A method for the differential determination of plasma antithrombins.

Musumeci, V; Vincenti, Albert Cilia; Bizzi, B

doi:10.1136/jcp.29.1.63

Cited by 5 publications

(6 citation statements)

References 12 publications

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“…The higher percentage observed in d-plasma can be explained by the absence of fibrin which is known to adsorb thrombin. These results are in apparent contrast to previous observations [4,11] that a 2-M accounts for about 30 % of the total antithrombin activity of plasma. The use in our experiments of undiluted plasma could be considered in order to explain this discrepancy.…”

Section: Discussioncontrasting

confidence: 56%

“…After cooling, the precipitate was removed by centrifugation at 3,000 g for 15 min. d-PIasma free of antithrombin 111 was prepared by Al(OH)3 adsorption [11], Heparinized plasma was prepared by adding heparin (Liquemin, Prodotti Roche SpA, Milano) to citrated plasma to the final concentration of 1 unit/ml.…”

Section: Methodsmentioning

confidence: 99%

“…It can be assumed that the anti thrombin activity of this sample is due only to the content of a 2-M [11]. A thrombin solu tion in T1CT buffer was mixed with adsorbed d-plasma to yield a sample containing final concentrations of 20 NIH units/ml of thrombin and 1 mg/ml of a 2-M.…”

Section: Specific Activity O F a -M-bound Thrombin With Respect To Fmentioning

confidence: 99%

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Amidolytic Assay of Thrombin Bound to α<sub>2</sub>-Macroglobulin in Plasma

Musumeci¹,

Landolfi²,

Bizzi³

1977

Pathophysiol Haemos Thromb

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A method for the determination in plasma of α₂-macroglobulin-bound thrombin is described. α₂-Macroglobulin-bound thrombin is precipitated from plasma by 13% polyethyleneglycol, and its amidolytic activity is assayed by using the chromogenic substrate benzoyl-Phe-Val-Arg-p-nitroanilide (S 2160). After thrombin addition to plasma, only about 1.7 % of the added thrombin activity was recovered in the α₂-macroglobulin precipitate. It is suggested that the contribution of α₂-macroglobulin to the antithrombin activity of normal plasma is of little relevance.

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Section: Discussioncontrasting

confidence: 56%

Section: Methodsmentioning

confidence: 99%

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Amidolytic Assay of Thrombin Bound to α<sub>2</sub>-Macroglobulin in Plasma

Musumeci¹,

Landolfi²,

Bizzi³

1977

Pathophysiol Haemos Thromb

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“…To measure the second-order association rate constant (K ass) values for serum A1AT with NE or PR3, it was first necessary to inactivate A2M. This was achieved by treating the serum samples with 0.1 M methylamine (Fisher Scientific, Loughborough, United Kingdom) for 10 min at 37°C (33). The inactivation of A2M was confirmed indirectly by measuring NE and PR3 inhibition by serum samples with and without treatment with methylamine.…”

Section: Methodsmentioning

confidence: 99%

α-1-Antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease

Sinden

Baker

Smith

et al. 2015

American Journal of Physiology-Lung Cellular and Molecular Phys

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The excessive activities of the serine proteinases neutrophil elastase and proteinase 3 are associated with tissue damage in chronic obstructive pulmonary disease. Reduced concentrations and/or inhibitory efficiency of the main circulating serine proteinase inhibitor ␣-1-antitrypsin result from point mutations in its gene. In addition, ␣-2-macroglobulin competes with ␣-1-antitrypsin for proteinases, and the ␣-2-macroglobulin-sequestered enzyme can retain its catalytic activity. We have studied how serine proteinases partition between these inhibitors and the effects of ␣-1-antitrypsin mutations on this partitioning. Subsequently, we have developed a three-dimensional reaction-diffusion model to describe events occurring in the lung interstitium when serine proteinases diffuse from the neutrophil azurophil granule following degranulation and subsequently bind to either ␣-1-antitrypsin or ␣-2-macroglobulin. We found that the proteinases remained uninhibited on the order of 0.1 s after release and diffused on the order of 10 m into the tissue before becoming sequestered. We have shown that proteinases sequestered to ␣-2-macroglobulin retain their proteolytic activity and that neutrophil elastase complexes with ␣-2-macroglobulin are able to degrade elastin. Although neutrophil elastase is implicated in the pathophysiology of emphysema, our results highlight a potentially important role for proteinase 3 because of its greater concentration in azurophil granules, its reduced association rate constant with all ␣-1-antitrypsin variants studied here, its greater diffusion distance, time spent uninhibited following degranulation, and its greater propensity to partition to ␣-2-macroglobulin where it retains proteolytic activity.␣-1-antitrypsin; chronic obstructive pulmonary disease; reaction-diffusion model; enzyme kinetics; serine proteinase CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) is a major cause of morbidity and mortality worldwide (40) and creates a significant economic burden (60). Several pulmonary phenotypes have been recognized, including chronic bronchitis, which affects the large airways and is associated with chronic mucus hypersecretion (28a), and emphysema, which is associated with destruction of alveolar walls and enlargement of airspaces distal to the terminal bronchioles (38). Although the main risk factor for the development of COPD is cigarette smoking (10), only around 20% of smokers develop clinically significant disease (49), indicating that other environmental and genetic risk factors are important in the etiology. To date, deficiency of the serine proteinase inhibitor (serpin) ␣-1-antitrypsin (A1AT) is the only widely recognized genetic predisposition.A1AT is secreted by hepatocytes (16), enters the lungs primarily by passive diffusion, and inhibits neutrophil serine proteinases (NSPs) irreversibly with 1:1 stoichiometry. Circulating levels of A1AT are in the range of 20 -53 M in individuals with the normal (protease inhibitor MM, PiMM) genotype (5). Over 100 naturally occurring genetic variants...

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“…This was achieved by treating the serum samples with 0.1 M methylamine (Fisher Scientific, UK) for 10 minutes at 37°C [11]. The inactivation of A2M was confirmed indirectly by measuring NE and PR3 inhibition by serum samples with and without treatment with methylamine.…”

Section: Methodsmentioning

confidence: 99%

The significance of the F variant of alpha-1-antitrypsin and unique case report of a PiFF homozygote

Sinden

Koura²,

Stockley

2014

BMC Pulm Med

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BackgroundInheritance of the F variant of alpha-1-antitrypsin is associated with normal circulating protein levels, but it is believed to be dysfunctional in its ability to inhibit neutrophil elastase and therefore has been implicated as a susceptibility factor for the development of emphysema. In this study, its functional characteristics were determined following the identification of a unique patient with the PiFF phenotype, and the implications as a susceptibility factor for emphysema are considered both in homozygotes and heterozygotes.MethodsSecond order association rate constants were measured for M, Z, S and F variants of alpha-1-antitrypsin with neutrophil elastase and proteinase 3. Clinical characteristics of the PiFF homozygote and six PiFZ heterozygote subjects were studied.ResultsThe F variant had a reduced association rate constant with neutrophil elastase (5.60 ± 0.83 × 106 M-1 s-1) compared to the normal M variant (1.45 ± 0.02 × 107 M-1 s-1), indicating an increased time to inhibition that was comparable to that of the Z variant (7.34 ± 0.03 × 106 M-1 s-1). The association rate constant for the F variant and proteinase 3 (1.06 ± 0.22 × 106 M-1 s-1) was reduced compared to that with neutrophil elastase, but was similar to that of other alpha-1-antitrypsin variants. Of the six PiFZ heterozygotes, five had airflow obstruction and radiological evidence of emphysema. The PiFF homozygote had airflow obstruction but no emphysema. None of the patients had clinical evidence of liver disease.ConclusionsThe F variant may increase susceptibility to elastase-induced lung damage but not emphysema, whereas co-inheritance with the Z deficiency allele may predispose to emphysema despite reasonable plasma concentrations of alpha-1-antitrypsin.

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A method for the differential determination of plasma antithrombins.

Cited by 5 publications

References 12 publications

Amidolytic Assay of Thrombin Bound to α<sub>2</sub>-Macroglobulin in Plasma

Amidolytic Assay of Thrombin Bound to α<sub>2</sub>-Macroglobulin in Plasma

α-1-Antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease

The significance of the F variant of alpha-1-antitrypsin and unique case report of a PiFF homozygote

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