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BACKGROUND Leukoreduction of blood components was implemented to reduce transfusion‐associated risks. The detection level for residual white blood cells (rWBCs) required to demonstrate leukoreduction was originally considered too low for hematology analyzers. Developments enabling cell counts in body fluids have, however, renewed interest in rWBC counting. An assessment of Sysmex XN hematology analyzers with software offering automated rWBC enumeration intended for use on blood components was performed. STUDY DESIGN AND METHODS Performance characteristics were determined using platelet, red blood cell (RBC), and plasma samples spiked with WBCs. Subsequently, components (platelets, n = 1367; and plasma, n = 80) were tested and results compared with flow cytometry, to monitor leukoreduction efficiency to a level of less than 1 × 106/unit. Components identified by flow cytometry as having poor leukoreduction, exceeding this limit, were also tested (platelets, n = 3; and RBCs, n = 10). RESULTS Linearity studies up to 32 WBCs/μL showed good correlation between observed and expected results (R2 > 0.9996). Precision analysis gave an average limit of quantitation of 2 WBCs/μL with coefficients of variation less than 20%. Average carryover was 0.1%. Plain sample tubes were a source of aberrant results with routine components. Using ethylenediaminetetraacetic acid tubes the analyzer gave results greater than 1 × 106/unit in 2.7% of cases compared with 1.4% by flow cytometry, but overall results were within specification, with more than 90% of components having rWBC values below the limit. All incidences of poor leukoreduction, with flow cytometry results greater than 13 rWBCs/μL were correctly identified, with an excellent correlation between results (R2 = 0.9818). CONCLUSION The analyzer demonstrated acceptable performance characteristics for enumeration of rWBCs; consequently, additional multisite evaluations are warranted., http://onlinelibrary.wiley.com/doi/10.1111/trf.15642/full

Aims To determine the effects of in vivo S-nitrosoglutathione (GSNO) infusion on cardiovascular function, platelet function, proteinuria and biomarker parameters in early-onset pre-eclampsia. Methods We performed an open-label dose-ranging study of GSNO in early-onset pre-eclampsia. Six women underwent GSNO infusion whilst receiving standard therapy. The dose of GSNO was increased incrementally to 100 μg min−1 whilst maintaining blood pressure of >140/80 mmHg. Aortic augmentation index, aortic pulse wave velocity, blood pressure and maternal–fetal Doppler parameters were measured at each dose. Platelet P-selectin, protein-to-creatinine ratio and soluble anti-angiogenic factors were measured pre- and postinfusion. Results Augmentation index fell at 30 μg min−1 S-nitrosoglutathione (−6%, 95% confidence interval 0.6 to 13%), a dose that did not affect blood pressure. Platelet P-selectin expression was reduced [mean (interquartile range), 6.3 (4.9–7.6) vs. 4.1 (3.1–5.7)% positive, P = 0.03]. Soluble endoglin levels showed borderline reduction (P = 0.06). There was a borderline significant change in pre-to-postinfusion protein-to-creatinine ratio [mean (interquartile range), 0.37 (0.09–0.82) vs. 0.23 (0.07–0.49) g mmol−1, P = 0.06]. Maternal uterine and fetal Doppler pulsatility indices were unchanged. Conclusions In early-onset pre-eclampsia, GSNO reduces augmentation index, a biomarker of small vessel tone and pulse wave reflection, prior to affecting blood pressure. Proteinuria and platelet activation are improved at doses that affect blood pressure minimally. These effects of GSNO may be of therapeutic potential in pre-eclampsia, a condition for which no specific treatment exists. Clinical studies of GSNO in early-onset pre-eclampsia will determine whether these findings translate to improvement in maternal and/or fetal outcome.

Background Studies show that 1 in 1200 neonates have a low platelet (PLT) count due to alloimmunization against human PLT antigen (HPA)-1a (β3-L33). This mainly occurs in HPA-1a–negative mothers who are positive for the human leukocyte antigen (HLA)-DRB3*01:01 allele, but only about one-third of cases will mount an effective alloimmune response. The development of specific treatment modalities requires that the mechanisms driving the maternal alloimmune response against the fetal PLTs be further explored. An antibody reagent that has a different binding affinity to HLA-DRA/DRB3*01:01 with and without the β3-L33 peptide would be a valuable reagent to study peptide presentation on maternal antigen-presenting cells. Study Design and Methods To identify such antibodies, HLA-DRA/DRB3*01:01 was recombinantly expressed in Drosophila S2 cells. To delineate the epitope of interesting antibodies, seven mutant HLA-DRA/DRB3*01:01 molecules were generated by site-directed mutagenesis introducing naturally occurring amino acid changes encoded by DRB3*02 and DRB3*03 alleles. Results The murine monoclonal antibody (MoAb) DA2 showed robust binding by enzyme-linked immunosorbent assay to recombinant HLA-DRA/DRB3*01:01, but binding was reduced in the presence of β3-L33 peptide. The binding affinity of DA2 to the mutant HLA-DRA/DRB3*0101 in which serine at Position 60 of the β1-chain was replaced by tyrosine was greatly enhanced. Interestingly the binding of DA2 to the mutant was not reduced by the presence of β3-L33 peptide. Conclusion The results of this study generate a molecular model of the interaction of the HLA-DRA/DRB3*01:01 molecule with MoAb DA2. This will inform functional studies with the recombinant Class II molecules.

Fetomaternal alloimmune thrombocytopenia, caused by the maternal generation of antibodies against fetal human platelet antigen-1a (HPA-1a), can result in intracranial hemorrhage and intrauterine death. We have developed a therapeutic human recombinant high-affinity HPA-1a antibody (B2G1Δnab) that competes for binding to the HPA-1a epitope but carries a modified constant region that does not bind to Fcγ receptors. In vitro studies with a range of clinical anti–HPA-1a sera have shown that B2G1Δnab blocks monocyte chemiluminescence by >75%. In this first-in-man study, we demonstrate that HPA-1a1b autologous platelets (matching fetal phenotype) sensitized with B2G1Δnab have the same intravascular survival as unsensitized platelets (190 hours), while platelets sensitized with a destructive immunoglobulin G1 version of the antibody (B2G1) are cleared from the circulation in 2 hours. Mimicking the situation in fetuses receiving B2G1Δnab as therapy, we show that platelets sensitized with a combination of B2G1 (representing destructive HPA-1a antibody) and B2G1Δnab survive 3 times as long in circulation compared with platelets sensitized with B2G1 alone. This confirms the therapeutic potential of B2G1Δnab. The efficient clearance of platelets sensitized with B2G1 also opens up the opportunity to carry out studies of prophylaxis to prevent alloimmunization in HPA-1a–negative mothers.

The effect of variation in platelet function in platelet donors on patient outcome following platelet transfusion is unknown. This trial assessed the hypothesis that platelets collected from donors with highly responsive platelets to agonists in vitro assessed by flow cytometry (high-responder donors) are cleared more quickly from the circulation than those from low-responder donors, resulting in lower platelet count increments following transfusion. This parallel group, semirandomized double-blinded trial was conducted in a single center in the United Kingdom. Eligible patients were those 16 or older with thrombocytopenia secondary to bone marrow failure, requiring prophylactic platelet transfusion. Patients were randomly assigned to receive a platelet donation from a high- or low-responder donor when both were available, or when only 1 type of platelet was available, patients received that. Participants, investigators, and those assessing outcomes were masked to group assignment. The primary end point was the platelet count increment 10 to 90 minutes following transfusion. Analysis was by intention to treat. Fifty-one patients were assigned to receive platelets from low-responder donors, and 49 from high-responder donors (47 of which were randomized and 53 nonrandomized). There was no significant difference in platelet count increment 10 to 90 minutes following transfusion in patients receiving platelets from high-responder (mean, 21.0 × 109/L; 95% confidence interval [CI], 4.9-37.2) or low-responder (mean, 23.3 × 109/L; 95% CI, 7.8-38.9) donors (mean difference, 2.3; 95% CI, -1.1 to 5.7; P = .18). These results support the current policy of not selecting platelet donors on the basis of platelet function for prophylactic platelet transfusion.

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.\ud Materials and Methods Platelet function was assessed by flow cytometry, measuring fibrinogen binding and P-selectin expression after stimulation with either\ud cross-linked collagen-related peptide or adenosine 50-diphosphate. Eighty-nine donors from the Cambridge Platelet Function Cohort whose platelet responses\ud were initially within the lower or upper decile of reactivity were retested between 4 months and five and a half years later.\ud 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).\ud 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.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) inhibits platelet response to collagen and may also inhibit two other major platelet agonists ADP and thrombin although this has been less well explored. We hypothesized that the combined effect of inhibiting these three platelet activating pathways may act to significantly inhibit thrombus formation. We demonstrate a negative relationship between PECAM-1 surface expression and platelet response to cross-linked collagen related peptide (CRP-XL) and ADP, and an inhibitory effect of PECAM-1 clustering on platelet response to CRP-XL, ADP and thrombin. This combined inhibition of multiple signaling pathways results in a marked reduction in thrombus formation.

Platelet response to activation varies widely between individuals but shows interindividual consistency and strong heritability. The genetic basis of this variation has not been properly explored. We therefore systematically measured the effect on function of sequence variation in 97 candidate genes in the collagen and adenosine-diphosphate (ADP) signaling pathways. Resequencing of the genes in 48 European DNA samples nearly doubled the number of known single nucleotide polymorphisms (SNPs) and informed the selection of 1327 SNPs for genotyping in 500 healthy Northern European subjects with known platelet responses to collagen-related peptide (CRP-XL) and ADP. This identified 17 novel associations with platelet function (P < .005) accounting for approximately 46% of the variation in response. Further investigations with platelets of known genotype explored the mechanisms behind some of the associations. SNPs in PEAR1 associated with increased platelet response to CRP-XL and increased PEAR1 protein expression after platelet degranulation. The minor allele of a 3′ untranslated region (UTR) SNP (rs2769668) in VAV3 was associated with higher protein expression (P = .03) and increased P-selectin exposure after ADP activation (P = .004). Furthermore the minor allele of the intronic SNP rs17786144 in ITPR1 modified Ca2+ levels after activation with ADP (P < .004). These data provide novel insights into key hubs within platelet signaling networks.

Human Domain Antibodies (dAbs) that bind to and inhibit the function of platelet glycoprotein VI (GPVI) have been isolated from phage display libraries and their efficacy demonstrated using in vitro models of platelet activation. Here we describe the properties of one such antibody, BLO8-1, which has been shown to specifically inhibit the binding of recombinant human GPVI to cross-linked collagen related peptide (CRP-XL) in vitro. BLO8-1 specifically binds to the platelet cell surface and prevents CRP-XL induced platelet aggregation in platelet-rich plasma, as well as inhibiting thrombus formation in whole blood under arterial shear conditions. Using a series of mutant GPVI molecules, BLO8-1 was shown to recognize an epitope within the collagen binding domain of GPVI, therefore the anti-thrombotic effect of this dAb is predicted to be due to direct blocking of the collagen-GPVI interaction. These data, together with the desirable properties of Domain Antibodies, show that dAbs could potentially be used to generate novel biopharmaceuticals with anti-thrombotic properties.

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin and a P53L mutation in glycoprotein (GP) Ibalpha. We show that GPIbalpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta3-H723 causes constitutive, albeit partial, activation of the alphaIIbbeta3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the alphaIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alphaIIbbeta3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alphaIIbbeta3-H723, but not wild-type alphaIIbbeta3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alphaIIbbeta3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree.

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.

We report the development of an expression system for the production of soluble, calmodulin (CaM)-tagged proteins in Drosophila Schneider S2 cells and the subsequent use of these proteins for the selection of phage displayed antibodies. The CaM-tag permitted the purification of recombinant protein to >90% purity in a single step at yields of >20 mg/l. Using platelet glycoprotein VI (GP6) as a model, we demonstrated that the recombinant CaM-tagged protein was post-translationally N-glycosylated and had identical ligand specificity to native protein. A novel selection strategy, exploiting the CaM tag, was then used to isolate four single chain Fv fragments (scFvs) specific for GP6 from a non-immune phage display library. In contrast to other selection methods, which can result in antibodies that do not recognise native protein, all of the scFvs we selected bound cell surface expressed GP6. In conclusion, the production of CaM-tagged proteins in Drosophila Schneider S2 cells and the selection strategy reported here offer advantages over previously published methods, including simple culture conditions, rapid protein purification, specific elution of phage antibodies and preferential selection of phage antibodies that recognise native, cell surface expressed protein.

Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)–like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVI-specific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled.

Glycoprotein (GP) Ib 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 GPIb is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIb 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 GPIb 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 GPIb in vWF binding and indicate that recombinant GPIb may be used to detect HPA-2 antibodies. (Blood. 2000;95:205-211)

Glycoprotein (GP) Ib 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 GPIb is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIb 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 GPIb 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 GPIb in vWF binding and indicate that recombinant GPIb may be used to detect HPA-2 antibodies. (Blood. 2000;95:205-211)

A previously transfused female patient, known to have a platelet defect, was transfused with platelets prior to surgery. After the 18th unit she felt unwell, developed fever, rigor, became nauseous, and vomited. Her blood pressure decreased from 140/90 to 80/50mm Hg. Passive transfer of donor granulocytes or red cell antibodies were excluded as a cause. Therefore, a serum sample from the patient was investigated for the presence of antibodies to human leukocyte antigens (HLA) using a standard National Institutes of Health (NIH) lymphocytotoxicity test, but antibodies were not detected. However, an extended incubation cytotoxicity test demonstrated the presence of an anti-HLA-A2, and indirect immunofluorescence flow cytometry showed the presence of an IgG1 antibody reacting with 50 percent of cells in a random pool of lymphocytes. One week later, multispecific HLA antibodies were detectable by both NIH and extended incubation cytotoxicity tests. Flow cytometry showed a 16-fold increase in the amount of IgG antibodies and the appearance of an IgM component. Such clinically important HLA antibodies can be detected by extended incubation cytotoxicity and flow cytometric assays prior to becoming reactive in a standard NIH cytotoxicity technique.

There are only a few reports of thrombocytopenia associated with clinical doses of teicoplanin, a glycopeptide antibiotic used against Gram-positive bacteria. We investigated 39 patients receiving teicoplanin; 31 were thrombocytopenic with platelet counts between 1-105 x 10(9)/l and 8 were not thrombocytopenic. We identified 14 thrombocytopenic cases (45%) and two (25%) non-thrombocytopenic cases with IgG teicoplanin-dependent platelet-reactive antibodies. Use of glycoprotein (GP) capture enzyme-linked immunosorbent assay with platelets and GPIIb/IIIa transfected Chinese Hamster Ovary cells as well as flow cytometry with GP-deficient platelets indicated that the GPIIb/IIIa complex is a major target antigen of these antibodies.

To determine the effects of in vivo S-nitrosoglutathione (GSNO) infusion on cardiovascular function, platelet function, proteinuria and biomarker parameters in early-onset pre-eclampsia.We performed an open-label dose-ranging study of GSNO in early-onset pre-eclampsia. Six women underwent GSNO infusion whilst receiving standard therapy. The dose of GSNO was increased incrementally to 100 μg min(-1) whilst maintaining blood pressure of140/80 mmHg. Aortic augmentation index, aortic pulse wave velocity, blood pressure and maternal-fetal Doppler parameters were measured at each dose. Platelet P-selectin, protein-to-creatinine ratio and soluble anti-angiogenic factors were measured pre- and postinfusion.Augmentation index fell at 30 μg min(-1) S-nitrosoglutathione (-6%, 95% confidence interval 0.6 to 13%), a dose that did not affect blood pressure. Platelet P-selectin expression was reduced [mean (interquartile range), 6.3 (4.9-7.6) vs. 4.1 (3.1-5.7)% positive, P = 0.03]. Soluble endoglin levels showed borderline reduction (P = 0.06). There was a borderline significant change in pre-to-postinfusion protein-to-creatinine ratio [mean (interquartile range), 0.37 (0.09-0.82) vs. 0.23 (0.07-0.49) g mmol(-1) , P = 0.06]. Maternal uterine and fetal Doppler pulsatility indices were unchanged.In early-onset pre-eclampsia, GSNO reduces augmentation index, a biomarker of small vessel tone and pulse wave reflection, prior to affecting blood pressure. Proteinuria and platelet activation are improved at doses that affect blood pressure minimally. These effects of GSNO may be of therapeutic potential in pre-eclampsia, a condition for which no specific treatment exists. Clinical studies of GSNO in early-onset pre-eclampsia will determine whether these findings translate to improvement in maternal and/or fetal outcome.

Studies show that 1 in 1200 neonates have a low platelet (PLT) count due to alloimmunization against human PLT antigen (HPA)-1a (β3 -L33). This mainly occurs in HPA-1a-negative mothers who are positive for the human leukocyte antigen (HLA)-DRB3*01:01 allele, but only about one-third of cases will mount an effective alloimmune response. The development of specific treatment modalities requires that the mechanisms driving the maternal alloimmune response against the fetal PLTs be further explored. An antibody reagent that has a different binding affinity to HLA-DRA/DRB3*01:01 with and without the β3 -L33 peptide would be a valuable reagent to study peptide presentation on maternal antigen-presenting cells.To identify such antibodies, HLA-DRA/DRB3*01:01 was recombinantly expressed in Drosophila S2 cells. To delineate the epitope of interesting antibodies, seven mutant HLA-DRA/DRB3*01:01 molecules were generated by site-directed mutagenesis introducing naturally occurring amino acid changes encoded by DRB3*02 and DRB3*03 alleles.The murine monoclonal antibody (MoAb) DA2 showed robust binding by enzyme-linked immunosorbent assay to recombinant HLA-DRA/DRB3*01:01, but binding was reduced in the presence of β3 -L33 peptide. The binding affinity of DA2 to the mutant HLA-DRA/DRB3*0101 in which serine at Position 60 of the β1-chain was replaced by tyrosine was greatly enhanced. Interestingly the binding of DA2 to the mutant was not reduced by the presence of β3 -L33 peptide.The results of this study generate a molecular model of the interaction of the HLA-DRA/DRB3*01:01 molecule with MoAb DA2. This will inform functional studies with the recombinant Class II molecules.

To investigate if the prediction of hemolytic disease of the newborn (HDN) in infants of RhD immunized women has been improved by introduction of AutoAnalyzer quantitation and may be further improved by the use of a functional assay.Manual antibody titration, AutoAnalyzer quantitation and chemiluminescence test (CLT) were compared by testing 42 sera from 38 RhD immunized mothers. The sera were also screened for the presence of monocyte-reactive antibodies which have the potential to protect the unborn.Among the 42 infants there were 19 unaffected by HDN and 23 affected by HDN. Manual titration correctly predicted the occurrence of HDN in 29/42 (69%), AutoAnalyzer was correct in 28/42 (67%) and CLT showed correct predictions with 30/42 (71%). In babies born without signs of HDN, maternal monocyte-reactive antibodies were found in 13/18 cases. The majority (9/13) were HLA class I-specific, the remaining four antibodies were either HLA class II or monocyte-specific. In affected HDN group, 8/16 monocyte-reactive anti- bodies were HLA class II or monocyte-specific.AutoAnalyzer and CLT improve the ability to discriminate unaffected babies from those affected by RhD HDN, when compared to manual titration. A protocol for the laboratory management of RhD immunized women is proposed that includes these tests to further improve the prediction of HDN. This study has also highlighted the need for more investigations into the protective role of monocyte-reactive antibodies in HDN.

Monocyte ADCC assays are helpful indicators of the severity of hemolytic disease of the newborn (HDN) due to anti-D. It would be particularly useful if the assays also accurately predicted the ability of antibodies to high-frequency antigens (UFA) to cause HDN. To investigate this possibility, 14 antenatal sera containing antibodies to HFA were tested and the results correlated with the severity of HDN. Antibody titers were determined using an indirect antiglobulin test (IAT). Eight sera (anti-Hy, -Dib, -Jra -ScI, -Inb, -Wrb, and-U [n =2]) had low ADCC activity (< 28%) and did not cause HDN. The IAT titers ranged from 4 to 128. Two antibodies (anti-Ena and anti-Rh29) had high ADCC activity (≥ 72%) and high IAT titers (≥ 1,024), and both caused severe HDN. The remaining four antibodies (anti-Cra, -Inb, and -U [n = 2]) had ADCC activity ≥ 40 percent and titers ≥ 128, but they did not cause HDN. The inability of anti-Cra and anti-Inb to cause HDN is presumed to be related to fetal antigen expression and tissue distribution. The anti-Inb was further investigated using monocyte binding and inhibition studies. The absence of HDN in the presence of potent anti-U remains unexplained. In general, antibodies with low ADCC activity do not cause HDN, but high ADCC activity does not always indicate severe HDN. Immunohematology 1996;12:20-26.

Item does not contain fulltext Anaemia is a chief determinant of global ill health, contributing to cognitive impairment, growth retardation and impaired physical capacity. To understand further the genetic factors influencing red blood cells, we carried out a genome-wide association study of haemoglobin concentration and related parameters in up to 135,367 individuals. Here we identify 75 independent genetic loci associated with one or more red blood cell phenotypes at P < 10(-8), which together explain 4-9% of the phenotypic variance per trait. Using expression quantitative trait loci and bioinformatic strategies, we identify 121 candidate genes enriched in functions relevant to red blood cell biology. The candidate genes are expressed preferentially in red blood cell precursors, and 43 have haematopoietic phenotypes in Mus musculus or Drosophila melanogaster. Through open-chromatin and coding-variant analyses we identify potential causal genetic variants at 41 loci. Our findings provide extensive new insights into genetic mechanisms and biological pathways controlling red blood cell formation and function.

Autoimmune thrombocytopenia is generally caused by autoantibodies against glycoprotein (GP) IIb-IIIa or GPIb-IX and occasionally against GPIa-IIa or GPV. By investigating 38 rheumatoid arthritis (RA) patients on gold therapy, 10 with profound thrombocytopenia and 28 nonthrombocytopenic controls, we showed that in all 10 patients with thrombocytopenia, the platelet autoantibodies preferentially targeted GPV but the presence of gold was not required for their reactivity. Elevated levels of platelet-associated IgG (PAIgG) were observed in 8 of the 10 patients in whom the tests were performed. In 5 patients with sufficient autologous platelets, the GPV specificity of PAIgG was confirmed. Tests with GPV transfectants revealed that the antibodies reacted with GPV independent of GPIbα, GPIbβ, or GPIX. Autoantibodies recognizing GPV were not seen in the 28 nonthrombocytopenic control RA patients. Thus, GPV seems to be targeted in gold-induced autoimmune thrombocytopenia.

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count-3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.

The number and volume of cells in the blood affect a wide range of disorders including cancer and cardiovascular, metabolic, infectious and immune conditions. We consider here the genetic variation in eight clinically relevant hematological parameters, including hemoglobin levels, red and white blood cell counts and platelet counts and volume. We describe common variants within 22 genetic loci reproducibly associated with these hematological parameters in 13,943 samples from six European population-based studies, including 6 associated with red blood cell parameters, 15 associated with platelet parameters and 1 associated with total white blood cell count. We further identified a long-range haplotype at 12q24 associated with coronary artery disease and myocardial infarction in 9,479 cases and 10,527 controls. We show that this haplotype demonstrates extensive disease pleiotropy, as it contains known risk loci for type 1 diabetes, hypertension and celiac disease and has been spread by a selective sweep specific to European and geographically nearby populations.

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis. ispartof: BLOOD vol:113 issue:19 pages:4754-4762 ispartof: location:United States status: published

Mean platelet volume (MPV) and platelet count (PLT) are highly heritable and tightly regulated traits. We performed a genome-wide association study for MPV and identified one SNP, rs342293, as having highly significant and reproducible association with MPV (per-G allele effect 0.016 ± 0.001 log fL; P < 1.08 × 10−24) and PLT (per-G effect −4.55 ± 0.80 109/L; P < 7.19 × 10−8) in 8586 healthy subjects. Whole-genome expression analysis in the 1-MB region showed a significant association with platelet transcript levels for PIK3CG (n = 35; P = .047). The G allele at rs342293 was also associated with decreased binding of annexin V to platelets activated with collagen-related peptide (n = 84; P = .003). The region 7q22.3 identifies the first QTL influencing platelet volume, counts, and function in healthy subjects. Notably, the association signal maps to a chromosome region implicated in myeloid malignancies, indicating this site as an important regulatory site for hematopoiesis. The identification of loci regulating MPV by this and other studies will increase our insight in the processes of megakaryopoiesis and proplatelet formation, and it may aid the identification of genes that are somatically mutated in essential thrombocytosis.

BACKGROUND: Process-induced platelet (PLT) activation occurs with all production methods, including apheresis. Recent studies have highlighted the range and consistence of interindividual variation in the PLT response, but little is known about the contribution of a donors' inherent PLT responsiveness to the activation state of the apheresis PLTs or the effect of frequent apheresis on donors' PLTs. STUDY DESIGN AND METHODS: The relationship between the donors' PLT response on the apheresis PLTs was studied in 47 individuals selected as having PLTs with inherently low, intermediate, or high responsiveness. Whole-blood flow cytometry was used to measure PLT activation (levels of bound fibrinogen) before donation and in the apheresis PLTs. The effects of regular apheresis on the activation status of donors' PLTs were studied by comparing the in vivo activation status of PLTs from apheresis (n = 349) and whole-blood donors (n = 157), before donation. The effect of apheresis per se on PLT activation was measured in 10 apheresis donors before and after donation. RESULTS: The level of PLT activation in the apheresis packs was generally higher than in the donor, and the most activated PLTs were from high-responder donors. There was no significant difference in PLT activation before donation between the apheresis and whole-blood donors (p = 0.697), and there was no consistent evidence of activation in the donors immediately after apheresis. CONCLUSION: The most activated apheresis PLTs were obtained from donors with more responsive PLTs. Regular apheresis, however, does not lead to PLT activation in the donors.

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.

To assess removal of cytomegalovirus (CMV) by leukocyte depletion (LD) filters, we developed a spiking model of latent virus using peripheral blood mononuclear cells (PBMCs) infected by coculture with CMV-infected human fibroblasts. Infected PBMCs were purified by dual magnetic column selection and then spiked into whole blood units or buffy coat pools prior to LD by filtration. CMV load and fibroblast contamination were assessed using quantitative CMV DNA real-time PCR and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA encoding the fibroblast-specific splice variant of prolyl-4-hydroxylase, respectively. After correcting for fibroblast-associated CMV, the mean CMV load was reduced in whole blood by LD from 7.42 × 102 to 1.13 copies per microliter (2.8110log reduction) and from 3.8 × 102 to 4.77 copies per microliter (1.910log reduction) in platelets. These results suggest that LD by filtration reduces viral burden but does not completely remove CMV from blood components. (Blood. 2004;103:1137-1139)

BACKGROUND: Implementation of WBC reduction of the blood supply increases the importance of measurement of residual WBC subtypes responsible for immunologic and infectious complications of transfusion. STUDY DESIGN AND METHODS: Real-time RT-PCR assays were developed to detect mRNA encoding lineage-specific WBC markers. Primers and fluorescent probes were designed for CD45 (pan-WBC), CD3 (T-lymphocyte), CD19 (B-lymphocyte), CD14 (monocyte), and CD66 (granulocyte), and the specificity was assessed by comparison with flow cytometric analysis of enriched cell populations. WBC subsets were examined in WBC-reduced whole blood prepared with filters (WBF2, Pall; and RZ2000, Baxter) and in platelet concentrates prepared with other filters (Autostop, Pall; and PLX-5, Baxter) and apheresis (COBE Spectra LRS, Gambro). RESULTS: All real-time RT-PCR assays were linear over >5 log concentration range, allowing pre-WBC-reduction and post-WBC-reduction comparisons. Sensitivity limits ranged from 10 cells per mL (CD45) to 200 cells per mL (CD19). Assay specificity was confirmed by the close correlation of real-time RT-PCR and immunophenotyping results by flow cytometry. For all subsets, >3.8 log and >3.1 log reduction was obtained during WBC reduction of whole blood and platelets, respectively. CONCLUSION: Real-time RT-PCR assays are suitable for analysis of subset removal during WBC reduction. There was no significant difference between the two whole-blood filters or between platelet filtration and apheresis in the removal of any WBC subset.

Genetic variation is known to account for up to 70% of the wide variation in platelet function observed in healthy individuals. We have shown that platelet concentrates (PC) that are highly activated when collected by apheresis are produced from donors whose platelets are highly responsive to adenosine diphosphate (ADP) and collagen related peptide (CRP-XL) in vitro(high responder donors) with activation being measured by P-selectin expression and fibrinogen binding. The effect of this variation in donors on the outcome from platelet transfusion is unknown. The aim of the PROmPT trial (Platelet Responsiveness and Outcome from Platelet Transfusion, ISRCTN 56366401) was to test the hypothesis that PC following transfusion from high responder donors are cleared more rapidly from the circulation than PC from low responder donors. Method We established a cohort of 1,000 blood donors phenotyped for responsiveness to CRP (0.1-0.5 µg/mL) or ADP (10-7M) by flow cytometry. A sub-set of these individuals (26 high and 19 low responder donors) who reproducibly had global responses, i.e. not outliers for just one agonist, in the top and bottom 10% of the cohort were selected to supply apheresis platelets collected on the Terumo Trima system for the trial. The trial was designed as a parallel group, semi-randomised double-blinded trial in stable haematology patients who required prophylactic platelet transfusions. Based on 80% power, a 5% significance level, and a presumed 10% dropout, 100 patients (with the aim of 50 receiving a unit from high responders and 50 from low responders) were required to detect a mean difference of 7x109/L in the primary endpoint of the 1 hour (10-90 min) count increment (CI) between the high and low responder PC. Patients were randomised to receive a single PC from either a high or low responder donor when both were available. If only one type of PC was available then patients received that PC. Patients with factors known to affect platelet CI were excluded. In addition, 30 patients acted as controls, receiving non-trial PC from random donors. PCs were ABO and Rh compatible. Patients were followed up for 5 days or until their next platelet transfusion. Secondary outcomes included an assessment of bleeding score (modified WHO score) both by the patient and the clinician, 24 hour (12-36 hours) CI, as well as time to next platelet transfusion, and red cell transfusion requirements. CIs were adjusted for confounding factors that affect platelet increments, these were age and dose of PC, and body surface area. Results Baseline characteristics of patients were adequately balanced between groups. Fifty one patients received a PC from low responder donors, and 49 from high responder donors (47 of which were randomised and 53 non-randomised) and 30 patients received non-trial units (2 non trial transfusions were excluded from final analysis). There was no significant difference in the adjusted 1 hour CI, in patients receiving platelets from high or low responder donors (high response 21.04 versus low response 23.34x109/L; difference 2.30, 95%CI -1.09 to 5.69, p = 0.18) or the 24 hour platelet CI (high response 12.9 versus low response 14.31x109/L; difference 1.41, 95% CI -1.96 to 4.78, p = 0.41). There were no significant differences in all other secondary outcomes between patients receiving PC from high or low responder donors, including maximum bleeding scores (odds ratio 0.78, 95%confidence interval 0.29 to 2.16, p=0.64) and bleeding rates (number of days with Grade 2-4 bleed) (rate ratio was 0.70, 95% confidence interval 0.16 to 2.97, p = 0.63). In addition, there was no significant difference in 1 hour CI between patients receiving non-trial units and those from high (p = 0.28) or low responders (p = 0.95) or in any secondary outcomes. Conclusions Results from this trial suggest that in a setting of prophylactic transfusion, the platelet responsiveness of the donor does not affect platelet count increments, bleeding scores, inter-transfusion interval or red cell usage in recipients. Further studies will be required to determine whether PC derived from high responders may be more efficacious in patients who are actively bleeding at the time of transfusion. Disclosures No relevant conflicts of interest to declare.

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. Aim To characterize the first UK case of GPVI deficiency. Methods Numerous platelet function tests and immunophenotyping assays, western blotting and genomic sequencing. Results A female patient of 33 years with mild thrombocytopenia (80–120 × 109 L−1), had experienced moderate bleeding with atypical locations, including bruises and petechiae on neck and chest, and a perineal haematoma and postnatal bleeding. Bone marrow examination revealed plentiful megakaryocytes, compatible with a state of compensated thrombocytolysis. PFA 100 response with the collagen/epinephrine cartridge was >295 seconds, and aggregometry showed a profoundly reduced response to collagen and collagen-related peptide. Response to ADP and PAR1 peptide was moderately reduced, but ristocetin induced agglutination was normal. By flow cytometry, normal surface expression was observed to many platelet receptors, including GPIIbIIIa, GPIaIIa and PECAM-1. Expression of PAR-1 was mildly reduced, whereas for GPVI a 90% reduction in surface expression was observed. Total GPVI levels as measured by blot densitometry were also reduced, although not as profoundly. The sequence of the GP6 exons was identical to the reference sequence. A potent GPVI autoantibody was detected in the patients’ plasma by a sandwich ELISA (MAIPA), and reactivity was mapped to the collagen-binding domains using recombinant GPVI fragments. Conclusions The patient has GPVI autoantibodies of the IgG class, resulting in an acquired GPVI deficiency and with a bleeding phenotype apparently more severe than other reported cases of GPVI deficiency.

Haematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell surface receptors. In order to further understand haematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B-cells, cytotoxic and helper T-cells, Natural Killer cells, granulocytes and monocytes using whole genome microarrays. A bioinformatics analysis of this data was performed focusing on transcription factors, immunoglobulin superfamily members and lineage specific transcripts. We observed that the numbers of lineage specific genes varies by two orders of magnitude, ranging from five for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel co-expression patterns for key transcription factors involved in haematopoiesis (eg. GATA3–GFI1 and GATA2–KLF1). This study represents the most comprehensive analysis of gene expression in haematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which is freely accessible, will be invaluable for future studies on haematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.

The variation in blood cell indices between individuals is to a large extent genetically controlled. In the normal population mean platelet volume (MPV) is inversely correlated with platelet count. We undertook a genome-wide association analysis of MPV on 2.5 million imputed genotypes in 1,475 individuals from the UK Blood Services Common Controls, followed by replication in an additional 7,098 samples from four independent collections. We identified a novel locus on chromosome 7q22.3, in a region frequently deleted in myeloid malignancies, where the lead single nucleotide polymorphism (SNP) had a highly significant association with MPV (average G allele effect size 0.15 log fl, 95% CI 0.0118–0.0174, P-value = 9.5×10−24) and an opposite effect on platelet counts (G allele effect −4.51 109/l, 95% CI −6.112 - −2.900, P-value = 1.46×10−7), but the SNP did not exert an effect on red cell indices. The lead SNP with a minor allele frequency of 0.46 is intergenic between the hypothetical gene FLJ36031 and the PIK3CG gene. There are 6 genes in the 1-Mb window centred around the lead SNP and all but HBP1 are transcribed in megakaryocytes (MKs) and to a variable degree in the other seven blood cell lineages. The formation and regulation of volume of platelets is critically dependent on the interaction of MK-expressed integrins with extracellular matrix proteins like fibrinogen and collagen. This prompted us to analyze, in our platelet function cohort of 500 healthy individuals, the association between the lead SNP and binding of both fibrinogen and annexin V to platelets following activation with the collagen mimetic CRP-XL, both associations were significant, with P-values of 0.05 and 0.003, respectively. In summary, we describe a common SNP associated with differences in volume, count and function of platelets from healthy individuals. Intriguingly, our results indicate that the same 7q22.3 region which is frequently deleted in myeloid malignancies harbours important regulatory elements of several platelet phenotypic traits.

The extent of platelet response to agonists varies between individuals and is to a large extent genetically controlled. Here we present the results of a systems biology study designed to define the association between sequence variation and platelet functional phenotype. The exons and flanking intronic regions of 98 genes, with known function in platelets, were sequenced in 48 Caucasoid CEPH DNA reference samples, identifying 1949 SNPs, approximately half of which were novel. From this enriched data set 1340 SNPs, comprising tagging and non-synonymous validated SNPs, were mounted on an Illumina Golden Gate array and typed in 500 DNA samples from a cohort of healthy subjects whose platelet functional phenotype was measured by flow cytometry, using fibrinogen binding and P-selectin expression in response to pathway-specific ADP (10−7M), or CRP-XL (0.1μg/ mL) stimulation. Thirty-six SNPs in 16 genes showed an association (p0.2, respectively), did not show significant effect on the level of ITPR1 protein, but there was increased mobilisation of [Ca2+]i in response to ADP (but not CRP-XL) with the minor allele (p=0.004). By applying systems biology to platelets we have identified SNPs in 16 genes which together account for up to 60% of the genetic variation underlying platelet response to ADP and CRP-XL. These data provide novel insights into key “hubs” in well-studied platelet signalling networks where sequence variation modifies the response phenotype.

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. Method The activation status of the platelets in the donors was determined from the percentage on platelets with bound fibrinogen, by whole-blood flow cytometry. The immediate effect of apheresis on circulating platelets was studied in 10 donors in samples taken from the arm, pre and post donation, measuring platelet-bound fibrinogen in unstimulated blood samples and in response to in vitro stimulation with ADP (10-7M). The long-term effect of apheresis was assessed by measuring fibrinogen binding to platelets in unstimulated blood, and in response to ADP (10-7M) and cross-linked collagen related peptide (XL-CRP, 0.1μg/mL) activation prior to donation. Results Apheresis did not have a consistent immediate effect on resting platelet activation: pre-donation bound fibrinogen levels ranged from 2.69–7.63% positive platelets (mean 4.41%) and post donation levels ranged from 1.46–9.66% (mean 4.71) (p=0.71). ADP induced fibrinogen binding levels ranged from 11.6–41.4% (mean 30.7%) prior to donation and decreased post donation in 8/10 cases, with values ranging from 6.74–43.6% (mean 23.45%) (p=0.024). The long-term effect of apheresis was assessed by comparing the results of apheresis and whole blood donors. For all donors resting bound fibrinogen levels ranged from 0.51% to 6.96% positive, with no significant difference between the whole blood and apheresis donors (p=0.69). Responses to ADP did not differ between the two groups, but the apheresis donors showed a 4.01±1.8% (P=0.03) increase in fibrinogen binding in response to XL-CRP. Conclusion Apheresis does not lead to short- or long-term platelet activation. However, prior to donation, apheresis donors show slightly higher responses to XL-CRP than whole blood donors. This may reflect an increase in circulating younger, more active platelets.

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. Method The study consisted of three phases. Phase 1 evaluated a range of platelet function assays in 46 individuals to determine reproducibility and feasibility for a large cohort study. Calcium flux, flow cytometric detection of fibrinogen and Annexin V binding and P-selectin expression, and aggregometry were used to measure responses to three concentrations of adenosine diphosphate (ADP) and cross linked collagen related peptide (XL-CRP). From this study, whole blood flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single sub-maximal dose of ADP (10–7 M) and XL-CRP (0.1 μg mL-1) were selected for use in phase 2, during which the platelet responses were measured in 506 blood donors attending the Cambridge Centre. In these assays inhibitors of thrombin (hirudin), thromboxane (aspirin), and ADP (apyrase–XL-CRP stimulated samples only) were included to ensure activation was pathway specific. Results There was a wide range of response to both XL-CRP (fibrinogen binding 3.1%–84.4% +ve; P-selectin expression 6.2%–90.3% +ve) and ADP (fibrinogen binding 3.5%–78.8% +ve; P-selectin expression 2.8%–54.1% +ve). Three groups of individuals were selected; two groups with responses to both agonists and both endpoints that were at the ‘Extreme Ends’ of the normal range, (15 low and 16 high responders) and a third group of 16 identified as having mid-range responses. In phase 3 these 47 donors were re-tested to confirm the reproducibility of the original classification. There was a high level of concordance between the two visits for the fibrinogen-CRP, fibrinogen-ADP, P-Selectin-CRP, and P-Selectin-ADP assays (P = 0.851, 0.794, 0.855 and 0.527 respectively). Conclusion We have demonstrated that different measures of the platelet response to two separate agonists are highly variable between healthy subjects, but are consistent over time within an individual. This standard method for defining platelet functional phenotype is being used in the BLOODOMICS programme to define the association between genetic variation and the risk of atherothrombosis.

Background Glanzmann's 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. Methods and Results The exons and flanking intronic sequences of ITGB3, ITGA2B GPIBA, GPIBB, GP9 and GP5 genes were sequenced. Heterozygous, non-synonymous single nucleotide polymorphisms (SNP) were identified in the ITGB3 and the GPIBA genes. These result in the substitution of Histidine for Aspartate at residue 723 (D723H) in ITGB3 and Leucine for Proline at residue 69 (P69L) in GPIBA. Asp723 in ITGB3 is known to form a critical salt-bridge with ITGA2B and charge reversal at residue 723 causes constitutive activation of the integrin in vitro. In agreement with this, mutant alphaIIb/β3-His723 expressing CHO cell lines showed an increased expression of activation dependent epitopes relative to wild-type as measured by mAb PAC-1 binding. Increased PAC-1 binding was also observed on resting platelets of affected individuals compared with non-affected family members. Further investigation of three generations of the propositus’ family identified several individuals with both SNPs each of whom had an identical platelet phenotype. However, in the one family member identified who carried the GPIBA mutation alone, normal platelet size and count were observed suggesting that the ITGB3 D723H substitution is the causative mutation. Genotyping a panel of 1127 unrelated controls for the two SNPs identified five individuals with the GPIBA mutation and none with the ITGB3 mutation. Conclusions We report a novel autosomal dominant cause of a mild form of macrothrombocytopenia caused by a D723H substitution in the beta3 integrin. We postulate that the low platelet count is due to increased consumption of platelets due to enhanced levels of surface expressed, activated GPIIbIIIa.

Introduction Numerous epidemiological and clinical studies have established a firm link between infection, the inflammatory response and cardiovascular disease. Furthermore, it is known that individuals differ in their responses to immune stimuli such as Lipopolysaccharide (LPS) that stimulates monocytes through Toll-like receptor 4 (TLR4). In this study we set out to identify healthy individuals with extremes of monocyte response to LPS stimulation. Once identified these individuals will be studied further to identify genes responsible for the observed differences. Methods Small volumes of whole blood from 208 healthy blood donors were incubated with 0, 1 or 5 ng mL−1E. coli LPS for 4 hr at 37°C. Cell free plasma was collected both pre- and post-incubation and the production of the pro-inflammatory cytokine IL-6 measured by ELISA. In addition, TaqMan genotyping was performed on two TLR4 SNPs, (rs4986790 and rs4986791, encoding D299G and T399I respectively) to determine whether these SNPs correlated with the observed functional responses. Results The range of IL-6 produced was 0–26 ng mL−1 in response to 1 ng mL−1 LPS, and 2–56 ng mL−1 in response to 5 ng mL−1 LPS. Frequency distributions of IL-6 levels show one-tailed distributions for both stimuli with more outlying high extremes than low extremes. Quality control assays showed that both inter- and intra-assay variability had a CV