Blood group A1 and A2 revisited: an immunochemical analysis

Background and Objective  The basis of blood group A₁ and A₂ phenotypes has been debated for many decades, and still the chemical basis is unresolved. The literature generally identifies the glycolipid chemical differences between blood group A₁ and A₂ phenotypes as being poor or no expression of A type 3 and A type 4 structures on A₂ red cells, although this assertion is not unanimous.
Materials and Methods  Using purified glycolipids and specific monoclonal antibodies, we revisited the glycolipid basis of the A₁ and A₂ phenotypes. Purified glycolipids were extracted from four individual A₁ and four individual A₂ blood units. One blood unit from an A weak subgroup was also included. Monoclonal anti-A reagents including those originally used to define the basis of A₁ and A₂ phenotypes were used in a thin layer chromatography – enzyme immunoassay to identify the presence of specific glycolipids.
Results  A type 3 glycolipid structures were found to be present in large amounts in all phenotypes. In contrast, the A type 4 glycolipid structure was virtually undetectable in the A₂ phenotype, but was present in the A₁ and A subgroup samples.
Conclusion  The major glycolipid difference between the A₁ and A₂ phenotypes is the dominance of A type 4 glycolipids in the A₁ phenotype.     

Studies with Ferritin-Labelled Dolichos biflorus Lectin on the Numbers and Distribution of A Sites on A1 and A2 Erythrocytes, and on the Nature of its Specificity and Enhancement by Enzymes

S ummary . The conjugation of Dolichos biflorus extract with ferritin produced an electron dense anti-A reagent that was used to study the number and distribution of A sites on A₁ and A₂ red cells; there were five times as many ferritin-labelled lectin molecules on A₁ as on A₂ cells (about 800,000: 150,000). The arrangement of sites on both A₁ and A₂ cells was diffuse but not random, as pattern analysis of the ferritin molecules revealed a contagious distribution of the sensitized A sites. It is suggested that the anti-A₁ saline test activity shown by the Dolichos reagent is due to the inefficiency of the small lectin molecules to bring about agglutination of the A₂ cells. A₂ cells have fewer sites and consequently a larger mean distance between their A sites than have A₁ cells. Thus the probability of 'bridging' between the A sites of adjacent cells is reduced. Enzyme treatment increased lectin uptake and the A₂ and B cells both bound over 8 million molecules per cell. The A₂ cells gave agglutination reactions like untreated A₁ cells but the B cells were not agglutinated. In areas of agglutination between the cells lectin molecules probably occurred mainly as a monolayer; however, in the other areas they occurred in multiple layers and only the first layer was in contact with cell membrane sites.     

Serology and Genetics of the A1 High H Subgroup

Abstract. Tests on 1,000 A group Indians in Bombay showed high H content in 5%. This incidence was the same in Maharashtrians and Gujarathis. A₁ subjects with high H had a mean score for A₁ antigen of 22.54 ( Dolichos ) which was slightly more than or equivalent to A₁ individuals (19.66). Ulex gave a score of 9.7 in comparison to 0.55 for A₁ and 16.5 for A₂. Absorption studies confirmed higher A and H antigen expression than in A₂ and A₁ individuals, respectively. Family studies of eight A₁/high H individuals showed no specific inheritance pattern. Various hypotheses to explain the irregular inheritance pattern have been discussed.     

Signal transduction involved in the platelet adenylate cyclase sensitization associated with PGH2/TxA2 receptor desensitization

The exposure of human platelets to prostaglandin H₂ analogue (PGH2, U46619) induces homologous desensitization and a concomitant adenylate cyclase (AC) sensitization.
We demonstrate the involvement of phospholipase C (PLC) in this enzyme sensitization. Pre-incubation of platelets with neomycin, a PLC activity inhibitor, prevented AC sensitization but not PGH₂/thromboxane (Tx)A₂ receptor desensitization. PGH₂/TxA₂ receptor desensitization, although necessary, is not sufficient to induce AC sensitization, since neomycin, which prevents AC sensitization, failed to prevent receptor desensitization. Inositol phosphate formation, determined in parallel, was also inhibited. Interestingly, no guanylate cyclase sensitization was noted, suggesting a specific relationship between PGH₂/TxA₂ receptor desensitization and AC sensitization. In addition, using alkaline phosphatase, a dephosphorylating enzyme, and the tyrosine kinase inhibitor erbstatin, we examined the role of phosphorylation–dephosphorylation on AC sensitization. Effectively, alkaline phosphatase, which has no effect by itself, enhances the cAMP production triggered by prostacyclin in control but not in desensitized platelets. In contrast, erbstatin failed to modify this synthesis, indicating the non-involvement of tyrosine kinase pathway in this process.
Our results indicate that the AC sensitization was mediated by PLC and also suggest the participation of other mechanisms, including phosphorylation–dephosphorylation processes. This specific enzyme sensitization may be relevant for the in vivo modulation of platelet activation, in different thrombotic diseases with an increased TxA₂ generation.     

Some Notes on the Specificity of Anti-A1 Reagents

Abstract. The agglutinability of red blood cells of A₁, A₂, A₃, A_x and cis-AB phenotypes by several anti-A₁ reagents was quantitatively evaluated and found to correlate with the number of A antigenic sites/cell determined by using ¹²⁵I-labelled IgG anti-A. Moreover, anti-A₁ sera could be absorbed by some A₂ red blood cells and were inhibited by concentrated saliva from A₂ secretor individuals. The significance of these findings is discussed in the light of recent data from the literature.     

The Development of Haemoglobin A2 in Normal Negro Infants and in Sickle Cell Disease

The development of haemoglobin A₂ levels from birth to 3 years has been compared in normal, β-thalassaemia trait, sickle cell (SS) disease, and S-β-thalassaemia genotypes. Hb A₂ levels were almost identical in normals and in children with SS disease at 1, 2 and 3 years. The most rapid increases in Hb A₂ levels occurred before 6 months but levels were still rising at the end of the third year. Sickle cell-β⁺ thalassaemia could be differentiated from SS disease by the higher Hb A₂ levels between 6 months and 1 year. Insufficient data were available on S-β° thalassaemia but since Hb A₂ levels in this condition are generally higher than those in S-β⁺ thalassaemia, differentiation from SS disease may also be possible from the age of 6 months.     

The Radioimmunoassay of Serum and Salivary Blood Group A and Lea Glycoproteins

S ummary . A radioimmunoassay was developed for the detection of A and Le^a blood group activities in glycoprotein fractions extracted with perchloric acid (PCA) from normal serum. The technique was also applied to studies of salivary blood group glycoproteins.
Blood group A activity was detected in all group A sera tested. Higher levels of A activity were found in sera from group A₁ than from A₂ donors, and sera from both group A₁ and A₂ secretor donors gave higher levels of A activity than sera from non-secretor donors. No difference was found in the levels of A activity in group A₁ and A₂ secretor salivas but only small numbers were assayed. Parallel radioinimunoassay inhibition lines, indicating immunological identity, were obtained with PCA-extracts of A₁ and A₂ secretor plasma and with extracts of A₁ and A₂ secretor salivas.
Le^a activity was detected in all sera tested. The levels of Le^a activity were higher in sera from Le(a + b -) donors than from h(a - b +) or Le(a - b -) donors and were unaffected by ABO group. Radioimmunoassay inhibition lines indicated qualitative differences in the Le^a antigen in salivas from secretors and non-secretors, consistent with a lower density of Le^a antigen on salivary glycoprotein from secretor donors.     

Possible Existence of Hybrid Glycosyltransferase in Heterozygous Blood Group AB Subjects

Abstract. The human blood group glycosyltransf erases A and B have a dimeric structure, i.e., the A enzyme is an aa dimer and the B enzyme is a bb dimer. Considering the fact that the ABO blood group determinant are not x-linked, i.e. both A and/or B genes are expressed in a given cell, a hybrid enzyme (ab dimer) may exist in heterozygous A₁B subjects. Because the A enzyme, but not the B enzyme, adsorbs with Sepharose 4-B, the adsorption characteristics of the A and B enzymes from plasma of various phenotypes were examined to look for this hybrid enzyme. The A enzyme activity from A₁ plasma and from a mixture of A₁ and B plasma was completely adsorbed to Sepharose 4-B, while 25–50% of A enzyme activity from heterozygous A₁B plasma was not adsorbed. The results indicated that heterozygous A₁B plasma contains an additional enzyme component which does not exist in a mixture of A₁ and B plasma, suggesting the existence of a hybrid heterodimer (ab) in heterozygous A₁B subjects.     

Quantitative Measurments Concerning A and B Antigen Sites

Using ¹²⁵I-labelled anti-A and anti-B, the number of A and B antigen sites per red cell for samples obtained from adults of different phenotypes was estimated to be: on A₁ cells, 0.81—1.17×10⁶; on A₁B cells, 0.46—0.85×10⁵; on A₂ cells, 0.24—0.29×10⁶; on A₂B cells, 0.12×10⁶. The number of sites on A₁ cells obtained from cord blood was 0.25—0.37×10⁶, and on the single example of A₂ cord cells were 0.14×10⁸ sites. The number of B sites on adult cells were as follows: on B cells, 0.61—0.83×10⁶; on A₁B cells, 0.31—0.56×10⁶ sites.
The equilibrium constants and the rates of dissociation of the reaction between anti-A and A₁ and A₂ cells were also determined. The value of the equilibrium constant was approximately three times as great and the rate of dissociation three times as slow with A₁ cells compared to A₂ cells. This is consistant with the view that theere is a small difference in the molecular structure between the A₁ and A₂ antigen.     

A family in which Ax is transmitted through a person of the blood group A2B

A family is described in which the rare kind of blood known as A_x, A₄, A_z or A_o occurs. The father's phenotype is A_x, that of his son is A₂B and that of the son's daughter is A_x. That an A₂B person can transmit A_x emphasizes our ignorance of the genetic background of this kind of blood.

Résumé

Etude d'une famille dans laqueue plusieurs exemples du groupe rare A_x, dgalement désigné sous le nom de A₄, A_z ou A_o, ont été trouvés. Le phénotype du père est A_x, cdui de son fils est A₂B, et celui de la fille de ce dernier est aussi A_x. Le fait que le groupe A_x se trouve chez la descendante d'une personne du groupe A₂B sert à souligner notre ignorance du mode de transmission du groupe A_x.

Zusammenfassung

Eine Familie, bei welcher die seltene, als A_x, A₄, A_z oder A_o bezeichnete Blutgruppe vorkommt, wird beschrieben. Der Phänotyp des Vaters ist A_x, der seines Sohnes A₂B und derjenige der Tochter dieses Sohnes A_x. Die Tatsache, daß eine Person der Gruppe A₂B A_x übertragen kann, unterstreicht nur unsere mangelnde Kenntnis über die Erbweise dieser Eigenschaft.     

Molecular Genetic Analysis of the ABO Blood Group System: 1. Weak Subgroups: A3 and B3 Alleles

We have determined the nucleotide sequences of the coding region in the last two coding exom of ABO genes (which occupy 91% of the soluble form of A¹ transferase) from 7 individuals with weak subgroup phenotypes. Four of the individuals had an A₃ phenotype and 3 individuals had a B₃ phenotype. We determined the nucleotide sequences based on PCR followed by subcloning and DNA sequencing of the amplified fragments. Two cases of the A³ allele and 1 case of the B³ allele were found to contain a single-base substitution which resulted in an amino acid substitution. However, no other cases of A³ and B³ alleles were found to contain differences in this region. This finding demonstrates for the first time heterogeneity among these weak subgroups at the nucleotide level.     

Molecular Genetic Analysis of the ABO Blood Group System: 1. Weak Subgroups: A3 and B3 Alleles

We have determined the nucleotide sequences of the coding region in the last two coding exom of ABO genes (which occupy 91% of the soluble form of A¹ transferase) from 7 individuals with weak subgroup phenotypes. Four of the individuals had an A₃ phenotype and 3 individuals had a B₃ phenotype. We determined the nucleotide sequences based on PCR followed by subcloning and DNA sequencing of the amplified fragments. Two cases of the A³ allele and 1 case of the B³ allele were found to contain a single-base substitution which resulted in an amino acid substitution. However, no other cases of A³ and B³ alleles were found to contain differences in this region. This finding demonstrates for the first time heterogeneity among these weak subgroups at the nucleotide level.     

An Example of Auto-Anti-A1 Agglutinins

Abstract. The serum of an elderly man, group A₁ Le(a+b−), contained an IgM antibody that agglutinated his own cells and the cells of random group A₁ donors. Over a period of 5 months, the titre of these auto-anti-A₁ agglutinins was 4 at 22°C.     

Dose-Dependent Destruction of A1 Cells by Anti-A1

Abstract. In a patient of subgroup A₂ the serum contained an unusually potent anti-A₁, giving the following reactions with A₁ red cells in vitro: agglutination of saline-suspended cells up to a temperature of 32°C; a positive indirect antiglobulin test (complement only) at 37°C and lysis of enzyme-treated cells at 37°C. A series of tests was carried out to estimate the ability of the antibody to destroy varying amounts of A₁ red cells in vivo . When about 0.55 ml of red cells was injected, about 65% of the cells were destroyed within 30 min; 2 days later when 18.9 ml of cells were injected, only about 45% were destroyed within 30 min; 5 days after this when a whole unit of A₁ red cells was transfused, survival at 24 h was about 90%. This last figure may indicate that destruction of red cells by anti-A₁ was negligible since at the time of the transfusion of the whole unit the patient was bleeding into her gastrointestinal tract. On the other hand, the titre of anti-A₁ appeared to be declining spontaneously during the period in which tests were carried out so that, if the whole unit of A₁ blood had been transfused at the beginning of this period, survival might have been less good. Nevertheless, from the observed difference in survival between the 0.55 ml and 18.9 ml doses it seems safe to conclude that, even if the unit had been transfused at the time when the antibody concentration was maximal, the percentage of cells destroyed would have been small.     

Siedler: An Antibody which Reacts with A1Le(a-b+) Red Cells

Summary. An antibody, discovered on routine testing in a man of group A₁B Le(a-b-), is described. This antibody reacts with A₁ Le(b+) cells but not with cells possessing the A₁ or Le^b antigen alone.     

The A System of Cattle Blood Groups

(1) The strength of the A antigen was found to be increased by as much as a factor of two in the presence of a double dose of the A gene.
(2) The subgroups A₁ and A₂ were detected by only some A antisera.
(3) No qualitative difference was found between the A₁ and A₂ antigens.
(4) Quantitatively, the A₁ cells carried 6–7 times the amount of effective antigen than that found on A₂ cells.

Résumé

1° La force de l'antigbne A a été trouvée augmentée plus de deux fois lorsque le gène A était en double dose.
2° Les sous-groupes A₁ et A₂ ne sont mis en évidence que par certains anti-sérurns A.
3° Il n'a pas été mis en évidence de différence qualitative entre les antigènes A₁ ou A₂.
4° Quantitativement, les érythrocytes A₁ comportent 6 à 7 fois plus d'antigènes que les érythrocytes A₂.

Zusammenfassung

1. Das A-Gen bewirkt im Falle der Homozygotie eine starke bis zur Verdoppelung reichende quantitative Vermehrung des A-Antigens.
2. Nur wenige Anti-A-Seren sind zum Nachweis der A-Untergruppen A₁ und A₂ geeignet.
3. Ein qualitativer Unterschied zwischen A₁ und A₂ wurde nicht gefunden.
4. Bei A₁-Zellen war der effektive Antigenbestand 6-7mal größer als bei A₂-Zellen.     

Studies on the Human Red Cell Agglutinins of the Swan Mussel (Anodonta cygnea)

Summary. Extracts of the swan mussel Anodonta cygnea specifically agglutinated enzyme treated human erythrocytes of phenotypes A₁, A₂, B, A₁B and A₂B. Absorption with A₂ or B cells left a specific agglutinin for A₁ cells, but separable agglutinins against A and B cells could not be demonstrated. Of 29 sugars tested in parallel with anti-A from Helix aspersa , only lactose inhibited the A. cygnea agglutinins, whereas the H. aspersa anti-A was inhibited by N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, raffinose and stachyose.     

Assay of α-N-Acetylgalactosaminyltransferases in Human Sera: Further Evidence for Several Types of Am Individuals

Abstract. The study of the α - N -acetylgalactosaminyltransferase in the sera of 19 individuals belonging to the rare A_m blood group makes it possible to confirm the heterogeneity of this phenotype established on genetical and immunological criteria. Two groups of subjects, Am and A_y, can be distinguished.
For the individuals of the first group, named A_m, 15 samples (7 families) have been studied, the phenotype is inherited as an allele at the ABO locus. 14 of these subjects, have an α - N -acetylgalactosaminyltransferase whose kinetic properties were similar to those of A₁ subjects. In one family, however, the A transferase detected is of the A₂ type. On a quantitative level, the enzyme activities of these sera only reached 30–50% of the average value observed for A₁ or A₂ subjects, respectively.
These facts suggest the existence of a genetic inhibitor, possibly linked to the ABO locus, preventing either an A₁ or A₂ gene from acting at the level of some cellular lines and leading therefore to the recognition of phenotypes named A^A₁^m and A^A₂^m
On the contrary, under the experimental conditions used, no α - N -acetylgalactosaminyltransferase activity was detected among the four individuals of the second group, named A_y by W einer et al. [37], and whose appeareance in siblings results from the action of a recessive modifying y^A gene.     

Anti-A1 Leb in Serum of a Person of a Blood Group A1h

Abstract. An antibody, anti-A₁ Le^b, has been found in the serum of a person belonging to group A₁h, with complete depression of H on the red cells as well as in the saliva. It differs from earlier examples of the antibody in that it can be neutralized by A₁ nL, secretor saliva as well as O Le^b secretor saliva in addition to A₁ Le^b secretor saliva. Although the proposita secretes A substance, she only secretes Le^a substance and not Le^b substance. No such abnormalities were found in the family investigated. An interpretation of the case is given according to the classical hypothesis of ABO and Lewis system development.     

Loss of Rh Antigen Activity Following the Action of Phospholipase A2 on Red Cell Stroma

Abstract. The degradation of red cell stroma phospholipids by phospholipase A₂ is accompanied by a concomitant fall in the activity of the Rh antigens, c, D and e. The action of phospholipase C on stroma also brings about a fall in D antigen activity. Anti-D bound to the red cells protects the D antigen from inactivation by phospholipase A₂.