A. Rankin

Loss of function of a lupus-associated FcγRIIb polymorphism through exclusion from lipid rafts

Dysfunction of receptors for IgG (FcγRs) has been thought to be involved in the pathogenesis of systemic lupus erythematosus (SLE). We show that a recently described SLE-associated polymorphism of FcγRIIb (FcγRIIbT232), encoding a single transmembrane amino acid substitution, is functionally impaired. FcγRIIbT232 is unable to inhibit activatory receptors because it is excluded from sphingolipid rafts, resulting in the unopposed proinflammatory signaling thought to promote SLE.

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

Summary.  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 [Ca2+]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 (r2 = 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 ∼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.

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

Summary.  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.

Severe fetomaternal alloimmune thrombocytopenia due to anti-human platelet antigen (HPA)-1a in a mother with a rare and silenced ITGB3*0101 (GPIIIa) allele

Background and Objectives  Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal antibodies against a human platelet antigen (HPA) present on fetal, but absent from maternal platelets. We identified and characterized a case of FMAIT due to anti‐HPA‐1a in a mother with an HPA‐1a1b genotype.

Platelet biology: collagen activation mechanisms and their relevance to transfusion medicine

Platelets are the second most important cellular product issued by the National Blood Service of England. Per annum ∼ 220 000 adult units are prescribed with ∼ 50% for the prophylactic treatment to reduce the risk of bleeding in haemato-oncology and the other half for the treatment of bleeding in patients with a wide range of conditions. The trigger for prophylactic platelet transfusion is 10 × 10 9 /l. A further lowering of the trigger to 5 × 10 9 /l has been considered but there are concerns about a possible increase in the risk of severe bleeding. It would be attractive to use differential transfusion triggers informed by the patients’ estimated risk for bleeding at a low platelet count. Patients who are predetermined to ‘easy bleeding’ could be transfused at a higher trigger than patients who have a more robust haemostasis. We postulate that an individual’s bleeding tendency at a low platelet count is to a large extent genetically determined. Although studies on the heritability of ‘global haemostasis’ are lacking, there is evidence from the Framingham study that the response of platelets to activation by agonist is in part genetically controlled. With the completion of the sequencing of the human genome it becomes feasible to answer the question to which extent the risk of bleeding on the one hand and the risk of atherothrombosis on the other is genetically determined. The capacity to sequence large numbers of genes to define common sequence variation together with the availability of new genotyping platforms to interrogate DNA samples for thousands of single nucleotide polymorphisms (SNPs) in a single test provides unique opportunities for exciting clinical studies on the relation between sequence variation and haemostasis. Here we review the way we are conducting such studies using the interaction of platelets and collagen as a model.

SI09 Analysing Platelet Function in Blood Donors: Selection of Subjects for Functional Genetic Studies

Introduction  The objective of this study; performed as part of the BLOODOMICS project, was to develop a methodology to characterise the platelet response phenotype following agonist induced activation, which would then be used to test a cohort of healthy blood donors.

P03 The Effect of Apheresis on the Donors’ Platelets

Introduction  Many studies have investigated the effects of donation processes on the activation state of platelet concentrates. Apheresis platelets are often found to be the least activated products. However, little is known about the immediate or long‐term effects of frequent apheresis on donors’ platelets. This question was addressed in a cohort of donors in the BLOODOMICS research project, comprising 157 whole blood donors who had donated twice on average (range 1–4), and 349 apheresis donors with an average of 10 donations (range 1–25), in the previous year.

P49 First UK Report of a Case of Glycoprotein VI Deficiency in a 33‐Year‐Old Patient with Moderate Thrombocytopenia and Autoantibodies Against the Immunoglobulin‐Like Domains of the Receptor

Background  Studies of cases of acquired and inherited GPVI deficiency have made critical contributions to the understanding of the pivotal role of GPVI in collagen mediated platelet signaling.

P47 Inherited Familial Thrombocytopenia due to an Autosomal Dominant Mutation in the Cytoplasmic Domain of Platelet β3-Integrin that is Associated with the Expression of Activation-Dependent Epitopes

Background  Glanzmanns Thrombasthenia (GT) and Bernard Soulier Syndrome (BSS) are the two most common inherited forms of thrombasthenia. They are caused by alterations in expression or function of integrin alphaIIb/β3 or glycoprotein (GP) Ib/IX/V respectively. We identified an individual with chronic mild macrothrombocytopenia (range 59–100 × 109 L−1) but no overt bleeding diathesis. The propositus was found to have atypical binding of monoclonal antibodies (mAb) to β3 integrin and increased expression of GPIbVIX.