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

Dysfunction of receptors for IgG (FcgammaRs) has been thought to be involved in the pathogenesis of systemic lupus erythematosus (SLE). We show that a recently described SLE-associated polymorphism of FcgammaRIIb (FcgammaRIIbT(232)), encoding a single transmembrane amino acid substitution, is functionally impaired. FcgammaRIIbT(232) is unable to inhibit activatory receptors because it is excluded from sphingolipid rafts, resulting in the unopposed proinflammatory signaling thought to promote SLE.

A novel variant on chromosome 7q22.3 associated with mean platelet volume, counts, and function.

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 x 10(-24)) and PLT (per-G effect -4.55 +/- 0.80 10(9)/L; P < 7.19 x 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.

A functional genomics approach reveals novel quantitative trait loci associated with platelet signaling pathways.

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 Ca(2+) levels after activation with ADP (P < .004). These data provide novel insights into key hubs within platelet signaling networks.

Alloantibodies against low-frequency human platelet antigens do not account for a significant proportion of cases of fetomaternal alloimmune thrombocytopenia: evidence from 1054 cases.

BACKGROUND: Maternal alloantibodies against the five common human platelet antigen (HPA) systems (HPA-1 to -3, -5, and -15) are found in only 20% of cases referred for fetal and neonatal thrombocytopenia (FMAIT) investigations. The question asked was whether mismatches for the remaining 11 low-frequency HPAs (HPA-4 and -6bw to -17bw) might in part explain the remaining 80% of cases. STUDY DESIGN AND METHODS: A total of 1054 paternal DNA samples from referred FMAIT cases (among which 223 cases where antibodies against a common HPA were found) were genotyped for 11 low-frequency HPAs as well as a recently discovered polymorphism (ITGA2B-C2320T). The initial genotyping was carried out by TaqMan and potential heterozygotes were confirmed by DNA sequencing. Clinical and serologic data were collected for each case with a heterozygote father. RESULTS: In total, eight heterozygous fathers were identified: four for HPA-6w, one each for HPA-10w and -11w, and two for HPA-12w. Maternal antibodies against the corresponding antigen were identified in four of the eight cases. In two of these cases, antibodies against HPA-1a and HPA-1b were also found. CONCLUSION: It was concluded that the minor alleles of HPA-4 and -6bw to -17bw are exceptionally rare in the Caucasian population and therefore do not explain the large number of FMAIT referrals which test negative for the common HPA antibodies.

Identification of variation in the platelet transcriptome associated with glycoprotein 6 haplotype.

Platelet Glycoprotein VI (GPVI) is the activatory collagen signalling receptor that transmits an outside-in signal via the FcR gamma-chain. In Caucasians two GP6 haplotypes have been identified which encode GPVI isoforms that differ by five amino-acids. The minor haplotype is associated with a modest but statistically significant reduction in GPVI abundance and reduced downstream signalling events. As GPVI is also expressed on megakaryocytes, different GPVI isoforms may imprint on the platelet transcriptome. We investigated the association of GP6 haplotype with transcription by comparing the transcriptomes of platelets from individuals homozygous for the major ('a') and minor ('b') haplotypes to identify differentially expressed (DE) transcripts. Platelet RNA was isolated from apheresis concentrates from 16 'aa' donors and eight 'bb' donors. mRNA was amplified using a template-switching PCR based protocol and fluorescently labelled. Samples were randomly paired both within and between haplotypes and compared on a cDNA microarray. No consistently DE transcripts were identified within the 'aa' haplotype but 52 significantly DE transcripts were observed between haplotypes. Generally the fold differences were low (two to four-fold) but were confirmed by qRT-PCR for selected transcripts (TUBB1, P = 0.0004; VWF, P = 0.0126). The results of this study indicate that there are subtle differences between the platelet transcriptomes of individuals who differ by GP6 haplotype. The identification of DE genes may identify critical pathways and nodes not previously known to be involved in platelet development and function.

PECAM-1 expression and activity negatively regulate multiple platelet signaling pathways.

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.

A single-nucleotide polymorphism in the human ITGB3 gene is associated with the platelet-specific alloantigen Va (HPA-17bw) involved in fetal maternal alloimmune thrombocytopenia.

BACKGROUND: The previously reported platelet (PLT)-specific antigen, Va(a), was defined by an alloantibody detected in the serum sample of a mother who delivered an infant displaying symptoms of severe fetal maternal alloimmune thrombocytopenia (FMAIT). This PLT antigen was localized to the integrin alphaIIbbeta3 (GPIIbIIIa) but its genetic basis was not defined. STUDY DESIGN AND METHODS: Genomic ITGA2B (alphaIIb) and ITGB3 (beta3) DNA from a Va(a)-positive individual were sequenced to identify potential polymorphisms underlying the Va(a) antigen. Recombinant beta3 integrin carrying the putative mutation was then used to assess serologic reactivity with the original maternal Va(a) antiserum. RESULTS: A single-nucleotide polymorphism (SNP; C622>T) in exon 5 of ITGB3 resulting in the replacement of threonine with methionine at residue 195 of the mature beta3 was identified. Calmodulin-tagged soluble recombinant beta3 encoding Met195 was produced in S2 cells and found to react specifically with the Va(a) antiserum. CONCLUSION: With the use of a combination of DNA sequencing and recombinant antigen expression, the molecular basis of the PLT-specific Va(a) antigen (HPA-17bw), a low-frequency antigen implicated in FMAIT, has been resolved. These data further demonstrate the value of using recombinant beta3 peptides for the detection of rare but clinically relevant antibodies.

A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the alphaIIbbeta3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia.

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.

A naturally occurring LeuVal mutation in beta3-integrin impairs the HPA-1a epitope: the third allele of HPA-1.

BACKGROUND: Single-amino-acid substitution Leu33Pro in the beta3-integrin is responsible for the formation of the human platelet antigen (HPA)-1. Alloimmunization against HPA-1a (beta3-Leu33) is the most frequent cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura. STUDY DESIGN AND METHODS: While HPA-1 genotyping a large cohort of patients with thromboembolic disease with a thermal cycler (LightCycler), one patient was identified with a unique HPA-1a melting curve. RESULTS: Sequence analysis revealed a C-to-G transversion at nucleotide 175 in the beta3-integrin (ITGB3) gene that alters the Leu33 codon to Val33. Further genotyping of healthy blood donors (n = 2950) identified one nonrelated Pro33Val33-positive individual. To examine whether the presence of Val33 affected the binding pattern of HPA-1 alloantibodies, transfectants were generated expressing recombinant beta3-Leu33 or beta3-Val33. Interestingly, differences in the reactivity of anti-HPA-1a were observed, with some HPA-1a alloantibodies showing diminished reactivity with beta3-Val33 compared to beta3-Leu33 and others reacting equally with both types. Similar findings were observed with recombinant human HPA-1a antibodies, with one of the three not binding to beta3-Val33. CONCLUSIONS: Our results demonstrate that the naturally occurring Leu33Val mutation in the beta3-integrin can disrupt some HPA-1a epitopes. These findings provide evidence for a heterogeneous humoral response against HPA-1a that may have potential clinical implications for alloimmune thrombocytopenia disorders.

The low-frequency allele of the platelet collagen signaling receptor glycoprotein VI is associated with reduced functional responses and expression.

Interaction of platelets with collagen under conditions of blood flow is a multi-step process with tethering via glycoprotein IbIXV (GPIbIXV) over von Willebrand factor, adhesion by direct interaction with the integrin GPIaIIa, and signaling via GPVI. GPVI can be specifically agonized by cross-linked collagen-related peptide (CRP-XL), which results in a signaling cascade very similar to that evoked by native collagen. The GPVI gene has 2 common alleles that differ by 3 replacements in the glycosylated stem and 2 in the cytoplasmic domain. We used CRP-XL to elucidate the variation in responses observed in platelet function in different individuals. We observed a 3-fold difference in the response to CRP-XL in platelet aggregation when comparing platelets from 10 high-frequency allele homozygotes with 8 low-frequency ones (2-way analysis of variance [ANOVA], P <.0001). The difference in functional responses was reflected in fibrinogen binding and in downstream signaling events as measured by tyrosine phosphorylation, the expression of P-selectin, and the binding of annexin V and the generation of thrombin on the platelet surface (2-way ANOVA, P <.001). Platelets homozygous for the low-frequency allele tended to be less able to form a thrombus on a collagen surface in flowing whole blood or in the platelet function analyzer-100 (t test, P =.065 and P =.061, respectively). The functional difference was correlated to a difference in total and membrane-expressed GPVI measured by monoclonal and polyclonal antibodies. This study demonstrates for the first time that platelet function may be altered by allelic differences in GPVI.