New approach to 'top-and-bottom' whole blood separation using the multiunit TACSI WB system: quality of blood components.

BACKGROUND AND OBJECTIVES: TACSI whole blood system is designed to combine primary and secondary processing of six whole blood bags into plasma units, buffy coat and red blood cell concentrates. The aim of this study was to investigate the specifications and in vitro storage parameters of blood components compared with standard centrifugation and separation processing. MATERIALS AND METHODS: Whole blood bags, collected in CRC kits, were treated on a TACSI whole blood system. They were compared with whole blood bags collected in Composelect kits. In addition to routine quality control analyses, conservation studies were performed on red blood cell concentrates for 42 days and on plasma for 6 months. Platelets pools with five buffy coats were also created, and cellular contamination was evaluated. RESULTS: Red blood cell concentrates produced from TACSI whole blood met European quality requirements. For white blood cell count, one individual result exceeded 1 × 10(6) cells/unit. All plasma units fell within specifications for residual cellular contamination and storage parameters. The performances of the TACSI whole blood system allow for the preparation of low volume buffy coats with a recovery of 90% of whole blood platelets. Haemoglobin losses in TACSI BC are smaller, but this did not result in higher haemoglobin content of red cells. These BC are suitable for the production of platelet concentrates. CONCLUSION: From these in vitro data, red blood cell concentrates produced using TACSI whole blood are suitable for clinical use with a quality at least equivalent to the control group.

Degradation of human gastrin and CCK by endopeptidase 24.11: differential behaviour of the sulphated and unsulphated peptides.

The degradation of human sulphated heptadecapeptide gastrin (G17s) by human endopeptidase 24.11 was studied in vitro. The products of degradation were characterized by HPLC, region-specific gastrin radioimmunoassay and amino acid analysis. The enzyme cleaved G17s at four sites, Trp4-Leu5, Ala11-Tyr12, Gly13-Trp14 and Asp16-Phe17. The patterns of fragments produced when sulphated and unsulphated G17s are hydrolysed by endopeptidase 24.11 indicate that the enzyme cleaves both substrates at the same four bonds. However, the sulphated G17 was 3-times less rapidly degraded than the unsulphated G17 (G17ns). In contrast, the rate of cleavage of the octapeptide cholecystokinin (CCK8) was faster when the peptide was sulphated. The kinetic data of endopeptidase 24.11 indicated similar Km values for sulphated or unsulphated gastrin and CCK; sulphated CCK8 exhibited a 2-fold higher kcat/Km value compared to unsulphated CCK8, whereas G17s exhibited a 2-fold lower kcat/Km value compared to G17ns. The results indicate that the presence of a sulphate group causes a marked reduction in the rate of hydrolysis of gastrin by endopeptidase 24.11, whereas sulphation enhances cholecystokinin degradation by the same enzyme. They also suggest that endopeptidase 24.11 may be responsible for the difference in metabolism of sulphated and unsulphated G17, previously observed in human circulation.

Hydrolysis of alpha-human atrial natriuretic peptide in vitro by human kidney membranes and purified endopeptidase-24.11. Evidence for a novel cleavage site.

alpha-Human atrial natriuretic peptide (hANP) is secreted by the heart and acts on the kidney to promote a strong diuresis and natriuresis. In vivo it has been shown to be catabolized partly by the kidney. Crude microvillar membranes of human kidney degrade 125I-ANP at several internal bonds generating metabolites among which the C-terminal fragments were identified. Formation of the C-terminal tripeptide was blocked by phosphoramidon, indicating the involvement of endopeptidase-24.11 in this cleavage. Subsequent cleavages by aminopeptidase(s) yielded the C-terminal dipeptide and free tyrosine. Using purified endopeptidase 24.11, we identified seven sites of hydrolysis in unlabelled alpha-hANP: the bonds Arg-4-Ser-5, Cys-7-Phe-8, Arg-11-Met-12, Arg-14-Ile-15, Gly-16-Ala-17, Gly-20-Leu-21 and Ser-25-Phe-26. However, the bonds Gly-16-Ala-17 and Arg-4-Ser-5 did not fulfil the known specificity requirements of the enzyme. Cleavage at the Gly-16-Ala-17 bond was previously observed by Stephenson & Kenny [(1987) Biochem. J. 243, 183-187], but this is the first report of an Arg-Ser bond cleavage by this enzyme. Initial attack of alpha-hANP by endopeptidase-24.11 took place at a bond within the disulphide-linked loop and produced a peptide having the same amino acid composition as intact ANP. The bond cleaved in this metabolite was determined as the Cys-7-Phe-8 bond. Determination of all the bonds cleaved in alpha-hANP by endopeptidase-24.11 should prove useful for the design of more stable analogues, which could have therapeutic uses in hypertension.

Effect of human endopeptidase 24.11 ("enkephalinase") on IL-1-induced thymocyte proliferation activity.

Endopeptidase 24.11 (enkephalinase) is a membrane bound protease involved in the degradation of neuropeptides and hormones. Its presence on cells of the thymus and lymph nodes suggests a possible role in the inactivation of immune system mediators. IL-1 (both purified IL-1 beta and an IL-1-rich supernatant) bioactivity, as measured in the thymocyte proliferation assay, was found to disappear upon incubation with endopeptidase 24.11. This inactivation was dependent on both incubation time and enzyme concentration. IL-1 beta was protected by the presence in the incubation medium of phosphoramidon, a specific inhibitor of endopeptidase 24.11. After incubation of IL-1-rich supernatant with the enzyme, the thymocyte proliferation activity could be restored by adding purified IL-1 beta to the samples, indicating that neither the enzyme nor the buffer had any toxic effect on thymocyte proliferation. In the same experimental conditions, IL-2 activity was not destroyed by endopeptidase 24.11.

In vitro and in vivo degradation of human gastrin by endopeptidase 24.11.

The degradation of human unsulfated heptadecapeptide gastrin (G-17) by human kidney endopeptidase 24.11 has been studied in vitro, and some of the products of degradation have been identified in plasma after in vivo infusion of G-17. The enzyme cleaved G-17 at four peptide bonds: Trp4Leu5, Ala11Tyr12, Gly13Trp14, and Asp16Phe17. The cleavage at Gly-Trp was rapid and 1-13 G-17 was an important intermediate. All the products of cleavage of synthetic 1-13 G-17 were also found after degradation of intact G-17. When normal human volunteers received infusions of G-17, there appeared in their blood peptides with the properties of 1-11, 1-13, 1-16, and 5-17 G-17 on the basis of immunochemical and high-performance liquid chromatographic properties. These observations provide evidence that endopeptidase 24.11 is involved in gastrin metabolism in humans, and may be responsible for the generation of G-17 fragments in the peripheral circulation.

Degradation of cholecystokinin octapeptide by the neutral endopeptidase EC 3.4.24.11 and design of proteolysis-resistant analogues of the peptide.

Inactivation of cholecystokinin octapeptide in vitro involves a metalloendopeptidase (EC 3.4.24.11) also called enkephalinase that inactivated the peptide both by a sequential pathway of hydrolysis (removal of Phe-NH(2) followed by cleavage of Trp-Met-Asp) and by an endopeptidase action (production of the tetrapeptides). As enkephalinase cleaved CCK-8 at the Gly(4)-Trp(5), Trp(5)-Met(6) and Asp(7)-Phe(8) bonds, we investigated the stability of analogues having: (1) substitutions of l amino acids by a d stereoisomer, (2) a substitution of Asp(7) by a ? Ala residue and (3) modifications of the Trp residue obtained by replacing the nitrogen atom in the indol ring by either an oxygen ([Bfa(5)]CCK-8) or a sulphur atom ([Bta(5)]CCK-8). Among these different CCK derivatives, [?Ala(7)], [dMet(6)] and [dTrp(5)]CCK-8 were not hydrolyzed by enkephalinase: [dAla(d)]CCK-8 was rapidly cleaved by the enzyme. [Bta(5)] and [Bfa(5)]CCK-8 did not prove to be quite resistant; however the C-terminal tetrapeptides having the same modifications on the Trp residue were not cleaved although they interacted with the enzyme binding site. The stability and biological activity of the peptidase-resistant analogues of CCK-8 remain to be determined in vivo.

Hydrolysis of the C-terminal octapeptide of cholecystokinin by rat kidney membranes: characterization of the cleavage by solubilized endopeptidase-24.11.

Rat kidney membranes were solubilized by Triton X-100 and the CCK-8 degrading peptidases were resolved by chromatography on DEAE-cellulose. Four proteases were detected: two phosphoramidon-sensitive endopeptidases (EC 3.4.24.11), a bestatin-sensitive aminopeptidase and an unidentified enzyme. The pattern of cleavage of CCK-8 and shorter C-terminal fragments by endopeptidase 24.11 was investigated and indicated that the Gly29-Trp30, Trp30-Met31 and Asp32-Phe33 were scissile bonds. However, the cleavage pattern differed markedly from one CCK peptide to another: in the penta- and hexapeptide of CCK the bonds hydrolyzed were either Asp-Phe and Trp-Met or, Asp-Phe and Gly-Trp, respectively. The presence of the sulfate group on the tyrosine residue of CCK-8 influence markedly the nature of the major cleavage fragments produced by the endopeptidase. The major bonds cleaved were Asp-Phe, Trp-Met and Gly-Trp for unsulfated CCK-8, whilst for the sulfated octapeptide, the Trp-Met bond became a minor cleavage site.