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D C Shields

Publications and source records attributed to D C Shields.

At least 19 recordsLinked to original sources
PubMed2006

Monitoring modulators of platelet aggregation in a microtiter plate assay.

Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.

Amino Acid Sequence↗
PubMed2006

The impact on coronary artery disease of common polymorphisms known to modulate responses to pathogens.

There are two distinct models to explain how genetic variants contributing to cardiovascular disease may have arisen. Firstly, variants may result from random, initially neutral, mutations whose effects are largely revealed in post-reproductive individuals in industrialized societies. Alternatively, the introduced variants may confer an adaptive advantage in certain circumstances. Resistance to pathogens is one of the strongest selection pressures on human proteins. To determine whether this evolutionary pressure has made a large contribution to heart disease we tested whether seventeen polymorphisms in fourteen innate-immunity genes, with documented evidence of modulating response to pathogens, had an impact on heart disease. Genotyping was performed in 1,598 CAD subjects (ACS or stable angina) and 332 controls. The TLR4 399Ile allele had the greatest impact on ACS risk (uncorrected p = 0.006); however there was no evidence overall that the resistance alleles cumulatively influenced the risk of ACS compared to controls or stable angina patients (p = 0.12, and p = 0.40, respectively). We did note a significant interaction between age at onset of disease and combined resistance allele carriership when the ACS and non-thrombotic, stable angina groups were compared (p = 0.04, 16 d.f.). This suggests that innate immunity factors could have a greater impact on thrombus formation among younger CAD patients.

Acute Disease↗
PubMed2006

Comparative RNA expression analyses from small-scale, single-donor platelet samples.

BACKGROUND: Comparisons of platelet RNAs could provide crucial information on platelet function, thrombopoiesis and the etiology of megakaryocyte (MK) or platelet disorders. OBJECTIVES: We developed a method for stringent purification of platelets from small blood samples from single donors. Purity of the platelet preparations was verified by an RT-PCR assay. We tested three methods to identify the differences in RNA between platelet sources. METHODS: Differential hybridization to cDNA macro-arrays and suppressive-subtractive hybridization PCR (SSH-PCR) were used to compare RNAs from normal platelets to those from a Bernard-Soulier syndrome (BSS) patient. Affymetrix GeneChip U133 plus 2.0 arrays were used to compare male and female platelet RNAs. RESULTS: Macroarrays identified approximately 7500 platelet transcripts, but failed to identify differentially expressed transcripts with confidence. SSH-PCR produced libraries almost exclusively of mitochondrial-derived transcripts, but included nuclear-encoded genes that could not be confirmed by immunoblotting of normal and BSS platelet lysates. The Affymetrix platform gave reproducible profiles from our small-scale purified platelet preparations, whereas a partially purified platelet preparation produced a drastically skewed transcript profile. The microarray analysis identified the heparanase precursor transcript as overexpressed in female platelets, and we observed variable yet consistently higher levels of heparanase protein in female platelets compared with male platelets in four independent donor pairs. CONCLUSIONS: This demonstrates for the first time that differential platelet transcript levels can identify changes in expression level of platelet proteins. Combined with our small-scale platelet preparation method, this establishes a system to compare platelets from the limited clinical sources to help elucidate molecular bases for platelet or megakaryocyte pathologies.

Blood Platelets↗
PubMed2005

Cyclooxygenase-1 haplotype modulates platelet response to aspirin.

BACKGROUND: Aspirin (acetylsalicylic acid) irreversibly inhibits platelet cyclooxygenase (COX)-1, the enzyme that converts arachidonic acid (AA) to the potent platelet agonist thromboxane (TX) A2. Despite clear benefit from aspirin in patients with cardiovascular disease (CAD), evidence of heterogeneity in the way individuals respond has given rise to the concept of 'aspirin resistance.' AIMS: To evaluate the hypothesis that incomplete suppression of platelet COX as a consequence of variation in the COX-1 gene may affect aspirin response and thus contribute to aspirin resistance. PATIENTS AND METHODS: Aspirin response, determined by serum TXB2 levels and AA-induced platelet aggregation, was prospectively studied in patients (n = 144) with stable CAD taking aspirin (75-300 mg). Patients were genotyped for five single nucleotide polymorphisms in COX-1 [A-842G, C22T (R8W), G128A (Q41Q), C644A (G213G) and C714A (L237M)]. Haplotype frequencies and effect of haplotype on two platelet phenotypes were estimated by maximum likelihood. The four most common haplotypes were considered separately and less common haplotypes pooled. RESULTS: COX-1 haplotype was significantly associated with aspirin response determined by AA-induced platelet aggregation (P = 0.004; 4 d.f.). Serum TXB2 generation was also related to genotype (P = 0.02; 4 d.f.). CONCLUSION: Genetic variability in COX-1 appears to modulate both AA-induced platelet aggregation and thromboxane generation. Heterogeneity in the way patients respond to aspirin may in part reflect variation in COX-1 genotype.

Adult↗
PubMed2003

Integration of proteomics and genomics in platelets: a profile of platelet proteins and platelet-specific genes.

Platelets, while anucleate, contain RNA, some of which is translated into protein upon activation. Hypothesising that the platelet proteome is reflected in the transcriptome, we identified 82 proteins secreted from activated platelets and compared these, as well as published proteomic data, to the transcriptional profile. We also compared the transcriptome of platelets to other tissues to identify platelet-specific genes and used ontology to determine gene categories over-represented in platelets. RNA was isolated from highly pure platelet preparations for hybridization to Affymetrix oligonucleotide arrays. We identified 2,928 distinct messages as being present in platelets. The platelet transcriptome was compared with the proteome by relating both to UniGene clusters. Platelet proteomic data correlated well with the transcriptome, with 69% of secreted proteins detectable at the mRNA level, and similar concordance was obtained using two published datasets. While many of the most abundant mRNAs are for known platelet proteins, messages were detected for proteins not previously reported in platelets. Some of these may represent residual megakaryocyte messages; however, proteomic analysis confirmed the expression of many previously unreported genes in platelets. Transcripts for well-described platelet proteins are among the most platelet-specific messages. Ontological categories related to signal transduction, receptors, ion channels, and membranes are over-represented in platelets, while categories involved in protein synthesis are depleted. Despite the absence of gene transcription, the platelet proteome is mirrored in the transcriptome. Conversely, transcriptional analysis predicts the presence of novel proteins in the platelet. Transcriptional analysis is relevant to platelet biology, providing insights into platelet function and the mechanisms of platelet disorders.

Blood Platelets↗
PubMed2003

A palmitylated peptide derived from the glycoprotein Ib beta cytoplasmic tail inhibits platelet activation.

The platelet receptor GPIb/IX/V mediates a crucial role in hemostasis, yet the signaling mechanisms involved are incompletely understood. The complex consists of four polypeptides GPIb alpha, GPIb beta, GPIX and GPV. We identified an amino acid sequence in the cytoplasmic tail of the GPIb beta subunit between residues R151 and A161 that is highly conserved across species and hypothesized that it has functional importance. To target this motif, we synthesized a corresponding cell-permeable palmitylated peptide (Pal-RRLRARARARA) and investigated its effect on platelet function. Pal-RRLRARARARA completely inhibited low dose thrombin- and ristocetin-induced aggregation in washed platelets but only partially inhibited collagen- and U46619-induced aggregation. Thromboxane production in platelets stimulated with thrombin was significantly reduced by Pal-RRLRARARARA compared with collagen. Activation of the integrin alpha IIb beta 3 in response to thrombin was significantly reduced when platelets were preincubated with Pal-RRLRARARARA. The adhesion of washed platelets to von Willebrand factor (VWF) under static conditions was significantly reduced by Pal-RRLRARARARA. Under conditions of high shear, the velocity of platelets rolling on VWF was significantly increased when platelets are preincubated with Pal-RRLRARARARA. This study defines a novel function for the RRLRARARARA motif of GPIb beta in platelet activation.

Blood Platelets↗
PubMed2002

The contribution of genetic factors to thrombotic and bleeding outcomes in coronary patients randomised to IIb/IIIa antagonists.

Genetic variants are risk factors for coronary disease, but their role in recurrent events and in response to treatment is less clear. We genotyped genetic variants implicated in primary coronary disease in 924 Caucasians with acute coronary syndromes participating in the OPUS-TIMI16 trial of the GPIIb/IIIa antagonist orbofiban. These were the platelet glycoprotein (GP) receptors GPIIIa, GPIa, GPIbalpha; platelet ligands beta-fibrinogen and von Willebrand Factor (vWF); interleukins (IL) IL-1RN, and IL-6; adhesion proteins E-selectin and P-selectin; and metalloproteinase MMP-9. Cox modelling of all genetic variants demonstrated no significant impact on the composite endpoint (P = 0.88), which included myocardial infarction (MI), death, recurrent ischemia, urgent revascularisation and stroke, but a significant impact on recurrent myocardial infarction alone (chi(2) = 20.4, 10 df, P = 0.04). There was a significant interaction of the polymorphisms with orbofiban treatment influencing bleeding outcomes (P = 0.004). Thus, genetic polymorphisms may be associated with subsequent myocardial infarction, and may also be associated with treatment-associated bleeding among coronary patients.

Alanine↗
PubMed2001

Genetic variation in glycoprotein IIb/IIIa (GPIIb/IIIa) as a determinant of the responses to an oral GPIIb/IIIa antagonist in patients with unstable coronary syndromes.

This study examined the influence of the Pl(A) polymorphism of glycoprotein IIIa (GPIIIa) in determining the response to an oral GPIIb/IIIa antagonist, orbofiban, in patients with unstable coronary syndromes. Genotyping for the Pl(A) polymorphism was performed in 1014 patients recruited into the OPUS-TIMI-16 (orbofiban in patients with unstable coronary syndromes-thrombolysis in myocardial infarction 16) trial, in which patients were randomized to low- or high-dose orbofiban or placebo for 1 year. The primary end point (n = 165) was a composite of death, myocardial infarction (MI), recurrent ischemia requiring rehospitalization, urgent revascularization, and stroke. Overall, orbofiban failed to reduce ischemic events when compared with placebo, but increased the rate of bleeding. In the whole population, Pl(A2) carriers had a significant increase in MI (n = 33) during follow up, with a relative risk (RR) of 2.71 (95% CI, 1.37 to 5.38; P =.004). There was a significant interaction between treatment (placebo and orbofiban) and the Pl(A) polymorphism for bleeding (n = 187; P =.05). Thus, while orbofiban increased bleeding in noncarriers (RR = 1.87, 1.29 to 2.71; P <.001) in a dose-dependent fashion, it did not increase bleeding events in Pl(A2) carriers (RR = 0.87, 0.46 to 1.64). There was no interaction between treatment (placebo and orbofiban) and the Pl(A) polymorphism for the primary end point (P =.10). However, in the patients receiving orbifiban there was a higher risk of a primary event (RR = 1.55, 1.03 to 2.34; P =.04) and MI (RR 4.27, 1.82 to 10.03; P <.001) in Pl(A2) carriers compared with noncarriers. In contrast, there was no evidence that Pl(A2) influenced the rate of recurrent events in placebo-treated patients. In patients presenting with an acute coronary syndrome, the Pl(A) polymorphism of GPIIb/IIIa may explain some of the variance in the response to an oral GPIIb/IIIa antagonist.

Aged↗
PubMed2001

No association between 5HT-2A and bipolar disorder irrespective of genomic imprinting.

Recent evidence that 5HT-2A may be subjected to genomic imprinting prompted us to examine a collection of Irish family trios (an affected individual and both parents) for evidence of an association between 5HT-2A and bipolar disorder. Family trios offer an advantage over case control studies in regard to genomic imprinting since with family trios it is possible to trace the path of alleles from the parents to the offspring. Using haplotype-based haplotype relative risk (HHRR) and transmission/disequilibrium (TDT) analyses, no evidence was found for an association of 5HT-2A with bipolar affective disorder under the assumption of no imprinting and of imprinting.

Alleles↗
PubMed2001

Mechanism of myelin breakdown in experimental demyelination: a putative role for calpain.

Although calpain has been extensively studied, its physiological function is poorly understood. In contrast, its role in the pathophysiology of various diseases has been implicated, including that of experimental allergic encephalomyelitis (EAE), an animal model of the demyelinating disease multiple sclerosis (MS). In EAE, calpain degrades myelin proteins, including myelin basic protein (MBP), suggesting a role for calpain in the breakdown of myelin in this disease. Subsequent studies revealed increased calpain activity and expression in the glial and inflammatory cells concomitant with loss of axon and myelin proteins. This suggested a crucial role for calpain in demyelinating diseases.

Animals↗
PubMed2001

Thermolabile MTHFR genotype and retinal vascular occlusive disease.

BACKGROUND: Raised levels of total plasma homocysteine (tHcy) are associated with an increased risk of retinal vascular occlusive disease. A thermolabile form of a pivotal enzyme in homocysteine metabolism, methylenetetrahydrofolate reductase (MTHFR), has been associated with vascular occlusive disease and raised tHcy levels. The relation between thermolabile MTHFR genotype, tHcy, and retinal vascular occlusive disease has not been determined. METHODS: A retrospective case-control study involving hospital based controls and cases with retinal vascular occlusions in whom tHcy levels had been determined was undertaken. Genotyping for the MTHFR 677 C-T mutation that specifies the thermolabile form of the enzyme was performed by established methods in all subjects. The relation between homozygosity for thermolabile MTHFR genotype (TT), raised tHcy levels, and risk of retinal vascular occlusive disease was examined. RESULTS: 87 cases of retinal vascular occlusive disease (mean age 68.7 years) comprising 26 cases of retinal artery occlusion and 61 of retinal vein occlusion were compared with 87 controls (mean age 70.2 years). The TT genotype did not confer a significantly increased risk of retinal vascular occlusive disease. The mean tHcy level was significantly higher in the cases than in the controls (p<0.0001). Overall, and in both the cases and controls, the frequency of the TT genotype was higher in those with normal tHcy levels than in those with increased levels of tHcy. However, the TT genotype did not significantly alter the risk of increased tHcy levels in these patients. CONCLUSIONS: The TT genotype is not associated with an increased risk of retinal vascular occlusive disease or increased tHcy levels in this group of elderly patients. In older patients, nutritional rather than genetic factors may be more important in increasing tHcy levels, a known risk factor for retinal vascular occlusive disease.

Aged↗
PubMed2000

Calpain activity and expression increased in activated glial and inflammatory cells in penumbra of spinal cord injury lesion.

Following traumatic injury of the spinal cord, cells adjacent to the lesion are subject to ischemic cell death as a result of vascular disruption and secondary inflammatory responses. Proteases such as calcium-activated neutral proteinase (calpain) have been implicated in axon and myelin destruction following injury since they degrade structural proteins in the axon-myelin unit. To examine the role of calpain in cell death following spinal cord injury (SCI), calpain activity and translational expression were evaluated using Western blotting techniques. Calpain activity (as measured by specific substrate degradation) was significantly increased in and around the lesion site as early as 4 hr following injury with continued elevation at 48 hr compared to sham controls. Likewise, calpain expression was significantly increased in both the lesion site and penumbra at 4 and 48 hr after injury. Using double immunofluorescent labeling for calpain and cell-specific markers, this increase in calpain expression was found to be due in part to activated glial/inflammatory cells such as astrocytes, microglia, and infiltrating macrophages in these areas. Thus, since calpain degrades many myelin and axonal structural proteins, the increased activity and expression of this enzyme may be responsible for destruction of myelinated axons adjacent to the lesion site following SCI.

Animals↗
PubMed2000

Association between historically high frequencies of neural tube defects and the human T homologue of mouse T (Brachyury).

The human T developmental gene has been implicated in the etiology of neural tube defects (NTDs) on the basis both of mouse studies of its homologue, T (Brachyury), and of allelic association in a Caucasian population. We have investigated the frequency of the T allelic variant TIVS7-2 in 218 Irish NTD case-parent triads. This population showed the same trend as previously reported, with an excess of the TIVS7-2 allele among cases. Log-linear modeling of case and maternal genotypic effects within families indicated that TIVS7-2 was elevated in cases (relative risk, RR = 1.36) but not in mothers (RR = 0.91). The TIVS7-2 allele is markedly associated with cases born before 1980 (RR = 2.09; CI = 1.23-3.55; corrected p = 0.030), but not with more recent cases (RR = 0.92). Cases carrying a TIVS7-2 allele did not show any increased tendency to be homozygous for the thermolabile variant of the folate-dependent enzyme 5,10-methylene tetrahydrofolate reductase, which is an established genetic risk factor for NTDs. Since the incidence of NTDs has declined markedly in Ireland over the last few decades, we suggest that the T-associated risk is potentiated by nutritional or environmental risk factor(s), the impact of which have been diminishing over time.

Adolescent↗
PubMed2000

Gene conversion among chemokine receptors.

It has been proposed that proteins which are involved in host defence and susceptibility undergo accelerated evolution. Chemokine receptors have roles as pro-inflammatory agents acting in response to infection, and in addition are receptors for entry of viruses and other pathogens into cells. Consistent with this, their rate of evolution is higher than that for other members of the seven-transmembrane domain receptor family. The pattern of evolution of the chemokine receptors was examined in detail. Both chromosomal clusters of chemokine receptors (CC and CXC) showed evidence of a number of gene conversions. These are likely to have resulted in protein sequence changes, which could possibly alter function. 45% of a control group of clustered genes also showed evidence of conversion. Thus, the fixation of a gene conversion is not in itself sufficiently unusual in tandemly repeated genes and cannot be taken as strong evidence of a selection for a novel function. However, the degree of amino acid difference between the chemokine receptors CCR1 and CCR3 was greater than that for any of the control genes. Such changes could have functional implications for inter-species differences in chemokine receptor interactions with pathogens.

Amino Acid Sequence↗
PubMed2000

Combined TUNEL and double immunofluorescent labeling for detection of apoptotic mononuclear phagocytes in autoimmune demyelinating disease.

Apoptosis is usually associated with genomic DNA fragmentation which can be detected in situ by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. We describe a combined TUNEL and double immunofluorescent labeling technique to determine the fate of inflammatory infiltrates and resident glial cells in the central nervous system following the onset of an autoimmune demyelinating disease such as experimental allergic encephalomyelitis (EAE) in rats. Anti-digoxigenin (anti-DIG) antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) emitting blue fluorescence was used to detect apoptotic cell DNA, which was already labeled by modified TUNEL using alkali-stable DIG-11-dUTP. Anti-mouse IgG secondary antibody conjugated with Texas Red emitting red fluorescence was used to detect anti-rat CD11b primary antibody (clone OX-42) directed to the surface antigen of mononuclear phagocytes including microglia. Using this technique, we detected apoptotic mononuclear phagocytes (co-labeled with blue and red fluorescences) in the spinal cord sections of rats with EAE.

Animals↗