Platelet responses to agonists in a cohort of highly characterised platelet donors are consistent over time.

BACKGROUND AND OBJECTIVES: Platelet function shows significant inheritance that is at least partially genetically controlled. There is also evidence that the platelet response is stable over time, but there are few studies that have assessed consistency of platelet function over months and years. We aimed to measure platelet function in platelet donors over time in individuals selected from a cohort of 956 donors whose platelet function had been previously characterised. MATERIALS AND METHODS: Platelet function was assessed by flow cytometry, measuring fibrinogen binding and P-selectin expression after stimulation with either cross-linked collagen-related peptide or adenosine 5'-diphosphate. Eighty-nine donors from the Cambridge Platelet Function Cohort whose platelet responses were initially within the lower or upper decile of reactivity were retested between 4 months and five and a half years later. RESULTS: There was moderate-to-high correlation between the initial and repeat platelet function results for all assays (P ≤ 0·007, r2 0·2961-0·7625); furthermore, the range of results observed in the initial low and high responder groups remained significantly different at the time of the second test (P ≤ 0·0005). CONCLUSION: Platelet function remains consistent over time. This implies that this potential influence on quality of donated platelet concentrates will remain essentially constant for a given donor.

Sensitivity of assays for the detection of HPA-1a antibodies: results of an international workshop demonstrating the impact of cation chelation from integrin αIIbß3 on three widely used assays.

BACKGROUND AND OBJECTIVES: HPA-1a antibodies account for 70-80% of cases of fetal-neonatal alloimmune thrombocytopenia (FNAIT) in Caucasians. However, numerous workshops have demonstrated variability in their detection. We recently showed that exposure of αIIbß3 to ethylene diamine tetraacetic acid (EDTA) affected binding of many anti-αIIbß3 monoclonal, and HPA-1a allo-, antibodies; this adversely affected sensitivity of the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay and indirect platelet immunofluorescence test (PIFT). This study presents results from an international workshop studying the impact of cation chelation on HPA-1a antibody detection in routine diagnostic laboratories. MATERIALS AND METHODS: Serum and EDTA-anticoagulated plasma samples containing anti-HPA-1a were distributed to 39 laboratories. Participants were asked to detect and identify any HPA antibodies present. RESULTS: 2/39 (5.1%) participants were able to detect and identify anti-HPA-1a in the serum, but not in the plasma sample. EDTA plasma reduced MAIPA assay sensitivity by ≥ 20% in 17/24 (70.8%) laboratories and by ≥ 50% in 9/24 (37.5%) when using HPA-1a1a platelets (mean: 27.7%, range 0-85.1%); when using HPA-1a1b platelets 3/4 (75%), participants reported ≥ 50% loss of sensitivity (mean 65.6%, range 0-96.6%). A small but significant increase in optical densities was observed in antigen capture ELISA assays when using plasma (mean difference: 0.081, P < 0.01). Insufficient PIFT data were returned to draw firm conclusions. CONCLUSION: Use of EDTA plasma significantly affects the sensitivity of the MAIPA assay and can affect detection of even potent, FNAIT-causing examples of anti-HPA-1a. These data highlight the importance of use of αIIbß3 in an appropriate conformation for the sensitive detection of anti-HPA-1a.

An International Reference Reagent (minimum sensitivity) for the detection of anti-human platelet antigen 1a.

BACKGROUND AND OBJECTIVES: The platelet-specific alloantibody anti-human platelet antigen (HPA) 1a is involved in feto-maternal alloimmune thrombocytopenia, post-transfusion purpura and platelet refractoriness. The existing minimum potency preparation for the detection of anti-HPA-1a (NIBSC code 93/710) was established by World Health Organization in 1997 and is used by laboratories to validate new assays or to calibrate 'in-house' controls. However, it has been well-used and a replacement is required. This report describes the production and comparative evaluation of a freeze-dried preparation of pooled human plasma, coded 05/106, containing anti-HPA-1a. MATERIALS AND METHODS: Plasma containing anti-HPA-1a was obtained and 2974 1-ml aliquots were prepared and freeze-dried in glass ampoules. In order to characterize the material and compare it to the existing reference material, three collaborative studies were organized, involving a total of 50 different laboratories in 23 countries. RESULTS: As expected only anti-HPA-1a could be detected in the plasma and no additional HPA or human leucocyte antigen antibodies were detected. When tested in titration, there was a wide variation in the sensitivity of antibody detection by different laboratories, irrespective of the technique used. However, there was no significant difference between the two materials when compared using a t-test. CONCLUSIONS: When diluted 1 in 2, most laboratories were able to detect the presence of anti-HPA-1a in both materials and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. In October 2007, the World Health Organization Expert Committee on Biological Standardization approved the material 05/106 as an International Reference Reagent.

NOX1 loss-of-function genetic variants in patients with inflammatory bowel disease.

Genetic defects that affect intestinal epithelial barrier function can present with very early-onset inflammatory bowel disease (VEOIBD). Using whole-genome sequencing, a novel hemizygous defect in NOX1 encoding NAPDH oxidase 1 was identified in a patient with ulcerative colitis-like VEOIBD. Exome screening of 1,878 pediatric patients identified further seven male inflammatory bowel disease (IBD) patients with rare NOX1 mutations. Loss-of-function was validated in p.N122H and p.T497A, and to a lesser degree in p.Y470H, p.R287Q, p.I67M, p.Q293R as well as the previously described p.P330S, and the common NOX1 SNP p.D360N (rs34688635) variant. The missense mutation p.N122H abrogated reactive oxygen species (ROS) production in cell lines, ex vivo colonic explants, and patient-derived colonic organoid cultures. Within colonic crypts, NOX1 constitutively generates a high level of ROS in the crypt lumen. Analysis of 9,513 controls and 11,140 IBD patients of non-Jewish European ancestry did not reveal an association between p.D360N and IBD. Our data suggest that loss-of-function variants in NOX1 do not cause a Mendelian disorder of high penetrance but are a context-specific modifier. Our results implicate that variants in NOX1 change brush border ROS within colonic crypts at the interface between the epithelium and luminal microbes.

NH2-terminal globular domain of human platelet glycoprotein Ib alpha has a methionine 145/threonine145 amino acid polymorphism, which is associated with the HPA-2 (Ko) alloantigens.

The glycoprotein (GP) Ib/IX complex, a prominent platelet GP complex, is the primary receptor for vWF. Previously, we have established that an antigenic polymorphism of platelets, the HPA-2 or Ko alloantigen system, is located on the 45-kD amino-terminal globular domain of GPIb alpha. With the polymerase chain reaction, we have amplified two segments of the GPIb alpha gene coding for the first 382 amino acids of two HPA-2a and two HPA-2b homozygous individuals. Nucleotide sequence analysis revealed as the only difference a C-T polymorphism at position 434 of the coding region for the mature protein. This base change results in a substitution of threonine (ACG) in HPA-2a (Kob) to methionine (ATG) in HPA-2b (Koa) at amino acid position 145. The C-T polymorphism is reflected in a difference in restriction enzyme recognition, resulting in an Aha 2-site in the HPA-2b allele and a SfaN1 site in the HPA-2a allele. Restriction fragment length polymorphism analysis of the amplified DNA of 3 HPA-2(a-,b+), 2 HPA-2(a+,b+), and 11 HPA-2(a+,b-) donors showed that these restriction sites were associated with the HPA-2 alleles. DNA-typing for the HPA-2 alloantigen system on genomic DNA obtained from a small number of cells may be applied for determining the genotype of a fetus from an immunized mother or of severely thrombocytopenic patients.

Platelet genomics and the risk of atherothrombosis.

Platelets play a pivotal role in atherothrombosis after coronary artery plaque rupture. The extent of response of platelets to such an event varies between individuals. This variation is for a large extent genetically controlled. A comprehensive study of sequence variation that modifies the platelet response to agonists is, however, lacking. We set out to discover the regulatory nodes of platelet function by an integrated systems biology approach. The high density genotyping of 110 genes in a cohort of more than 500 individuals, in whom the platelet response to ADP and collagen-related peptide was determined, allowed the robust definition of the first set of regulatory nodes. Microarray and proteomics studies on platelets from individuals with a so-called 'extreme end' response phenotype provided further insight into key regulators of platelet function. In addition, the completion of the HapMap project allows the comprehensive surveying of the genome for sequence variation by the testing of a limited number of single nucleotide polymorphisms (SNPs). With the advent of high density (i.e. 500,000 SNPs) genotyping arrays large number of case and control samples can be tested at an affordable cost. The recently completed Wellcome Trust Case Control Consortium (WTCCC) study has allowed us to address the question of whether common sequence variation confers risk for seven common diseases, one being myocardial infarction. The results of the WTCCC genome-wide association study and issues of case-control study design, particularly the selection of suitable controls, will be reviewed. In conclusion the integration of the results from the platelet systems biology study with those of the WTCCC project enhances our understanding of the mechanisms underlying common conditions such as atherothrombosis and provides pointers to novel cellular mechanisms and pathways.

Mapping the platelet profile for functional genomic studies and demonstration of the effect size of the GP6 locus.

BACKGROUND: Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response. OBJECTIVE: To develop a methodology suitable for measuring signaling pathway-specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene. METHODS: Three established platelet assays were evaluated: mobilization of [Ca(2+)](i), aggregometry and flow cytometry, each in response to adenosine 5'-diphosphate (ADP) or the glycoprotein (GP) VI-specific crosslinked collagen-related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter-individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways. RESULTS: Individuals were identified who were hypo- or hyper-responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r(2) = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall. The effect of sequence variation at the GP6 locus accounted for approximately 35% of the variation in the CRP-XL response. CONCLUSION: Genotyping-phenotype association studies in a well-characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.

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.

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.

Leucine33-proline33 substitution in human platelet glycoprotein IIIa determines HLA-DRw52a (Dw24) association of the immune response against HPA-1a (Zwa/PIA1) and HPA-1b (Zwb/PIA2).

Alloantibody formation against HPA-1a (Zwa/PIA1) has, to date, only been found in HLA-DRw52(a+) (Dw24) individuals. Alloimmunization against the product of the other HPA-1 allele, HPA-1b, is rare. We have been able to evaluate ten cases of HPA-1b alloimmunization in Europe in order to study whether there is an association between HLA phenotype and anti-HPA-1b antibody formation. HLA typing of these patients was performed with particular attention to the DRw52a specificity using specific T-cell clones. No association with DRw52a or any other known HLA phenotype was found. This finding implies that the amino acid substitution leucine33-proline33 in GPIIIa, responsible for HPA-1a/b, is of primary importance for the association of anti-HPA-1a antibody formation with DRw52a. These data show that the amino acid polymorphism affects the presentation of the immunogenic oligopeptides of HPA-1a and -1b in the HLA class-II groove.

A fatal case of autoimmune thrombocytopenia with an IgM anti-GPIb/IX following one antigen mismatched unrelated donor bone marrow transplantation.

We report the case of a 32-year-old patient with ALL who developed autoimmune thrombocytopenia 2 months following allogeneic bone marrow transplantation. An IgM autoantibody against the platelet glycoprotein Ib/IX complex was observed. Treatment with high-dose steroids and intravenous immunoglobulin G failed to produce any benefit and the thrombocytopenia led to fatal gastrointestinal haemorrhage. The possible factors contributing to post-allograft thrombocytopenia and potential management strategies are discussed.

Human antibody fragments specific for human blood group antigens from a phage display library.

We have isolated human antibody fragments with binding specificities against the blood group antigens of the ABO and I blood group systems (B and HI), of the Rh system (D and E) and of the Kell system (Kpb), by selecting a phage-antibody library on human red cells. The fragments, expressed in bacteria, were antigen-specific and effective in assays including agglutination and immunohistochemistry. Selection of phage-antibody libraries using intact cells seems to offer a promising alternative to hybridoma technology for the production of antibodies against cell surface molecules.

Inhibition of MT1-MMP activity using functional antibody fragments selected against its hemopexin domain.

The membrane associated MMP, MT1-MMP, is a critical pericellular protease involved in tumour cell invasion and angiogenesis and is highly up-regulated in numerous human cancers. It therefore represents an exciting new therapeutic cancer-specific target. We have generated recombinant human scFv antibodies against the non-catalytic, hemopexin domain of MT1-MMP that modulate its interactions with collagen. One of these is an effective inhibitor of the invasive capacity of cancer cells and of angiogenesis in model systems. This demonstrates that targeting sites outside the catalytic domain presents a potential novel approach to proteinase inhibition that could have applications in cancer therapeutics.

The discovery of genes implicated in myocardial infarction.

The era of Genome-Wide Association Studies (GWAS) commenced in 2007 with the study of the Wellcome Trust Case Control Consortium (WTCCC) which for the first time ever showed that risk loci can be identified by scanning the complete genome for sequence variation in large numbers of cases of disease and healthy controls. We and others have expanded on this effort and successfully identified the first 11 risk loci for myocardial infarction (MI) and coronary artery disease (CAD). Studies on quantitative traits provide an alternative approach to identify MI/CAD risk loci. This review captures the early successes in the emerging field of disease genomics.

Overexpression of the partially activated alpha(IIb)beta3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet-like extensions in Chinese hamster ovary cells.

BACKGROUND: We have recently reported a novel mutation in the beta3 subunit of the platelet fibrinogen receptor (alpha(IIb)beta3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. RESULTS: Here we demonstrate that the partially activated alpha(IIb)beta3D723H or alpha(IIb)beta3D723A salt bridge mutants, but not fully activated alpha(IIb)beta3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of alpha(IIb)beta3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant alpha(IIb)beta3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type alpha(IIb)beta3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO alpha(IIb)beta3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. CONCLUSIONS: CHO alpha(IIb)beta3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions.

Three novel beta3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency beta3-HPA antibodies.

BACKGROUND: Antibodies against human platelet antigens (HPA) are clinically important in fetal-maternal alloimmune thrombocytopenia, refractoriness to platelet transfusions and post-transfusion purpura. Of the 16 HPAs, nine are located on the beta3 subunit of the alphaIIb beta3 integrin. Antibody detection is generally based on platelet-derived alphaIIb beta3 from HPA-genotyped donors. Recombinant allelic beta3 peptides, expressed at high levels would improve consistency in antibody detection, but the expression of soluble and monomeric integrins expressing complex dependent epitopes has previously proved challenging. OBJECTIVES: We aimed to generate three recombinant beta3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency beta3 alloantigens. METHODS: The removal of the specificity-determining loop from the betaA domain and fusion of truncated beta3 to calmodulin was exploited to obtain expression of monomeric protein. Using site-directed mutagenesis, the mutations for HPA-4b and HPA-8bw were introduced in the ITGB3*001 haplotype. A third peptide for the detection of antibodies against HPA coded by non-synonymous single nucleotide polymorphisms of low frequency was generated by the introduction of five mutations forming the basis of HPA-6bw, -7bw, -10bw, -11bw, and -16bw antigens. RESULTS: Reactivity of the three peptides with beta3-specific murine monoclonal antibodies and human HPA-1a phage antibodies confirmed the structural integrity of the recombinant fragments, and reactivity with a unique panel of polyclonal anti-HPA sera confirmed expression of the relevant HPA epitopes. CONCLUSIONS: These data demonstrate that beta3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.