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Wadie F Bahou

Publications and source records attributed to Wadie F Bahou.

15 recordsLinked to original sources
PubMed2006

Proteomic approaches to dissect platelet function: Half the story.

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.

Animals↗
PubMed2005

The organomercurial 4-aminophenylmercuric acetate, independent of matrix metalloproteinases, induces dose-dependent activation/inhibition of platelet aggregation.

Matrix metalloproteinases (MMPs) play an important role in many biological and pathological processes including tissue remodeling, wound healing, inflammation, atherosclerosis, and cancer. Numerous publications have supported the concept that activated MMP-2 enhances agonist-induced platelet aggregation and activated MMP-9 inhibits platelet aggregation. In this study, we demonstrated that the organomercurial compound, 4-aminophenyl mercuric acetate (APMA), which is routinely employed to activate latent MMPs at a concentration of 1000 microM, induces platelet aggregation at low concentration (5 microM) and inhibits agonist-induced platelet aggregation at concentrations >or= 50 microM. Activated MMP-2, MMP-1, and MMP-9, following removal of APMA by ultrafiltration through an anisotropic membrane, exert no independent effect on platelet aggregation. Acetylsalicylic acid and BAPTA inhibited APMA-induced platelet aggregation indicating that the APMA mediated pathway of platelet activation is dependent upon thromboxane and calcium signaling. Zinc chelation with 1,10-phenanthroline, which inhibits zinc-dependent proteins including metalloproteinases, also abrogated platelet functional responses to APMA. Additional studies will be required to clarify the mechanism of the biphasic effect of APMA on platelet aggregation.

Calcium Signaling↗
PubMed2005

Platelets express steroidogenic 17beta-hydroxysteroid dehydrogenases. Distinct profiles predict the essential thrombocythemic phenotype.

Human blood platelets have important, regulatory functions in diverse hemostatic and pathological disorders, including vascular remodeling, inflammation, and wound repair. Microarray analysis was used to study the molecular basis of essential thrombocythemia, a myeloproliferative disorder with quantitative and qualitative platelet defects associated with cardiovascular and thrombohemorrhagic symptoms, not infrequently neurological. A platelet-expressed gene (HSD17B3) encoding type 3 17beta-hydroxysteroid dehydrogenase (previously characterized as a testis-specific enzyme catalyzing the final step in gonadal synthesis of testosterone) was selectively down-regulated in ET platelets, with reciprocal induction of the type 12 enzyme (HSD17B12). Functional 17beta-HSD3 activity corresponding to approximately 10% of that found in murine testis was demonstrated in normal platelets. The induction of HSD17B12 in ET platelets was unassociated with a concomitant increase in androgen biosynthesis, suggesting distinct functions and/or substrate specificities of the types 3 and 12 enzymes. Application of a molecular assay distinguished ET from normal platelets in 20 consecutive patients (p < 0.0001). These data provide the first evidence that distinct subtypes of steroidogenic 17beta-HSDs are functionally present in human blood platelets, and that the expression patterns of HSD17B3 and HSD17B12 are associated with an uncommon platelet disorder manifest by quantitative and qualitative platelet defects.

17-Hydroxysteroid Dehydrogenases↗
PubMed2004

A shear-restricted pathway of platelet procoagulant activity is regulated by IQGAP1.

Circulating blood platelets regulate the initial phase of the hemostatic response through adhesive and aggregatory events and by providing the necessary procoagulant surface for prothrombinase complex assembly and thrombin generation. The signaling pathway(s) that regulate platelet procoagulant activity are largely unknown, although they are distinct from platelet aggregatory signals linked to fibrinogen ligation to the conformationally active alpha(IIB)beta(3) integrin. We describe a novel intracellular signaling mechanism involving platelet IQGAP1 that specifically regulates the development of platelet procoagulant activity under conditions of mechanical shear stress. Murine platelets that are deficient in IQGAP1 demonstrate increased prothrombinase activity compared with wild-type littermate controls when activated by a physiological shear stress of 16 dynes/cm(2) (shear rates of 1600 s(-1)) (p < 0.0001), corresponding to approximately 2.5 times the normal shear stress, or approximately 40% degree of stenosis in coronary arteries. The exaggerated prothrombinase activity is not associated with enhanced platelet microvesiculation (cytoskeletal proteolysis) and occurs independently of the intracellular calcium release, [Ca(2+)](i), but it is specifically coupled to the alpha-granule exocytic pathway without concomitant effects on aminophospholipid exposure. These observations identify platelet IQGAP1 as an important modulator of normal hemostasis and as an appropriate pharmacological target for control of platelet procoagulant function.

Animals↗
PubMed2004

Distinct roles for the catalytic and hemopexin domains of membrane type 1-matrix metalloproteinase in substrate degradation and cell migration.

Substrate degradation and cell migration are key steps in cancer metastasis. Membrane-type 1-matrix metalloproteinase (MT1-MMP) has been linked with these processes. Using the fluorescein isothiocyanate (FITC)-labeled fibronectin degradation assay combined with the phagokinetic cell migration assay, structure-function relationships of MT1-MMP were studied. Our data indicate that MT1-MMP initiates substrate degradation and enhances cell migration; cell migration occurs as a concurrent but independent event. Using recombinant DNA approaches, we demonstrated that the hemopexin-like domain and a nonenzymatic component of the catalytic domain of MT1-MMP are essential for MT1-MMP-mediated cell migration. Because the cytoplasmic domain of MT1-MMP was not required for MT1-MMP-mediated fibronectin degradation and cell migration, it is proposed that cross-talk between the hemopexin domain of MT1-MMP and adjacent cell surface molecules is responsible for outside-in signaling. Employing cDNAs encoding dominant negative mutations, we demonstrated that Rac1 participates in the MT1-MMP signal transduction pathway. These data demonstrated that each domain of MT1-MMP plays a distinct role in substrate degradation and cell migration.

Animals↗
PubMed2004

Distinct PAR/IQGAP expression patterns during murine development: implications for thrombin-associated cytoskeletal reorganization.

Thrombin has a critical role in many adult and embryologic cellular processes, exerting its effects through two high-affinity thrombin receptor systems: protease-activated receptor 1 (PAR1) and the PAR3/PAR4 system. Both hPAR1 and hPAR3 are coclustered in the human genome, with hPAR3 encompassed within hIQGAP2, a putative GTPase activating protein with actin polymerizing functions linked to cytoskeletal reorganization. Since hPARs colocalize with hIQGAP2 in the human genome and function coordinately with this protein in platelet thrombin signaling pathways, we have further characterized these genes in developing embryonic and adult tissues. We confirmed the presence of a mIQGAP2/ mPAR gene cluster on murine Chromosome 13 and showed it to be organized similarly to that in humans, except that murine PAR3 is translated off the forward (sense) strand. Northern analysis demonstrated limited mPAR3 expression in adult tissues, although its expression during embryogenesis was evident at E15 in cartilage, brain, and keratinocytes. mIQGAPs 1 and 2 had congruent expression patterns in 11 of 15 adult tissues studied. In contrast, whole embryos demonstrated predominant mIQGAP1 expression starting at E7 and evident to E17. In situ hybridization of whole embryos (E9-E16) demonstrated distinct patterns of tissue-dependent mIQGAP1/ mIQGAP2 expression. Concordant expression (absence or presence) of mPAR1 with either mIQGAP1 or mIQGAP2 was seen in the majority (12 of 15) of adult tissues studied. Similarly, there was no evidence for mPAR3 expression during embryogenesis in the absence of either mIQGAP1 or mIQGAP2. These data provide a panoramic survey of PAR/ IQGAP expression as an initial approach to dissect thrombin signaling pathways linked to cytoskeletal reorganization.

Animals↗
PubMed2004

Expression of protease activated receptor 3 (PAR3) is upregulated by induction of megakaryocyte phenotype in human erythroleukemia (HEL) cells.

OBJECTIVE: Two major protease-activated receptors (PARs), PAR1 and PAR4, are involved in the activation of human platelets by thrombin. A third, PAR3, is preferentially expressed by tissues of hematopoietic origin and megakaryocytes. Although PAR3 is also a thrombin substrate, its low-level expression on human platelets suggests a function distinct from that of PAR1, the major receptor involved in thrombin-mediated platelet activation. We studied the expression of PARs during megakaryocyte differentiation of human erythroleukemia (HEL) cells in order to determine the role of PAR3 in megakaryocytopoiesis. METHODS: HEL cells exposed to phorbol 12-myristate 13-acetate (PMA) to induce megakaryocyte differentiation were examined by light microscopy and flow cytometry (DNA ploidy, surface expression of PAR1, PAR3, GPIIb-IIIa). Northern blot, RT-PCR, and quantitative RT-PCR were used to evaluate the expression of PARs 1, 3, and 4 mRNA. HEL cells were also exposed to thrombin and thrombopoietin (TPO). RESULTS: In baseline studies, unstimulated HEL cells were found to express comparable levels of PAR1 and PAR3 by Northern blot. Minimal expression of PAR4 was detected by RT-PCR, but not by Northern analysis. Exposure to PMA, but not thrombin or TPO, resulted in megakaryocytic differentiation as evident by increased cell size and nuclear complexity, increased ploidy, and enhanced expression of GPIIb-IIIa, a specific marker of megakaryocytes/platelets. PMA-stimulated HEL cells showed enhanced PAR3 cell-surface expression (approximately threefold increase by day 2) by flow cytometry. In contrast, there was no change in cell-surface PAR1 expression. Northern blot analysis (approximately 10-fold) and quantitative RT-PCR (approximately threefold) confirmed the upregulation of PAR3 mRNA expression (by 24 hours) in cells exposed to PMA. This did not occur with exposure to TPO. CONCLUSION: These data demonstrate increased expression of PAR3 mRNA and protein in HEL cells undergoing megakaryocytic maturation following PMA exposure, suggesting a developmental role for PAR3. Furthermore, regulation of PAR3 expression appears to be specifically coupled to the protein kinase C system, but independent of the Ras/Raf/MAP kinase pathway.

Cell Differentiation↗
PubMed2004

Platelet transcriptome: the application of microarray analysis to platelets.

Human blood platelets are intimately involved in the regulation of thrombosis, inflammation, and wound repair. These cells retain megakaryocyte-derived cytoplasmic mRNA and functionally intact protein translational capabilities, although very little is known about normal or pathological mRNA profiles. Microarray analysis has demonstrated a clear and reproducible molecular signature unique to platelets. There is a relative paucity of expressed transcripts compared with those found in other eukaryotic cells, most likely related to mRNA decay in these anucleate cells. In contrast, a complementary methodology for transcript profiling (serial analysis of gene expression [SAGE]) demonstrates that 89% of tags represent mitochondrial (mt) transcripts (enriched in 16S and 12S ribosomal RNAs), presumably related to persistent mt-transcription in the absence of nuclear-derived transcripts. The abundance of nonmitochondrial SAGE tags parallels relative expression for the most abundant transcripts as determined by microarray analysis, establishing the concordance of both techniques for platelet profiling. These observations establish the validity of transcript analysis as a tool for identifying novel platelet genes that may regulate normal and pathologic platelet (and/or megakaryocyte) functions. The potential application of platelet-specific microarrays in scientific and clinical settings related to platelet production, cardiovascular, and cerebrovascular diseases is reviewed.

Animals↗
PubMed2004

Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus.

We have generated an E1a/E1b/E3-deleted adeno/adeno-associated (Ad/AAV) hybrid virus driven by a small nuclear RNA (pHU1-1) promoter for expression of a B domain-deleted (Thr761-Asn1639) factor VIII transgene (FVIIIDelta761-1639). Productive replication of Ad/AAV/FVIIIDelta761-1639 in AAV rep-expressing cells resulted in generation of monomeric and dimeric mini-adenoviral (mAd) replicative forms that retained the AAV integration elements (mAd/FVIIIDelta761-1639). In vitro studies using Ad/AAV/FVIIIDelta761-1639 generated approximately 2-logs greater FVIII activity than mAd/FVIIIDelta761-1639. To determine its capacity for in vivo excision and/or genomic integration, Ad/AAV/FVIIIDelta761-1639 was injected by tail vein into three groups of hemophilia A mice (2 x 10(11) vp [n = 3]; 4 x 10(11) vp [n = 3]; 8 x 10(11) vp [n = 3]), with clear concentration-dependent increase in FVIII activity (range 160-510 mU/ml; plasma activity 16%-51% of normal). Peak activity was seen by Day (D) 5, with slow return to baseline by D28 (0.1-0.9% activity); in only 3/9 mice was loss of FVIII activity associated with development of anti-FVIII antibodies. Quantitative-PCR using genomic DNA isolated from D28 liver, spleen, heart, lungs, and kidney demonstrated the highest concentration in liver (approximately 10 genomes/cell), with little to no organ toxicity at early (D5 or 6) or late (D28) post-infusion time points. There was no evidence for spontaneous transgene excision or genomic integration in vivo as evaluated by quantitative PCR and genomic blotting. These data establish (i) the feasibility and applicability of developing high-titer Ad/AAV hybrid viruses for FVIII delivery using a small cellular promoter, (ii) the potential utility of this virus for generation of "gutted" monomeric and dimeric mAD/FVIII retaining AAV integration elements, and (iii) that the development of strategies for regulated Rep68/78 co-expression may provide a novel approach for excision, integration, and long-term FVIII transgene expression.

Adenoviridae↗
PubMed2004

Proteomics in platelet science.

Proteomics, the rapid large-scale analysis of cellular proteins, is becoming an important tool in the study of human platelet biology. A number of investigators have used proteomics techniques to study platelets including two-dimensional gel electrophoresis, protein separation, and mass spectrometry protein identification. Most efforts have focused on: 1) cataloging the spectrum of proteins that comprise the normal platelet "proteome", 2) characterizing proteins released from activated platelets, and 3) identifying phosphoproteins generated upon platelet stimulation. The incorporation of modern proteomics technology to platelet studies will further our understanding of platelet activation, adhesion, and aggregation at the protein level. Moreover, proteomics offers hope that novel biomarkers can be discovered that diagnose inherited and/or acquired platelet disorders, or predict the susceptibility of such individuals to bleeding or thrombosis. Finally, proteomics could lead to identification of novel agents that have therapeutic value. This review discusses the current status and future opportunities of proteomics technologies in platelet science.

Blood Platelets↗
PubMed2002

IQGAP2 functions as a GTP-dependent effector protein in thrombin-induced platelet cytoskeletal reorganization.

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.

Actin-Related Protein 2↗
PubMed2002

Transcript profiling of human platelets using microarray and serial analysis of gene expression.

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.

Base Sequence↗
PubMed2002

Genomic signature tags (GSTs): a system for profiling genomic DNA.

Genomic signature tags (GSTs) are the products of a method we have developed for identifying and quantitatively analyzing genomic DNAs. The DNA is initially fragmented with a type II restriction enzyme. An oligonucleotide adaptor containing a recognition site for MmeI, a type IIS restriction enzyme, is then used to release 21-bp tags from fixed positions in the DNA relative to the sites recognized by the fragmenting enzyme. These tags are PCR-amplified, purified, concatenated, and then cloned and sequenced. The tag sequences and abundances are used to create a high-resolution GST sequence profile of the genomic DNA. GSTs are shown to be long enough for use as oligonucleotide primers to amplify adjacent segments of the DNA, which can then be sequenced to provide additional nucleotide information or used as probes to identify specific clones in metagenomic libraries. GST analysis of the 4.7-Mb Yersinia pestis EV766 genome using BamHI as the fragmenting enzyme and NlaIII as the tagging enzyme validated the precision of our approach. The GST profile predicts that this strain has several changes relative to the archetype CO92 strain, including deletion of a 57-kb region of the chromosome known to be an unstable pathogenicity island.

Binding Sites↗