A unique elastase in human blood platelets.

James, Harold L.; Wachtfogel, YT; James, Pranee; Zimmerman, M.D.; Colman, Roberta F.; Cohen, Allen B.

doi:10.1172/jci112244

Cited by 21 publications

(13 citation statements)

References 31 publications

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“…Platelets were obtained from whole blood of normal volunteers essentially as described by Mustard et al (1972), but the method was modified to more efficiently remove leucocytes (James et al, 1985). Examination of the washed platelet preparations revealed no neutrophils or monocytes, and only occasional lymphocytes.…”

Section: Methodsmentioning

confidence: 99%

Cellular and molecular regulation of factor V expression in human megakaryocytes

Gewirtz

Shapiro

Shen

et al. 1992

Journal Cellular Physiology

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The mechanisms responsible for regulating FV expression in normal human megakaryocytes are unknown. To test the hypothesis that they are related to cell maturation events, we correlated human megakaryocyte FV antigen content with several putative maturation markers including cell size, morphologic stage of development, and ploidy level. Mature megakaryocytes were isolated from normal marrow by counterflow centrifugal elutriation. The cells were immunofluorescently labeled with a monoclonal antibody probe (B10) directed against the FV connecting peptide (150 kDa) and then reacted with Chromomycin A3 to allow for simultaneous DNA quantitation in the same cell. After processing, individual cells were stages and sized. FV antigen and nuclear DNA levels (ploidy) were measured by microphotometric measurements of total cytoplasmic or nuclear fluorescence. A total of 1,006 cells were examined, of which 12% were stage I, 8% were stage II, 35% were stage III, and 45% were stage IV. The geometric mean diameter (+/- SD) of cells in these stages was 48.3 +/- 11.8 microns2, 54.9 +/- 14.4 microns2, 61.7 +/- 20.02 microns2, and 56.7 +/- 13.2 microns2, respectively. Respective ploidy values in arbitrary fluorescence units, where 2 N = 5%, were 28.2 +/- 18.2%, 31.4 +/- 19.3%, 54.3 +/- 26.6%, and 33.2 +/- 22.7%. Calculated correlation coefficients (r) and coefficient of determination (r2) values suggested that FV antigen levels varied independently of any of the maturation markers studied. However, FV antigen levels could be upregulated by 24 h exposure to 8 nM phorbol myristate acetate (PMA). Presence of FV mRNA in a pure population of megakaryocytes was demonstrated by in situ hybridization and the polymerase chain reaction. Relative levels of megakaryocyte FV mRNA, as assessed by in situ hybridization, failed to reveal a detectable change after PMA exposure in spite of an increase in detectable protein. These data suggest that FV synthesis may be regulated at the post-transcriptional level and that it is subject to regulatory influences which are not coordinately linked to development of other terminal maturation markers.

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Section: Methodsmentioning

confidence: 99%

Cellular and molecular regulation of factor V expression in human megakaryocytes

Gewirtz

Shapiro

Shen

et al. 1992

Journal Cellular Physiology

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“…Molecular isoforms of CTAP-I11 in plasma. CTAP-I11 released from the platelet a-granule into the plasma may be enzymatically converted to smaller isoforms by proteolytic enzymes, including the tryptic, chymotryptic, and elastase-like enzymes found in the platelets themselves (38,39). To evaluate this we examined platelet-pack plasma, which contained platelet releasate 5-6 days of age, as well as fresh, conventional plasma samples.…”

Section: Resultsmentioning

confidence: 99%

Connective tissue activation. xxxvi. the origin, variety, distribution, and biologic fate of connective tissue activating peptide–iii isoforms: characteristics in patients with rheumatic, renal, and arterial disease

Castor

Andrews

Swartz

et al. 1993

Arthritis & Rheumatism

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Objective. To determine the origin, distribution, and biologic fate of platelet‐derived connective tissue activating peptide–III (CTAP‐III), to define the relative amounts of the antigen forms (CTAP‐III, betathromboglobulin [β‐TG], neutrophil activating peptide–2 [NAP‐2]) in plasma of normal persons and those with rheumatic or end‐stage renal disease, and to define the isoforms of CTAP‐III in platelets, plasma, transudates, and tissue deposits. Methods. CTAP‐III in plasma was measured by enzyme‐linked immunosorbent assay, and growth promoting activity of CTAP‐III isoforms was tested in synovial and peritoneal cell cultures by measuring increased synthesis of 14C‐glycosaminoglycan (14C‐GAG) and 3H‐DNA. Isolated CTAP‐III was characterized by Western blotting, microsequencing, and mass spectrometry. Results. CTAP‐III was the primary isoform of this antigen family in normal platelets and platelet‐rich plasma; β‐TG and NAP‐2 accounted for <1% of CTAP‐III isoforms. Previously undescribed isoforms, i.e., CTAP‐III des 1, des 1–2, des 1–3, and a phosphate adduct of CTAP‐III, were observed in varying amounts. Elevated plasma levels of CTAP‐III antigen were found in a substantial fraction of rheumatic disease patients: 24% of those with rheumatoid arthritis, 36% of those with systemic sclerosis, and 15% of those with systemic lupus erythematosus. All 10 patients with end‐stage kidney disease had marked elevations of plasma CTAP‐III levels, which stimulated DNA and GAG synthesis by peritoneal cells in culture. Only large isoforms (such as CTAP‐III) were detected in venous plasma of normal subjects, rheumatic disease patients, and patients receiving long‐term dialysis. Normal human spleen and kidney contained substantial (μ/gm) amounts of CTAP‐III and traces of an isoform with the electrophoretic mobility of CTAP‐III des 1–15/NAP‐2. Liver, lung, and urine contained lesser (ng/gm) amounts of CTAP‐III. Conclusion. These data show that, among the 10 known isoforms, intact CTAP‐III itself was the major circulating isoform (>90%), and β‐TG was the most rare (0–1%). Deposition of CTAP‐III in tissues, such as synovium, spleen, and kidney, is associated with partial processing to NAP‐2–like isoforms and the potential to induce neutrophil and fibroblast activation in patients with rheumatic or end‐stage renal disease.

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“…Platelets were prepared free of neutrophils by a modification 11 of the washing procedure of Mustard and colleagues. 15 Subcellular marker pro tein assays used were: (1) for the cytosol, lactate dehydrogenase, 2 by change in A (340 nm) due to oxidation of reduced nicotinamide-adenine dinucleotide at 23°C, pH 7.4; (2) for alpha-granules, beta-thromboglobulin, 3 using an Amersham radio immunoassay (RIA) kit (code TM.88); (3) for membranes, 125 I-surface-labeled protein assay for detection of glycoprotein IIIa, 8 measured in parallel experiments to avoid interference with the betathromboglobulin RIA; (4) for lysosomes, acid phos phatase, 4 by change in A (405 nm) due to hydrolysis of p-nitrophenyl phosphate at 37°C, pH 4.5; and (5) for peroxisomes, peroxidase, 16 by change in A (450 nm) using o-phenylene diamine and hydrogen peroxide at 37°C, pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, we prepared platelets free of detec table neutrophils and compared the elastase activity in lysed platelets with that of purified neutrophil elastase. 11 The platelet enzyme differed from neutrophil elastase in substrate specificities and ef fects of inhibitors but also was shown to be a serine protease. Additionally, the platelet enzyme was not adsorbed by an immunoadsorbent column specific for neutrophil elastase.…”

mentioning

confidence: 99%

“…Additionally, the platelet enzyme was not adsorbed by an immunoadsorbent column specific for neutrophil elastase. Furthermore, the platelet enzyme was inhibited by α 2 -macroglobulin but was not inhibited by α 1 -protease inhibitor, 11 one of the most potent natural inhibitors of neutrophil elas tase. These findings indicated that a unique elastase exists in platelets, and the present study was then undertaken to verify its subcellular localization and whether it participates in the platelet release reaction.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Subcellular Localization of Platelet Elastase and Its Retention During the Release Reaction

James¹,

James²,

Painter³

et al. 1986

Semin Thromb Hemost

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A unique elastase in human blood platelets.

Cited by 21 publications

References 31 publications

Cellular and molecular regulation of factor V expression in human megakaryocytes

Cellular and molecular regulation of factor V expression in human megakaryocytes

Connective tissue activation. xxxvi. the origin, variety, distribution, and biologic fate of connective tissue activating peptide–iii isoforms: characteristics in patients with rheumatic, renal, and arterial disease

Subcellular Localization of Platelet Elastase and Its Retention During the Release Reaction

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