◽

Plasminogen Activator Inhibitor 1 ◽

Apolipoprotein A ◽

Activator Inhibitor ◽

Pai 1

SummaryFibrates are used to lower plasma triglycerides and cholesterol levels in hyperlipidemic patients. In addition, fibrates have been found to alter the plasma concentrations of fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and apolipoprotein A-I (apo A-I). We have investigated the in vitro effects of fibrates on fibrinogen, PAI-1 and apo A-I synthesis and the underlying regulatory mechanisms in primary monkey hepatocytes.We show that fibrates time- and dose-dependently increase fibrinogen and apo A-I expression and decrease PAI-1 expression in cultured cynomolgus monkey hepatocytes, the effects demonstrating different potency for different fibrates. After three consecutive periods of 24 h the most effective fibrate, ciprofibrate (at 1 mmol/l), increased fibrinogen and apo A-I synthesis to 356% and 322% of control levels, respectively. Maximum inhibition of PAI-1 synthesis was about 50% of control levels and was reached by 1 mmol/l gemfibrozil or ciprofibrate after 48 h. A ligand for the retinoid-X-receptor (RXR), 9-cis retinoic acid, and specific activators of the peroxisome proliferator-activated receptor-α (PPARα), Wy14,643 and ETYA, influenced fibrinogen, PAI-1 and apo A-I expression in a similar fashion, suggesting a role for the PPARα/RXRα heterodimer in the regulation of these genes. When comparing the effects of the various compounds on PPARα trans-activation activity as determined in a PPARα-sensitive reporter gene system and the ability of the compounds to affect fibrinogen, PAI-1 and apo A-I antigen production, a good correlation (r = 0.80; p <0.01) between PPARα transactivation and fibrinogen expression was found. Apo A-I expression correlated only weakly with PPARα transactivation activity (r = 0.47; p = 0.24), whereas such a correlation was absent for PAI-1 (r = 0.03; p = 0.95). These results strongly suggest an involvement of PPARα in the regulation of fibrinogen gene expression.

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