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The MAPK-interacting kinase (Mnk) family includes Mnk1 and Mnk2, which are phosphorylated and activated in response to extracellular stimuli. Mnk1 contributes to cellular responses by regulating messenger RNA (mRNA) translation, and mRNA translation influences platelet production and function. However, the role of Mnk1 in megakaryocytes and platelets has not previously been studied. The present study investigated Mnk1 in megakaryocytes and platelets using both pharmacological and genetic approaches. We demonstrate that Mnk1, but not Mnk2, is expressed and active in human and murine megakaryocytes and platelets. Stimulating human and murine megakaryocytes and platelets induced Mnk1 activation and phosphorylation of eIF4E, a downstream target of activated Mnk1 that triggers mRNA translation. Mnk1 inhibition or deletion significantly diminished protein synthesis in megakaryocytes as measured by polysome profiling and [35S]-methionine incorporation assays. Depletion of Mnk1 also reduced megakaryocyte ploidy and proplatelet forming megakaryocytes in vitro and resulted in thrombocytopenia. However, Mnk1 deletion did not affect the half-life of circulating platelets. Platelets from Mnk1 knockout mice exhibited reduced platelet aggregation, α granule secretion, and integrin αIIbβ3 activation. Ribosomal footprint sequencing indicated that Mnk1 regulates the translation of Pla2g4a mRNA (which encodes cPLA2) in megakaryocytes. Consistent with this, Mnk1 ablation reduced cPLA2 activity and thromboxane generation in platelets and megakaryocytes. In vivo, Mnk1 ablation protected against platelet-dependent thromboembolism. These results provide previously unrecognized evidence that Mnk1 regulates mRNA translation and cellular activation in platelets and megakaryocytes, endomitosis and thrombopoiesis, and thrombosis.

Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease-activated receptor (PAR) amino termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies.

Background Acquired von Willebrand syndrome (AVWS) has been associated with monoclonal gammopathy of undetermined significance (MGUS), with limited data on its management. Methods We conducted a systematic literature search in Medline (Ovid), Embase, and Scopus up to September 11, 2019, for studies reporting on the management of AVWS associated with MGUS (AVWS‐MGUS). Data on patient characteristics, laboratory parameters at presentation, and clinical and laboratory outcomes were extracted. Objectives To describe the clinical presentation and outcomes of different therapeutic approaches. Results Seventy‐five studies were included in the final review, for a total of 137 patients. Most patients had von Willebrand factor ristocetin cofactor activity

The objectives of this study were firstly to determine the prevalence of overweight/obesity in adult persons with hemophilia in Utah, and to explore the association between age, disease severity and race with body mass index (BMI), and secondly to provide recent data on the prevalence of overweight/obesity in the hemophilia population via a review of the literature. We conducted a retrospective cross section study of adult persons with hemophilia who were seen at a Utah hemophilia treatment center from 1 January 2017 to 31 December 2019. The electronic database PubMed was searched for studies with observation periods from 1 January 2012 to 31 December 2019. The age-adjusted prevalence for overweight/obesity in the adult Utah hemophilia population was higher than the overall Utah population and the general US population. After adjusting for race and age, mild hemophilia was associated with a 7.7% higher BMI (95% confidence interval, 0.023-15.98%, P < 0.05). Review of the literature demonstrated high levels of overweight/obesity in hemophilia communities globally with considerable heterogeneity between studies. Despite increasing awareness, prevalence of overweight/obesity in the hemophilia population remains high in comparison with the general population. There is a critical need to address this issue acutely at hemophilia treatment centers due to the considerable burden of obesity.

The purpose of this research was to assess the effects of a microRNA (miRNA) cluster on platelet production. Human chromosome 19q13.41 harbors an evolutionarily conserved cluster of three miRNA genes (MIR99B, MIRLET7E, MIR125A) within 727 base-pairs. We now report that levels of miR-99b-5p, miR-let7e-5p and miR-125a-5p are strongly correlated in human platelets, and all are positively associated with platelet count, but not white blood count or hemoglobin level. Although the cluster regulates hematopoietic stem cell proliferation, the function of this genomic locus in megakaryocyte (MK) differentiation and platelet production is unknown. Furthermore, studies of individual miRNAs do not represent broader effects in the context of a cluster. To address this possibility, MK/platelet lineage-specific Mir-99b/let7e/125a knockout mice were generated. Compared to wild type littermates, cluster knockout mice had significantly lower platelet counts and reduced MK proplatelet formation, but no differences in MK numbers, ploidy, maturation or ultra-structural morphology, and no differences in platelet function. Compared to wild type littermates, knockout mice showed similar survival after pulmonary embolism. The major conclusions are that the effect of the Mir-99b/let7e/125a cluster is confined to a late stage of thrombopoiesis, and this effect on platelet number is uncoupled from platelet function.

Rationale:Longitudinal studies are required to distinguish within versus between-individual variation and repeatability of gene expression. They are uniquely positioned to decipher genetic signal from environmental noise, with potential application to gene variant and expression studies. However, longitudinal analyses of gene expression in healthy individuals—especially with regards to alternative splicing—are lacking for most primary cell types, including platelets.Objective:To assess repeatability of gene expression and splicing in platelets and use repeatability to identify novel platelet expression quantitative trait loci (QTLs) and splice QTLs.Methods and Results:We sequenced the transcriptome of platelets isolated repeatedly up to 4 years from healthy individuals. We examined within and between individual variation and repeatability of platelet RNA expression and exon skipping, a readily measured alternative splicing event. We find that platelet gene expression is generally stable between and within-individuals over time—with the exception of a subset of genes enriched for the inflammation gene ontology. We show an enrichment among repeatable genes for associations with heritable traits, including known and novel platelet expression QTLs. Several exon skipping events were also highly repeatable, suggesting heritable patterns of splicing in platelets. One of the most repeatable was exon 14 skipping ofSELP. Accordingly, we identify rs6128 as a platelet splice QTL and define an rs6128-dependent association betweenSELPexon 14 skipping and race. In vitro experiments demonstrate that this single nucleotide variant directly affects exon 14 skipping and changes the ratio of transmembrane versus soluble P-selectin protein production.Conclusions:We conclude that the platelet transcriptome is generally stable over 4 years. We demonstrate the use of repeatability of gene expression and splicing to identify novel platelet expression QTLs and splice QTLs. rs6128 is a platelet splice QTL that altersSELPexon 14 skipping and soluble versus transmembrane P-selectin protein production.

Accurate and consistent sequence variant interpretation is critical to the correct diagnosis and appropriate clinical management and counseling of patients with inherited genetic disorders. To minimize discrepancies in variant curation and classification among different clinical laboratories, the American College of Medical Genetics and Genomics (ACMG), along with the Association for Molecular Pathology (AMP), published standards and guidelines for the interpretation of sequence variants in 2015. Because the rules are not universally applicable to different genes or disorders, the Clinical Genome Resource (ClinGen) Platelet Disorder Expert Panel (PD-EP) has been tasked to make ACMG/AMP rule specifications for inherited platelet disorders. ITGA2B and ITGB3, the genes underlying autosomal recessive Glanzmann thrombasthenia (GT), were selected as the pilot genes for specification. Eight types of evidence covering clinical phenotype, functional data, and computational/population data were evaluated in the context of GT by the ClinGen PD-EP. The preliminary specifications were validated with 70 pilot ITGA2B/ITGB3 variants and further refined. In the final adapted criteria, gene- or disease-based specifications were made to 16 rules, including 7 with adjustable strength; no modification was made to 5 rules; and 7 rules were deemed not applicable to GT. Employing the GT-specific ACMG/AMP criteria to the pilot variants resulted in a reduction of variants classified with unknown significance from 29% to 20%. The overall concordance with the initial expert assertions was 71%. These adapted criteria will serve as guidelines for GT-related variant interpretation to increase specificity and consistency across laboratories and allow for better clinical integration of genetic knowledge into patient care. ispartof: BLOOD ADVANCES vol:5 issue:2 pages:414-431 ispartof: location:United States status: published

Apoptosis is a recognized limitation to generating large numbers of megakaryocytes in culture. The genes responsible have been rigorously studied in vivo in mice, but are poorly characterized in human culture systems. As CD34-positive (+) cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity (LLG), and smaller size, higher granularity (SHG). The LLG cells were CD41aHigh CD42aHigh phosphatidylserineLow, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and displayed a signaling response to platelet agonists. The SHG cells were CD41aLowCD42aLowphosphatidylserineHigh, had a distinctly apoptotic morphology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte apoptosis regulator. Lentiviral BCL2L2 overexpression decreased megakaryocyte apoptosis, increased CD41a+ LLG cells, and increased proplatelet formation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet BCL2L2 mRNA levels. This finding was consistent with the observed increase in platelet-like particles derived from cultured megakaryocytes over-expressing BCL2L2. BCL2L2 also induced small, but significant increases in thrombin-induced platelet-like particle αIIbβ3 activation and P-selectin expression. Thus, BCL2L2 restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation, and is associated with platelet number. BCL2L2 is a novel target for improving megakaryocyte and platelet yields in in vitro culture systems.

Glanzmann thrombasthenia (GT) is an autosomal recessive disorder of platelet aggregation caused by quantitative or qualitative defects in integrins αIIb and β3. These integrins are encoded by the ITGA2B and ITGB3 genes and form platelet glycoprotein (GP)IIb/IIIa, which acts as the principal platelet receptor for fibrinogen. Although there is variability in the clinical phenotype, most patients present with severe mucocutaneous bleeding at an early age. A classic pattern of abnormal platelet aggregation, platelet glycoprotein expression and molecular studies confirm the diagnosis. Management of bleeding is based on a combination of hemostatic agents including recombinant activated factor VII with or without platelet transfusions and antifibrinolytic agents. Refractory bleeding and platelet alloimmunization are common complications. In addition, pregnant patients pose unique management challenges. This review highlights clinical and molecular aspects in the approach to patients with GT, with particular emphasis on the significance of multidisciplinary care. ispartof: HAEMATOLOGICA vol:105 issue:4 pages:888-894 ispartof: location:Italy status: published

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk.

Supplemental Digital Content is available in the text., Objective— Platelet activation after stimulation of PAR (protease-activated receptor) 4 is heightened in platelets from blacks compared with those from whites. The difference in PAR4 signaling by race is partially explained by a single-nucleotide variant in PAR4 encoding for either an alanine or threonine at amino acid 120 in the second transmembrane domain. The current study sought to determine whether the difference in PAR4 signaling by this PAR4 variant is because of biased Gq signaling and whether the difference in PAR4 activity results in resistance to traditional antiplatelet intervention. Approach and Results— Membranes expressing human PAR4-120 variants were reconstituted with either Gq or G13 to determine the kinetics of G protein activation. The kinetics of Gq and G13 activation were both increased in membranes expressing PAR4-Thr120 compared with those expressing PAR4-Ala120. Further, inhibiting PAR4-mediated platelet activation by targeting COX (cyclooxygenase) and P2Y12 receptor was less effective in platelets from subjects expressing PAR4-Thr120 compared with PAR4-Ala120. Additionally, ex vivo thrombus formation in whole blood was evaluated at high shear to determine the relationship between PAR4 variant expression and response to antiplatelet drugs. Ex vivo thrombus formation was enhanced in blood from subjects expressing PAR4-Thr120 in the presence or absence of antiplatelet therapy. Conclusions— Together, these data support that the signaling difference by the PAR4-120 variant results in the enhancement of both Gq and G13 activation and an increase in thrombus formation resulting in a potential resistance to traditional antiplatelet therapies targeting COX-1 and the P2Y12 receptor.

Genome wide association studies (GWAS) have associated mitochondria related loci with platelet numbers, function, and CVD. However, causality has not been established for many of these variants, and their mechanism and functional consequences are unknown. One such variant, MFN2 eQTL rs1474868 (T/T), has been associated with reduced platelet counts and reduced expression (5 fold) of MFN2 RNA in platelets. We show here that the MFN2 T/T variant corresponds with significantly reduced MFN2 protein in platelets. This difference contributes to a significant correlation between MFN2 RNA levels and mitochondrial load and potential in platelets. MFN2 RNA is also reduced by T/T in human cord blood derived megakaryocytes resulting in unfused mitochondria and impaired megakaryopoiesis. Using platelet/megakaryocyte specific Mfn2-/- (Mfn2 KO) mice, we show that Mfn2 impacts platelet numbers, activation and function by regulating mitochondrial energetics. Platelets without Mfn2 had reduced mitochondrial membrane potential and significantly reduced platelet lifespan (P Disclosures Rondina: Novartis: Research Funding; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Transcriptomics: Patents & Royalties.

There is heritability to interindividual variation in platelet count, and better understanding of the regulating genetic factors may provide insights for thrombopoiesis. MicroRNAs (miRs) regulate gene expression in health and disease, and megakaryocytes (MKs) deficient in miRs have lower platelet counts, but information about the role of miRs in normal human MK and platelet production is limited. Using genome-wide miR profiling, we observed strong correlations among human bone marrow MKs, platelets, and differentiating cord blood–derived MK cultures, and identified MK miR-125a-5p as associated with human platelet number but not leukocyte or hemoglobin levels. Overexpression and knockdown studies showed that miR-125a-5p positively regulated human MK proplatelet (PP) formation in vitro. Inhibition of miR-125a-5p in vivo lowered murine platelet counts. Analyses of MK and platelet transcriptomes identified LCP1 as a miR-125a-5p target. LCP1 encodes the actin-bundling protein, L-plastin, not previously studied in MKs. We show that miR-125a-5p directly targets and reduces expression of MK L-plastin. Overexpression and knockdown studies show that L-plastin promotes MK progenitor migration, but negatively correlates with human platelet count and inhibits MK PP formation (PPF). This work provides the first evidence for the actin-bundling protein, L-plastin, as a regulator of human MK PPF via inhibition of the late-stage MK invagination system, podosome and PPF, and PP branching. We also provide resources of primary and differentiating MK transcriptomes and miRs associated with platelet counts. miR-125a-5p and L-plastin may be relevant targets for increasing in vitro platelet manufacturing and for managing quantitative platelet disorders.

RATIONALE: Longitudinal studies are required to distinguish within versus between-individual variation, and repeatability of gene expression. They are uniquely positioned to decipher genetic signal from environmental noise, with potential application to gene variant and expression studies. However, longitudinal analyses of gene expression in healthy individuals—especially with regards to alternative splicing—are lacking for most primary cell types, including platelets. OBJECTIVE: To assess repeatability of gene expression and splicing in platelets and use repeatability to identify novel platelet eQTLs and sQTLs. METHODS AND RESULTS: We sequenced the transcriptome of platelets isolated repeatedly up to 4 years from healthy individuals. We examined within and between-individual variation and repeatability of platelet RNA-expression and exon skipping, a readily measured alternative splicing event. We find that platelet gene expression is generally stable between and within individuals over time—with the exception of a subset of genes enriched for the inflammation gene ontology. We show an enrichment among repeatable genes for associations with heritable traits, including known and novel platelet eQTLs. Several exon skipping events were also highly repeatable, suggesting heritable patterns of splicing in platelets. One of the most repeatable was exon 14 skipping of SELP. Accordingly, we identify rs6128 as a platelet sQTL and define an rs6128-dependent association between SELP exon 14 skipping and race. In vitro experiments demonstrate that this single nucleotide variant directly affects exon 14 skipping and changes the ratio of transmembrane versus soluble P-selectin protein production. CONCLUSIONS: We conclude that the platelet transcriptome is generally stable over 4-years. We demonstrate the use of repeatability of gene expression and splicing to identify novel platelet eQTLs and sQTLs. rs6128 is a platelet sQTL that alters SELP exon 14 skipping and soluble versus transmembrane P-selectin protein production.

ispartof: JOURNAL OF THROMBOSIS AND HAEMOSTASIS vol:17 issue:8 pages:1253-1260 ispartof: location:England status: published

Acute myocardial infarction is primarily due to coronary atherosclerotic plaque rupture and subsequent thrombus formation. Platelets play a key role in the genesis and progression of both atherosclerosis and thrombosis. Since platelets are anuclear cells that inherit their mRNA from megakaryocyte precursors and maintain it unchanged during their life span, gene expression profiling at the time of an acute myocardial infarction provides information concerning the platelet gene expression preceding the coronary event. In ST-segment elevation myocardial infarction (STEMI), a gene-by-gene analysis of the platelet gene expression identified five differentially expressed genes: FKBP5, S100P, SAMSN1, CLEC4E and S100A12. The logistic regression model used to combine the gene expression in a STEMI vs healthy donors score showed an AUC of 0.95. The same five differentially expressed genes were externally validated using platelet gene expression data from patients with coronary atherosclerosis but without thrombosis. Platelet gene expression profile highlights five genes able to identify STEMI patients and to discriminate them in the background of atherosclerosis. Consequently, early signals of an imminent acute myocardial infarction are likely to be found by platelet gene expression profiling before the infarction occurs.

SummaryPlatelet activation in response to stimulation of the Protease Activated Receptor 4 (PAR4) receptor differs by race. One factor that contributes to this difference is the expression level of Phosphatidylcholine Transfer Protein (PCTP), a regulator of platelet PAR4 function. We have conducted an expression Quantitative Trait Locus (eQTL) analysis that identifies single nucleotide polymorphisms (SNPs) linked to the expression level of platelet genes. This analysis revealed 26 SNPs associated with the expression level of PCTP at genome-wide significance (p < 5×10–8). Using annotation from ENCODE and other public data we prioritised one of these SNPs, rs2912553, for functional testing. The allelic frequency of rs2912553 is racially-dimorphic, in concordance with the racially differential expression of PCTP. Reporter gene assays confirmed that the single nucleotide change caused by rs2912553 altered the transcriptional potency of the surrounding genomic locus. Electromobility shift assays, luciferase assays, and overexpression studies indicated a role for the megakaryocytic transcription factor GATA1. In summary, we have integrated multi-omic data to identify and functionalise an eQTL. This, along with the previously described relationship between PCTP and PAR4 function, allows us to characterise a genotype-phenotype relationship through the mechanism of gene expression.

Atherosclerotic vessel injury induces recruitment of both platelets and neutrophils where multiple proteases induce platelet activation and aggregation. Platelets contain two protease activated receptors, PAR1 and PAR4, the cleavage of which results in exposure of a new amino terminus to serve as a tethered ligand. Released neutrophil cathepsin G (CatG) has been shown to be a physiologic modulator of platelet thrombus formation in mice. CatG activates PAR4 and not PAR1, presumably because CatG cleaves PAR1 by removing its tethered ligand. However, neither the CatG biochemical cleavage of PAR4 nor the resulting tethered ligands have been reported. The goals of the current study are to (1) identify the CatG-PAR4 cleavage sites and resulting tethered ligands and (2) determine how CatG-stimulated PAR4 signaling is altered by the PAR4 Ala120Thr variant. We synthesized two portions of the PAR4 extracellular N-terminus: amino acids Asp38-Ser58 (PAR4-B) and Asp57-Arg78 (PAR4-C) and exposed each peptide to purified CatG. Mass spectrometry identified two major cleavage sites for PAR4-B: the previously documented CatG and thrombin site Arg47-Gly48 and a novel Cys54-Ala55. Analysis of PAR4-C digestion yielded an additional three novel CatG cleavage sites, two major: Arg68-Ala69 and Leu71-Leu72, and one minor: Leu70-Leu71. Neither concentration or time of exposure appeared to alter the CatG cleavage sites. To assess functionality, we generated peptides based on the novel cleavage sites produced by CatG cleavage. Human washed platelets were treated with each peptide, and platelet activation was assessed by PAC-1 binding. As expected, the known tethered ligand sequence GYPGQV showed a statistically significant increase in PAC-1 binding (p=.02) compared to resting platelets. Three of the remaining four novel peptides generated no significant change in PAC-1 binding compared to baseline. However, peptide 3, representing novel tethered ligand ALLLGW, induced a substantial increase (462%) in PAC-1 binding compared to resting platelets. To assess the effect of the PAR4Ala120Thr variant on CatG-stimulated platelet reactivity, human washed platelets were collected from donors homozygous for Ala120 or Thr120 and stimulated with CatG. Platelets expressing the Thr120 variant displayed a significant increase in PAC-1 compared to Ala120 platelets (54%; p=.004). Addition of a CatG inhibitor caused a significant decrease in platelet activation triggered by CatG for both groups (Thr: -81%, p=.000001; Ala: -78%, p=.00017), and abrogated the significant increase in platelet activation displayed by Thr120 platelets (p=.99). To further examine the relationship between the PAR4 Ala120Thr variant and CatG, platelets from each variant were subjected to increasing amounts of CatG. Platelet activation was measured by PAC-1 binding and P-selectin expression. Both PAC-1 binding and P-selectin expression were significantly increased in platelets from Thr120 donors compared to Ala120 platelets (PAC-1, p=.026; P-selectin, p=.025). Overall, our study identified a previously unidentified CatG cleavage site in PAR4, which produced a novel tethered ligand capable of activating platelets. Because cleavage resulting in the ALLLGW ligand is downstream of the thrombin cleavage site, CatG may modulate in vivo thrombin-induced signaling in platelets or other cell types. Our findings also indicate the hyperactive response of 120Thr platelets is not dependent on a specific PAR4 protease. These new insights into PAR4 biology may provide targets for future antithrombotic therapies. Disclosures No relevant conflicts of interest to declare.

Human platelets express two thrombin-activated G-protein coupled receptors, PAR1 and PAR4, that activate upon protease cleavage of the amino-terminus of the receptor. Thrombin induces rapid and transient platelet activation through PAR1, while PAR4 mediates slower and sustained activation, as well as greater phosphatidylserine (PS) exposure and thrombin generation under shear stress. Because PAR1 blockade is contraindicated in treating stroke, PAR4 has been considered as a novel potential anti-platelet target. Human PAR4 has a common Ala120Thr variant that is much more common in individuals of sub-Saharan African ancestry. The Thr120 variant induces platelet hyperreactivity, and is associated with increased stroke risk and less bleeding. Because human PAR4 (hPAR4) differs significantly from mouse Par4 (mPar4), in vivo studies of hPAR4 and the Ala120Thr variant have not been possible. The goals of the current study were to (1) express and characterize hPAR4 in vivo and (2) determine specifically if the PAR4 Thr120 variant results in platelet hyperactivity. We generated humanized PAR4 Ala120 (hPAR4Ala) and PAR4 Thr120 (hPAR4Thr) mouse lines using CRISPR/Cas9 to replace the mouse F2rl3 with either human variant gene (F2RL3). Both hPAR4Ala and hPAR4Thr lines are viable, display germline transmission of hPAR4 and produce expected numbers of offspring. Genomic DNA from both strains contained human F2RL3 and not murine F2rl3, and whole genome sequencing confirmed the successful insertion of a single copy of F2RL3 in the proper location. hPAR4 mRNA was abundantly expressed in platelets from hPAR4Ala and hPAR4Thr, but not wild type mice. Our engineering strategy retained murine F2rl3 regulatory sequences, and hPAR4 transcripts were readily detected in brain, liver and lung in hPAR4Ala and hPAR4Thr mice. Importantly for our goals, hPAR4 mRNA and protein was expressed at similar levels in both hPAR4Ala and hPAR4Thr lines. Platelet counts and mean platelet volume from hPAR4Ala (635±149 K/μL and 4.2±0.1) and hPAR4Thr (617±145 K/μL and 4.0±0.3) mice were similar to wild type mPar4 mice (705±142 K/μL and 4.2±0.1). When washed platelets were stimulated with PAR4 activation peptides (APs) AYPGKF (artificial) or GYPGQV (natural tethered ligand), a dose-dependent activation of integrin aIIbb3 (by Jon/A binding) was observed that was significantly higher on hPAR4Thr platelets compared to hPAR4Ala platelets (p Disclosures No relevant conflicts of interest to declare.

Platelets carry a diverse repertoire of coding and noncoding RNAs that together make up the platelet transcriptome. Because most RNAs are known to be made from DNA in the nucleus, and platelets are born without a nucleus or nuclear DNA, the origin, purpose, and fate of platelet RNAs have been the subject of investigation for more than 70 years. Classic RNA analysis techniques and next-generation technologies have yielded significant insights into the types of RNAs that are expressed by platelets, their source, their relationship to proteins, and how they change according to genetics, environment, and disease. Analysis of RNA expression and structure, in association with platelet physiology in health, and hematologic or non-hematologic disease has garnered new knowledge regarding the diverse physiological roles of platelets, the genetics and mechanisms of platelet function, and how platelets function in health and disease. Here we review the history and impact of platelet RNA on hematologic research. Our review is divided into three main sections that include: (1) The history of RNA-based studies that have contributed to our understanding of the platelet transcriptome; (2) The basics of gene expression that underlie the origins and properties of megakaryocyte and platelet RNAs; and (3) Knowledge gained by defining the platelet transcriptome in health and disease.

Evolving evidence indicates that platelets and megakaryocytes (MKs) have unexpected activities in inflammation and infection; whether viral infections upregulate biologically active, antiviral immune genes in platelets and MKs is unknown, however. We examined antiviral immune genes in these cells in dengue and influenza infections, viruses that are global public health threats. Using complementary biochemical, pharmacological, and genetic approaches, we examined the regulation and function of interferon-induced transmembrane protein 3 (IFITM3), an antiviral immune effector gene not previously studied in human platelets and MKs. IFITM3 was markedly upregulated in platelets isolated from patients during clinical influenza and dengue virus (DENV) infections. Lower IFITM3 expression in platelets correlated with increased illness severity and mortality in patients. Administering a live, attenuated DENV vaccine to healthy subjects significantly increased platelet IFITM3 expression. Infecting human MKs with DENV selectively increased type I interferons and IFITM3. Overexpression of IFITM3 in MKs was sufficient to prevent DENV infection. In naturally occurring, genetic loss-of-function studies, MKs from healthy subjects harboring a homozygous mutation in IFITM3 (rs12252-C, a common single-nucleotide polymorphism in areas of the world where DENV is endemic) were significantly more susceptible to DENV infection. DENV-induced MK secretion of interferons prevented infection of bystander MKs and hematopoietic stem cells. Thus, viral infections upregulate IFITM3 in human platelets and MKs, and IFITM3 expression is associated with adverse clinical outcomes. These observations establish, for the first time, that human MKs possess antiviral functions, preventing DENV infection of MKs and hematopoietic stem cells after local immune signaling.

Apoptosis is a recognized limitation to generating large numbers of megakaryocytes in culture. The genes responsible have been rigorously studied in vivo in mice, but are poorly characterized in human culture systems. As CD34-positive (+) cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity (LLG), and smaller size, higher granularity (SHG). The LLG cells were CD41aHigh CD42aHigh phosphatidylserineLow, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and displayed a signaling response to platelet agonists. The SHG cells were CD41aLowCD42aLowphosphatidylserineHigh, had a distinctly apoptotic morphology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 apoptosis genes identified BCL2L2 as a novel candidate megakaryocyte apoptosis regulator. Lentiviral BCL2L2 overexpression decreased megakaryocyte apoptosis, increased CD41a+ LLG cells, and increased proplatelet formation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet BCL2L2 mRNA levels. This finding was consistent with the observed increase in platelet-like particles derived from cultured megakaryocytes over-expressing BCL2L2. BCL2L2 also induced small, but significant increases in thrombin-induced platelet-like particle αIIbβ3 activation and P-selectin expression. Thus, BCL2L2 restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation, and is associated with platelet number. BCL2L2 is a novel target for improving megakaryocyte and platelet yields in in vitro culture systems.

Essentials The action of microRNAs (miRs) in human megakaryocyte signaling is largely unknown. Cord blood-derived human megakaryocytes (MKs) were used to test the function of candidate miRs. miR-15a-5p negatively regulated MK GPVI-mediated αIIbβ3 activation and α-granule release. miR-15a-5p acts as a potential "master-miR" regulating genes in the MK GPVI signaling pathway. SUMMARY: Background Megakaryocytes (MKs) invest their progeny platelets with proteins and RNAs. MicroRNAs (miRs), which inhibit mRNA translation into protein, are abundantly expressed in MKs and platelets. Although platelet miRs have been associated with platelet reactivity and disease, there is a paucity of information on the function of miRs in human MKs. Objective To identify MK miRs that regulate the GPVI signaling pathway in the MK-platelet lineage. Methods Candidate miRs associated with GPVI-mediated platelet aggregation were tested for functionality in cultured MKs derived from cord blood. Results An unbiased, transcriptome-wide screen in 154 healthy donors identified platelet miR-15a-5p as significantly negatively associated with CRP-induced platelet aggregation. Platelet agonist dose-response curves demonstrated activation of αIIbβ3 in suspensions of cord blood-derived cultured MKs. Overexpression and knockdown of miR-15a-5p in these MKs reduced and enhanced, respectively, CRP-induced αIIbβ3 activation but did not alter thrombin or ADP stimulation. FYN, SRGN, FCER1G, MYLK. and PRKCQ, genes involved in GPVI signaling, were identified as miR-15a-5p targets and were inhibited or de-repressed in MKs with miR-15a-5p overexpression or inhibition, respectively. Lentiviral overexpression of miR-15a-5p also inhibited GPVI-FcRγ-mediated phosphorylation of Syk and PLCγ2, GPVI downstream signaling molecules, but effects of miR-15a-5p on αIIbβ3 activation did not extend to other ITAM-signaling receptors (FcγRIIa and CLEC-2). Conclusion Cord blood-derived MKs are a useful human system for studying the functional effects of candidate platelet genes. miR-15a-5p is a potential "master-miR" for specifically regulating GPVI-mediated MK-platelet signaling. Targeting miR-15a-5p may have therapeutic potential in hemostasis and thrombosis.

Thrombin activates human platelets via 2 protease-activated receptors (PARs), PAR1 and PAR4, both of which are antithrombotic drug targets: a PAR1 inhibitor is approved for clinical use, and a PAR4 inhibitor is in trial. However, a common sequence variant in human PAR4 (rs773902, encoding Thr120 in place of Ala120) renders the receptor more sensitive to agonists and less sensitive to antagonists. Here, we develop the first human monoclonal function-blocking antibody to human PAR4 and show it provides equivalent efficacy against the Ala120 and Thr120 PAR4 variants. This candidate was generated from a panel of anti-PAR4 antibodies, was found to bind PAR4 with affinity (KD ≈ 0.4 nM) and selectivity (no detectable binding to any of PAR1, PAR2, or PAR3), and is capable of near-complete inhibition of thrombin cleavage of either the Ala120 or Thr120 PAR4 variant. Platelets from individuals expressing the Thr120 PAR4 variant exhibit increased thrombin-induced aggregation and phosphatidylserine exposure vs those with the Ala120 PAR4 variant, yet the PAR4 antibody inhibited these responses equivalently (50% inhibitory concentration, 4.3 vs 3.2 µg/mL against Ala120 and Thr120, respectively). Further, the antibody significantly impairs platelet procoagulant activity in an ex vivo thrombosis assay, with equivalent inhibition of fibrin formation and overall thrombus size in blood from individuals expressing the Ala120 or Thr120 PAR4 variant. These findings reveal antibody-mediated inhibition of PAR4 cleavage and activation provides robust antithrombotic activity independent of the rs773902 PAR4 sequence variant and provides rationale for such an approach for antithrombotic therapy targeting this receptor.

F2RL3 encodes protease-activated receptor 4 (PAR4) and harbors a A/G SNP (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP), AYPGKF, which does not occur in vivo. Platelet function studies from healthy human donors demonstrated that compared to rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation to low concentrations (2+ signaling, increased granule secretion and decreased PAR4 desensitization. The increased thrombin sensitivity of rs773902 AA platelets required both PAR4 and PAR1. Whereas receptor cleavage, dimerization, or lipid raft localization were not different between platelets from the two F2RL3 genotypes, rs773902 AA platelets required more than 3-fold amounts of PAR4-AP for receptor desensitization. PAR4-AP stimulation of PAR4-transfected COS-7 cells increased PAR4 levels on the cell surface. Thrombin-stimulated platelets from rs773902 AA donors displayed reduced inhibition by P2Y12 and PAR1 blockers, but not aspirin. Finally, the association of rs773902 alleles with stroke was assessed using the Stroke Genetics Network (SiGN) study. Patients carrying at least one copy of the rs773902 A allele had an increased risk of both large vessel stroke and diabetes in the SiGN dataset. In summary, both PAR4 and PAR1 are required for platelet activation to sub-nanomolar thrombin concentrations. Compared to rs773902 GG platelets, rs773902 AA platelets have a lower EC50 for thrombin and are relatively resistant to receptor desensitization, perhaps due to anterograde trafficking of internal PAR4 to the plasma membrane post-stimulation. The association of the rs773902 A variant with large vessel stroke, diabetes and partial resistance to PAR1 and P2Y12 inhibition must be considered for development of personalized anti-platelet agents.

Platelet activation and thrombus formation play a central role in ischemic vascular disease. Thrombin, an especially potent physiologic agonist mediating in vivo activation of platelets, acts via a unique family of G-protein-coupled receptors called protease-activated receptors (PARs) with a broad tissue expression. This review focuses on current antiplatelet therapies as well as innovative approaches to targeting PARs in patients with atherothrombotic vascular disease.

Variation in platelet response to thrombin may affect the safety and efficacy of PAR antagonism. The Thr120 variant of the common single nucleotide polymorphism (SNP) rs773902 in the protease-activated receptor (PAR) 4 gene is associated with higher platelet aggregation compared to the Ala120 variant. We investigated the relationship between the rs773902 SNP with major bleeding and ischemic events, safety, and efficacy of PAR1 inhibition in 6177 NSTE ACS patients in the TRACER trial. There was a lower rate of GUSTO moderate/severe bleeding in patients with the Thr120 variant. The difference was driven by a lower rate in the smaller homozygous group (recessive model, HR 0.13 [0.02–0.92] P = 0.042). No significant differences were observed in the ischemic outcomes. The excess in bleeding observed with PAR1 inhibition was attenuated in patients with the Thr120 variant, but the interactions were not statistically significant. In summary, lower major bleeding rates were observed in the overall TRACER cohort with the hyperreactive PAR4 Thr120 variant. The increase in bleeding with vorapaxar was attenuated with the Thr120 variant, but we could not demonstrate an interaction with PAR1 inhibition. These findings warrant further exploration, including those of African ancestry where the A allele (Thr120) frequency is ~65%.

Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function-a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.

Objective— Black individuals are at an increased risk of myocardial infarction and stroke, 2 vascular diseases with strong thrombotic components. Platelet activation is a key step in platelet clot formation leading to myocardial infarction and stroke, and recent work supports a racial difference in platelet aggregation through the thrombin protease-activated receptors (PARs). The underlying mechanism for this racial difference, however, has not been established. Determining where in the signaling cascade these racial differences emerge will aid in understanding why individuals of differing racial ancestry may possess an inherent difference in their responsiveness to antiplatelet therapies. Approach and Results— Washed human platelets from black volunteers were hyperaggregable in response to PAR4-mediated platelet stimulation compared with whites. Interestingly, the racial difference in PAR4-mediated platelet aggregation persisted in platelets treated ex vivo with aspirin and 2MeSAMP (2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate), suggesting that the racial difference is independent of secondary feedback. Furthermore, stimulation of platelets from black donors with PAR4-activating peptide showed a potentiated level of activation through the G q pathway compared with platelets from white donors. Differences in signaling included increased Ca 2+ mobilization, Rap1 (Ras-related protein 1) activation, and integrin α IIb β 3 activation with no observed difference in platelet protein expression between the groups tested. Conclusions— Our study is the first to demonstrate that the G q pathway is differentially regulated by race after PAR4 stimulation in human platelets. Furthermore, the racial difference in PAR4-mediated platelet aggregation persisted in the presence of cyclooxygenase and P2Y 12 receptor dual inhibition, suggesting that current antiplatelet therapy may provide less protection to blacks than whites.

There is little data considering relationships among human RNA, demographic variables, and primary human cell physiology. The platelet RNA and expression-1 study measured platelet aggregation to arachidonic acid, ADP, protease-activated receptor (PAR) 1 activation peptide (PAR1-AP), and PAR4-AP, as well as mRNA and microRNA (miRNA) levels in platelets from 84 white and 70 black healthy subjects. A total of 5911 uniquely mapped mRNAs and 181 miRNAs were commonly expressed and validated in a separate cohort. One hundred twenty-nine mRNAs and 15 miRNAs were differentially expressed (DE) by age, and targets of these miRNAs were over-represented among these mRNAs. Fifty-four mRNAs and 9 miRNAs were DE by gender. Networks of miRNAs targeting mRNAs, both DE by age and gender, were identified. The inverse relationship in these RNA pairs suggests miRNAs regulate mRNA levels on aging and between genders. A simple, interactive public web tool (www.plateletomics.com) was developed that permits queries of RNA levels and associations among RNA, platelet aggregation and demographic variables. Access to these data will facilitate discovery of mechanisms of miRNA regulation of gene expression. These results provide new insights into aging and gender, and future platelet RNA association studies must account for age and gender.

The concept of race based on physical appearance, social grouping, or self-identification has been challenged for decades. With the recent sequencing of thousands of human genomes, this concept has been further redefined. In this issue of Blood, Heenkenda et al show that the A allele frequency of the F2RL3 rs773902 A/G dimorphism associated with increased platelet reactivity is significantly lower in Somalians than what has been previously described for blacks in the United States, underscoring the importance of genetics and geographic ancestry for determining disease risk.

Introduction: Clotting factor replacement therapy in the management of persons with hemophilia A (PwHA) is typically dosed using the following weight-based formula: Factor VIII (FVIII) Dose = Body weight (BW) in kg X desired FVIII increase (%) 2 This equation is based upon a FVIII in vivo recovery (IVR) of 2, which assumes that each FVIII unit per kg of BW infused will increase the circulating FVIII concentration by 2%. There have been a few studies demonstrating that IVR increases with increasing BW. A pharmacokinetic study performed using recombinant FVIII concentrate for pediatric PwHA 29.6 kg/m2, each group had a significantly different and increasing IVR of 1.60, 2.14 and 2.70, respectively [Henrard S, et al. Haematologica 2013; 98: 1481-6]. As there are no guidelines for dosing of factor replacement therapy for obese PwHA, these studies suggest that ideal body weight (IBW) could be considered instead of actual body weight (ABW) in the dose calculations. With the prevalence of obesity in the hemophilia population now approaching that of the general population, dosing using IBW has potential economic ramifications [Wong TE, et al. Am J Prev Med 2011; 41: S369-75, Majumdar S, et al. Haemophilia 2011; 17: 717-8]. Objectives: 1) To estimate the prevalence of overweight and obese adult persons with hemophilia (PwH) in the state of Utah; 2) To estimate the cost savings for annual prophylactic clotting factor prescriptions if they were to be written using IBW vs. ABW for adult PwHA that are obese in the state of Utah. Methods: Adult PwH (≥18 years of age) who have been seen at least once in the past 3 years (January 1, 2016 to December 31, 2018) were identified from the Utah Center for Bleeding & Clotting Disorders Registry. The following information was collected: Demographics; type of hemophilia; severity of hemophilia; height; weight; BMI. If BMI was not available, this was calculated based on the following equation, BMI = kilograms/meters2. Overweight was defined as a BMI 25 - 29.9 kg/m2. Obese was defined as a BMI ≥ 30 kg/m2. As there is limited data on the relationship between increasing BMI and IVR for factor IX concentrates, persons with hemophilia B were excluded in our cost savings analysis. For PwHA on clotting factor prophylaxis who were obese, the IBW was calculated using their height and the following equation for men: IBW (in kg) = 50 + 2.3 kg per each inch over 5 feet Using the prophylaxis schedule for each obese PwHA, an estimate of the annual cost was calculated using Wholesale Acquisition Cost and compared using both ABW and IBW. Results: A total of 87 adult PwH were seen with documented measurements of height and weight within the past 3 years. The patient characteristics are presented in Table 1. The mean and median age was 37.1 and 34 years, respectively (range: 19 - 75 years). The mean value for BMI was 28.1 and the median was 27.2 (range 17.3 - 46.0). Two patients had class III obesity (BMI ≥40 kg/m2). Almost two-thirds of adult PwH were overweight or obese (58/87; 65.5%). The prevalence of overweight and obese adult PwH in Utah based on age and severity of disease is shown in Figure 1. The estimated annual cost for each of the 12 adult obese PwHA based on their ABW vs IBW is shown in Figure 2. The difference in the total annual cost of treatment for the 12 adult PwHA on prophylaxis factor replacement therapy would be reduced by a total of roughly $2,100,000 if IBW was used rather than ABW; an average savings of $175,000 per adult PwHA per year. Conclusion: The relatively high prevalence of overweight and obesity in the Utah hemophilic population is similar to what has been reported in other states across the United States. The use of IBW in dose calculations has significant economic ramifıcations. Further studies performing individualized pharmacokinetic analysis for PwHA using IBW dosing must be coupled with close clinical monitoring to ensure safety of this dosing strategy before it can be adopted into routine clinical practice. Disclosures Rodgers: Pfizer: Consultancy; Octapharma: Consultancy; Novartis: Consultancy; Sanofi: Consultancy; AstraZeneca: Consultancy.

Platelet count (PC) and mean platelet volume (MPV) are critical measurements when considering the role of platelets in hemostasis and thrombosis, abnormalities in PC and MPV have been shown to be predictors of ischemic cardiovascular disease. Ischemic vascular disease has a known genetic component, and we and others have shown high heritability for both PC and MPV. Many inherited, and most immune, thrombocytopenias show an inverse relationship between PC and MPV, and such a relationship has also been reported in healthy subjects. Although genome-wide association studies have identified numerous single nucleotide variants in protein-coding genes associated with PC and MPV (Geiger Nat Genet 2011), non-coding genes have had rather little consideration. The goal of our studies was to identify microRNAs (miRs) regulating PC and MPV and to begin to understand the molecular mechanisms of this inverse platelet number-volume relationship. Initially, we queried our Platelet RNA and Expression-1 data set and observed that PC and MPV are inversely correlated in these 154 healthy subjects (r=-0.45, p=3.98E-09). We next determined which of the 734 platelet-expressed miRs were significantly (p To begin to address the potential functionality of this miR cluster, we overexpressed and knocked down their expression in megakaryocytes (MKs), since MK proplatelet formation (PPF) is a necessary step in the generation of peripheral blood platelets. We utilized human umbilical cord blood derived CD34-positive hematopoietic stem and progenitor cells differentiated along the MK lineage with thrombopoietin. Quantification of miR levels with qPCR demonstrated all 3 miRs in the cluster progressively increased from culture day 6 to day 9 to day 13 (the time during which MKs mature in culture). Compared to a scrambled control, when miR activity was knocked down with locked nucleic acids (LNAs), we observed a 61% reduction in PPF for miR-125a-5p (p Disclosures No relevant conflicts of interest to declare.

Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. Platelet aggregation and calcium mobilization induced by the PAR4 thrombin receptor were significantly greater in black subjects. Numerous differentially expressed RNAs were associated with both race and PAR4 reactivity, including PCTP (encoding phosphatidylcholine transfer protein), and platelets from black subjects expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked PAR4- but not PAR1-mediated activation of platelets and megakaryocytic cell lines. miR-376c levels were differentially expressed by race and PAR4 reactivity and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. miR-376c regulated the expression of PC-TP in human megakaryocytes. A disproportionately high number of microRNAs that were differentially expressed by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus and were expressed at lower levels in platelets from black subjects. These results suggest that PC-TP contributes to the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race and emphasize a need to consider the effects of race when developing anti-thrombotic drugs.

Recent work by the Encyclopedia of DNA Elements project showed that non-protein-coding RNAs account for an unexpectedly large proportion of the human genome. Among these non-coding RNAs are microRNAs (miRNAs), which are small RNA molecules that modulate protein expression by degrading mRNA or repressing mRNA translation. MiRNAs have been shown to play important roles in hematopoiesis including embryonic stem cell differentiation, erythropoiesis, granulocytopoiesis/monocytopoiesis, lymphopoiesis, and megakaryocytopoiesis. Additionally, disordered miRNA biogenesis and quantitative or qualitative alterations in miRNAs and their targets are associated with hematological pathologies. Platelets contain machinery to process pre-miRNAs into mature miRNAs, and specific platelet miRNA levels have been found to correlate with platelet reactivity. This review summarizes the current state of knowledge of miRNAs in megakaryocytes and platelets, and the exciting possibilities for future megakaryocyte-platelet transcriptome research.

Noncoding RNAs (ncRNA) account for the majority of the human cellular transcriptome. Advances in sequencing technologies and analytic approaches continue to identify novel ncRNAs. A large amount of research has investigated the role of microRNAs (miRNAs) in megakaryopoiesis and platelet biology, whereas relatively little is known about other ncRNAs. miRNAs affect basic development and physiology by regulating protein translation, and in doing so miRNAs regulate hematopoiesis and megakaryopoiesis. To date, platelet miRNA studies have focused on their use as biomarkers and as tools for understanding basic mechanisms of megakaryocyte/platelet gene expression, but there is a rapidly expanding appreciation for miRNAs as mediators or modifiers of disease, the potential for platelet miRNAs to affect gene expression in other tissues, and as therapeutic targets.

Objective— The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). Approach and Results— HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin–platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2 − /− mice with transgenic FcγRIIA +/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2 − /− murine platelets. Compared with wild-type mice, TULA-2 − /− mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2 − /− and TULA-2 +/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2 +/− mice, whose platelets contained 50% as much protein as the TULA-2 +/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2 +/+ mice. Conclusions— Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

Most physicians believe they practiced personalized medicine prior to the genomics era that followed the sequencing of the human genome. The focus of personalized medicine has been primarily genomic medicine, wherein it is hoped that the nucleotide dissimilarities among different individuals would provide clinicians with more precise understanding of physiology, more refined diagnoses, better disease risk assessment, earlier detection and monitoring, and tailored treatments to the individual patient. However, to date, the “genomic bench” has not worked itself to the clinical thrombosis bedside. In fact, traditional plasma-based hemostasis-thrombosis laboratory testing, by assessing functional pathways of coagulation, may better help manage venous thrombotic disease than a single DNA variant with a small effect size. There are some new and exciting discoveries in the genetics of platelet reactivity pertaining to atherothrombotic disease. Despite a plethora of genetic/genomic data on platelet reactivity, there are relatively little actionable pharmacogenetic data with antiplatelet agents. Nevertheless, it is crucial for genome-wide DNA/RNA sequencing to continue in research settings for causal gene discovery, pharmacogenetic purposes, and gene-gene and gene-environment interactions. The potential of genomics to advance medicine will require integration of personal data that are obtained in the patient history: environmental exposures, diet, social data, etc. Furthermore, without the ritual of obtaining this information, we will have depersonalized medicine, which lacks the precision needed for the research required to eventually incorporate genomics into routine, optimal, and value-added clinical care.

Platelets play a central role in ischemic cardiovascular events. Cardiovascular disease (CVD) is a major cause of death worldwide. Numerous genome-wide association studies (GWASs) have identified loci associated with CVD risk. However, our understanding of how these variants contribute to disease is limited. Using data from the platelet RNA and expression 1 (PRAX1) study, we analyzed cis expression quantitative trait loci (eQTLs) in platelets from 154 normal human subjects. We confirmed these results in silico by performing allele-specific expression (ASE) analysis, which demonstrated that the allelic directionality of eQTLs and ASE patterns correlate significantly. Comparison of platelet eQTLs with data from the Genotype-Tissue Expression (GTEx) project revealed that a number of platelet eQTLs are platelet specific and that platelet eQTL peaks localize to the gene body at a higher rate than eQTLs from other tissues. Upon integration with data from previously published GWASs, we found that the trait-associated variant rs1474868 coincides with the eQTL peak for mitofusin 2 (MFN2). Additional experimental and computational analyses revealed that this eQTL is linked to an unannotated alternate MFN2 start site preferentially expressed in platelets. Integration of phenotype data from the PRAX1 study showed that MFN2 expression levels were significantly associated with platelet count. This study links the variant rs1474868 to a platelet-specific regulatory role for MFN2 and demonstrates the utility of integrating multi-omic data with eQTL analysis in disease-relevant tissues for interpreting GWAS results.

Women are underrepresented in cardiovascular studies, even as their preponderance in the aging population steadily increases. Although concerns have been raised about the differential benefit of antiplatelet medications for women, the propensity for increased bleeding among women has also been recognized. A better understanding of the factors contributing to the observed sex-related differences in platelet biology is warranted. These factors include differences in the frequency and expression of genetic polymorphisms affecting platelet responsiveness to agonists (with and without antiplatelet therapies), which might be obtained through population-based studies and in large controlled clinical trials; inflammatory marker levels and their influence on atherothrombotic risk, and the role of specific hormones in mediating platelet activation and function. Knowledge gained about these mechanistic factors might inform the development of sex-specific antithrombotic treatment regimens that confer optimized safety and efficacy.