A Sensitive Bioimmunoassay for Thrombin-cleaved Two-chain Urokinase-type Plasminogen Activator in Human Body Fluids

Braat, E.A.M.; Nauland, U; Dooijewaard, G.; Rijken, D.C.

doi:10.1055/s-0038-1650393

Cited by 10 publications

(10 citation statements)

References 19 publications

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“…4C) supports this hypothesis. It has previously been shown that thrombin can cleave scuPA both in purified system [13] and in vivo: in synovial and intravascular spaces [18]. Thrombin cleaved scuPA could be activated and reliably detected in the complex protein mixtures, including biological fluids, as demonstrated in these studies.…”

Section: Discussionsupporting

confidence: 57%

“…While cleavage of scuPA is a putative mechanism by which fibrin is protected from early fibrinolysis, the role of this pathway in the regulation of intrapleural fibrinolysis has not been investigated. Thrombin-cleaved uPA has previously been detected in plasma of patients with sepsis [18] and in synovial fluids of patients with rheumatoid arthritis [18], leading us to infer that thrombin-TM-mediated cleavage of scuPA contributes to intrapleural regulation of fibrinolysis during evolving pleurodesis or other pleural diseases. We also inferred that this mechanism may be involved in cleavage of exogenous scuPA, which prevents intrapleural loculations and lung trapping induced by tetracycline and is being evaluated as a candidate for future clinical use [16].…”

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confidence: 99%

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Thrombin–thrombomodulin inhibits prourokinase-mediated pleural mesothelial cell-dependent fibrinolysis

Iakhiaev

Nalian

Koenig

et al. 2007

Thrombosis Research

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SummaryFibrin deposition is a hallmark of pleural inflammation and loculation but understanding of mechanisms by which mesothelial cells regulate intrapleural fibrinolysins remains incomplete. We speculated that pleural mesothelial cells regulate local fibrinolytic capacity via processing of single chain urokinase type plasminogen activator (scuPA). Pretreatment of human pleural mesothelial (MeT-5A) cells with TGF-β or thrombin, either alone or in combination, inhibited urokinase (uPA)-mediated fibrinolysis by MeT-5A. Thrombin, unlike TGF-β, inhibited fibrinolysis without induction of PAI-1, suggesting that thrombin-mediated cleavage of scuPA inhibits the fibrinolytic capacity of MeT-5A cells. Thrombin cleaves both purified scuPA as well as that secreted by MeT-5A cells and cell surface thrombomodulin accelerates thrombin-mediated cleavage of scuPA to inhibit cellular fibrinolytic activity. Molecular dynamics analyses demonstrated that thrombin-cleaved scuPA (uPAt) do not acquire a catalytically active conformation and that secondary plasminogen binding sites of uPA implicated in plasminogen activation are distorted in uPAt, explaining, at least in part, why uPAt is a poor enzyme. uPAt was detectable in transudative and exudative pleural effusions from patients. Intrapleural administration of scuPA generated increased levels of uPAt in PF of rabbits with pleural injury and loculation induced by tetracycline in vivo. This pathway is operative in diverse forms of pleural injury, restricts the urokinase-dependent fibrinolytic capacity of pleural mesothelial cells and contributes to local control of fibrinolytic activity via processing of endogenous or exogenous scuPA within the pleural compartment. Keywords fibrinolysis; single chain urokinase type plasminogen activator (scuPA); thrombin; thrombomodulin Fibrin deposition in the pleural space is a hallmark of inflammatory pleural disease [1,2]. Fibrin is deposited as a result of excessive activation of coagulation and insufficient fibrinolysis. Under physiological conditions, coagulation and fibrinolysis are precisely regulated and molecular links between these systems permit timely removal of ongoing or acutely induced fibrin deposits [3,4]. The major fibrinolytic protease -plasmin, is generated by activation of plasminogen (PLG) by both tissue type PLG activator (tPA) as well as by urokinase (uPA) [5,6,7]. Plasmin cleaves both tPA and uPA, transforming them from single chain forms to more Please address all correspondence to: Alexei Iakhiaev Ph.D., Assistant Professor of Biochemistry, The University of Texas Health Center at Tyler, 11937 US HWY 271, Tyler, TX 75708, Telephone: (903) FAX: (903) 877-5627, Email: alexei.iakhiaev@uthct.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable for...

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Section: Discussionsupporting

confidence: 57%

mentioning

confidence: 99%

Thrombin–thrombomodulin inhibits prourokinase-mediated pleural mesothelial cell-dependent fibrinolysis

Iakhiaev

Nalian

Koenig

et al. 2007

Thrombosis Research

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“…The inactivation of scu-PA has been postulated as a mechanism for protecting a fresh blood clot from early fibrinolysis [3,9]. The inactivation does indeed take place in vivo, as we have demonstrated the presence of tcu-PA/T in human body fluids under pathological conditions that involve the production of large amounts of thrombin [10][11][12].Thrombomodulin accelerates the inactivation of scu-PA by thrombin, both in a purified system and in a plasma milieu [13][14][15]. In addition, it has been shown that endogenous thrombomodulin enhances the inactivation of scu-PA by thrombin in a perfused rabbit heart model, suggesting that this potentiating effect is of significance in vivo [14].…”

supporting

confidence: 54%

Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator

Schenk-Braat¹,

Morser²,

Rijken³

2001

Eur J Biochem

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Single-chain urokinase-type plasminogen activator (scu-PA) can be cleaved by thrombin into a virtually inactive form called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), a process accelerated by thrombomodulin, which contains six epidermal growth factor (EGF)-like domains. In this study, we identified the EGFlike domains of thrombomodulin required for the acceleration of the inactivation of scu-PA by thrombin using various forms of thrombomodulin (TM). scu-PA was treated with thrombin in the absence and presence of full-length rabbit TM (containing EGF1-6), recombinant TM comprising all of the extracellular domains including EGF1-6 (TM LEO ) and recombinant TM comprising EGF4-6 plus the interconnecting region between EGF3 and EGF4 (TM E i4-6), and the tcu-PA/T generated was quantitated in each case. Rabbit TM accelerated the inactivation of scu-PA < 35-fold, while both recombinant forms accelerated it only threefold due to the absence of a critical chondroitin sulfate moiety. Subsequently, TM E 5-6 was prepared by cyanogen bromide digestion of TM E i4-6. TM E 5-6 bound to thrombin but did not accelerate the activation of protein C. In contrast, the inactivation of scu-PA by thrombin was accelerated to the same extent as that induced by TM LEO and TM E i4-6. This study demonstrates that, in addition to the chondroitin sulfate moiety, only EGF-like domains 5 and 6 are essential for the acceleration of the inactivation of scu-PA by thrombin. This differs from the domains that are critical for activation of protein C (EGF-like domains i4 -6) and thrombin activatable fibrinolysis inhibitor (EGF-like domains 3-6).Keywords: single-chain urokinase-type plasminogen activator; thrombin; inactivation; thrombomodulin; epidermal growth factor-like domains.Single-chain urokinase-type plasminogen activator (scu-PA) is the precursor form of two-chain urokinase-type plasminogen activator (tcu-PA), which plays a role in fibrinolysis and extracellular proteolysis by converting plasminogen into plasmin [1]. Activation of scu-PA into tcu-PA occurs after cleavage at Lys158 -Ile159 by plasmin or other activators. Cleavage of scu-PA at another site can result in inactivation. Thrombin cleaves scu-PA at Arg156 -Phe157, two residues prior to the activation site, in this way generating an inactivated tcu-PA form called thrombincleaved tcu-PA (tcu-PA/T) [2]. In vitro, tcu-PA/T appears to have little amidolytic and fibrinolytic activity [2,3]. However, in a rabbit jugular vein thrombosis model, tcu-PA/T was found to be a potent and fibrin-specific thrombolytic agent [4,5], which may be explained by re-activation of tcu-PA/T by dipeptidyl peptidase I (cathepsin C) and by platelets [6,7], or by the promoting effect of fibrin fragment E-2 on plasminogen activation by tcu-PA/T [8]. The inactivation of scu-PA has been postulated as a mechanism for protecting a fresh blood clot from early fibrinolysis [3,9]. The inactivation does indeed take place in vivo, as we have demonstrated the presence of tcu-PA/T in human bod...

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“…Previous studies (6)(7)(8)(9) have demonstrated rapid increases in circulating PA levels, as well as increased pulmonary concentrations of uPA after endotoxemia or bacterial infections. These studies document a close temporal relationship between the development of LPS-induced ALI and uPA expression.…”

mentioning

confidence: 97%

Post-Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor Expression in Lipopolysaccharide-induced Acute Lung Injury

Bhandary¹,

Velusamy²,

Shetty³

et al. 2009

Am J Respir Crit Care Med

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Rationale: Urokinase-type plasminogen activator (uPA) receptor (uPAR) is required for the recruitment of neutrophils in response to infection. uPA induces its own expression in lung epithelial cells, which involves its interaction with cell surface uPAR. Regulation of uPAR expression is therefore crucial for uPA-mediated signaling in infectious acute lung injury (ALI). Objectives: To determine the role of uPA in uPAR expression during ALI caused by sepsis. Methods: We used Western blot, Northern blot, Northwestern assay, and immunohistochemistry. Phosphate-buffered saline-and lipopolysaccharide (LPS)-treated wild-type and uPA 2/2 mice were used. Measurements and Main Results: Biological activities of uPA, including proteolysis, cell adhesion, migration, proliferation, and differentiation, are dependent on its association with uPAR. Bacterial endotoxin (LPS) is a major cause of pulmonary dysfunction and infectionassociated mortality. The present study shows that LPS induces uPAR expression both in vitro and in vivo, and that the mechanism involves post-transcriptional stabilization of uPAR mRNA by reciprocal interaction of phosphoglycerate kinase (PGK) and heterogeneous nuclear ribonucleoprotein C (hnRNPC) with uPAR mRNA coding region and 39 untranslated region determinants, respectively. The process involves tyrosine phosphorylation of PGK and hnRNPC. uPA 2/2 mice failed to induce uPAR expression after LPS treatment. In these mice, LPS treatment failed to alter the binding of PGK and hnRNPC protein with uPAR mRNA due to lack of tyrosine phosphorylation. Conclusions: Our study shows that induction of LPS-mediated uPAR expression is mediated through tyrosine phosphorylation of PGK and hnRNPC. This involves expression of uPA as an obligate intermediary.

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A Sensitive Bioimmunoassay for Thrombin-cleaved Two-chain Urokinase-type Plasminogen Activator in Human Body Fluids

Cited by 10 publications

References 19 publications

Thrombin–thrombomodulin inhibits prourokinase-mediated pleural mesothelial cell-dependent fibrinolysis

Thrombin–thrombomodulin inhibits prourokinase-mediated pleural mesothelial cell-dependent fibrinolysis

Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator

Post-Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor Expression in Lipopolysaccharide-induced Acute Lung Injury

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