Page 1876 - Williams Hematology ( PDFDrive )

1850 Part XII: Hemostasis and Thrombosis Chapter 112: Platelet Morphology, Biochemistry, and Function 1851

increasing the “noise” in genetic epidemiology studies testing for asso- 11 of the genes as novel regulators of blood cell formation using gene
ciations with complex phenotypes. The use of intermediate phenotypes silencing in Danio rerio and Drosophila melanogaster.
as outcomes in genetic association studies has enhanced power to detect
gene associations because the number of genes potentially responsible
for the phenotype is reduced, thereby increasing the fraction of the vari- PLATELET GENE EXPRESSION
ance explained by any single factor or gene. Despite large interindivid-
ual variability in platelet reactivity, light transmission aggregometry has TRANSCRIPTOMICS
been shown to be reproducible and heritable, with the reproducibility
persisting for years. 556,557 The Homo sapiens genome includes approximately 21,000 protein-
coding genes (genome build GRCh38). To date, more than 10 times this
number of protein-coding transcripts have been identified, primarily
Candidate Functional Platelet Genes as a result of alternate exon splicing, and more are being continually
The Leu33Pro variant of integrin β (rs5918 of ITGB3) is responsible discovered. Platelets from healthy subjects contain approximately 2.20
3
A1
for human platelet alloantigen 1a/b (Pl /Pl ). Fibrinogen and proth- femtograms (fg) of total RNA per cell, which is approximately 1000-
A2 558
33
rombin binding is enhanced to the Pro isoform of purified integrin fold less than nucleated blood cells. Platelets can splice pre-mRNA into
α β . Compared to cell lines expressing Leu33 variant of integrin, mature mRNA, which is translated into proteins. 574,575 Characterization
559
IIb 3
Pro33 cells have increased adhesion, spreading, actin cytoskeletal reor- of the transcriptome enables quantitative assessment of gene expression
562
ganization and migration under static 560,561 and shear conditions. This in the tissue of interest and identification of alternately spliced tran-
prothrombotic phenotype of Pro33 is mediated by enhanced outside-in scripts. Genome-wide transcriptome studies have enabled dissection of
platelet signaling through integrin α β . 563,564 Notably, this variant does the molecular basis of inherited platelet disorders and a better under-
IIb 3
not affect inside-out signaling, as assessed by standard platelet light standing of the relationship between gene expression and megakaryo-
565
transmission aggregometry of human platelets. Additional support cyte and platelet differentiation. In addition, platelet RNA profiles may
for the prothrombotic nature of the Pro33 variant of integrin β comes have utility as biomarkers. 576
3
from mice made homozygous for Pro33. These animals have reduced Technologic advances have greatly facilitated understanding the
bleeding, increased in vivo thrombosis and enhanced outside-in integ- platelet transcriptome. Early studies using serial analysis of gene expres-
rin α β signaling, but normal inside-out signaling. 566 sion and microarrays estimated approximately 6000 mRNAs in the
IIb 3
Laboratory evidence for functional effects of genetic variants in the human platelet. 577,578 Platelet RNA-sequencing (RNA-seq) has demon-
gene encoding GPIbβ has been inconsistent. Variants in the two plate- strated an unexpected complexity to the transcriptome and substantive
579
let collagen receptors, GPVI and integrin α subunit (of integrin α β ) differences between the human and mouse platelet transcriptome. The
2 1
2
alter receptor expression and adhesion to collagen using in vitro per- exquisite sensitivity of RNA-seq provided estimates of approximately
fusion assays. 567–569 Functional variants in the genes encoding FcγRIIA 9,000 protein-coding genes in platelets (Fig. 112–8), 580,581 although only
582
(FCGR2A), P2Y (P2RY12), GPIV (CD36), and PAR-1 (F2R) have also approximately 7800 are commonly expressed in human platelets.
12
been reported. Approximately half of the transcripts in platelets encode mitochondrial
581
Associations between SNPs in 97 hematopoietic cell genes were genes. Platelet mitochondrial mRNAs are inversely correlated with
582
tested and 17 novel associations with platelet responses to crosslinked subject age, and mitochondrial function may regulate platelet apopto-
584
583
collagen-related peptide (CRP) and ADP were identified, including sis and support optimal platelet function during storage, but plate-
genes encoding cell surface receptors (CD36, GP6, ITGA2, PEAR1, and let mitochondria diseases have not been described. The S-shaped curves
P2Y12), kinases (JAK2, MAP2K2, MAP2K4, and MAPK14), and other in Fig. 112–8 illustrate several features of the human platelet transcrip-
signaling molecules (GNAZ, VAV3, ITPR1, and FCERG1). Variants tome: (1) estimates of expressed protein-coding genes are more simi-
570
at the Chr9p21.3 locus are associated with the platelet aggregation lar amongst different subjects for high abundance genes (leftwards in
response to low (0.5 mcg/mL) but not higher concentrations of collagen Fig. 112–8) and (2) there is substantial interindividual variation in total
in a large cohort with two replication studies. 571 transcript estimates when considering the less abundant genes (right-
ward in Fig. 112–9). Furthermore, it is not known what is the biologi-
cally relevant copy number of transcripts in any cell, and the arbitrary
Genome-Wide Association Studies choice of “threshold” could dramatically affect the number of reported
The first GWAS reported for platelet reactivity tested association of 2.5 genes expressed in platelets. Transcriptomes of primary megakaryo-
million SNPs with platelet aggregation responses to ADP, collagen, and cytes have not been determined, but RNA profiling of megakaryocytes
epinephrine. The primary cohorts were generally healthy, European- derived from cultured CD34+ hematopoietic stem cells has identified
565
ancestry populations from the Framingham Heart Study (FHS) (n = transcripts that are differentially expressed upon differentiation and
2753) and the GeneSTAR cohorts (n = 1238). SNPs at seven loci (PEAR1, between normal subjects and patients with essential thrombocytosis
MRVI1, SHH, ADRA2A, PIK3CG, JMJD1C, and GP6) met genome- (ET). 585,586
wide statistical significance and were replicated in an African-ancestry
cohort (n = 840). A second platelet function GWAS identified SNPs in
SVIL (encodes supervillin) as associated with closure time in the in vitro Platelet mRNAs Associated with Disease
platelet function analyzer PFA-100. Human platelet gene expression Platelet mRNA profiling in patients with acute ST-segment-elevation
572
studies and data with Svil –/– mice demonstrated an inhibitory role for MI and stable CAD demonstrated that S100A9 (myeloid-related
supervillin in platelet adhesion and thrombus formation under high- protein-14 [MRP-14]) was expressed at higher levels in patients than
587
shear but not low-shear conditions. A meta-analysis by the HaemGen controls. This discovery was validated in the Women’s Health Study
consortium of 66,867 individuals identified 43 and 25 loci associated and PROVE IT-TIMI 22 trials. 587,588 Platelet mRNA expression profiling
573
with platelet number and mean platelet volume (MPV), respectively. can distinguish essential thrombocythemia (ET) patients from healthy
These loci accounted for 4.8 percent of the phenotypic variance in plate- subjects and levels of HIST1H1A, SRP72, C20orf103, and CRYM can
589
590
let number and 9.9 percent in MPV and included well-known platelet predict JAK2 V617F–negative ET in 87 percent of patients. mRNA
regulators (ITGA2B, GP1BA, and F2R). These investigators identified expression profiling identified reduced MYL9 transcripts in platelets of
Kaushansky_chapter 112_p1829-1914.indd 1851 17/09/15 3:27 pm