Tissue factor inflammatory response regulated by promoter genotype and p38 MAPK in neonatal vs. adult microvascular endothelial cells.

OBJECTIVE AND DESIGN: Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18-basepair deletion (D) or insertion (I) at -1,208. We sought to determine the relationship between these haplotypes and interleukin-1α (IL-1α)-induced TF expression in neonatal versus adult HMVEC. RESULTS: IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed threefold higher levels of TF mRNA, twofold higher TF protein, and fourfold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates, but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC. CONCLUSIONS: Upregulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants.

Mean platelet volume and integrin alleles correlate with levels of integrins α(IIb)ß(3) and α(2)ß(1) in acute coronary syndrome patients and normal subjects.

OBJECTIVE: The interindividual variation in platelet α(2)ß(1) exceeds a 2-fold variance in platelet α(IIb)ß(3) level. Our objective was to parse the contribution of mean platelet volume (MPV) and integrin gene alleles to this variation in large cohorts of patients with acute coronary syndrome (ACS) and normal subjects. METHODS AND RESULTS: Platelet α(IIb)ß(3) and α(2)ß(1) levels were measured by flow cytometry in whole blood from 320 ACS patients and 128 normal subjects and compared with MPV, platelet count, ITGA2 rs1126643, and ITGB3 rs5918 alleles. In all subjects, a strong direct correlation was found between MPV and α(IIb)ß(3) level (P<0.001). Neither MPV nor α(IIb)ß(3) level correlated with ITGB3 rs5918 alleles. In the case of α(2)ß(1) level, MPV contributed modestly, whereas ITGA2 rs1126643 exerted a greater effect. An inverse correlation was found between MPV and the rs1126643 minor allele. CONCLUSIONS: MPV is the major effector of platelet α(IIb)ß(3) level, whereas the ITGA2 rs1126643 alleles influence α(2)ß(1) level more than MPV does. The rs1126643 minor allele, associated with lower MPV, likely exerts this effect via the influence of α(2)ß(1) on megakaryocyte maturation. Because of the hyperactivity of larger platelets, MPV is an accurate metric of risk for adverse outcome in ACS.

Genetic variants that affect platelet function.

PURPOSE OF REVIEW: This review summarizes our current knowledge of common gene variants (polymorphisms) that have small individual effects on platelet function in humans, but can cumulatively lead to hyperreactive platelets and increase risk for negative outcomes in thrombotic disorders. RECENT FINDINGS: Candidate gene association and genome-wide association studies (GWAS) have identified loci that include single nucleotide polymorphisms, which exert a cumulative effect on platelet function by modifying basic platelet parameters, such as mean platelet volume (MPV) or platelet count, by altering the expression or activity of key platelet receptors, or by influencing downstream effector pathways utilized by these receptors. SUMMARY: Variation in MPV between normal individuals is responsible for roughly a two-fold range in platelet protein content, including key surface receptors and reactive granule constituents, the association of ADRA2, GP1BA, GP6, ITGA2 and P2Y12 variants with platelet reactivity, initially identified by candidate gene analyses, has now been validated by genome-wide approaches in much larger individual cohorts, and GWAS have identified novel gene variants, most notably PEAR1, that participate in variation in platelet reactivity among normal individuals, all of which contribute to a genetic basis for differences in platelet reactivty among normal individuals.

The genetics of normal platelet reactivity.

Genetic and environmental factors contribute to a substantial variation in platelet function seen among normal persons. Candidate gene association studies represent a valiant effort to define the genetic component in an era where genetic tools were limited, but the single nucleotide polymorphisms identified in those studies need to be validated by more objective, comprehensive approaches, such as genome-wide association studies (GWASs) of quantitative functional traits in much larger cohorts of more carefully selected normal subjects. During the past year, platelet count and mean platelet volume, which indirectly affect platelet function, were the subjects of GWAS. The majority of the GWAS signals were located to noncoding regions, a consistent outcome of all GWAS to date, suggesting a major role for mechanisms that alter phenotype at the level of transcription or posttranscriptional modifications. Of 15 quantitative trait loci associated with mean platelet volume and platelet count, one located at 12q24 is also a risk locus for coronary artery disease. In most cases, the effect sizes of individual quantitative trait loci are admittedly small, but the results of these studies have led to new insight into regulators of hematopoiesis and megakaryopoiesis that would otherwise be unapparent and difficult to define.

Platelet adhesion to decorin but not collagen I correlates with the integrin α2 dimorphism E534K, the basis of the human platelet alloantigen (HPA)-5 system.

A single nucleotide polymorphism in the integrin α2 gene ITGA2 (rs1801106; G1600A) creates the non-conservative amino acid substitution E534K, the basis of the human platelet alloantigen system HPA-5. Yet HPA-5 alleles do not influence binding of α2ß1 to its primary ligand collagen I, and the effect of HPA-5 on platelet function has not been determined. We used a direct platelet adhesion assay to evaluate whether differential inheritance of HPA-5 alleles influences platelet adhesion to collagen I or an alternative ligand, decorin. Platelets from donors bearing one or more minor allele HPA-5b showed attenuated adhesion to purified decorin but not collagen I. Adhesion to decorin was significantly inhibited by human alloantibodies specific for HPA-5a but not by the collagen I sequence GFOGER or α2-specific inhibitory monoclonal antibodies. The minor allele 534K attenuates platelet adhesion to decorin but not collagen I, providing the first evidence of a functional effect of HPA-5 alleles.

The low-frequency isoform of platelet glycoprotein VIb attenuates ligand-mediated signal transduction but not receptor expression or ligand binding.

The 2 most common haplotypes of human GP6, GP6a and GP6b, generate the allelic isoforms glycoprotein VI (GPVI)a and GPVIb that differ by 5 amino acids: S219P, K237E, and T249A in the ectodomains, and Q317L and H322N in the cytoplasmic domain. By quantitative Western blot, we found no association between GP6 genotype and total platelet GPVI content among 132 normal subjects. When expressed as soluble products or as membrane-associated receptors, GPVIa and GPVIb have identical affinities for type I collagen, collagen-related peptide, or convulxin. However, the cytoplasmic domain substitutions in GPVIb have a significant effect on GPVI-dependent subcellular associations and ligand-induced signal transduction. L317 increases binding to calmodulin, whereas N322 attenuates binding to Fyn/Lyn. Consistent with the latter finding, convulxin-induced Syk phosphorylation is significantly attenuated in Dami cells stably transfected with GPVIb, relative to GPVIa. This represents direct evidence that haplotype-related GPVI functional differences are inherent in the cytoplasmic domain substitutions, whereby GPVIb binds less strongly to Fyn/Lyn and attenuates the rate and extent of Syk phosphorylation. These allelic differences in GP6a and GP6b explain functional differences in the respective isoforms, but the molecular basis for the several-fold range in GPVI levels of human platelets remains to be determined.

Distinct spatio-temporal Ca2+ signaling elicited by integrin alpha2beta1 and glycoprotein VI under flow.

We studied how integrin alpha2beta1 and glycoprotein VI (GPVI) contribute to collagen-induced platelet activation under flow conditions by evaluating stable adhesion and intracellular Ca(2+) concentration ([Ca(2+)](i)) of FLUO 3-AM-labeled platelets perfused over acid-soluble type I or microfibrillar type VI collagen. Adhering platelets displayed 2 kinds of [Ca(2+)](i) oscillations. Rapid alpha-like peaks were unaffected by the membrane-impermeable Ca(2+) chelator ethyleneglycoltetraacetic acid but abolished by membrane-permeable BAPTA-AM. Longer-lasting gamma-like peaks were always preceded by at least one alpha-like peak and abolished by intracellular or extracellular Ca(2+) chelation. Inhibition of phosphatidylinositol 3-kinase or phospholipase C and modulation of cyclic nucleotides, but not blockage of adenosine diphosphate receptors, prevented both Ca(2+) responses. Human or mouse platelets lacking GPVI function exhibited alpha-like but not gamma-like Ca(2+) peaks, whereas those lacking alpha2beta1 showed markedly reduced to absent alpha-like and no gamma-like Ca(2+) peaks. Specific alpha2beta1 ligation induced alpha-like but not gamma-like peaks. Thus, alpha2beta1 may generate Ca(2+) signals that are reinforced by GPVI and required for subsequent longer-lasting Ca(2+) oscillation mediated by GPVI through transmembrane ion flux. Our results delineate a GPVI-independent signaling role of alpha2beta1 in response to collagen stimulation.

Lack of association between aspirin responsiveness and seven candidate gene haplotypes in patients with symptomatic vascular disease.

We studied the effect of prophylactic aspirin (ASA) ingestion on platelet function in 463 patients with stroke, transient ischemic attack (TIA) or acute coronary disease (ACD), using the Platelet Function Analyzer-100 (PFA-100). We correlated ASA responsiveness with haplotypes of seven candidate genes, selected for their documented role in platelet function, namely, the genes for integrins alpha2beta1and alphaIIbbeta3 (ITGA2, ITGA2B, and ITGB3), platelet glycoproteins Ibalpha and VI (GPIBA and GP6), the purinergic receptor P2Y1 (P2RY1), and prostaglandin H synthase 1 (PTGS1 = COX1). Non-responsiveness to ASA was defined as the failure of prior ASA ingestion to prolong the PFA-100 closure time (CT) when blood was perfused through cartridges coated with collagen plus epinephrine (CEPI-CT). ASA non-responsiveness was observed in 114 of 463 patients (24.6 %), but was not associated with haplotypes of any of the seven candidate genes. There was also no association between any haplotypes and the CT when blood was perfused through cartridges coated with collagen plus ADP (CADP-CT). The ASA non-responsive cohort had significantly increased whole blood platelet counts (p = 0.03) and plasma von Willebrand Factor antigen levels (p < 0.001), which likely contributes to resistance to the inhibitory effects of ASA in the PFA-100.

Transcriptional and epigenetic regulation of the integrin collagen receptor locus ITGA1-PELO-ITGA2.

The integrin collagen receptor locus on human chromosome 5q11.2 includes the integrin genes ITGA1 and ITGA2, and the cell cycle regulation gene PELO, embedded within ITGA1 intron 1. ITGA1 contains a CArG box that is bound by serum response factor (SRF), while PELO contains two Sp1 binding elements. A comparison of mRNA levels in megakaryocytic (MK) and non-megakaryocytic (non-MK) cell lines and an analysis of the transcriptional activity of promoter-LUC reporter gene constructs in transfected cells revealed that ITGA1 is selectively suppressed in the MK lineage. Sodium bisulfite genomic sequencing established that a CpG-rich ITGA1 promoter region (-209/+115) is fully methylated at 19 CpG sites in MK cells that do not express alpha1beta1, but completely demethylated in expressing cells. In vitro methylation of ITGA1 suppresses transcription, while treatment of megakaryocytic cells with 5-aza-2'-deoxycytidine, but not Trichostatin A, resulted in de novo expression of ITGA1. During thrombopoietin-induced in vitro differentiation of primary human cord blood mononuclear cells into megakaryocytes, we observed rapid, progressive CpG methylation of ITGA1, but not PELO or ITGA2. Thus, selective CpG methylation of the ITGA1 promoter is a specific feature of alpha1beta1 regulation that coincides with the initiation of megakaryocyte differentiation.

Characterization of a patient with atypical amegakaryocytic thrombocytopenia.

We report a 6-year-old girl with amegakaryocytic thrombocytopenia, the first case of this rare congenital disorder not to have an MPL gene mutation. Although no mutations were identified in MPL, Mpl protein was absent in the platelets and TPO induced phosphorylation of the Janus tyrosine kinase 2 (Jak2) was not detected. In addition to the defect of Mpl, the patient demonstrated markedly reduced expression of glycoprotein VI (GPVI) in contrast to normal expression of other platelet-specific proteins GPIb alpha, GPIb beta, and GPIIb. To explore the causes for the absence of Mpl, the entire coding region of Jak2 and AML1 were sequenced and no mutations were identified. To our knowledge, this is the first report that describes a case of amegakaryocytic thrombocytopenia that is not caused by a mutation in MPL and demonstrates the severe impairment of GPVI expression on platelets.

The influence of platelet collagen receptor polymorphisms in hemostasis and thrombotic disease.

Extracellular collagens modulate the rate of platelet activation and thereby markedly influence hemostasis and thrombosis. Platelet receptors for collagens, such as the integrin alpha(2)beta(1), platelet glycoprotein (GP) VI or, indirectly, the GPIb complex, are unexploited targets of pharmacological control, and polymorphisms of these receptors have recently become factored into the genetic risk for thrombosis. Seemingly contradictory findings already exist with regard to the contribution of GPIbalpha and integrin alpha(2) polymorphisms, but these discrepancies will be resolved once there is better standardization of clinical studies. There is already substantial evidence that GPIbalpha VNTR A or B alleles, the GPIbalpha-5C allele, and integrin alpha(2) allele 1 (T(807)) each contribute to increased risk for morbidity in thrombotic disease. However, larger, prospective genetic and epidemiological studies are needed to clarify the role of each of these polymorphisms, the contribution of other platelet receptor polymorphisms, and the synergistic effects of combinations of these factors. In addition, in vitro studies that establish the functional relevance of these polymorphisms will provide sound biological explanations for the results of clinical correlation studies.

Characterization of human glycoprotein VI gene 5' regulatory and promoter regions.

OBJECTIVE: Platelet glycoprotein VI is a collagen receptor belonging to the immunoglobulin-like protein family that is essential for platelet interactions with collagen and is exclusively expressed in the megakaryocytic lineage. The objective of this study was to characterize the human glycoprotein VI gene (GP6) 5' regulatory and promoter regions. METHODS AND RESULTS: We first used 5' RACE to establish experimentally that the major transcription start site lies 28 bp upstream from the start codon. We next subcloned the 5' regulatory region of GP6 into pGL3-basic [pGL3(-1576)] and used deletion mutagenesis to identify important regulatory regions, comparing the activity of transiently expressed promoter-luciferase constructs in Dami and HeLa cells. We found that megakaryocyte lineage-specific transcription is largely controlled within the segment -191/-39. By site-directed mutagenesis, we confirmed that a GATA-1 site at -176 and an Ets-1 site at -45 play important roles in the regulation of GP6 transcriptional activity. CONCLUSIONS: We have determined that the GP6 sequence -191 to -39 represents the core promoter and that transcription is driven largely by GATA-1 (-176) and c-Ets-1 (-45) sites within this segment.

Influence of platelet collagen receptor polymorphisms on risk for arterial thrombosis.

CONTEXT: Collagens are major components of the vascular subendothelium, and the interaction of platelets with collagens initiates normal hemostasis or pathologic arteriothrombosis. Genetic factors that affect the interaction of platelets with collagens could represent risk factors for either arteriothrombosis or excessive hemorrhage. In this regard, we first found that platelet levels of one of the major platelet collagen receptors, integrin alpha(2)beta(1), vary up to 10-fold in normal healthy individuals and that the higher-level phenotype is associated with allele 1 (807T) of the integrin alpha(2) gene. More recently, we found that there is roughly a fivefold range in platelet glycoprotein VI content among normal individuals, which may also influence risk for thromboembolism. OBJECTIVE: To determine if genetic polymorphisms of platelet glycoproteins involved in collagen-related function are associated with higher risk for thrombotic disorders, such as coronary heart disease, myocardial infarction, or stroke. METHODS: We examined the genetic mechanisms responsible for variation in expression levels of the collagen receptor integrin alpha(2)beta(1) and the potential influence of this variation on risk for thrombotic diseases. RESULTS: We found that patients with arteriothrombotic diseases have a higher frequency of alpha(2) allele 1 (associated with higher levels of platelet integrin alpha(2)beta(1)). We further found that platelet glycoprotein VI content directly correlates with platelet prothrombinase activity, suggesting that a higher phenotype of platelet glycoprotein VI also may contribute to increased risk of arteriothrombotic diseases. CONCLUSION: Genetic polymorphisms that influence the level or function of platelet collagen receptors need to be seriously considered as genetic risk factors for arteriothrombotic diseases.

α2ß1 integrin, GPVI receptor, and common FcRγ chain on mouse platelets mediate distinct responses to collagen in models of thrombosis.

OBJECTIVE: Platelets express the α2ß1 integrin and the glycoprotein VI (GPVI)/FcRγ complex, both collagen receptors. Understanding platelet-collagen receptor function has been enhanced through use of genetically modified mouse models. Previous studies of GPVI/FcRγ-mediated collagen-induced platelet activation were perfomed with mice in which the FcRγ subunit was genetically deleted (FcRγ-/-) or the complex was depleted. The development of α2ß1-/- and GPVI-/- mice permits side-by-side comparison to address contributions of these collagen receptors in vivo and in vitro. APPROACH AND RESULTS: To understand the different roles played by the α2ß1 integrin, the GPVI receptor or FcRγ subunit in collagen-stimulated hemostasis and thrombosis, we compared α2ß1-/-, FcRγ-/-, and GPVI-/- mice in models of endothelial injury and intravascular thrombosis in vivo and their platelets in collagen-stimulated activation in vitro. We demonstrate that both the α2ß1 integrin and the GPVI receptor, but not the FcRγ subunit influence carotid artery occlusion in vivo. In contrast, the GPVI receptor and the FcRγ chain, but not the α2ß1 integrin, play similar roles in intravascular thrombosis in response to soluble Type I collagen. FcRγ-/- platelets showed less attenuation of tyrosine phosphorylation of several proteins including RhoGDI when compared to GPVI-/- and wild type platelets. The difference between FcRγ-/- and GPVI-/- platelet phosphotyrosine levels correlated with the in vivo thrombosis findings. CONCLUSION: Our data demonstrate that genetic deletion of GPVI receptor, FcRγ chain, or the α2ß1 integrin changes the thrombotic potentials of these platelets to collagen dependent on the stimulus mechanism. The data suggest that the FcRγ chain may provide a dominant negative effect through modulating signaling pathways in platelets involving several tyrosine phosphorylated proteins such as RhoGDI. In addition, these findings suggest a more complex signaling network downstream of the platelet collagen receptors than previously appreciated.

Conditional knockout of integrin α2ß1 in murine megakaryocytes leads to reduced mean platelet volume.

We have engineered a transgenic mouse on a C57BL/6 background that bears a floxed Itga2 gene. The crossing of this mouse strain to transgenic mice expressing Cre recombinase driven by the megakaryocyte (MK)-specific Pf4 promoter permits the conditional knockout of Itga2 in the MK/platelet lineage. Mice lacking MK α2ß1 develop normally, are fertile, and like their systemic α2ß1 knockout counterparts, exhibit defective adhesion to and aggregation induced by soluble type I collagen and a delayed onset to low dose fibrillar collagen-induced aggregation, results consistent with blockade or loss of platelet α2ß1. At the same time, we observed a significant reduction in mean platelet volume, which is consistent with the reported role of α2ß1 in MK maturation and proplatelet formation in vivo. This transgenic mouse strain bearing a floxed Itga2 gene will prove valuable to distinguish in vivo the temporal and spatial contributions of α2 integrin in selected cell types.

Enhanced binding of poly(ADP-ribose)polymerase-1 and Ku80/70 to the ITGA2 promoter via an extended cytosine-adenosine repeat.

BACKGROUND: We have identified a cytosine-adenosine (CA) repeat length polymorphism in the 5'-regulatory region of the human integrin alpha2 gene ITGA2 that begins at -605. Our objective was to establish the contribution of this polymorphism to the regulation of integrin alpha2beta1 expression, which is known to vary several-fold among normal individuals, and to investigate the underlying mechanism(s). METHODOLOGY/PRINCIPAL FINDINGS: In combination with the SNP C-52T, previously identified by us as a binding site for the transcription factor Sp1, four ITGA2 haplotypes can be distinguished, in the order in which they enhance ITGA2 transcription: (CA)(12)/-52C>(CA)(11)/-52C>(CA)(11)/-52T>(CA)(10)/-52T. By DNA affinity chromatography and chromatin immunoprecipitation (ChIP) assays, we show that poly (ADP-ribose)polymerase-1 (PARP-1) and Ku80/70 bind specifically and with enhanced affinity to the longer (CA)(12) repeat alleles. CONCLUSIONS/SIGNIFICANCE: The increased binding of PARP-1 and Ku80/70, known components of transcription co-activator complexes, to the longer (CA)(12) alleles of ITGA2 coincides with enhanced alpha2beta1 expression. The most likely explanation for these findings is that PARP-1 and Ku80/70 contribute to the transcriptional regulation of ITGA2. These observations provide new insight into the mechanisms(s) underlying haplotype-dependent variability in integrin alpha2beta1 expression in human platelets and other cells.

The Modifier of hemostasis (Mh) locus on chromosome 4 controls in vivo hemostasis of Gp6-/- mice.

Platelet glycoprotein VI (GPVI) is a key receptor for collagens that mediates the propagation of platelet attachment and activation. Targeted disruption of the murine gene Gp6 on a mixed 129 x 1/SvJ x C57BL/6J background causes the expected defects in collagen-dependent platelet responses in vitro. The extent of this dysfunction in all Gp6(-/-) mice is uniform and is not affected by genetic background. However, the same Gp6(-/-) mice exhibit 2 diametrically opposed phenotypes in vivo. In some mice, tail bleeding times are extremely prolonged, and thrombus formation in an in vivo carotid artery ferric chloride-injury model is significantly impaired. In other littermates, tail bleeding times are within the range of wild-type mice, and in vivo thrombus formation is indistinguishable from that of control mice. Directed intercrosses revealed that these phenotypes are heritable, and a genome-wide single-nucleotide polymorphism scan revealed the most significant linkage to a single locus (8 megabases) on chromosome 4 (logarithm of the odds [LOD] score = 6.9, P < .0001) that we designate Modifier of hemostasis (Mh). Our results indicate that one or more modifier genes in Mh control the extent to which in vivo platelet thrombus formation is disrupted by the absence of platelet GPVI.