PubMed · 4488843
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Ringuier. 1973. [Hemolysis].. https://pubmed.ncbi.nlm.nih.gov/4488843/
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Electron microscopic (EM) studies were performed to clarify the interactions of membrane proteins in the red blood cell membrane structure in situ of a homozygous patient with total deficiency of protein 4.1 who carried a point mutation of the downstream translation initiation codon (AUG --> AGG) of the protein 4.1 gene [the 4.1 (-) Madrid; Dalla Venezia et al, J Clin Invest 90:1713, 1992]. Immunologically, as expected, protein 4.1 was completely missing in the red blood cell membrane structure in situ. A markedly disrupted skeletal network was observed by EM using the quick-freeze deep-etching method and the surface replica method, although the number of spectrin molecules was only minimally reduced (395 +/- 63/microm2; normal, 504 +/- 36/microm2). The number of basic units in the skeletal network was strikingly reduced (131 +/- 21/microm2; normal, 548 +/- 39/microm2), with decreased small-sized units (17 +/- 4/microm2; normal, 384 +/- 52/microm2) and increased large-sized units (64% +/- 14%; normal, 5% +/- 1%). Concomitantly, immuno-EM disclosed striking clustering of spectrin molecules with aggregated ankyrin molecules in the red blood cell membrane structure in situ. Although no quantitative abnormalities in the number and size distribution of the intramembrane particles were observed, there was a disappearance of regular distribution, with many clusters of various sizes, probably reflecting the distorted skeletal network. Therefore, protein 4.1 suggests by EM to play a crucial role in maintenance of the normal integrity of the membrane structure in situ not only of the skeletal network but also of the integral proteins.
The pathogenesis of two metabolic disorders caused by enzyme defects in the red blood cell leading to hemolytic anemia, and in some cases of glucose phosphate isomerase (GPI) deficiency additionally to neurological impairment was investigated. Rheological studies were performed to determine the influence of a shortage of energy on the deformability of the erythrocytes. The functions of the enzymes were determined by studying the enzyme kinetics, the temperature dependence of the enzyme activity and the migration of the proteins in an electric field. A detailed molecular genetic analysis of the gene encoding for the given protein allowed the detection of mutations involving amino acid exchanges which cause alterations of the protein structure. For both enzyme deficiencies, a good correlation was found between the structural changes (usually caused by single point mutations in the gene), the altered function of the enzymes and the severity of the clinical picture. The exchange of amino acids close to either the active site or the regulatory domain results in a decreased turnover as well as an alteration of the regulatory properties of the enzymes; this usually leads to an increased severity of the disease. Increased concentrations of glucose-6-phosphate (G-6-P), found in all red blood cells of patients suffering from hemolytic anemia caused by pyruvate kinase (PK) and GPI deficiency, correlate well with the severity of the clinical picture, apparently reflecting the degree of the perturbation of glycolysis. This results in a lack of the energy donor adenosine triphosphate (ATP); this leads then to a destabilization of the red cell membrane which causes earlier lysis of the red blood cell, which in turn gives rise to hemolytic anemia of variable degrees. One patient with neurological symptoms has been studied so far biochemically and at the molecular genetic level. The point mutations found in this patient's GPI gene support the idea that GPI may have a neurological function in addition to its role in the carbohydrate metabolism; this is due to the presence of a monomeric sequence analogue called neuroleukin (NLK). The mutations apparently lead to the incorrect folding of this neurotrophic factor, and thus destroy the neurological activity.
We have used our previously described baboon model of infusion of both a sublethal dose of Escherichia coli and C4b binding protein to assess the impact of inhibiting platelet function with the F(ab')2 fragment of the monoclonal antibody 7E3, directed against the platelet glycoprotein (GP)IIb/IIIa receptor, on the characteristic microvascular changes. At a dose of 0.25 to 0.35 mg/kg bolus plus an infusion of 0.25 to 0.35 mg/kg over 6 hours, c7E3 F(ab')2 had only a minimal impact on fibrinogen consumption and delayed but did not prevent, the development of thrombocytopenia. Treatment with 7E3 F(ab')2, however, produced significant protection from the development of microangiopathic hemolysis and renal insufficiency. Histologic examination supported these observations, with treated animals having fewer schistocytes on blood smear and less evidence of ischemic renal changes. Treated animals also had more rapid recovery of peripheral white blood counts, suggesting a possible protective effect of treatment on ischemic damage to the bone marrow. These data indicate that potent inhibition of platelet function via GPIIb/IIIa receptor blockade can decrease ischemic organ damage in this animal model that has features similar to those found in diffuse intravascular coagulation, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura.