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W H Ouwehand

Publications and source records attributed to W H Ouwehand.

At least 19 recordsLinked to original sources
PubMed2006

Definition of novel GP6 polymorphisms and major difference in haplotype frequencies between populations by a combination of in-depth exon resequencing and genotyping with tag single nucleotide polymorphisms.

BACKGROUND: Common genetic variants of cell surface receptors contribute to differences in functional responses and disease susceptibility. We have previously shown that single nucleotide polymorphisms (SNPs) in platelet glycoprotein VI (GP6) determine the extent of response to agonist. In addition, SNPs in the GP6 gene have been proposed as risk factors for coronary artery disease. METHODS: To completely characterize genetic variation in the GP6 gene we generated a high-resolution SNP map by sequencing the promoter, exons and consensus splice sequences in 94 non-related Caucasoids. In addition, we sequenced DNA encoding the ligand-binding domains of GP6 from non-human primates to determine the level of evolutionary conservation. RESULTS: Eighteen SNPs were identified, six of which encoded amino acid substitutions in the mature form of the protein. The single non-synonymous SNP identified in the exons encoding the ligand-binding domains, encoding for a 103Leu > Val substitution, resulted in reduced ligand binding. Two common protein isoforms were confirmed in Caucasoid with frequencies of 0.82 and 0.15. Variation at the GP6 locus was characterized further by determining SNP frequency in over 2000 individuals from different ethnic backgrounds. CONCLUSIONS: The SNPs were polymorphic in all populations studied although significant differences in allele frequencies were observed. Twelve additional GP6 protein isoforms were identified from the genotyping results and, despite extensive variation in GP6, the sequence of the ligand-binding domains is conserved. Sequences from non-human primates confirmed this observation. These data provide valuable information for the optimal selection of genetic variants for use in future association studies.

Amino Acid Sequence↗
PubMed2005

Gain- and loss-of-function mutants confirm the importance of apical residues to the primary interaction of human glycoprotein VI with collagen.

BACKGROUND: By site-directed mutagenesis of recombinant receptor fragments, we have previously identified residue lysine59 of the platelet collagen receptor glycoprotein VI (GPVI) as being critical for its interaction with the synthetic ligand collagen-related peptide (CRP) and the inhibitory phage antibody 10B12. Lysine59 is proposed to lie on the apical surface of the receptor near the linker joining the two immunoglobulin (Ig)-like extracellular domains. Recently, others have postulated the involvement of a portion of the first domain distant from the interdomain hinge as being involved in an extended collagen-binding site. AIM AND METHODS: To extend our knowledge of the primary collagen-binding site of GPVI, a number of neighboring residues on the apical surface of recombinant soluble GPVI were mutated to alanine and binding of these mutants, as well as the lysine59 mutant, to fibrillar collagen was measured. RESULTS: Binding of recombinant GPVI to collagen, like CRP, was dramatically reduced by the mutation of residue lysine59 to glutamate. Remarkably, the mutation of residues arginine60 in domain one and arginine166 in domain two, individually to alanine, which had no significant affect on CRP binding, reduced binding of recombinant GPVI to collagen. Mutation of the residue lysine41 to alanine dramatically increased binding to both CRP and collagen. This mutation abolished 10B12 binding, confirming its position in the epitope of our inhibitory phage antibody. CONCLUSIONS: Residues lysine59, arginine60, and arginine166, from both Ig-like domains of GPVI, are critical for collagen binding by the receptor. This provides additional evidence for a basic patch on the apical surface of the receptor as the primary collagen-binding site of GPVI.

Binding Sites↗
PubMed2005

Collaborative study to establish the first international standard for quantitation of anti-HPA-1a.

BACKGROUND AND OBJECTIVES: This report describes the production of a freeze-dried preparation of pooled human plasma, containing immunoglobulin G (IgG) antibodies against the human platelet antigen 1a (HPA-1a). The material, coded 03/152, is proposed as an International Standard containing 100 arbitrary units of anti-HPA-1a for use in quantitative assays to determine the anti-HPA-1a activity in clinical samples. MATERIALS AND METHODS: Plasma samples containing potent anti-HPA-1a were pooled and freeze dried in 1-ml ampoules. In addition, three individual plasma samples were selected which had varying levels of anti-HPA-1a activity. The anti-HPA-1a activity of these three samples was determined by using a variety of quantitative assays with the proposed standard as a reference. RESULTS: An international collaborative study, which was part of the 2004 ISBT Platelet Immunology Workshop, involved 39 laboratories in 24 countries and showed that the anti-HPA-1a activity in three test samples could be reliably determined by using the proposed standard. CONCLUSIONS: Laboratories can use this standard to measure the anti-HPA-1a activity in patient's samples. Further studies are required to determine the relationship between anti-HPA-1a activity and clinical outcome in patients with neonatal alloimmune thrombocytopenia (NAIT).

Antibodies, Monoclonal↗
PubMed2004

Molecular studies of anti-HLA-A2 using light-chain shuffling: a structural model for HLA antibody binding.

Human leukocyte antigen (HLA) A2 is one of the most immunodominant HLA antigens. Through a process of light-chain variable domain (VL) shuffling, we analyzed the VL domains' role in anti-HLA-A2/A28-binding site diversity. This was achieved by combining a VH3-30-encoded HLA-A2/A28-specific heavy-chain variable domain with 10(4) non-immune VL domains. Twelve HLA-A2/A28-specific antibodies were subsequently identified. VL gene analysis demonstrated an absence of Vlambda domains and that all have VkappaI-encoded light chains. The affinities correlated with the VkappaI gene present, with the seven highest affinity antibodies using Vkappa domains encoded by the O18 gene segment. A 300-fold difference in affinity was observed between the 12 antibodies, and homology modeling demonstrated a correlation between electrostatic surface potential of the antigen-binding site and affinity for HLA. Overlap between the T-cell receptor-binding site and that of the antibodies was indicated by inhibition of cytotoxic T-lymphocyte killing of peptide-pulsed target cells. A model of antibody binding to HLA-A2 suggested contact with both alpha helices of the HLA molecule, such that the antigen-binding site spans the peptide-binding groove. These data increase the understanding of antibody recognition of HLA and may facilitate the production of clonotypic antibodies with peptide-specific binding.

Antibodies, Monoclonal↗
PubMed2003

Collaborative studies to establish the first WHO Reference Reagent for detection of human antibody against human platelet antigen-5b.

BACKGROUND AND OBJECTIVES: This report describes the production of a freeze-dried preparation of pooled human plasma, coded 99/666, containing immunoglobulin G (IgG) antibodies against human platelet antigen 5b (HPA-5b). MATERIALS AND METHODS: The material is intended for use as a minimum sensitivity reagent in the assays currently used for detection of antibodies to HPA-5b. Laboratories can use it to assess the sensitivity of their 'in-house' assays for antibodies to HPA-5b and to calibrate local controls for routine use in each batch of tests. RESULTS: Two collaborative studies demonstrated that the two candidate materials contained antibodies to HPA-5b and that there were no other HPA or human leucocyte antigen (HLA) antibodies which might confuse the detection of antibodies to HPA-5b. The two samples were pooled and freeze-dried in 1-ml ampoules. CONCLUSIONS: The minimum dilution of the antibody against 5b required to yield a positive result was determined, by two international collaborative studies involving a total of 49 laboratories in 26 countries, to be 1 in 2.

Antibodies, Monoclonal↗
PubMed2002

Genotyping for platelet-specific antigens: techniques for the detection of single nucleotide polymorphisms.

Accurate typing of patients for platelet-specific (human platelet) antigens (HPA) is required in several different clinical situations, and blood services need to maintain panels of HPA-typed apheresis platelet donors and whole-blood donors to support HPA alloimmunized patients. Six clinically relevant HPA alloantigen systems have been described and, in addition, a significant number of HPA alloantigens with a highly skewed allele frequency or of very low immunogenicity have been reported. Certain well-characterized biallelic systems such as Gov have not as yet been included in the HPA nomenclature but are included in this review. Biochemical studies have identified the platelet membrane proteins on which the HPA antigens are localized. Cloning of the genes encoding these proteins and the realization that there is adequate mRNA in fresh platelets has led to identification of the molecular basis of HPA antigens over the last decade. All but one of the biallelic platelet-specific alloantigen systems are based on a single nucleotide polymorphism in the DNA sequence, corresponding to a single amino acid substitution in the encoded primary protein sequence. The discovery of the genetic basis of the alloantigens has allowed the development of polymerase chain reaction-based techniques for HPA genotyping using genomic DNA. The genetic basis of the HPA alloantigens, the most commonly used genome typing techniques and their pitfalls, and future developments, are discussed in this review.

Antigens, Human Platelet↗
PubMed2001

Expression of the amino-terminal domain of platelet glycoprotein Ib alpha: exploitation of a calmodulin tag for determination of its functional activity.

Platelet glycoprotein (GP) Ibalpha is a component of the GPIb-IX receptor complex, which is involved in multiple physiological and pathological processes, including platelet adhesion at sites of vascular injury, thrombin binding, Bernard-Soulier syndrome, platelet-type von Willebrand disease, and immune-mediated thrombocytopenias. The amino-terminal domain of approximately 300 residues of GPIbalpha mediates both normal biological function (by providing the sites for direct ligand interaction) and aberrant function (through amino acid substitutions). To investigate the molecular interactions mediated by this region of GPIbalpha, we have developed a recombinant baculovirus to facilitate its expression as a calmodulin fusion protein from insect cells. By employing the calmodulin tag, the fusion protein could be obtained at >90% purity after a single isolation step at yields of 8 mg/L of insect cell medium (purified fusion protein). The recombinant GPIbalpha fragment was shown to be posttranslationally sulfated and glycosylated, although its glycosylation differed from that of the equivalent GPIbalpha fragment isolated from human platelets. The differential glycosylation, however, did not affect the function of the recombinant GPIbalpha fragment in either von Willebrand factor (vWf) or thrombin binding as these were both found to be identical to those of the same-length GPIbalpha fragment derived from human platelets. The calmodulin tag was also exploited in the development of assays to measure directly vWf and thrombin binding, since it did not interfere with either, demonstrating the feasibility for the use of this soluble receptor fusion protein in detailed biophysical assays to investigate the molecular mode of binding of platelet glycoprotein Ibalpha to these ligands.

Animals↗
PubMed2001

Provision of platelet support for fetuses and neonates affected by severe fetomaternal alloimmune thrombocytopenia.

Severe fetomaternal alloimmune thrombocytopenia requires urgent treatment with compatible platelet concentrates. As prompt treatment is sometimes delayed owing to the unavailability of compatible platelets, we established an accredited platelet donor panel to provide effective and timely transfusion support for fetal and neonatal therapy. After a mass screening programme of over 60,000 blood donations, 45 HPA-1a-negative donors with no antibodies to HPA, HLA, red cell antigens and granulocytes/lymphocytes, and with low titre anti-A and/or -B were accredited. All accredited donors were fully genotyped for HPA-1, -2, -3 and -5 by PCR-SSP. Ninety-one per cent of the accredited donors were also negative for HPA-5b.

Antigens, Human Platelet↗
PubMed2000

Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes.

A serious complication in hemophilia care is the development of factor VIII (FVIII) neutralizing antibodies (inhibitors). The authors used V gene phage display technology to define human anti-FVIII antibodies at the molecular level. The IgG4-specific, variable, heavy-chain gene repertoire of a patient with acquired hemophilia was combined with a nonimmune, variable, light-chain gene repertoire for display as single-chain variable domain antibody fragments (scFv) on filamentous phage. ScFv were selected by 4 rounds of panning on immobilized FVIII light chain. Sequence analysis revealed that isolated scFv were characterized by V(H) domains encoded by germline genes DP-10, DP-14, and DP-88, all belonging to the V(H)1 gene family. All clones displayed extensive hypermutation and were characterized by unusually long CDR3 sequences of 20 to 23 amino acids. Immunoprecipitation revealed that all scFv examined bound to the C2 domain of FVIII. Furthermore, isolated scFv competed with an inhibitory murine monoclonal antibody for binding to the C2 domain. Even though scFv bound FVIII with high affinity, they did not inhibit FVIII activity. Interestingly, the addition of scFv diminished the inhibitory potential of patient-derived antibodies with C2 domain specificity. These results suggest that the epitope of a significant portion of anti-C2 domain antibodies overlaps with that of the scFv isolated in this study. (Blood. 2000;95:558-563)

Adult↗
PubMed2000

Threonine-145/methionine-145 variants of baculovirus produced recombinant ligand binding domain of GPIbalpha express HPA-2 epitopes and show equal binding of von Willebrand factor.

Glycoprotein (GP) Ibalpha is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex, with the von Willebrand factor (vWF) binding site residing on the amino-terminus. A threonine for methionine-145 replacement of GPIbalpha is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIbalpha were expressed as calmodulin fusion proteins in insect cells. Both recombinant proteins were recognized by their respective alloantibodies, independent of glycosylation or intactness of disulfide bonds, and gave similar results to platelet-derived GPIbalpha in antibody detection assays. Resonant mirror studies showed that vWF binding was not affected by the HPA-2 mutation; however, vWF binding was partially inhibited by IgG HPA-2 antibodies. Our data are compatible with an involvement of the leucine-rich repeat domain of GPIbalpha in vWF binding and indicate that recombinant GPIbalpha may be used to detect HPA-2 antibodies. (Blood. 2000;95:205-211)

Amino Acid Substitution↗
PubMed2000

The isolation and characterisation of human monoclonal HLA-A2 antibodies from an immune V gene phage display library.

Molecular cloning techniques and V gene phage display have revolutionised the production of human monoclonal antibodies. Antibodies of a defined specificity can be obtained by selecting phage display libraries on antigen in a process known as panning. We have applied these techniques to the isolation of three HLA-A2-specific single chain variable domain fragments (scFv) from a patient alloimmunised by blood transfusion. Analysis of specificity with cells of HLA genotyped donors revealed the following: i) in addition to the major reactivity with HLA-A2, cross-reactivity with the HLA-A28 epitope; and ii) inhibition of scFv binding to the antigen by the patients' antibodies. The heavy chain variable genes of all three were derived from the germline gene Cos-3, carry the hallmarks of somatic hypermutation, and are most likely derived from clonally related B cells. The light chain variable domains were encoded by DPK1 and DPK8 from the VkappaI family. These data show that phage display can be used to clone HLA-specific alloantibodies that recognise the native antigen from alloimmunised patients.

Amino Acid Sequence↗
PubMed2000

A rapid one-stage whole-blood HPA-1a phenotyping assay using a recombinant monoclonal IgG1 anti-HPA-1a.

Severe neonatal alloimmune thrombocytopenia and patients with HPA-1a-specific antibodies require transfusion of HPA-1a-negative platelets. Identifying HPA-1a-negative donors requires simple and reliable typing methods. Most existing techniques use polyclonal antibodies, are time consuming and involve platelet isolation. We have used a horseradish peroxidase (HRP)-conjugated recombinant IgG1 anti-HPA-1a (CAMTRAN007) to develop a rapid and reliable enzyme-linked immunosorbent assay (ELISA), which eliminates sample preparation and reduces the incubation and wash steps associated with traditional sandwich ELISAs. The assay uses simultaneous incubation of the monoclonal antibody RFGP56 to capture GPIIbIIIa from whole blood and the recombinant IgG1 antibody to detect captured HPA-1a antigen. It allows 96 samples to be typed in less than 1 h and can be used on stored samples. Initial testing of 85 samples of known HPA-1a genotype demonstrated that HPA-1a-negative samples had OD values of < 0.266, whereas HPA-1a-positive samples had OD values of > 0.6. Testing of 1862 random donor samples in two blood centres confirmed these OD cut-off values and identified 45 HPA-1a-negative samples (2.4%), all except one giving OD values of < 0.2. The remaining HPA-1a-negative sample had an OD value of 0.303. The HPA-1a status on all the negative samples and an equivalent number of randomly selected positive samples was confirmed by flow cytometry and polymerase chain reaction with sequence-specific primers (PCR- SSP).

Antigens, Human Platelet↗
PubMed2000

Detection of Gov system antibodies by MAIPA reveals an immunogenicity similar to the HPA-5 alloantigens.

The glycosylphosphatidylinositol-linked platelet protein CD109 carries the biallelic alloantigen system Gov. There is limited information on the incidence of Gov alloantibodies in neonatal alloimmune thrombocytopenia (NAITP), post-transfusion purpura (PTP) and platelet refractoriness. We adapted the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay to the detection of Gov antibodies and determined their incidence in 605 archived samples (112 with HPA antibodies) referred for the aforementioned conditions. Here, we show that CD109 expression was reduced upon platelet storage in saline or by cryopreservation, but was stable when stored as whole blood or therapeutic platelet concentrate. Fourteen of the 605 samples contained Gov alloantibodies (anti-Gova, n = 10; anti-Govb, n = 4), with the majority in platelet refractoriness (n = 9) and, of the remaining five, four in NAITP and one in PTP. In seven cases, no other HPA antibodies were detected, three being NAITP cases. The incidence of Gov antibodies was significantly lower than HPA-1 system antibodies (n = 87), but equalled the number of HPA-5 system antibodies (n = 14) and outnumbered HPA-2 and -3 system antibodies (10 altogether).

Alleles↗
PubMed2000

Introduction to antibody engineering and phage display.

The vertebrate immune system, capable of rapidly producing highly specific antibodies upon immunisation, has been used to produce murine monoclonal antibodies (mAbs) via immortalisation and isolation of antibody-secreting cells. These mAbs have had a profound impact in the fields of diagnostics and therapeutics. However, the therapeutic use of murine mAbs is complicated by their immunogenicity. To circumvent this immunogenicity, antibody engineering techniques which render murine mAbs more compatible with the human immune system have been devised. Over the last decade, the technique of antibody phage display, which facilitates the production of human mAbs, has been developed. This review serves as an introduction to antibody engineering, phage display and the development of antibody fragments into viable diagnostic and therapeutic reagents.

Animals↗