Determination of the vitronectin binding site on plasminogen activator inhibitor 1 (PAI‐1)

Meijer, Marja van; Gebbink, Raymond Klein; Preissner, Klaus T.; Pannekoek, Hans

doi:10.1016/0014-5793(94)00990-2

Cited by 52 publications

(45 citation statements)

References 33 publications

(28 reference statements)

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“…Higher concentrations of the antibody were not studied here, since, ®rstly, our objectives were to ascertain that there was sucient cross-reactivity between the two species for antithrombotic studies, and secondly, full concentration inhibition curves for MA33H1 have already been shown with human, murine and rabbit PAI by Debrock and Declerck (1997), where 480% inhibition was obtained with a molar ratio of antibody: antigen of 15. The binding site of MA33H1 partly comprises a region (Met110-Lys145 in human PAI-1) believed to be putative sites for the binding of vitronectin (Keijer et al, 1991) and ®brin (Van Meijer et al, 1994). Our studies with vitronectin, support the observation by Debrock and Declerck (1998) that MA33H1 binds to a region on human PAI-1 which is distinct from (albeit in close proximity to) the vitronectin binding site; however, in rat PAI-1 the two binding sites would appear to have some considerable degree of overlap.…”

Section: Discussionmentioning

confidence: 99%

Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis

Berry¹,

Lunven²,

Lechaire³

et al. 1998

British J Pharmacology

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1 Elevated plasminogen activator inhibitor 1 (PAI-1) is a risk factor for thrombosis, and inhibitors of the interaction between PAI-1 and tissue plasminogen activator (t-PA) have antithrombotic and prothrombolytic activity in animals. We describe the antithrombotic eects in the rat of a monoclonal antibody (MA33H1) which converts PAI-1 to a non-inhibitory substrate. 2 The activity of MA33H1 against rat PAI-1 was con®rmed using two-chain t-PA and a chromogenic substrate. MA33H1 was evaluated in rat venous (thromboplastin+stasis in the abdominal vena cava) and arterial (electric current applied to a carotid artery) thrombosis models. The eects on tailtransection bleeding time were studied. 3 MA33H1 at 100 ng ml 71 inhibited both human (44.1%) and rat PAI-1 (49.7%). This eect was concentration-dependent. Its eect on human PAI-1 was not signi®cantly inhibited by 1 mg ml 71 ®brin or a &7 fold molar excess of vitronectin (1 nM). Inhibition of rat PAI-1 was unchanged by ®brin, but vitronectin reduced inhibition from 0.5 nM. 4 In the venous thrombosis model, MA33H1 signi®cantly reduced thrombus weights by 38 and 58.6% at 50 and 100 mg kg 71 min 71 i.v. respectively. This eect was inhibited by tranexamic acid. In the arterial model, MA33H1 signi®cantly increased the delay to occlusive thrombus formation by 58 and 142% at 50 and 100 mg kg 71 min 71 i.v., and did not aect bleeding time at 300 mg kg 71 min 71 i.v. 5 Thus, a monoclonal antibody which transforms PAI-1 to a t-PA substrate prevents thrombus formation in the rat with no eect on bleeding time at a higher dose.

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Section: Discussionmentioning

confidence: 99%

Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis

Berry¹,

Lunven²,

Lechaire³

et al. 1998

British J Pharmacology

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“…Domain mapping studies using proteolysis, synthetic peptides, monoclonal antibodies, and site-directed mutagenesis have identified two discrete sites on vitronectin that may bind and stabilize PAI-1 (27,33,34). Similar approaches have delineated a single vitronectin-binding site on PAI-1 (35,36).…”

Section: I-labeled Fibrin Clots By T-pa This Effect Is Dependent On mentioning

confidence: 99%

Type 1 Plasminogen Activator Inhibitor Binds to Fibrin via Vitronectin

Podor¹,

Peterson²,

Lawrence³

et al. 2000

Journal of Biological Chemistry

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Type 1 plasminogen activator inhibitor (PAI-1), the primary inhibitor of tissue-type plasminogen activator (t-PA), circulates as a complex with the abundant plasma glycoprotein, vitronectin. This interaction stabilizes the inhibitor in its active conformation In this report, the effects of vitronectin on the interactions of PAI-1 with fibrin clots were studied. Confocal microscopic imaging of platelet-poor plasma clots reveals that essentially all fibrin-associated PAI-1 colocalizes with fibrin-bound vitronectin. Moreover, formation of platelet-poor plasma clots in the presence of polyclonal antibodies specific for vitronectin attenuated the inhibitory effects of PAI-1 on t-PA-mediated fibrinolysis. Addition of vitronectin during clot formation markedly potentiates PAI-1-mediated inhibition of lysis of 125 I-labeled fibrin clots by t-PA. This effect is dependent on direct binding interactions of vitronectin with fibrin. There is no significant effect of fibrin-associated vitronectin on fibrinolysis in the absence of PAI-1. The binding of PAI-1 to fibrin clots formed in the absence of vitronectin was characterized by a low affinity (K d ϳ 3.5 M) and rapid loss of PAI-1 inhibitory activity over time. In contrast, a high affinity and stabilization of PAI-1 activity characterized the cooperative binding of PAI-1 to fibrin formed in the presence of vitronectin. These findings indicate that plasma PAI-1⅐vitronectin complexes can be localized to the surface of fibrin clots; by this localization, they may modulate fibrinolysis and clot reorganization.Tissue-type plasminogen activator (t-PA) 1 initiates intravascular fibrinolysis by binding to fibrin, where it activates fibrinbound plasminogen (1-4). The major inhibitor of t-PA, type 1 plasminogen activator inhibitor (PAI-1), circulates in plasma and is released from platelet ␣-granules during blood clotting (5, 6). PAI-1 accumulates in thrombi, rendering them resistant to t-PA-mediated fibrinolysis (7-14). In purified systems, PAI-1 has been shown to bind directly to fibrin, with a K d of 3.7 M (15-18). Consequently, it has been hypothesized that PAI-1 accumulation in thrombi reflects a direct interaction of PAI-1 with fibrin. PAI-1 circulates in plasma (19,20) and platelets in complex with vitronectin (21, 22, 23, 24), a glycoprotein that binds PAI-1 with high affinity (25). The vitronectin interaction with PAI-1 stabilizes the inhibitor in its active conformation (26, 27), induces allosteric changes in vitronectin that expose cryptic epitopes (28, 29), and modulates vitronectin-dependent cell adhesion (25,30,31). Domain mapping studies using proteolysis, synthetic peptides, monoclonal antibodies, and site-directed mutagenesis have identified two discrete sites on vitronectin that may bind and stabilize 33,34). Similar approaches have delineated a single vitronectin-binding site on PAI-1 (35, 36).Recent studies from our laboratories have further characterized the PAI-1-vitronectin interaction. Analytical ultracentrifugation experiments indicate that PAI-1 and native vi...

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“…The PAI-1 interactive sites for vitronectin are at Gln-55, Phe-109, Met-110, Leu-116, Gln-123, and residues 128 -145 (43), heparin (Lys-65 to Lys-88) (44), a second tPA site (amino acids 128 -145) (43), and thrombin interaction at residues 128 -145 (7,30). The region between amino acid residues 110 -145 binds urokinase and fibrin (30).…”

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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

Journal of Biological Chemistry

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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.

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Determination of the vitronectin binding site on plasminogen activator inhibitor 1 (PAI‐1)

Cited by 52 publications

References 33 publications

Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis

Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis

Type 1 Plasminogen Activator Inhibitor Binds to Fibrin via Vitronectin

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

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