Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). A study in vitro and in two animal species.

Gurewich, Victor; Pannell, Ralph; Louie, Shirley C.; Kelley, Philip M.; Suddith, Robert L.; Greenlee, R

doi:10.1172/jci111381

Cited by 322 publications

(120 citation statements)

References 24 publications

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“…We resolved the paradox of PA remaining active in cells pretreated with DFP by showing that PA was stored in these cells in a pro-enzyme, single-chain form (Table 111). Pro-UK does not have detectable amidolytic activity when assayed with a specific, chromogenic substrate (14,22,24). It can be converted to an active form through catalytic activity of plasmin (14) .…”

Section: Resultsmentioning

confidence: 99%

“…Since PA is a serine protease, and therefore should be inhibited by DFP, this finding may seem paradoxical . However, our results (Table 111) showed that PA was present in these cells in the form of an amidolytically inactive proenzyme that could not bind DFP (14,22,24) and consequently was protected from inactivation by this compound .…”

mentioning

confidence: 77%

“…100-,l samples to be tested were mixed with synthetic, chromogenic substrate (S 244), which measures the amidolytic activity of UK . Pro-UK does not have any measurable, amidolytic activity, and only after activation with catalytic amounts of plasmin does it become active (14,22) . We tested the amidolytic activity of samples both in the presence and absence of plasmin, and also in the presence and absence of 10 U/ml of Trasylol .…”

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confidence: 99%

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Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis.

Heiple¹,

Ossowski²

1986

The Journal of Experimental Medicine

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Plasminogen activator (PA)' is produced and secreted by mouse and human monocytes and neutrophils (1-4). A role for the enzyme in cell migration associated with inflammation has been postulated (5). Recently, a specific PA receptor on the surface of monocytes has been described (6, 7). PA bound to this receptor remains active, providing a mechanism through which a cell can concentrate proteolytic activity on its membrane . This finding suggests PA participation in processes requiring direct, local proteolysis.It has been shown that PA activity of macrophages and neutrophils can be modulated by agents such as Con A, PMA, or glucocorticoids (1, 3, 5). In these experiments, long-term effects were studied and the enzyme was measured either in whole cell lysates or in the conditioned medium. In a number of other studies (8-12) involving thymocytes, macrophages, basophils, and several tumor cell lines, the subcellular localization of PA has been examined but no uniform conclusion has been reached, nor was a comparison of subcellular PA distribution in resting and activated states in neutrophils or any other cells attempted.Using a previously published method (13) of neutrophil fractionation, we obtained highly enriched fractions of azurophilic and specific granules and plasma membranes, and we determined PA activity in these fractions. We compared the localization of PA in resting and activated neutrophils.Our results provide evidence that in resting neutrophils most of PA is associated with the specific granule membranes, and upon activation, PA translocates to the plasma membrane, thus equipping the neutrophil with surface-associated proteolytic activity .

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Section: Resultsmentioning

confidence: 99%

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confidence: 77%

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confidence: 99%

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Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis.

Heiple¹,

Ossowski²

1986

The Journal of Experimental Medicine

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“…Binding to its GPI-anchored receptor (uPAR, CD87) on the cell surface, uPA activates plasminogen to plasmin which, in turn, degrades extracellular matrix proteins (1,2). The established role of uPA in breast cancer is to proteolytically cleave the extracellular matrix (3).…”

Section: Introductionmentioning

confidence: 99%

Urokinase-type plasminogen activator induces proliferation in breast cancer cells

Gandhari

Arens²,

Majety³

et al. 2006

Int J Oncol

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Abstract. Urokinase-type plasminogen activator (uPA) is implicated in various pathophysiological processes, including extracellular matrix turnover, cell migration and invasion. Our study aimed to determine the role of uPA in both proliferation and mitogen-activated protein kinase (MAPK) pathway. Hence, we analyzed the effects induced by exogeneous addition of domain-specific uPA antibodies and uPAinteracting molecules on proliferation of uPA-suppressed MDA-MB-231 breast cancer cells. uPA expression was reduced to 53% by stable transfection with an antisense/ vector construct and to 65% by siRNA transfection. Immunocytochemical Ki67 staining and flow cytometry (S-phase) analysis indicated a strong decrease of cellular proliferation activity (35% and 38%, respectively). Exogenous addition of high molecular weight-uPA (HMW-uPA) or incubation with the amino terminal fragment (ATF), which lacks the enzymatic activity of uPA, lead to increased cell proliferation. A strong increase of proliferation was absent when the monoclonal antiuPAR antibody IIIF10 (blocking uPA binding site), soluble uPAR (scavenger effect) and phosphatidyl-inositol-specific phopholipase C (PI-PLC, degrading uPAR) was added prior to the addition of HMW-uPA. In conclusion, HMW-uPA and ATF induce proliferation of breast cancer cells by binding to uPAR. Thereby, integrins situated adjacent to uPAR carry the signals into the cell, thus stimulating proliferation that is mediated via the MAPK pathway.

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“…Fibrin dissolution is also regulated by inhibitors of PLG activation, such as PLG activator inhibitor-1 (PAI-1), which is a major inhibitor of uPA and tPA in vivo and is produced by many cell types upon exposure to TGF-β, TNF-α and other cytokines [10,11]. Cells synthesize uPA as a single-chain precursor form (pro-uPA or scuPA) that can be converted to the two-chain uPA (tcuPA) after cleavage at Lys158-Ile159 by plasmin [8] or other activators [12]. Thrombin cleaves scuPA at Arg156-Phe157, two residues preceding the activation cleavage site, to generate a less active form of tcuPA called thrombincleaved tcuPA (uPAt) [13] The mechanism by which these two amino acid residues prevent conversion of zymogen scuPA into proteolytically active tcuPA is not known.…”

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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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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Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). A study in vitro and in two animal species.

Cited by 322 publications

References 24 publications

Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis.

Human neutrophil plasminogen activator is localized in specific granules and is translocated to the cell surface by exocytosis.

Urokinase-type plasminogen activator induces proliferation in breast cancer cells

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

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