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Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

ABSTRACT: Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.

SUBMITTER: Southan C

PROVIDER: S-EPMC1145117 | biostudies-other | 1985 Aug

REPOSITORIES: biostudies-other

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Project description:BackgroundShroom family member 3 (SHROOM3) encodes an actin-associated protein that regulates epithelial morphology during development. Several genome-wide association studies (GWAS) have identified genetic variances primarily in the 5' region of SHROOM3, associated with chronic kidney disease (CKD) and poor transplant outcomes. These genetic variants are associated with alterations in Shroom3 expression.ObjectiveCharacterize the phenotypic abnormalities associated with reduced Shroom3 expression in postnatal day 3-, 1-month and 3-month-old mice.MethodsThe Shroom3 protein expression pattern was determined by immunofluorescence. We generated Shroom3 heterozygous null mice (Shroom3Gt/+) and performed comparative analyses with wild type littermates based on somatic and kidney growth, gross renal anatomy, renal histology, renal function at postnatal day 3, 1 month, and 3 months.ResultsThe Shroom3 protein expression localized to the apical regions of medullary and cortical tubular epithelium in postnatal wild type kidneys. Co-immunofluorescence studies confirmed protein expression localized to the apical side of the tubular epithelium in proximal convoluted tubules, distal convoluted tubules, and collecting ducts. While Shroom3 heterozygous null mice exhibited reduced Shroom3 protein expression, no differences in somatic and kidney growth were observed when compared to wild type mice. Although, rare cases of unilateral hypoplasia of the right kidney were observed at postnatal 1 month in Shroom3 heterozygotes. Yet renal histological analysis did not reveal any overt abnormalities in overall kidney structure or in glomerular and tubular organization in Shroom3 heterozygous null mice when compared to wild type mice. Analysis of the apical-basolateral orientation of the tubule epithelium demonstrated alterations in the proximal convoluted tubules and modest disorganization in the distal convoluted tubules at 3 months in Shroom3 heterozygotes. Additionally, these modest abnormalities were not accompanied by tubular injury or physiological defects in renal and cardiovascular function.ConclusionTaken together, our results describe a mild kidney disease phenotype in adult Shroom3 heterozygous null mice, suggesting that Shroom3 expression and function may be required for proper structure and maintenance of the various tubular epithelial parenchyma of the kidney.

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Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

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