Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for ‘second-site’ interactions

Chmielewska, Joanna; Rånby, Mats; Wiman, Björn

doi:10.1042/bj2510327

Cited by 111 publications

(48 citation statements)

References 23 publications

(33 reference statements)

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“…20 Importantly, when bound to fibrin, tPA is protected from inhibition by PAI-1. 21,22 The inhibition of tPA by PAI-1 is decreased by 80% to 90% in the presence of fibrin because PAI-1 has no access to the catalytic domain of fibrin-bound tPA. 23 Moreover, the rate of inactivation of plasmin by ␣ 2 -antiplasmin slows down very significantly when plasmin is bound to fibrin.…”

Section: Discussionmentioning

confidence: 99%

Platelet Protease Nexin-1, a Serpin That Strongly Influences Fibrinolysis and Thrombolysis

et al. 2011

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Background-Protease nexin-1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma, but we have shown recently that PN-1 is present within the ␣-granules of platelets. Methods and Results-In this study, the role of platelet PN-1 in fibrinolysis was investigated with the use of human platelets incubated with a blocking antibody and platelets from PN-1-deficient mice. We showed by using fibrin-agar zymography and fibrin matrix that platelet PN-1 inhibited both the generation of plasmin by fibrin-bound tissue plasminogen activator and the activity of fibrin-bound plasmin itself. Rotational thromboelastometry and laser scanning confocal microscopy were used to demonstrate that PN-1 blockade or deficiency resulted in increased clot lysis and in an acceleration of the lysis front. Protease nexin-1 is thus a major determinant of the lysis resistance of platelet-rich clots. Moreover, in an original murine model in which thrombolysis induced by tissue plasminogen activator can be measured directly in situ, we observed that vascular recanalization was significantly increased in PN-1-deficient mice. Surprisingly, general physical health, after tissue plasminogen activator-induced thrombolysis, was much better in PN-1-deficient than in wild-type mice. Conclusions-Our results reveal that platelet PN-1 can be considered as a new important regulator of thrombolysis in vivo.Inhibition of PN-1 is thus predicted to promote endogenous and exogenous tissue plasminogen activator-mediated fibrinolysis and may enhance the therapeutic efficacy of thrombolytic agents.

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Section: Discussionmentioning

confidence: 99%

Platelet Protease Nexin-1, a Serpin That Strongly Influences Fibrinolysis and Thrombolysis

et al. 2011

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“…Purified HCF t-PA and HCF t-PA⅐PAI-1 complexes were kindly provided by Dr. A. Wittwer (Monsanto Co., St. Louis, MO). Human PAI-1 from HT 1080 cells was purchased from American Diagnostica Inc. and re-activated from its latent form by treatment with 4 M guanidinium chloride according to the method of Chmielewska, et al (26). Purified RAP was expressed in the Salmonella japonicum glutathione S-transferase/39-kDa expression vector and isolated as described previously (20).…”

Section: Methodsmentioning

confidence: 99%

Low Density Lipoprotein Receptor-related Protein Modulates the Expression of Tissue-type Plasminogen Activator in Human Colon Fibroblasts

Hardy

Feder

Wolfe

et al. 1997

Journal of Biological Chemistry

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Human colon fibroblasts (HCF) produce tissue-type plasminogen activator (t-PA) in culture, but after 24 -48 h, t-PA ceases to accumulate in the medium. Here, we report negative feedback regulation of t-PA expression, exerted by t-PA or complexes of t-PA with its physiological inhibitor, plasminogen activator inhibitor type 1 (PAI-1). Inhibition of t-PA expression could be induced by addition of exogenous t-PA or t-PA⅐PAI-1 complexes and reversed by monoclonal antibody directed against the active site of t-PA. Analysis of metabolically radiolabeled protein and cellular mRNA showed that both t-PA protein and mRNA levels declined considerably after 24 h. When 125 I-labeled t-PA or t-PA⅐PAI-1 complexes were incubated with HCF, monensin-inhibitable endocytosis and catabolism were observed. The low density lipoprotein receptor-related protein (LRP) was found to be expressed by HCF and to mediate these events. Addition of the 39-kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, removed the block to t-PA expression and restored its accumulation in the medium. Moreover, RAP completely prevented the degradation of exogenous 125 I-labeled t-PA by HCF, suggesting that LRP is the endocytic receptor for t-PA in these cells. These results demonstrate that cellular modulation of t-PA expression in HCF involves LRP receptor-mediated clearance of t-PA. This LRP receptor-mediated event results in down-regulation of t-PA expression at the mRNA level.Fibrinolysis is a complex process that requires precise regulation in vivo to ensure that it is neither deficient nor excessive. The plasminogen activator system is critical for thrombolysis, as well as other physiological processes including cell migration and tissue remodeling (1, 2). Human tissue-type plasminogen activator (t-PA) 1 (M r 68,000), a key serine protease in this system, is of particular pharmacological interest because of its value in the treatment of thromboembolic disorders. Regulation of the plasminogen activator system involves modulation by cofactors and inhibitors as well as controlled synthesis of the key components. It is well documented that t-PA expression is influenced by a variety of exogenous factors such as hormones, growth factors, and cytokines (3-6). Regulation by these factors appears to be receptor-mediated and is imposed at the transcriptional level (7-10). However, information on the role of t-PA in the local regulation of its own biosynthesis, release, and clearance is limited. Kadouri and Bohak (11) suggested a negative feedback-type control for t-PA expression in human lung fibroblasts but did not identify the mechanism by which expression was regulated. Although t-PA has been reported to undergo receptor-mediated binding to a variety of cultured cells (12-15), the biological significance of this phenomenon remains uncertain. Reports on the internalization and lysosomal degradation of t-PA have been largely confined to studies on hepatocytes, which have a known clearance function (for reviews, see Refs. 16,17)....

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“…The more prominent enhance ment of the activity with sctPA in the pres ence of fibrinogen and fibrin, therefore, indi cates that the alteration of the active site of sctPA by these effectors was also recognized by the large natural substrate plasminogen. This is of interest because the second-order rate constant for the inhibition of sctPA by plasminogen activator inhibitor type 1 was not modified by the presence of fibrin [29]. Such enhancement of the plasminogen activa tion activity of sctPA by either fibrin or fibrinogen without modification of its suscep tibility toward its specific inhibitor may play an important role in physiological fibrinolysis [29].…”

Section: Discussionmentioning

confidence: 99%

“…This is of interest because the second-order rate constant for the inhibition of sctPA by plasminogen activator inhibitor type 1 was not modified by the presence of fibrin [29]. Such enhancement of the plasminogen activa tion activity of sctPA by either fibrin or fibrinogen without modification of its suscep tibility toward its specific inhibitor may play an important role in physiological fibrinolysis [29].…”

Section: Discussionmentioning

confidence: 99%

“…As shown with SDS-PAGE, the a, (5 and y chains of fibrinogen remained fairly intact during the Plasminogen Activation by Tissue Plasminogen Activator The enhancement by either fibrinogen or fibrin was more prominent with sctPA than with tctPA. The amidolytic activity of sctPA is reported to be enhanced by both fibrinogen and fibrin [28][29][30][31][32] by binding to the D do main of fibrinogen [33], most likely due to an alteration of the tertiary structure of sctPA. Such enhancement, however, could not be seen in tctPA.…”

Section: Discussionmentioning

confidence: 99%

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Reexamination of the Extent of the Activation of Lys<sub>78</sub> Plasminogen by Tissue Plasminogen Activator in the Presence of Polymerized Fibrin

Urano

Takada²,

Ihara³

et al. 1996

Pathophysiol Haemos Thromb

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The rate of the conversion of Lys₇₈ plasminogen (Lys₇₈-plg) to Lys₇₈ plasmin by tissue plasminogen activator (tPA) was directly quantitated by SDS-PAGE. The apparent second-order rate constant (Kapp) was (0.27 ± 0.07) × 10³/M/s for single-chain tPA and Lys₇₈-plg, and was enhanced approximately 5-fold by fibrinogen and 17-fold by polymerized fibrin. Kapp was (1.57 ± 0.46) × 10³/M/s for two-chain tPA and Lys₇₈-plg, and was enhanced approximately 2.6-fold by fibrinogen and 3.6-fold by fibrin. The factor of the enhancement by polymerized fibrin was far less than in previous reports in which chro-mogenic substrate was commonly employed.

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Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for ‘second-site’ interactions

Cited by 111 publications

References 23 publications

Platelet Protease Nexin-1, a Serpin That Strongly Influences Fibrinolysis and Thrombolysis

Platelet Protease Nexin-1, a Serpin That Strongly Influences Fibrinolysis and Thrombolysis

Low Density Lipoprotein Receptor-related Protein Modulates the Expression of Tissue-type Plasminogen Activator in Human Colon Fibroblasts

Reexamination of the Extent of the Activation of Lys<sub>78</sub> Plasminogen by Tissue Plasminogen Activator in the Presence of Polymerized Fibrin

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