Immunochemical Studies Research Articles

Reconstituted Fusion Pore

Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles.

Structure and serological characterization of the O-antigen of Proteus mirabilis O18 with a phosphocholine-containing oligosaccharide phosphate repeating unit

A phosphorylated, choline-containing polysaccharide was obtained by O-deacylation of the lipopolysaccharide (LPS) of Proteus mirabilis O18 by treatment with aqueous 12% ammonia, whereas hydrolysis with dilute acetic acid resulted in depolymerisation of the polysaccharide chain by the glycosyl phosphate linkage. Treatment of the O-deacylated LPS with aqueous 48% hydrofluoric acid cleaved the glycosyl phosphate group but, unexpectedly, did not affect the choline phosphate group. The polysaccharide and the derived oligosaccharides were studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, 1H, 13C HMQC and HMQC–TOSCY experiments, along with chemical methods, and the following structure of the pentasaccharide phosphate repeating unit was established: Where ChoP=Phosphocoline Immunochemical studies of the LPS, O-deacylated LPS and partially dephosphorylated pentasaccharide using rabbit polyclonal anti- P. mirabilis O18 serum showed the importance of the glycosyl phosphate group in manifesting the serological specificity of the O18-antigen.

14-3-3 proteins are constituents of the insoluble glycoprotein framework of the chlamydomonas cell wall.

The cell wall of the unicellular green alga Chlamydomonas reinhardtii consists predominantly of Hyp-rich glycoproteins, which also occur in the extracellular matrix of multicellular green algae and higher plants. In addition to the Hyp-rich polypeptides, the insoluble glycoprotein framework of the Chlamydomonas cell wall contains minor amounts of 14-3-3 proteins, as revealed by immunochemical studies and mass spectroscopic analysis of tryptic peptides. Polypeptides immunologically related to the 14-3-3 proteins also were found in the culture medium of Chlamydomonas. The levels of two of these 14-3-3-related polypeptides were decreased in the culture medium of the wall-deficient mutant cw-15. These findings indicate that 14-3-3 proteins are involved in the cross-linking of Hyp-rich glycoproteins in the Chlamydomonas cell wall.

Alterations in myocardial cytoskeletal and regulatory protein expression following a single Doxorubicin injection.

Immunochemical and ultrastructural studies of the rat heart after a single injection of doxorubicin (2.2 or 0.44 mg/kg) were performed. Ventricles were taken for the study 2 h and 3 weeks after injection. The light and electron microscopy and immunohistochemical determination of collagens of I, III, and IV types and fibronectin using specific antibodies were implied. Quantitive immunoblotting was used to analyze the expression levels of cytoskeletal and extracellular matrix proteins such as desmin, tubulin, vinculin, fibronectin, kinase-related protein (KRP or telokin), and smooth muscle/nonmuscle myosin light-chain kinase (MLCK). Doxorubicin (2.2 mg/kg) did not influence the relative volume and structure of collagen network but distinctly reduced the density of fibronectin distribution and decreased the content of tubulin, fibronectin, MLCK, and KRP. After 3 weeks, an increased density and extension of collagen network were observed, indicating the development of diffuse fibrosis whereas the content of tubulin and KRP increased above control level by 50 +/- 2.3% and 20 +/- 5.2%, correspondingly. Similar but less pronounced alterations were observed following the administration of 0.44 mg/kg doxorubicin. The content of MLCK after both doses consistently remained about 30% below its level in untreated animals. Isolated chick embryo cardiomyocytes subjected to doxorubicin responded by a 26% increase in KRP expression 4 days after whereas the level of tubulin expression remained unchanged. Thus, the damage of myocardium after a single injection of a therapeutic dose of doxorubicin was followed by an increased expression of selected cytoskeletal and extracellular matrix proteins, suggesting their involvement in cardiac reparation.

Structural and serological studies of the O-antigen of Proteus mirabilis O-9

The following structure of the O-polysaccharide (O-antigen) of the lipopolysaccharide of Proteus mirabilis O-9 was determined by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods: where the degree of O-acetylation is ∼70%. Immunochemical studies using rabbit polyclonal anti- Proteus mirabilis O-9 serum showed the importance of the O-acetyl groups in manifesting the serological specificity of the O-9 antigen. Anti- P. mirabilis O-9 cross-reacted with the lipopolysaccharides (LPS) of P. vulgaris O-25 and Proteus penneri 14, which could be accounted for by a structural similarity of their O-polysaccharides.

Colocalization of tau and alpha-synuclein epitopes in Lewy bodies.

The major protein constituent of Lewy bodies (LBs), the pathological hallmark of Parkinson disease and dementia with Lewy bodies, is considered to be alpha-synuclein, but other proteins, in particular the microtubule-associated protein tau, have been implicated in the pathogenesis of LBs. Tau is the major structural component of neurofibrillary tangles (NFTs). Both direct immunochemical studies of partially purified LBs and indirect immunohistochemical studies have suggested that LBs may contain tau, but most of these studies were based upon a single tau antibody, and immunologic cross-reactivity was not completely excluded. To gain insight into the relation between tau and alpha-synuclein in LBs, double immunostaining was performed in Lewy body cases with a rabbit polyclonal antibody to alpha-synuclein and a panel of monoclonal antibodies to phospho- and nonphospho-tau epitopes (Alz50, CP9, CP13, PG5, TG3, PHFI) that spanned the length of the tau molecule. Tau-immunoreactive LBs were present in the medulla in 80% of the cases, irrespective of Braak stage. All tau antibodies recognized at least some LBs, arguing against nonspecific antibody cross-reactivity. In most lesions the tau immunostaining was present at the periphery of the LB. The phospho-tau antibody, TG3, detected more LBs than any of the other tau antibodies. The proportion of LBs with tau immunoreactivity was greatest in neurons vulnerable to NETs, such as those in the locus ceruleus and basal nucleus of Meynert, and least in neurons resistant to NFTs, such as the dorsal motor nucleus of the vagus in the medulla. The present results suggest that tau may coaggregate with alpha-synuclein in LBs, especially in neuronal populations vulnerable to both NFTs and LBs.

Structure and Composition of the Fusion Pore

Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. Fusion pores were stable structures at the cell plasma membrane where secretory vesicles dock and fuse to release vesicular contents. In the present study, transmission electron microscopy confirms the presence of fusion pores and reveals their detailed structure and association with membrane-bound secretory vesicles in pancreatic acinar cells. Immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, α-fodrin and the calcium channels α1c and β3 are associated with the fusion complex. The localization and possible arrangement of SNAREs at the fusion pore are further demonstrated from combined AFM, immunoAFM, and electrophysiological measurements. These studies reveal the fusion pore or porosome to be a cup-shaped lipoprotein structure, the base of which has t-SNAREs and allows for docking and release of secretory products from membrane-bound vesicles.

Cellular and subcellular distributions of translation initiation, elongation and release factors in rat hippocampus

Novel protein synthesis in the brain has been suggested to contribute to the formation of synapses and neural circuits during development and the modulation of long-term synaptic plasticity through life. However, cellular and subcellular distribution of neuronal translation machinery and regulator molecules has not yet been extensively characterized in rat brain. In this report, the distribution of translation factors in the developing hippocampus, a region which is highly plastic, was analyzed by immunohistochemistry and Western blotting. Western blot analysis revealed that the hippocampus expresses the factors in all three steps of translation, initiation factors, elongation factors and a release factor. Immunochemical studies of hippocampal slices and culture showed that all translation factors were observed not only in cell bodies but also in dendrites of hippocampal neurons. In addition, the levels of the individual translation factors differed between hippocampal subregions. The differential distribution of translation factors was also confirmed by Western blotting. These results suggest that regulated protein synthesis occurs in the hippocampus, with differences existing between different subregions such as CA1, CA3 and dentate gyrus.

Karyotype and Sex Expression in Trichosanthes dioica

Cytological and immunochemical studies were carried out on dioecious Trichosanthes dioica to understand the genetic control of sex expression. The karyotype shows high homogenity and the absence of heteromorphic pair of chromosome in diploid males negates the possibility of XY mechanism. Soluble protein profile from the tuberous roots of the male and female plants of T. dioica did not show any marked distinction; only a variation in the staining pattern was observed. However, in the immunoblot assay the cross reactivity of anti-p-22 (antibody raised against a female biased 22 kD protein of Momordica dioica) at 29 kD and 32 kD regions of the protein profiles in both sex forms establishes p-22 as a sex linked marker. The ratio of the intensity of 29/32 kD polypeptide is also an indicator of sex expression and the data has been analysed as well.

Beta-Lactam allergenic determinants: fine structural recognition of a cross-reacting determinant on benzylpenicillin and cephalothin.

An appreciation of the structural heterogeneity of allergenic determinants on penicillins and cephalosporins reveals the importance of side-chain groups and their involvement in many allergies to beta-lactam drugs. Although allergenic cross-reactions between penicillins and cephalosporins are known to occur, the precise molecular bases of such recognitions and cross-sensitivities have rarely been studied and identified. The unexpected finding of a high incidence of positive IgE antibody reactions with both benzylpenicillin and cephalothin prompted serological and immunochemical studies to identify the chemical basis of antibody recognition of these drugs from the two different families of beta-lactam antibiotics. Adsorption studies were employed to identify whether or not a single population of antibodies was involved in the recognition of benzylpenicillin and cephalothin. Identification of the fine structural features recognized by IgE antibodies was investigated by quantitative hapten inhibition studies employing carefully selected beta-lactam drugs, analogues and some other structurally related chemicals. Adsorption studies with penicilloic acid-solid phase clearly established that a single population of cross-reacting antibodies recognized both benzylpenicillin and cephalothin. Quantitative inhibition findings, especially with phenylacetic acid and 2-thiopheneacetic acid and with cephaloridine and cefoxitin, which have the same (2-thienyl)methyl side-chain as cephalothin, implicated the methylene group as the focus of the allergenic determinant recognized on benzylpenicillin and cephalothin. In addition to the methylene group, recognition graded into neighbouring structures including the amide group and extended weakly to the beta-lactam ring. Results confirmed that structural features as small as a methylene group may be allergenically important. In the present case, this group, making up only part of the different side-chains on benzylpenicillin and cephalothin, together with neighbouring structures extending toward the beta-lactam ring, accounted for the cross-reactivity seen between structures that, at first sight, appear to be not closely related. Such subtle, small, common structural features are likely to be immunologically recognized and implicated in allergic reactions to other drugs, including beta-lactam antibiotics.

Generation and Characterization of Human Hybrid Neurons Produced between Embryonic CNS Neurons and Neuroblastoma Cells

A human hybrid neuronal cell line A1 has been generated by somatic fusion between a human fetal cerebral neuron and a human neuroblastoma cell, and RT-PCR, immunochemical, and electrophysiological studies of the hybrid cells indicated that the cells express faithfully of morphological, immunochemical, physiological, and genetic features of human cerebral neurons. A1 hybrid neurons express neuron-specific markers such as neurofilament-L (NF-L), NF-M, NF-H, MAP-2, and β tubulin III. A1 human hybrid neurons express messages for various cytokines and cytokine receptors which are similar to parental human CNS neurons and different from the other parental cell line, SK-SH-SY5Y neuroblastoma. A1 hybrid neurons also express messages for choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and glutamic acid decarboxylase (GAD), indicating that they could differentiate into various subsets of neuronal types. Whole-cell patch clamp experiments showed that A1 hybrid neurons expressed Na+ currents, which were completely blocked by tetrodotoxin. In addition, depolarizing and hyperpolarizing voltage clamp steps evoked respective outward and inward K+ currents in these cells. When A1 hybrid neurons were exposed to β amyloid for 72 hr, there was three-fold increase in TUNEL positive cells over controls, indicating that β amyloid is neurotoxic to A1 hybrid neurons. The present study indicates that the A1 human hybrid neuronal cell line should serve as a valuable in vitro model for studies of biology, physiology, and pathology of human neurons in health and disease.

Cyclosporin A as a model antigen: immunochemical and structural studies.

The immunosuppressant drug cyclosporin (Cs) A is a cyclic undecapeptide which has been used as a model antigen because structural information and a large number of analogs, modified at each of its 11 positions, were available. This review summarizes immunochemical and crystallographic studies of the interaction between the Fab of monoclonal antibody R45-45-11 and Cs. Three points are discussed: (1) the different conformations of CsA and the question of its biologically active form; (2) the Fab-CsA recognition mechanism; and (3) the relationship between structure and binding properties of CsA analogs.

Cloning and sequencing of cytochrome P450 2X1 from channel catfish (Ictalurus punctatus)

Previous purification and immunochemical studies in livers of channel catfish indicated the presence of at least four cytochrome P450 (CYP) 2-like isoforms. Sequencing of the first 18 amino acids of one purified form indicated a CYP2 isoform. From this N-terminal sequence and other published CYP2 sequences from fish, primers were designed and a full-length CYP cDNA was identified from reverse-transcribed catfish liver mRNA. 5′ and 3′ RACE was used to obtain an open reading frame of 1470 bp encoding a 490 amino acid protein (approximately 57 kD). CYP2X1 was most identical to Fundulus heteroclitus CYP2P2 (41%); CYP2N2 (40%): and CYP2N1 (39%).

A new platelet polymorphism Duva+, localized within the RGD binding domain of glycoprotein IIIa, is associated with neonatal thrombocytopenia

We report here the identification and characterization of a new platelet alloantigen, Duv(a+), implicated in a case of neonatal thrombocytopenia. Immunochemical studies demonstrated that the epitope was localized on glycoprotein (GP) IIIa. Sequencing of the exons 2 to 15 of GP IIIa gene polymerase chain reaction products from both parents revealed a single base substitution 517C>T (complementary DNA) present in a heterozygous state in DNA from the father leading to amino acid substitution Thr140Ile (ACC>ATC) within the Arg-Gly-Asp binding domain of GP IIIa. Flow cytometry and immunoprecipitation studies of IIb-C517 or T517 IIIa transfected Cos cells allowed us to demonstrate this mutation was responsible for expression of the Duv(a+) epitope. By polymerase chain reaction-single-strand conformational-polymorphism analysis, the mutated allele could not be detected in a population of 100 healthy unrelated donors, indicating a low frequency of occurrence. The Thr140/Ile dimorphism, localized 3 amino acids upstream from the Arg143 involved in the expression of HPA-4a, did not interfere with the binding of an anti-HPA-4a antibody in flow cytometry. Results of functional analysis of wild-type or mutated transfected CHO cells-(1) aggregation in the presence of Ca(++) and soluble fibrinogen after complex activation by dithiothreitol, (2) adhesion on coated fibrinogen, (3) binding of monoclonal antibody PAC-1 or LIBS antibody D3, and (4) outside-in signaling-all suggest that the Thr140Ile polymorphism localized in the Arg-Gly-Asp binding domain of GP IIIa does not affect significantly, if at all, the integrin function. We have shown that the anti-Duv(a+) antibody may inhibit platelet GP IIb-IIIa function.

Milestones in understanding allergy and its diagnosis

SummaryThe prevalence of allergic conditions continues to increase, affecting up to 40% of the population in some countries. Our understanding of the processes of allergy began in 1869, when Blackley first connected hay fever with pollen. In 1911, Dale and Laidlaw discovered that histamine was the principal mediator of anaphylaxis. This led to the development of the first antihistamine, in 1942. In 1968 the WHO International Immunoglobulin Reference Centre officially declared the discovery of IgE based on the work of Bennich, Ishizaka and Johansson. The importance of IgE in allergy is such that the definition of allergic disease is based on the presence of allergen‐specific IgE. Access to IgE allowed experimental, immunochemical studies in vitro. Research activity in the field of immunology increased enormously and generated more important knowledge, including the discovery and characterization of leukotrienes, cytokines and histamine receptors. The involvement of other cells in the allergy process was described. In parallel with the elucidation of the processes involved in allergy came the development of new drug treatments. The first anti‐allergy agents were the antihistamines. Later came sodium cromoglycate and corticosteroids. Immunotherapy was first used in the early 1900s but became less popular with the development of anti‐allergy drugs. Now, however, there is a resurgence of interest in the technique. The discovery that there are two subtypes of T‐helper (Th) cells, which produce different cytokines, and that the Th2 subtype appears to produce cytokines associated with allergy has opened new avenues of research that hold promise for reducing the prevalence of allergy in the population, either by preventing its development or by treatments that can drive the immune system towards a Th1 bias.

Antibodies obtained by xenotransplantation of organ-cultured median eminence specifically recognize hypothalamic tanycytes.

Tanycytes are specialized ependymal cells lining the infundibular recess of the third ventricle of the cerebrum. Early and recent investigations involve tanycytes in the mechanism of gonadotropin-releasing hormone (GnRH) release to the portal blood. The present investigation was performed to obtain a specific immunological marker of tanycytes and to identify the compound(s) responsible for this labeling. After 30 days of organ culture, explants of bovine median eminence formed spherical structures mostly constituted by tanycytes. These tanycyte spheres were xenotransplanted to rats, and the antibodies raised by the host animals against the transplanted living tanycytes were used for immunochemical studies of the bovine and rat median eminence. This antiserum immunoreacted with two compounds of 60 kDa and 85 kDa present in extracts of bovine and rat median eminence. The individual immunoblotting analysis of rat medial basal hypothalami showed a decrease in the amount of the 85-kDa compound in castrated rats as compared to control rats processed at oestrus and dioestrus. The antiserum, labeled as anti-P85, when used for immunostaining of sections throughout the rat central nervous system, immunoreacted specifically with the hypothalamic tanycytes. Within tanycytes, P-85 immunoreactivity was exclusively present in the basal processes. It is suggested that the 85-kDa and 60-kDa compounds correspond to two novel proteins selectively expressed by tanycytes. The possibility that they are secretory proteins involved in GnRH release is discussed. Anti-P85 appears to be the first specific marker of hypothalamic tanycytes.

Correlation of thymidine phosphorylase staining and the Ki-67 labeling index to clinicopathologic factors and hepatic metastasis in patients with colorectal cancer.

Our aim was to investigate the thymidine phosphorylase (TdRPase) expression and Ki-67 labeling index (LI) of primary tumors of colorectal cancer and hepatic metastases immunochemically and to evaluate the relationship of these parameters to various clinicopathologic factors and hepatic metastasis. We performed immunochemical studies in 74 patients with colorectal cancer, using anti-TdRPase antibody and MIB-1 antibody. TdRPase expression and a Ki-67 LI > or =30% in primary lesions were significantly more common in patients with lymphatic invasion (ly), venous invasion (v), lymph node involvement (n), and hepatic metastasis. A Ki-67 LI > or = 30% of the primary tumor was associated with a significantly higher frequency of metachronous liver metastasis. In the same patients, the mean Ki-67 LI was 24.3+/-17.9 for primary lesions and 5.0+/-4.2 for hepatic metastases, this difference being significant. These results indicate that TdRPase expression and the Ki-67 LI are related to various clinicopathologic factors, suggesting their usefulness as indices of tumor malignancy. We suggest that the Ki-67 LI of primary colorectal cancer could be an important predictor of the future development of metachronous liver metastasis.

Alterations in hippocampal GAP-43, BDNF, and L1 following sustained cerebral ischemia

Alterations in factors involved in the regeneration of the neuronal network in the hippocampus of rats with microsphere embolism (ME) were examined. Nine hundred microspheres (48 μm in diameter) were injected into the right hemisphere, and immunochemical and immunohistochemical studies on the hippocampus were performed on the seventh day thereafter. Hematoxylin–eosin staining showed progressive and severe degeneration of the hippocampus after ME. The protein levels of brain-derived neurotrophic factor (BDNF), 43-kDa growth-associated protein (GAP-43), and adhesion protein L1 (L1) in the ipsilateral hippocampus of the ME animal, determined by Western blot analysis or enzyme immunoassay, were increased, unaltered, and decreased, respectively. In contrast, the immunohistochemical study showed increases in a marker of axonal sprouting GAP-43, and a neurotrophic factor BDNF, and a decrease in an adhesion molecule L1 in some areas of the hippocampal ischemic penumbra of such animals. These results suggest that some factors for regeneration of the neuronal network in the ischemic penumbra responded to sustained cerebral ischemia for a certain period, although functional network of the nerve cells in the microsphere-injected hemisphere would be unlikely established after ME.

Structure of the O-polysaccharide and classification of Proteus mirabilis strain G1 in Proteus serogroup O3.

The O-chain polysaccharide of the lipopolysaccharide (LPS) of a previously nonclassified strain of Proteus mirabilis termed G1 was studied by sugar analysis and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, rotating-frame NOE (ROESY), H-detected 1H,13C HMQC, and heteronuclear multiple-bond correlation (HMBC) experiments. The following structure of the polysaccharide was established: [carbohydrate structure: see text] where D-GalA6(L-Lys) stands for N(alpha)-(D-galacturonoyl)-L-lysine. The structure of the O-polysaccharide of P. mirabilis G1 is similar, but not identical, to that of P. mirabilis S1959 and OXK belonging to serogroup O3. Immunochemical studies with P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera revealed close LPS-based serological relatedness of P. mirabilis G1 and S1959, and therefore it was suggested to classify P. mirabilis G1 in serogroup O3 as a subgroup. P. mirabilis G1 and S1959 anti-(O-polysaccharide) sera also cross-reacted with LPS of P. mirabilis strains from two other serogroups containing D-GalA6(L-Lys) in the O-polysaccharide or in the core region.

A functional platelet fibrinogen receptor with a deletion in the cysteine-rich repeat region of the β3 integrin: the Oea alloantigen in neonatal alloimmune thrombocytopenia

This report describes a new low-frequency alloantigen, Oe(a), responsible for a case of neonatal alloimmune thrombocytopenia (NAIT). In a population study none of 600 unrelated blood donors was an Oe(a) carrier. By immunochemical studies the Oe(a) antigen could be assigned to platelet glycoprotein (GP) IIIa. Sequencing of GPIIIa complementary DNA from an Oe(a) (+) individual showed deletion of a lysine residue at position 611 (DeltaLys(611)). Analysis of 20 Oe(a) (-) and 3 Oe(a) (+) individuals showed that the DeltaLys(611) form of GPIIIa was related to the phenotype. Anti-Oe(a) reacted with the DeltaLys(611), but not with the wild-type isoforms on stable transfectants expressing GPIIIa, indicating that DeltaLys(611) directly induces the expression of Oe(a) epitopes. Under nonreducing conditions the Pro(33)DeltaLys(611) variant migrated with a slightly decreased molecular weight compared to the Pro(33)Lys(611) isoform suggesting that DeltaLys(611) has an influence on the disulfide bonds of GPIIIa. The Pro(33)DeltaLys(611) GPIIIa could undergo conformational changes and bind to fibrinogen in a similar manner as the Pro(33)Lys(611) isoform. No difference was found in the tyrosine phosphorylation of pp125(FAK), suggesting that DeltaLys(611) has no effect on integrin function. In contrast to all other low-frequency antigens, the DeltaLys(611) isoform was associated with the HPA-1b, but not with the high frequency HPA-1a allele. Comparison with GPIIIa DNA from nonhuman primates indicated that the HPA-1a allele represents the ancestral form of GPIIIa. It can be assumed that the Oe(a) form did arise as a result of a mutational event from an already mutated GPIIIa allele.