Metabolic Functions of Biliverdin IXß Reductase in Redox-Regulated Hematopoietic Cell Fate.

Cytoprotective heme oxygenases derivatize heme to generate carbon monoxide, ferrous iron, and isomeric biliverdins, followed by rapid NAD(P)H-dependent biliverdin reduction to the antioxidant bilirubin. Recent studies have implicated biliverdin IXß reductase (BLVRB) in a redox-regulated mechanism of hematopoietic lineage fate restricted to megakaryocyte and erythroid development, a function distinct and non-overlapping from the BLVRA (biliverdin IXα reductase) homologue. In this review, we focus on recent progress in BLVRB biochemistry and genetics, highlighting human, murine, and cell-based studies that position BLVRB-regulated redox function (or ROS accumulation) as a developmentally tuned trigger that governs megakaryocyte/erythroid lineage fate arising from hematopoietic stem cells. BLVRB crystallographic and thermodynamic studies have elucidated critical determinants of substrate utilization, redox coupling and cytoprotection, and have established that inhibitors and substrates bind within the single-Rossmann fold. These advances provide unique opportunities for the development of BLVRB-selective redox inhibitors as novel cellular targets that retain potential for therapeutic applicability in hematopoietic (and other) disorders.

Age-restricted functional and developmental differences of neonatal platelets.

BACKGROUND: Developmental ontogeny of neonatal thrombopoiesis retains characteristics that are distinct from adults although molecular mechanisms remain unestablished. METHODS: We applied multiparameter quantitative platelet responses with integrated ribosome profiling/transcriptomic studies to better define gene/pathway perturbations regulating the neonatal-to-adult transition. A bioinformatics pipeline was developed to identify stable, neonatal-restricted platelet biomarkers for clinical application. RESULTS: Cord blood (CB) platelets retained the capacity for linear agonist-receptor coupling linked to phosphatidylserine (PS) exposure and α-granule release, although a restricted block in cross-agonist activation pathways was evident. Functional immaturity of synergistic signaling pathways was due to younger ontogenetic age and singular underdevelopment of the protein secretory gene network, with reciprocal expansion of developmental pathways (E2F, G2M checkpoint, c-Myc) important for megakaryocytopoiesis. Genetic perturbations regulating vesicle transport and fusion (TOM1L1, VAMP3, SNAP23, and DNM1L) and PS exposure and procoagulant activity (CLCN3) were the most significant, providing a molecular explanation for globally attenuated responses. Integrated transcriptomic and ribosomal footprints identified highly abundant (ribosome-protected) DEFA3 (encoding human defensin neutrophil peptide 3) and HBG1 as stable biomarkers of neonatal thrombopoiesis. Studies comparing CB- or adult-derived megakaryocytopoiesis confirmed inducible and abundant DEFA3 antigenic expression in CB megakaryocytes, ~3.5-fold greater than in leukocytes (the most abundant source in humans). An initial feasibility cohort of at-risk pregnancies manifested by maternal/fetal hemorrhage (chimerism) were applied for detection and validation of platelet HBG1 and DEFA3 as neonatal thrombopoiesis markers, most consistent for HBG1, which displayed gestational age-dependent expression. CONCLUSIONS: These studies establish an ontogenetically divergent stage of neonatal thrombopoiesis, and provide initial feasibility studies to track disordered fetal-to-adult megakaryocytopoiesis in vivo.

Cytokine pathway variants modulate platelet production: IFNA16 is a thrombocytosis susceptibility locus in humans.

Inflammatory stimuli have divergent effects on peripheral platelet counts, although the mechanisms of thrombocytopenic and thrombocytotic responses remain poorly understood. A candidate gene approach targeting 326 polymorphic genes enriched in thrombopoietic and cytokine signaling pathways was applied to identify single nucleotide variants (SNVs) implicated in enhanced platelet responses in cohorts with reactive thrombocytosis (RT) or essential (myeloproliferative neoplasm [MPN]) thrombocytosis (ET). Cytokine profiles incorporating a 15-member subset, pathway topology, and functional interactive networks were distinct between ET and RT, consistent with distinct regulatory pathways of exaggerated thrombopoiesis. Genetic studies using aggregate (ET + RT) or ET-restricted cohorts identified associations with 2 IFNA16 (interferon-α16) SNVs, and the ET associations were validated in a second independent cohort (P = .0002). Odds ratio of the combined ET cohort (n = 105) was 4.92, restricted to the JAK2V617F-negative subset (odds ratio, 5.01). ET substratification analysis by variant IFNA16 exhibited a statistically significant increase in IFN-α16 levels (P = .002) among 16 quantifiable cytokines. Recombinantly expressed variant IFN-α16 encompassing 3 linked non-synonymous SNVs (E65H95P133) retained comparable antiviral and pSTAT signaling profiles as native IFN-α16 (V65D95A133) or IFN-α2, although both native and variant IFN-α16 showed stage-restricted differences (compared with IFN-α2) of IFN-regulated genes in CD34+-stimulated megakaryocytes. These data implicate IFNA16 (IFN-α16 gene product) as a putative susceptibility locus (driver) within the broader disrupted cytokine network evident in MPNs, and they provide a framework for dissecting functional interactive networks regulating stress or MPN thrombopoiesis.

Genetic pathways regulating hematopoietic lineage speciation: Factorial latent variable model analysis of single cell transcriptome.

Genetic pathways regulating hematopoietic lineage commitment at critical stages of development remain incompletely characterized.  To better delineate genetic sources of variability regulating cellular speciation during steady-state hematopoiesis, we applied a factorial single-cell latent variable model (f-scLVM) to decompose single-cell transcriptome heterogeneity into interpretable biological factors (refined pathway annotations or gene sets without annotation) dynamically regulating cell fate.  Hematopoietic single cell transcriptomic raw sequencing data extracted from 1,920 hematopoietic stem and progenitor cells (HSPCs) derived from 12-week-old female mice were used for data analysis and model development. These single cell RNA sequencing data were subsequently analyzed using the factorial single-cell latent variable model (f-scLVM), with their heterogeneity decomposed into interpretable biological factors. The top biological factors underlying the basal hematopoiesis were subsequently identified for the aggregate, and lineage-restricted (myeloid, megakaryocyte, erythroid) progenitor cells. For a subset of factors, data were independently verified experimentally in a companion research paper [1]. These data facilitate the identification of novel subpopulations and adjust gene sets to discover new marker genes and hidden confounding factors driving basal hematopoiesis.

Divergent erythroid megakaryocyte fates in Blvrb-deficient mice establish non-overlapping cytoprotective functions during stress hematopoiesis.

Cytoprotective mechanisms of heme oxygenases function by derivatizing heme to generate carbon monoxide, ferrous iron, and isomeric biliverdins, followed by rapid NAD(P)H-dependent biliverdin reduction to the antioxidant bilirubin using two non-overlapping biliverdin reductases that display biliverdin isomer-restricted redox activity. Although cytoprotective functions of heme oxygenases are widely recognized, concomitant effects of downstream biliverdin reductases remain incomplete. A computational model predicated on murine hematopoietic single-cell transcriptomic data identified Blvrb as a biological driver linked to the tumor necrosis factor stress pathway as a predominant source of variation defining hematopoietic cell heterogeneity. In vivo studies using Blvrb-deficient mice established the dispensable role of Blvrb in steady-state hematopoiesis, although model validation using aged Blvrb-deficient mice established an important cytoprotective function in stress hematopoiesis with dichotomous megakaryocyte-biased hematopoietic recovery. Defective stress erythropoiesis was evident in Blvrb-/- spleens and in bone marrow erythroid development, occurring in conjunction with defective lipid peroxidation as a marker of oxidant mishandling. Cell autonomous effects on megakaryocyte lineage bias were documented using multipotential progenitor assays. These data provide the first physiological function of murine Blvrb in a non-redundant pathway of stress cytoprotection. Divergent effects on erythroid/megakaryocyte lineage speciation impute a novel redox-regulated mechanism for lineage partitioning.

Shear-Induced Platelet Activation is Sensitive to Age and Calcium Availability: A Comparison of Adult and Cord Blood.

INTRODUCTION: Antiplatelet therapy for neonates and infants is often extrapolated from the adult experience, based on limited observation of agonist-induced neonatal platelet hypoactivity and poor understanding of flow shear-mediated platelet activation. Therefore, thrombotic events due to device-associated disturbed flow are inadequately mitigated in critically ill neonates with indwelling umbilical catheters and infants receiving cardiovascular implants. METHODS: Whole blood (WB), platelet-rich plasma (PRP), and gel-filtered platelets (GFP) were prepared from umbilical cord and adult blood, and exposed to biochemical agonists or pathological shear stress of 70 dyne/cm2. We evaluated α-granule release, phosphatidylserine (PS) scrambling, and procoagulant response using P-selectin expression, Annexin V binding, and thrombin generation (PAS), respectively. Activation modulation due to depletion of intracellular and extracellular calcium, requisite second messengers, was also examined. RESULTS: Similar P-selectin expression was observed for sheared adult and cord platelets, with concordant inhibition due to intracellular and extracellular calcium depletion. Sheared cord platelet Annexin V binding and PAS activity was similar to adult values in GFP, but lower in PRP and WB. Annexin V on sheared cord platelets was calcium-independent, with PAS slightly reduced by intracellular calcium depletion. CONCLUSIONS: Increased PS activity on purified sheared cord platelets suggest that their intrinsic function under pathological flow conditions is suppressed by cell-cell or plasmatic components. Although secretory functions of adult and cord platelets retain comparable calcium-dependence, PS exposure in sheared cord platelets is uniquely calcium-independent and distinct from adults. Identification of calcium-regulated developmental disparities in shear-mediated platelet function may provide novel targets for age-specific antiplatelet therapy.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Biochemical characterization of biliverdins IXß/δ generated by a selective heme oxygenase.

The pro-oxidant effect of free heme (Fe2+-protoporphyrin IX) is neutralized by phylogenetically-conserved heme oxygenases (HMOX) that generate carbon monoxide, free ferrous iron, and biliverdin (BV) tetrapyrrole(s), with downstream BV reduction by non-redundant NADPH-dependent BV reductases (BLVRA and BLVRB) that retain isomer-restricted functional activity for bilirubin (BR) generation. Regioselectivity for the heme α-meso carbon resulting in predominant BV IXα generation is a defining characteristic of canonical HMOXs, thereby limiting generation and availability of BVs IXß, IXδ, and IXγ as BLVRB substrates. We have now exploited the unique capacity of the Pseudomonas aeruginosa (P. aeruginosa) hemO/pigA gene for focused generation of isomeric BVs (IXß and IXδ). A scalable system followed by isomeric separation yielded highly pure samples with predicted hydrogen-bonded structure(s) as documented by 1H NMR spectroscopy. Detailed kinetic studies established near-identical activity of BV IXß and BV IXδ as BLVRB-selective substrates, with confirmation of an ordered sequential mechanism of BR/NADP+ dissociation. Halogenated xanthene-based compounds previously identified as BLVRB-targeted flavin reductase inhibitors displayed comparable inhibition parameters using BV IXß as substrate, documenting common structural features of the cofactor/substrate-binding pocket. These data provide further insights into structure/activity mechanisms of isomeric BVs as BLVRB substrates, with potential applicability to further dissect redox-regulated functions in cytoprotection and hematopoiesis.

Assessment of neonatal, cord, and adult platelet granule trafficking and secretion.

Despite the transient hyporeactivity of neonatal platelets, full-term neonates do not display a bleeding tendency, suggesting potential compensatory mechanisms which allow for balanced and efficient neonatal hemostasis. This study aimed to utilize small-volume, whole blood platelet functional assays to assess the neonatal platelet response downstream of the hemostatic platelet agonists thrombin and adenosine diphosphate (ADP). Thrombin activates platelets via the protease-activated receptors (PARs) 1 and 4, whereas ADP signals via the receptors P2Y1 and P2Y12 as a positive feedback mediator of platelet activation. We observed that neonatal and cord blood-derived platelets exhibited diminished PAR1-mediated granule secretion and integrin activation relative to adult platelets, correlating to reduced PAR1 expression by neonatal platelets. PAR4-mediated granule secretion was blunted in neonatal platelets, correlating to lower PAR4 expression as compared to adult platelets, while PAR4 mediated GPIIb/IIIa activation was similar between neonatal and adult platelets. Under high shear stress, cord blood-derived platelets yielded similar thrombin generation rates but reduced phosphatidylserine expression as compared to adult platelets. Interestingly, we observed enhanced P2Y1/P2Y12-mediated dense granule trafficking in neonatal platelets relative to adults, although P2Y1/P2Y12 expression in neonatal, cord, and adult platelets were similar, suggesting that neonatal platelets may employ an ADP-mediated positive feedback loop as a potential compensatory mechanism for neonatal platelet hyporeactivity.

Heme degradation enzyme biliverdin IXß reductase is required for stem cell glutamine metabolism.

Bioenergetic requirements of hematopoietic stem cells and pluripotent stem cells (PSCs) vary with lineage fate, and cellular adaptations rely largely on substrate (glucose/glutamine) availability and mitochondrial function to balance tricarboxylic acid (TCA)-derived anabolic and redox-regulated antioxidant functions. Heme synthesis and degradation converge in a linear pathway that utilizes TCA cycle-derived carbon in cataplerotic reactions of tetrapyrrole biosynthesis, terminated by NAD(P)H-dependent biliverdin reductases (IXα, BLVRA and IXß, BLVRB) that lead to bilirubin generation and cellular antioxidant functions. We now demonstrate that PSCs with targeted deletion of BLVRB display physiologically defective antioxidant activity and cellular viability, associated with a glutamine-restricted defect in TCA entry that was computationally predicted using gene/metabolite topological network analysis and subsequently validated by bioenergetic and isotopomeric studies. Defective BLVRB-regulated glutamine utilization was accompanied by exaggerated glycolytic accumulation of the rate-limiting hexokinase reaction product glucose-6-phosphate. BLVRB-deficient embryoid body formation (a critical size parameter of early lineage fate potential) demonstrated enhanced sensitivity to the pentose phosphate pathway (PPP) inhibitor 6-aminonicotinamide with no differences in the glycolytic pathway inhibitor 2-deoxyglucose. These collective data place heme catabolism in a crucial pathway of glutamine-regulated bioenergetic metabolism and suggest that early stages of lineage fate potential require glutamine anaplerotic functions and an intact PPP, which are, in part, regulated by BLVRB activity. In principle, BLVRB inhibition represents an alternative strategy for modulating cellular glutamine utilization with consequences for cancer and hematopoietic metabolism.

In silico and crystallographic studies identify key structural features of biliverdin IXß reductase inhibitors having nanomolar potency.

Heme cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles. The second step is regulated by two non-redundant biliverdin reductases (IXα (BLVRA) and IXß (BLVRB)), which retain isomeric specificity and NAD(P)H-dependent redox coupling linked to BR's antioxidant function. Defective BLVRB enzymatic activity with antioxidant mishandling has been implicated in metabolic consequences of hematopoietic lineage fate and enhanced platelet counts in humans. We now outline an integrated platform of in silico and crystallographic studies for the identification of an initial class of compounds inhibiting BLVRB with potencies in the nanomolar range. We found that the most potent BLVRB inhibitors contain a tricyclic hydrocarbon core structure similar to the isoalloxazine ring of flavin mononucleotide and that both xanthene- and acridine-based compounds inhibit BLVRB's flavin and dichlorophenolindophenol (DCPIP) reductase functions. Crystallographic studies of ternary complexes with BLVRB-NADP+-xanthene-based compounds confirmed inhibitor binding adjacent to the cofactor nicotinamide and interactions with the Ser-111 side chain. This residue previously has been identified as critical for maintaining the enzymatic active site and cellular reductase functions in hematopoietic cells. Both acridine- and xanthene-based compounds caused selective and concentration-dependent loss of redox coupling in BLVRB-overexpressing promyelocytic HL-60 cells. These results provide promising chemical scaffolds for the development of enhanced BLVRB inhibitors and identify chemical probes to better dissect the role of biliverdins, alternative substrates, and BLVRB function in physiologically relevant cellular contexts.

Integrated micro/messenger RNA regulatory networks in essential thrombocytosis.

Essential thrombocytosis (ET) is a chronic myeloproliferative disorder with an unregulated surplus of platelets. Complications of ET include stroke, heart attack, and formation of blood clots. Although platelet-enhancing mutations have been identified in ET cohorts, genetic networks causally implicated in thrombotic risk remain unestablished. In this study, we aim to identify novel ET-related miRNA-mRNA regulatory networks through comparisons of transcriptomes between healthy controls and ET patients. Four network discovery algorithms have been employed, including (a) Pearson correlation network, (b) sparse supervised canonical correlation analysis (sSCCA), (c) sparse partial correlation network analysis (SPACE), and, (d) (sparse) Bayesian network analysis-all through a combined data-driven and knowledge-based analysis. The result predicts a close relationship between an 8-miRNA set (miR-9, miR-490-5p, miR-490-3p, miR-182, miR-34a, miR-196b, miR-34b*, miR-181a-2*) and a 9-mRNA set (CAV2, LAPTM4B, TIMP1, PKIG, WASF1, MMP1, ERVH-4, NME4, HSD17B12). The majority of the identified variables have been linked to hematologic functions by a number of studies. Furthermore, it is observed that the selected mRNAs are highly relevant to ET disease, and provide an initial framework for dissecting both platelet-enhancing and functional consequences of dysregulated platelet production.

Enzymatic Activity and Thermodynamic Stability of Biliverdin IXß Reductase Are Maintained by an Active Site Serine.

Biliverdin reductase IXß (BLVRB) is a crucial enzyme in heme metabolism. Recent studies in humans have identified a loss-of-function mutation (Ser111Leu) that unmasks a fundamentally important role in hematopoiesis. We have undertaken experimental and thermodynamic modeling studies to provide further insight into the role of the cofactor in substrate accessibility and protein folding properties regulating BLVRB catalytic mechanisms. Site-directed mutagenesis with molecular dynamic (MD) simulations establish the critical role of NAD(P)H-dependent conformational changes on substrate accessibility by forming the "hydrophobic pocket", along with identification of a single key residue (Arg35) modulating NADPH/NADH selectivity. Loop80 and Loop120 block the hydrophobic substrate binding pocket in apo BLVRB (open), whereas movement of these structures after cofactor binding results in the "closed" (catalytically active) conformation. Both enzymatic activity and thermodynamic stability are affected by mutation(s) involving Ser111, which is located in the core of the BLVRB active site. This work 1) elucidates the crucial role of Ser111 in enzymatic catalysis and thermodynamic stability by active site hydrogen bond network; 2) defines a dynamic model for apo BLVRB extending beyond the crystal structure of the binary BLVRB/NADP+ complex; 3) provides a structural basis for the "encounter" and "equilibrium" states of the binary complex, which are regulated by NAD(P)H.

BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans.

Human blood cell counts are tightly maintained within narrow physiologic ranges, largely controlled by cytokine-integrated signaling and transcriptional circuits that regulate multilineage hematopoietic specification. Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin/cellular myeloproliferative leukemia virus liganding is dispensable for definitive thrombopoiesis, establishing that fundamentally important modifier loci remain unelucidated. In this study, platelet transcriptome sequencing and extended thrombocytosis cohort analyses identified a single loss-of-function mutation (BLVRB(S111L)) causally associated with clonal and nonclonal disorders of enhanced platelet production. BLVRB(S111L) encompassed within the substrate/cofactor [α/ß dinucleotide NAD(P)H] binding fold is a functionally defective redox coupler using flavin and biliverdin (BV) IXß tetrapyrrole(s) and results in exaggerated reactive oxygen species accumulation as a putative metabolic signal leading to differential hematopoietic lineage commitment and enhanced thrombopoiesis. These data define the first physiologically relevant function of BLVRB and implicate its activity and/or heme-regulated BV tetrapyrrole(s) in a unique redox-regulated bioenergetic pathway governing terminal megakaryocytopoiesis; these observations also define a mechanistically restricted drug target retaining potential for enhancing human platelet counts.

Unraveling the role of KIAA1199, a novel endoplasmic reticulum protein, in cancer cell migration.

BACKGROUND: Cell migration is a critical determinant of cancer metastasis, and a better understanding of the genes involved will lead to the identification of novel targets aimed at preventing cancer dissemination. KIAA1199 has been shown to be upregulated in human cancers, yet its role in cancer progression was hitherto unknown. METHODS: Clinical relevance was assessed by examining KIAA1199 expression in human cancer specimens. In vitro and in vivo studies were employed to determine the function of KIAA1199 in cancer progression. Cellular localization of KIAA1199 was microscopically determined. SNAP-tag pull-down assays were used to identify binding partner(s) of KIAA1199. Calcium levels were evaluated using spectrofluorometric and fluorescence resonance energy transfer analyses. Signaling pathways were dissected by Western blotting. Student t test was used to assess differences. All statistical tests were two-sided. RESULTS: KIAA1199 was upregulated in invasive breast cancer specimens and inversely associated with patient survival rate. Silencing of KIAA1199 in MDA-MB-435 cancer cells resulted in a mesenchymal-to-epithelial transition that reduced cell migratory ability in vitro (75% reduction; P < .001) and decreased metastasis in vivo (80% reduction; P < .001). Gain-of-function assays further demonstrated the role of KIAA1199 in cell migration. KIAA1199-enhanced cell migration required endoplasmic reticulum (ER) localization, where it forms a stable complex with the chaperone binding immunoglobulin protein (BiP). A novel ER-retention motif within KIAA1199 that is required for its ER localization, BiP interaction, and enhanced cell migration was identified. Mechanistically, KIAA1199 was found to mediate ER calcium leakage, and the resultant increase in cytosolic calcium ultimately led to protein kinase C alpha activation and cell migration. CONCLUSIONS: KIAA1199 serves as a novel cell migration-promoting gene and plays a critical role in maintaining cancer mesenchymal status.

Genetic dissection of platelet function in health and disease using systems biology.

Technological advances in protein and genetic analysis have altered the means by which platelet disorders can be characterized and studied in health and disease. When integrated into a single analytical framework, these collective technologies are referred to as systems biology, a unified approach that links platelet function with genomic/proteomic studies to provide insight into the role of platelets in broad human disorders such as cardiovascular and cerebrovascular disease. This article reviews the historical progression of these applied technologies to analyze platelet function, and demonstrates how these approaches can be systematically developed to provide new insights into platelet biomarker discovery.

Expression, function and cooperating partners of protease-activated receptor type 3 in vascular endothelial cells and B lymphocytes studied with specific monoclonal antibody.

Receptor-specific antibodies can both prevent ligand-receptor interaction and initiate receptor signaling. Previously we generated monoclonal antibody 8E8 (mAb 8E8) against protease-activated receptor type 3 (PAR3) which inhibited proliferation of B cell hybridoma. Here we used mAb 8E8 and PAR1-specific polyclonal antibody to reveal the functions and cooperating partners of PAR3 in endothelial cells and in B lymphocytes. MAb 8E8 or PAR1 agonist peptide stimulated IL-6 and IL-8 production and VCAM-1 expression in HPMEC-ST1.6R cells. PAR1 antibody stimulated only VCAM-1 expression, while ICAM-1 expression was stimulated with mAB 8E8 or PAR3 peptide. MAb 8E8 stimulated weak mitogenic response, while PAR1 antibody inhibited it in normal but not in malignant B lymphocytes. Sandwich ELISA assay demonstrated the interaction of PAR3 with PAR1 in malignant cell lines and with IgM in normal B lymphocytes. It is concluded that PAR3 cooperates with PAR1 to mediate the effect of thrombin on cytokine production and VCAM-1 expression in endothelial cells and on cell proliferation in malignant B cells. ICAM-1 expression in endothelial cells requires PAR3 without PAR1. The inhibitory effect of thrombin in normal B lymphocytes is mediated by PAR1 alone, while mitogenic and pro-survival signaling in B lymphocytes is provided through PAR3 in cooperation with BCR.

Platelet genetic biomarker quantification: comparison of fluorescent microspheres and PCR platforms.

The platelet transcriptome has been extensively characterised using distinct genetic profiling platforms, with evolving evidence for differential expression patterns between healthy individuals and subject cohorts with various haematologic and cardiovascular disorders. Traditional technological platforms for platelet genetic biomarker quantification have limited applicability for clinical molecular diagnostics due to inherent complexities related to RNA isolation and analysis. We have previously established the feasibility of fluorescent microspheres as a simple and reproducible strategy for simultaneous quantification of platelet mRNAs from small volume of blood using intact platelets. We now extend these observations by formally comparing in a 50-member normal cohort the cross-platform behaviour of fluorescent microspheres to the currently accepted Q-PCR standard, using a clinically relevant 15-biomarker gene subset able to discriminate among normal and thrombocytosis cohorts. When compared to Q-PCR, genetic biomarker quantification using fluorescent microspheres demonstrated lower coefficients of variation for low-abundant transcripts, better linearity in serially diluted samples, and good overall between-platform consistency via the geometric mean regression. Neither platform demonstrated age or gender effects for any of the 15 biomarkers studied. Binding site saturation for highly abundant transcripts using fluorescent microspheres can be readily eliminated using an optimal platelet number corresponding to 0.3 ml of peripheral blood, additionally applicable to thrombocytopenic cohorts. These data provide a detailed cross-platform analysis using a relevant biomarker subset, further highlighting the applicability of fluorescent microspheres as potentially superior to Q-PCR for platelet mRNA diagnostics.

Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms.

Posttranscriptional and translational controls mediated by microRNAs (miRNA) regulate diverse biologic processes. We dissected regulatory effects of miRNAs relevant to megakaryocytopoiesis and platelet biology by analyzing expression patterns in 79 subjects with thrombocytosis and controls, and integrated data with transcriptomic and proteomic platforms. We validated a unique 21-miRNA genetic fingerprint associated with thrombocytosis, and demonstrated that a 3-member subset defines essential thrombocythemia (ET). The genetic signature includes functional guide and passenger strands of the previously uncharacterized miR 490 (5p and 3p), which displayed restricted, low-level expression in megakaryocytes/platelets (compared with leukocytes), and aberrant expression during thrombocytosis, most profound in ET. Overexpression of miR 490 in a bilineage differentiation model of megakaryocyte/erythroid progenitor formation was insufficient for hematopoietic colony differentiation and/or lineage specification. Integration of transcriptomic and mass spectrometric datasets with functional reporter assays identified dishevelled associated activator of morphogenesis 1 (DAAM1) as a miR 490 5p protein target demonstrating decreased expression in ET platelets, putatively by translational control (and not by mRNA target degradation). Our data define a dysregulated miRNA fingerprint in thrombocytosis and support a developmentally restricted function of miR 490 (and its putative DAAM1 target) to conditions associated with exaggerated megakaryocytopoiesis and/or proplatelet formation.

Platelet systems biology using integrated genetic and proteomic platforms.

Platelets retain megakaryocyte-derived mRNA, an abundant and diverse array of miRNAs, and have evolved unique adaptive signals for maintenance of genetic and protein diversity. Quiescent platelets generally display minimal translational activity, although maximally-activated platelets retain the capacity for protein synthesis. Progressive data using multiple platelet activation models clearly demonstrate that platelet responses to the majority (if not all) agonists are highly variable within the population, demonstrating considerable heritability in siblings, twins, and families with premature coronary artery disease. Research from our laboratory has adapted global profiling strategies to close the knowledge gap currently existing between genetic variability and platelet phenotypic responsiveness. We have applied iterative algorithms for genetic biomarker discovery and class prediction models of platelet phenotypes, with the goal of systematically analyzing integrated mRNA/miRNA/proteomic datasets for identification of regulatory networks that define phenotypic variability in platelet responses. This approach has the potential to define platelet genetic biomarkers predictive of thrombohemorrhagic outcomes in both normal and widely disparate clinical conditions.