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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., (© 2022 International Society on Thrombosis and Haemostasis.)

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., (© 2020 by The American Society of Hematology.)

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/β dinucleotide NAD(P)H] binding fold is a functionally defective redox coupler using flavin and biliverdin (BV) IXβ 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., (© 2016 by The American Society of Hematology.)

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.

Criteria for distinguishing among etiologies of thrombocytosis are limited in their capacity to delineate clonal (essential thrombocythemia [ET]) from nonclonal (reactive thrombocytosis [RT]) etiologies. We studied platelet transcript profiles of 126 subjects (48 controls, 38 RT, 40 ET [24 contained the JAK2V(617)F mutation]) to identify transcript subsets that segregated phenotypes. Cross-platform consistency was validated using quantitative real-time polymerase chain reaction (RT-PCR). Class prediction algorithms were developed to assign phenotypic class between the thrombocytosis cohorts, and by JAK2 genotype. Sex differences were rare in normal and ET cohorts (< 1% of genes) but were male-skewed for approximately 3% of RT genes. An 11-biomarker gene subset using the microarray data discriminated among the 3 cohorts with 86.3% accuracy, with 93.6% accuracy in 2-way class prediction (ET vs RT). Subsequent quantitative RT-PCR analysis established that these biomarkers were 87.1% accurate in prospective classification of a new cohort. A 4-biomarker gene subset predicted JAK2 wild-type ET in more than 85% patient samples using either microarray or RT-PCR profiling, with lower predictive capacity in JAK2V(617)F mutant ET patients. These results establish that distinct genetic biomarker subsets can predict thrombocytosis class using routine phlebotomy.

Platelets play critical roles in diverse hemostatic and pathologic disorders and are broadly implicated in various biological processes that include inflammation, wound healing, and thrombosis. Recent progress in high-throughput mRNA and protein profiling techniques has advanced our understanding of the biological functions of platelets. Platelet proteomics has been adopted to decode the complex processes that underlie platelet function by identifying novel platelet-expressed proteins, dissecting mechanisms of signal or metabolic pathways, and analyzing functional changes of the platelet proteome in normal and pathologic states. The integration of transcriptomics and proteomics, coupled with progress in bioinformatics, provides novel tools for dissecting platelet biology. In this review, we focus on current advances in platelet proteomic studies, with emphasis on the importance of parallel transcriptomic studies to optimally dissect platelet function. Applications of these global profiling approaches to investigate platelet genetic diseases and platelet-related disorders are also addressed.

Proteomic technology has the potential to transform the way we analyze platelet biology, through the determination of platelet protein composition and its modification upon stimulation and with disease. We are a considerable way from achieving these goals, however, because of significant limitations in current methodology. It is therefore important to consider the extent to which these aims can be met and the way that proteomic data should be presented and used. These issues are discussed in the present paper by the Platelet Physiology Subcommittee of the ISTH Scientific Standardisation Committee (SSC). It is recommended that proteomic information be combined with data from other experimental approaches to establish a database on protein expression and function in platelets.

Human blood platelets are anucleate cells whose response to extracellular stimuli results in actin cytoskeleton rearrangements, thereby providing the critical initial step in the regulation of hemostasis. The serine protease alpha-thrombin, known to activate platelets by cleavage of a family of protease-activated receptors (PARs), is the most potent physiologic activator of human platelets, though downstream effector proteins uniquely linked to platelet cytoskeletal actin polymerization remain largely uncharacterized. The gene encoding the putative rac1/cdc42 effector protein IQGAP2 was identified within the PAR gene cluster at 5q13, flanked telomeric by PAR1 and encompassing PAR3. Immunofluorescence microscopy demonstrated IQGAP2 expression in filopodial extensions of activated platelets and colocalized with F-actin in lamellipodia and filopodia of IQGAP2-transfected COS1 cells. Platelet activation by alpha-thrombin, but not saturating concentrations of fibrillar collagen or adenosine 5'-diphosphate, uniquely assemble an IQGAP2/arp2/3-actin cytoplasmic complex, an association regulated by guanosine triphosphate rac1 ([GTP]rac1) but not by [GTP]cdc42. Likewise, only thrombin-activated platelets resulted in rapid translocation of IQGAP2 to the platelet cytoskeleton. These observations identify a physiologic scaffolding function for IQGAP2 and establish the presence of a functional genomic unit in humans uniquely evolved to regulate thrombin-induced platelet cytoskeletal actin reorganization.

Human platelets are anucleate blood cells that retain cytoplasmic mRNA and maintain functionally intact protein translational capabilities. We have adapted complementary techniques of microarray and serial analysis of gene expression (SAGE) for genetic profiling of highly purified human blood platelets. Microarray analysis using the Affymetrix HG-U95Av2 approximately 12 600-probe set maximally identified the expression of 2147 (range, 13%-17%) platelet-expressed transcripts, with approximately 22% collectively involved in metabolism and receptor/signaling, and an overrepresentation of genes with unassigned function (32%). In contrast, a modified SAGE protocol using the Type IIS restriction enzyme MmeI (generating 21-base pair [bp] or 22-bp tags) demonstrated that 89% of tags represented mitochondrial (mt) transcripts (enriched in 16S and 12S ribosomal RNAs), presumably related to persistent mt-transcription in the absence of nuclear-derived transcripts. The frequency of non-mt SAGE tags paralleled average difference values (relative expression) for the most "abundant" transcripts as determined by microarray analysis, establishing the concordance of both techniques for platelet profiling. Quantitative reverse transcription-polymerase chain reaction (PCR) confirmed the highest frequency of mt-derived transcripts, along with the mRNAs for neurogranin (NGN, a protein kinase C substrate) and the complement lysis inhibitor clusterin among the top 5 most abundant transcripts. For confirmatory characterization, immunoblots and flow cytometric analyses were performed, establishing abundant cell-surface expression of clusterin and intracellular expression of NGN. These observations demonstrate a strong correlation between high transcript abundance and protein expression, and they establish the validity of transcript analysis as a tool for identifying novel platelet proteins that may regulate normal and pathologic platelet (and/or megakaryocyte) functions.

The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)-dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2(Arg36 --> Ala36). Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3-deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of approximately 80 micromol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares approximately 70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of alpha- and beta-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr approximately 130, confirming the presence of secretable and functionally active homotetrameric alpha- and beta-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at approximately 3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2(Arg36 --> Ala36). These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.

Purpose: We studied the ability of recombinant adeno-associated virus (rAAV) vectors to achieve gene transfer in vivo to intact rat carotid arteries., Methods: Isolated segments of uninjured rat carotid arteries were incubated with (1) rAAV vectors that expressed a beta-galactosidase gene, (2) a related vector with no promoter, or (3) a normal saline solution. Gene transfer was evaluated with in situ polymerase chain reaction (PCR). Transgene expression was assessed at intervals that ranged from 24 hours to 2 months by measurement of beta-galactosidase activity and protein mass in tissue extracts with fluorometric and enzyme-linked immunosorbent assays, respectively. Dose dependence of expression was determined for virus concentrations that ranged from 5 x 10(4) to 5 x 10(5) infectious units (iu)/ml., Results: Light microscopic analysis of in situ PCR-stained histologic sections of transduced vessel walls showed approximately 90% of intimal and medial cell nuclei contained the beta-galactosidase gene, compared with none in control arteries. In vivo beta-galactosidase expression was (1) highest 24 hours after gene transfer, (2) elevated for 1 month, and (3) dose responsive., Conclusions: rAAV vectors can mediate focal gene transfer into the intact rat carotid artery with detectable levels of transgene expression for 1 month and are potentially useful agents for in vivo gene transfer into intact arteries.

Thrombin receptor (TR) activation by alpha-thrombin requires proteolytic cleavage, although synthetic peptides modeled after the new N-terminus directly effect receptor activation without cleavage, presumably by interacting with an unidentified region of the receptor. To further define critical residues responsible for receptor activation, we performed epitope mapping of anti-TR1-160, a previously described polyclonal antibody that inhibits peptide ligand-induced receptor activation in various cell types expressing a functional TR. An enzyme-linked immunosorbent assay (ELISA) using overlapping decapeptides derived from the TR extracellular domains identified four immunodominant peaks within the long N-terminal extension centered between amino acids 34-44, 48-67, 65-79, and 87-94. Soluble peptides derived from regions 83-94, but not those from other regions of the receptor, neutralized the ability of anti-TR1-160 to inhibit peptide ligand-induced platelet aggregation, suggesting that antibodies directed against this region of the TR are important in ligand-mediated activation. Thrombin receptor mutants lacking discrete regions of the TR were subsequently evaluated using microinjected Xenopus oocytes. Whereas a TR mutant lacking amino acid residues Thr67-Lys82 (TR delta 67-82) showed normal to exaggerated responses to either alpha-thrombin or synthetic peptide ligands, only TR mutants with limited deletions spanning the residues Gln83-Ser93 exhibited dysfunctional responses to either agonist (200 nmol/L alpha-thrombin or 200 mumol/L TR42-47). These data provide a model for receptor activation that implicates a discrete and previously uncharacterized sequence within the TR N-terminal extension that is necessary for initiation of signal transduction events independent of the initiating agonist.

The integrin VLA-2 (alpha 2 beta 1), generally considered to represent the specific collagen receptor on human endothelial cells, contains an alpha 2-subunit inserted I domain with structural similarity to the type A domains found within the recently described superfamily of receptor-ligand recognition proteins. This region of the cDNA has now been isolated and used for molecular and functional characterization of this heterodimeric receptor complex. Comparative sequence analysis with the porcine homologue revealed 93% amino acid sequence identity, suggestive of a developmentally conserved function. To complete structure/function studies, this region of the human cDNA was expressed as a chimeric protein in Escherichia coli, and a rabbit polyclonal antibody (anti-I domain) was used to study determinants of endothelial cell attachment and spreading in vitro. Quantifiable and visual disruption of endothelial cell attachment to gelatin, type I collagen, and laminin was evident using the specific anti-I domain antibody, with minimal inhibitory effects demonstrable using fibronectin or fibrinogen matrices. Therefore, these data would suggest that the alpha 2 beta 1 I domain confers ligand-binding specificity for both known alpha 2 beta 1 substrates (laminin and collagen), and that this region subserves a regulatory function in the molecular processes controlling endothelial cell attachment and spreading in vitro.

A functional thrombin receptor (TR) structurally related to other members of the seven-transmembrane receptor family has been isolated from diverse cellular types intimately involved in the regulation of the thrombotic response. This receptor recapitulates many of the previously identified sequelae of thrombin-mediated cell activation phenomenon, and requires proteolytic cleavage for downstream effector-response coupling events. Using two complementary approaches, we have now completed the chromosomal assignment of the human thrombin receptor gene. Discordancy analysis of polymerase chain reaction products from a human-rodent hybrid cell mapping panel assigned the sequence to human chromosome 5 with no observed discordancies. Cytogenetic localization using fluorescence in situ hybridization on human metaphase chromosomes specifically localized the human TR gene to region q13 of chromosome 5, confirming its presence as a single-locus gene in the human genome. The chromosomal localization of the human TR gene is at or contiguous with the proximal breakpoint site identified in the majority of patients with the 5q- syndrome (dysmegakaryocytopoiesis and refractory anemia).

von Willebrand disease (vWD), one of the most common bleeding disorders in humans, is manifested as a quantitative or qualitative defect in von Willebrand factor (vWF), an adhesive glycoprotein (GP) with critical hemostatic functions. Except for the rare severely affected patient with a gene deletion as etiology of the disease, the molecular basis for vWD is not known. We studied the molecular basis for vWD in a breeding colony of pigs with a disease closely resembling the human disorder. The porcine vWF gene is similar in size and complexity to its human counterpart, and no gross gene deletion or rearrangement was evident as the pathogenesis of porcine vWD. A restriction fragment-length polymorphism (RFLP) within the porcine vWF gene was identified with the restriction endonuclease HindIII, and 22/35 members of the pedigree were analyzed for the polymorphic site. Linkage between the vWF locus and the vWD phenotype was established with a calculated LOD score of 5.3 (1/200,000 probability by chance alone), with no crossovers identified. These findings indicate that porcine vWD is due to a molecular defect within (or near) the vWF locus, most likely representing a point mutation or small insertion/deletion within the vWF gene.