Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

Bangert, Kristian; Thorsen, Sixtus

doi:10.1055/s-0037-1614011

Cited by 4 publications

(3 citation statements)

References 33 publications

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“…Interestingly, these enzymes also activate chemerin (TABLE ONE). Although the enzyme concentrations required were higher (1000 -10,000-fold) than their observed plasma zymogen levels (9), the uPA concentration was similar to the level required to cleave its primary physiologic target, plasminogen (11)(12)(13). Both plasminogen activators display increased abilities to activate plasminogen when in the bound state (14,15), particularly tPA, which displays a kinetic acceleration of ϳ50-fold in plasminogen activation in the presence of fibrin.…”

Section: Resultsmentioning

confidence: 99%

Chemerin Activation by Serine Proteases of the Coagulation, Fibrinolytic, and Inflammatory Cascades

Zabel

Allen

Kulig

et al. 2005

Journal of Biological Chemistry

332

311

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Proteases function at every level in host defense, from regulating vascular hemostasis and inflammation to mobilizing the "rapid responder" leukocytes of the immune system by regulating the activities of various chemoattractants. Recent studies implicate proteolysis in the activation of a ubiquitous plasma chemoattractant, chemerin, a ligand for the G-protein-coupled receptor CMKLR1 present on plasmacytoid dendritic cells and macrophages. To define the pathophysiologic triggers of chemerin activity, we evaluated the ability of serum-and inflammation-associated proteases to cleave chemerin and stimulate CMKLR1-mediated chemotaxis. We showed that serine proteases factor XIIa and plasmin of the coagulation and fibrinolytic cascades, elastase and cathepsin G released from activated neutrophil granules and mast cell tryptase are all potent activators of chemerin. Activation results from cleavage of the labile carboxyl terminus of the chemoattractant at any of several different sites. Activation of chemerin by the serine protease cascades that trigger rapid defenses in the body may direct CMKLR1-positive plasmacytoid dendritic cell and tissue macrophage recruitment to sterile sites of tissue damage, as well as trafficking to sites of infectious and allergic inflammation.A network of serine proteases regulates the primary response to injury and infection in the host. Serine proteases of the coagulation and fibrinolytic cascades mediate the homeostatic response to blood vessel injury. Kallikrein and factor XIIa process kininogens to generate bradykinin, a potent vasodilator that triggers increased vascular permeability during inflammation. Serine proteases termed convertases release multiple pathogen-neutralizing components of activated complement. Serine protease cascades also regulate the recruitment of phagocytic and antigen-presenting cells to sites of inflammation and tissue damage. The complement cascade, for example, releases active components C5a and C3a, potent attractants for many leukocytes, including neutrophils and monocytes (1, 2). Thus serine proteases are critical participants in rapid defense mechanisms in the body.We and others have recently identified chemerin as a potent chemoattractant for cells expressing the G-protein-linked receptor chemokine-like receptor 1 (CMKLR1), 5 also known as ChemR23 or DEZ (3-5). CMKLR1 is expressed in vitro by monocyte-derived macrophages and dendritic cells (3,5,6) and in vivo by circulating plasmacytoid dendritic cells (pDCs) (5) and tissue macrophages.6 pDCs are major producers of ␣-interferons and can differentiate into antigen-presenting cells capable of triggering T effector or suppressor responses (7). Tissue macrophages have a major role as phagocytes but, similar to pDCs, are also implicated in bridging innate and adaptive immune responses and in regulating immunity in sterile versus infectious tissue injury (8). Importantly, chemerin is widely expressed and circulates in human plasma in an inactive state (5). Active forms of chemerin have been isolated from hu...

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Section: Resultsmentioning

confidence: 99%

Chemerin Activation by Serine Proteases of the Coagulation, Fibrinolytic, and Inflammatory Cascades

Zabel

Allen

Kulig

et al. 2005

Journal of Biological Chemistry

332

311

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“…Plasminogen was quantified with a functional activity assay as previously described (with modifications) by Bangert and Thorson (41). Briefly, plasmas were diluted 1:50 in assay buffer (phosphate 80 mM, NaCl 50 mM, bovine serum albumin 2 mg/ ml), supplemented with 75 IU/ml urokinase, 1 mM tranexamic acid and 0.75 mM of the plasmin-selective chromogenic substrate in a final volume of 50 ml.…”

Section: Study Of Plasminogen Activation/fragmentation On Fibrin-nets...mentioning

confidence: 99%

DNA‐bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma

et al. 2019

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Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism via a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin-NETs matrices by seeding and activating neutrophils onto a fibrin surface and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE-DNA complexes is protected from inhibition by plasma antiproteases and sustains its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains (K 1+2+3 , k 1+2+3+4 ) and the serine protease (SP) region (K 5 -SP). We further demonstrate that patients with septic shock with disseminated intravascular coagulation have circulating HNE-DNA complexes, HNE-derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE-DNA complexes reduce plasminogen into fragments, thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin, and localized plasmin formation.

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“…Transformation of CSF plasminogen into plasmin was measured using uPA (5 IU/ml) and a plasmin-selective chromogenic substrate (0.75 mM CBS0065), as previously described [ 26 ]. In this system, the initial velocity of p -nitroaniline released from CBS0065 is proportional to the amount of plasmin(ogen).…”

Section: Methodsmentioning

confidence: 99%

Plasminogen in cerebrospinal fluid originates from circulating blood

Mezzapesa

Orset

Laurent

et al. 2014

J Neuroinflammation

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BackgroundPlasminogen activation is a ubiquitous source of fibrinolytic and proteolytic activity. Besides its role in prevention of thrombosis, plasminogen is involved in inflammatory reactions in the central nervous system. Plasminogen has been detected in the cerebrospinal fluid (CSF) of patients with inflammatory diseases; however, its origin remains controversial, as the blood–CSF barrier may restrict its diffusion from blood.MethodsWe investigated the origin of plasminogen in CSF using Alexa Fluor 488–labelled rat plasminogen injected into rats with systemic inflammation and blood–CSF barrier dysfunction provoked by lipopolysaccharide (LPS). Near-infrared fluorescence imaging and immunohistochemistry fluorescence microscopy were used to identify plasminogen in brain structures, its concentration and functionality were determined by Western blotting and a chromogenic substrate assay, respectively. In parallel, plasminogen was investigated in CSF from patients with Guillain-Barré syndrome (n = 15), multiple sclerosis (n = 19) and noninflammatory neurological diseases (n = 8).ResultsEndogenous rat plasminogen was detected in higher amounts in the CSF and urine of LPS-treated animals as compared to controls. In LPS-primed rats, circulating Alexa Fluor 488–labelled rat plasminogen was abundantly localized in the choroid plexus, CSF and urine. Plasminogen in human CSF was higher in Guillain-Barré syndrome (median = 1.28 ng/μl (interquartile range (IQR) = 0.66 to 1.59)) as compared to multiple sclerosis (median = 0.3 ng/μl (IQR = 0.16 to 0.61)) and to noninflammatory neurological diseases (median = 0.27 ng/μl (IQR = 0.18 to 0.35)).ConclusionsOur findings demonstrate that plasminogen is transported from circulating blood into the CSF of rats via the choroid plexus during inflammation. Our data suggest that a similar mechanism may explain the high CSF concentrations of plasminogen detected in patients with inflammation-derived CSF barrier impairment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0154-y) contains supplementary material, which is available to authorized users.

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Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

Cited by 4 publications

References 33 publications

Chemerin Activation by Serine Proteases of the Coagulation, Fibrinolytic, and Inflammatory Cascades

Chemerin Activation by Serine Proteases of the Coagulation, Fibrinolytic, and Inflammatory Cascades

DNA‐bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma

Plasminogen in cerebrospinal fluid originates from circulating blood

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