Degradation of a chromogenic substrate by α<sub>2</sub>‐macroglobulin from plasma of patients with rheumatoid arthritis

Teodorescu, Marius; Gaspar, Alexandre; Spear, Greg T.; Skosey, John L.; Ganea, Doina

doi:10.1002/art.1780271007

Cited by 4 publications

(3 citation statements)

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“…Plasmin is known to be present as a contaminant in IgG preparations (16), presumably being generated during storage from its precursor, plasminogen. Plasmin acts on most synthetic substrates of trypsin, including 2-Val-Gly-Arg-NHPhN02 (6). Contamination of IgG by serine proteinase activity was also reported by Harpel and Hayes (9), who overcame the problem by treatment of the IgG preparation with inhibitor before coupling it to the AffiGel beads.…”

Section: Resultsmentioning

confidence: 79%

“…Solid-phase assay of cuzM-proteinase complexes. The assay was done exactly as described by Teodorescu et al (6). Plasma samples were diluted fourfold in PBS, pH 7.2, to a final volume of 100 pl, mixed with 50 pl of antibody-coated bead suspension and 100 pl of BBS, and incubated with continuous roller action mixing for 2 hours at 22-23°C.…”

Section: Methodsmentioning

confidence: 99%

“…The enzymic activity of the adsorbed complexes is then measured as the hydrolysis of the low M , synthetic substrate, benzyloxycarbonyl-lvalylglycyl-L-arginine p-nitroanilide (Z-Val-Gly-Arg-NEIPhNOZ), also known as Chromozym-Try . The activity detected in the arthritis plasma samples (5,6) was not fully characterized and was described as trypsinlike because of its action on Chromozym-Try, abroadspectrum trypsin substrate.…”

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confidence: 99%

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Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α₂‐macroglobulin–proteinase complexes

Davies

Coughlan

Barrett

1987

Arthritis & Rheumatism

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We measured the levels of az-macroglobulin (azM)-proteinase complexes in the plasma of 18 patients with classic rheumatoid arthritis, 11 age-matched patients with noninflammatory back pain and osteoarthritis, and 8 healthy volunteers. In contrast with previous reports, we found no evidence of a2M-proteinase complexes in the plasma samples from individuals in any of the groups. In our assays, all activity that might have been the result of the presence of such complexes in the plasma samples proved instead to be an artifact attributable to contamination of the anti-a2M antibody immobilized on the AffiGel solid phase with a trypsin-like proteinase. When the contaminating activity was eliminated by pretreatment of the antibody with 1 mM diisopropyl fluorophosphate, no degradation of substrate was detected with any of the plasma samples. However, the ability of the solid-phase assay to detect and quantitate a2M-proteinase complexes when they are present was confirmed in control experiments with plasma samples to which preformed a2M-trypsin complexes had been added, or in which a2M-kallikrein complexes had been generated by activation of Hageman factor (coagulation factor XII). We therefore conclude that neither normal plasma nor that from rheumatoid arthritis patients contains measurable amounts of aZMproteinase complexes.It has been reported by Teodorescu and coworkers (1-6) that patients with classic seropositive rheumatoid arthritis (RA) consistently have abnormally high plasma concentrations of complexes of a2-macroglobulin ( a2M) with a trypsin-like proteinase. Such concentrations were not found in healthy individuals or patients with nonrheumatoid inflammatory arthritides. If confirmed, this observation could form the basis of a valuable laboratory test to distinguish patients with RA from those with other inflammatory arthritides. The report was surprising, however, in that complexes between proteinases and azM are known to be cleared very rapidly from the circulation, and so have not previously been found to accumulate to appreciable concentrations in the plasma (7,8).Teodorescu et a1 (6) used a modification of the solid-phase immunosorbent assay which was initially developed by Harpel and Hayes (9) for measuring a2M-proteinase complexes. In this assay, AffiGel agarose beads coated with monospecific antibodies against a2M are used to adsorb the complexes from the plasma. The enzymic activity of the adsorbed complexes is then measured as the hydrolysis of the low M , synthetic substrate, benzyloxycarbonyl-lvalylglycyl-L-arginine p-nitroanilide (Z-Val-Gly-ArgNEIPhNOZ), also known as Chromozym-Try . The activity detected in the arthritis plasma samples (5,6) was not fully characterized and was described as trypsinlike because of its action on Chromozym-Try, abroadspectrum trypsin substrate.In the present study, we attempted to repro-

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Section: Resultsmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α₂‐macroglobulin–proteinase complexes

Davies

Coughlan

Barrett

1987

Arthritis & Rheumatism

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α₂‐macroglobulin‐proteinase complexes as correlated with α₁‐proteinase inhibitor‐elastase complexes in synovial fluids of rheumatoid arthritis patients

Borth

Dunky

Kleesiek

1986

Arthritis & Rheumatism

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The levels of q-proteinase inhibitor-elastase and cu2-macroglobulin (cu2M)-proteinase complexes were measured in synovial fluids from arthritis patients by use of specific immunosorbent assays. Both types of proteinase inhibitor-proteinase complexes were significantly correlated with each other as well as with the total neutrophil count in synovial fluids of rheumatoid arthritis patients but were discordant in synovial fluids of patients with osteoarthritis. One synovial fluid sample showed active (inhibitory) a2M as well as active collagenase. We purified a2M from pooled synovial fluids obtained from patients with rheumatoid arthritis. This a2M retained approximately 90% of its proteinase binding (inhibiting) capacity, compared with that of normal plasma a@. We found no evidence that cvzM was inactivated by means other than proteinases.The role of proteinases in joint destruction has attracted considerable attention. However, studies of inactivation of antiproteinases as well as of proteolytic

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Granulocyte elastase as a new biochemical marker in the diagnosis of chronic joint diseases

et al. 1986

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Human granulocyte elastase (EC 3.4.21.37) is released from granulocytes in large amounts in chronic inflammatory joint diseases and is therefore of special pathogenic and diagnostic importance. In order to examine the diagnostic significance of this enzyme as a clinico-chemical parameter, we determined the concentration of granulocyte elastase in complex with alpha 1-proteinase inhibitor by an enzyme immunoassay in synovial fluids and plasma of patients with chronic joint diseases. In inflammatory synovial fluids the concentration of complexed elastase correlates well with the granulocyte number and may increase to an extremely high level. In 90% of patients with manifest rheumatoid arthritis increased elastase levels are also observed in the plasma, probably due to the large gradient between the synovial fluid and plasma concentration, whereas in osteoarthrosis normal plasma concentrations were observed. Thus, these results indicate that normal plasma concentrations in patients with chronic joint diseases exclude the diagnosis of rheumatoid arthritis with high probability. The simultaneous determination of complexed elastase in plasma and synovial fluid improves the nosological differentiation of chronic joint diseases. Elastase activity on a specific chromogenic substrate, which was found in many inflammatory synovial fluids, is mainly attributed to elastase alpha 2-macroglobulin complexes. In some purulent synovial fluids, however, we were able to detect free elastase, which has been shown to play an important role in the destruction of articular cartilage.

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Degradation of a chromogenic substrate by α₂‐macroglobulin from plasma of patients with rheumatoid arthritis

Cited by 4 publications

References 20 publications

Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α₂‐macroglobulin–proteinase complexes

Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α₂‐macroglobulin–proteinase complexes

α₂‐macroglobulin‐proteinase complexes as correlated with α₁‐proteinase inhibitor‐elastase complexes in synovial fluids of rheumatoid arthritis patients

Granulocyte elastase as a new biochemical marker in the diagnosis of chronic joint diseases

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Degradation of a chromogenic substrate by α2‐macroglobulin from plasma of patients with rheumatoid arthritis

Cited by 4 publications

References 20 publications

Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α2‐macroglobulin–proteinase complexes

Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α2‐macroglobulin–proteinase complexes

α2‐macroglobulin‐proteinase complexes as correlated with α1‐proteinase inhibitor‐elastase complexes in synovial fluids of rheumatoid arthritis patients

Granulocyte elastase as a new biochemical marker in the diagnosis of chronic joint diseases

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Degradation of a chromogenic substrate by α₂‐macroglobulin from plasma of patients with rheumatoid arthritis

Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α₂‐macroglobulin–proteinase complexes

Plasma from rheumatoid arthritis patients does not contain abnormally high levels of α₂‐macroglobulin–proteinase complexes

α₂‐macroglobulin‐proteinase complexes as correlated with α₁‐proteinase inhibitor‐elastase complexes in synovial fluids of rheumatoid arthritis patients