Effect of Human Granulocytic Elastase on Isolated Human Antithrombin III

Jochum, Marianne; Lander, S.; Heimburger, N.; Fritz, Hans

doi:10.1515/bchm2.1981.362.1.103

Cited by 116 publications

(40 citation statements)

References 11 publications

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“…Similarly, we found that antithrombin could not form a covalent complex with NSP4 in protease-rich neutrophil lysates. In line with our observations, it has been reported that NE inactivates the inhibitory activity of antithrombin within ,5 min (20,21).…”

Section: Production Of An Nsp4-reactive Serpin Forming Covalent Complsupporting

confidence: 93%

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NSP4 Is Stored in Azurophil Granules and Released by Activated Neutrophils as Active Endoprotease with Restricted Specificity

Perera

Wiesmüller

Larsen

et al. 2013

The Journal of Immunology

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Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer-based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1', P2', P3, P4, and P2 positions were tested. This enabled us to construct an alpha(1)-proteinase inhibitor variant (Ile-Lys-Pro-Arg-/-Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefevre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils

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Section: Production Of An Nsp4-reactive Serpin Forming Covalent Complsupporting

confidence: 93%

“…Antithrombin was previously shown to inactivate purified NSP4, but was not suited to trap NSP4 in the presence of other neutrophil proteases. Antithrombin complexes and free antithrombin were rapidly degraded, which was most likely caused by NE (20,21).…”

Section: Discussionmentioning

confidence: 99%

NSP4 Is Stored in Azurophil Granules and Released by Activated Neutrophils as Active Endoprotease with Restricted Specificity

Perera

Wiesmüller

Larsen

et al. 2013

The Journal of Immunology

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“…Elastase can degrade antithrombin III (30), and this effect appears to be operative in the new Factor IXa assay. Therefore, the elastase-specific chloromethyl ketone or al-antitrypsin were used to control for this effect.…”

Section: Resultsmentioning

confidence: 99%

Cleavage and inactivation of Factor IX by granulocyte elastase.

Takaki¹,

Enfield²,

Thompson³

1983

J. Clin. Invest.

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A B S T R A C T Radioiodinated Factor IX was cleaved by a crude sonicate from leukocytes. In the absence of calcium, fragments of <15,000 mol wt were seen from reduced samples on gel electrophoresis. After digestion in 2 mM calcium, however, electrophoresis of reduced samples showed, in addition to low molecular weight fragments, protein bands corresponding in size to heavy and light chains of Factor XIa-activated Factor IX. The cleaving activity in leukocyte sonicates was inhibited by soybean trypsin inhibitor, but only to a small extent by aprotinin.Granulocyte elastase was isolated from purified polymorphonuclear leukocyte granules by affinity chromatography on soybean trypsin inhibitor-agarose and further chromatography on carboxymethyl cellulose. The purified fraction contained two isozymes on acidic gels which cleaved both an ester sensitive to elastase and radiolabeled Factor IX. These two activities were inhibited by elastase-specific chloromethyl ketone. The isolated protease fraction rapidly inactivated apparent Factor IX activity in a coagulant assay system. The degree of inactivation correlated with the amount of intact, radiolabeled Factor IX cleaved. As with the crude sonicate, generation of the larger heavy and light chain-sized fragments was dependent upon calcium.To assess directly the effect of elastase on Factor IX, an immunospecific, active site-directed assay was developed. In this assay, the sample was incubated with solid-phase antibody to Factor IX and the amount of activated product was detected as that which had com-

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“…4). Moreover, the unspecific proteolytic degradation of AT III, which is also susceptible to PMN elastase cleavage (Jochum et al 1981;Jordan et al 1989), could be pointed out by an indirect method comparing the amount of AT III antigen with its inhibitory activity (Fig. 5).…”

Section: Plasma Levels Of Polymorphonuclear Granulocyte Elastase-indumentioning

confidence: 99%

Pathophysiology of Shock, Sepsis, and Organ Failure

Schlag¹,

Redl²

1993

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The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature.Typesetting: K+V Fotosatz GmbH, Beerfelden 23/3145-5 4 3 2 1 0 -Printed on acid-free paper

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Effect of Human Granulocytic Elastase on Isolated Human Antithrombin III

Cited by 116 publications

References 11 publications

NSP4 Is Stored in Azurophil Granules and Released by Activated Neutrophils as Active Endoprotease with Restricted Specificity

NSP4 Is Stored in Azurophil Granules and Released by Activated Neutrophils as Active Endoprotease with Restricted Specificity

Cleavage and inactivation of Factor IX by granulocyte elastase.

Pathophysiology of Shock, Sepsis, and Organ Failure

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