Stable transfectants of human MCF-7 breast cancer cells with increased levels of the human folate receptor exhibit an increased sensitivity to antifolates.

A major problem in cancer therapy is tumor drug resistance such as is found with antifolates (e.g., methotrexate [MTX]). We are specifically interested in the role of the human folate receptor (hFR) in MTX resistance. To investigate whether transfection of hFR results in increased MTX uptake and increased drug sensitivity, human mammary carcinoma (MCF-7) cells and Chinese hamster ovary cells (CHO) (cells which do not express detectable levels of hFR) were transfected with hFR cDNA. Stable human mammary carcinoma and Chinese hamster ovary transfectants expressing high levels of hFR were selected for further analysis. Transfected cells which express increased levels of hFR grow more rapidly than mock transfected or wild type cells in media containing physiologic concentrations of folates. The hFR expressed by these cells is sorted to the plasma membrane and is functional as determined by cell surface binding of a radiolabeled folic acid derivative and by internalization of [3H]methotrexate. The stable transfectants that express increased levels of hFR are also more sensitive to MTX in physiologic concentrations of folates. We conclude that increased expression of hFR by human mammary carcinoma and Chinese hamster ovary cells cultured under these conditions results in an enhanced growth rate, increased folic acid binding, and increased MTX uptake and cytotoxicity.

Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells.

The influence of extracellular folate concentration on cellular levels of the folate transport protein and its soluble product was studied directly in cultured human nasopharyngeal carcinoma (KB) cells. As determined by radioimmunoassay, levels of the folate transport protein and the soluble folate-binding protein were 58 +/- 17 (mean +/- SD) and 5 +/- 2 pmol/mg cell protein, respectively, in KB cells maintained in standard medium (containing 2,300 nM folic acid). These levels significantly increased to 182 +/- 34 and 26 +/- 6 pmol/mg cell protein, respectively, in KB cells serially passaged in low folate medium (containing 2-10 nM 5-methyltetrahydrofolate). Increases in folate-binding protein levels occurred more rapidly in KB cells serially passaged in very low folate medium containing less than 2 nM folate and were prevented by the addition of 100 nM 5-methyltetrahydrofolate or 0.1-1 microM 5-formyltetrahydrofolate to this medium. When KB cells which had been passaged in low folate medium were passaged back into either standard medium or low folate medium supplemented with reduced folates, the levels of both folate-binding proteins fell linearly towards the levels in KB cells continuously maintained in standard medium. The folate transport protein was identified in and underwent similar changes in human and mouse mammary tumor cells. These studies indicate that the folate transport system is probably regulated by the extracellular folate concentration through changes in intracellular metabolite levels.

CNS involvement in primary mediastinal large B-cell lymphoma.

PURPOSE: The risk of CNS involvement by non-Hodgkin's Lymphoma (NHL) has been associated with bone marrow and/or testicular involvement; however, it was recently reported that the number of extranodal sites is a more reliable predictor of CNS disease. Because primary mediastinal thymic large B-cell lymphoma (PMLCL) has a high propensity for involving extranodal sites, we investigated the frequency and pattern of CNS involvement in PMLCL. PATIENTS AND METHODS: The medical records of 219 patients with aggressive NHL, consecutively entered onto protocols at the National Cancer Institute between 1987 and 1998, were retrospectively reviewed. RESULTS: Twenty-three patients (11%) had clinical and pathologic features of PMLCL. These patients were young (median age, 29 years), female (61%), and presented with massive mediastinal adenopathy (70%). Extranodal disease occurred at presentation in 70% and at relapse in 93% of patients and involved contiguous intrathoracic structures and/or distant sites, including the lungs, kidneys, liver, adrenals, ovaries, pancreas, and bone. Six patients (26%) developed CNS involvement, two (9%) at presentation and four (27%) at relapse. All had extranodal disease, but only one had bone marrow involvement. Parenchymal and leptomeningeal CNS disease occurred in four and three patients, respectively. CONCLUSION: CNS involvement in PMLCL is associated with extranodal involvement other than bone marrow and may reflect the unique biology of this disease. The propensity to involve the CNS parenchyma raises the concern that intrathecal prophylaxis may not be effective and suggests that CNS imaging should be considered in patients with extranodal disease.

Expression of the human placental folate receptor transcript is regulated in human tissues. Organization and full nucleotide sequence of the gene.

The primary structures of the human KB cell (FR-KB1) folate receptor (FR) and of a human placental (FR-P2) FR, proteins important in cellular accumulation of folates, have been deduced from cDNA sequences. Herein, we report a novel human FR cDNA (FR-P3) isolated from a placental library and the chromosomal organization of the human FR-P3 gene. Compared to the FR-P2 cDNA, the composite 1084 base-pair (bp) FR-P3 cDNA is homologous, but contains a unique 5' terminus and sequence differences within the open reading frame (ORF) and at the exon I-II junction. Polymerase chain reaction and RNase protection assays demonstrate that the FR-P3 cDNA represents the major transcript, and suggest that the FR-P2 cDNA is encoded by an independent FR gene. The nucleotide sequences of two non-overlapping human genomic clones contain the FR-P3 gene, which spans 5148 bp, is composed of five exons, and is polymorphic relative to 5' restriction sites. The transcript size (1084 bp) predicted from structural analysis of the FR-P3 gene correlates with the size (1100 bp) determined by Northern blots. Based on RNase protection assays, both FR-P3 and FR-KB1 transcripts are expressed in human fetal and adult tissues, and the abundance of each transcript varies among the tissues studied. These results indicate that the FR transcripts are products of independent, conserved genes; that neither FR gene is preferentially expressed during fetal development; and that specific FR transcripts are differentially expressed in human tissues, suggesting that transcription of each FR gene is regulated independently. The isolation of the FR-P3 gene will permit functional analysis of the cis and trans regulatory elements of the FR-P3 gene and the mechanisms involved in tissue-specific FR gene expression.

Long term effect of breast feeding: cognitive function in the Caerphilly cohort.

STUDY OBJECTIVE: There is evidence suggesting that artificial feeding is associated with a reduction in cognitive function in infants and children, in contrast with breast feeding, but the available evidence suffers from confounding by social and educational factors. An opportunity arose in the Caerphilly cohort study to examine relations between cognitive function in older men and their feeding as infants, when breast feeding was usual. DESIGN: A prospective cohort study. SETTING: Caerphilly, South Wales, UK, was a deprived coal mining community when the men had been born in 1920-35. Most had been breast fed as infants. PARTICIPANTS: 779 men aged 60-74 years when tested. The men had earlier been asked to obtain from their mothers their birth weight, and how they had been fed as infants. RESULTS: Complete data were obtained for 779 men. In those whose birth weight had been at or above the median, the adjusted mean cognitive function was only slightly and non-significantly lower in those who had been artificially fed. In the men whose birth weight had been below the median, having been artificially fed was associated with significantly lower results in both a test of reasoning (the AH4) and word power (the national adult reading test (NART)). Two standard deviations below the median birth weight, artificial feeding was associated with a reduction of six points (70% of a SD) on word power (the NART). CONCLUSIONS: In men whose birth weight had been low, having been artificially fed is associated with poorer cognitive function in late adult life.

Milk consumption, stroke, and heart attack risk: evidence from the Caerphilly cohort of older men.

OBJECTIVE: To examine associations between milk consumption and incident heart disease and stroke. DESIGN: A representative population sample of men was asked to weigh and record their food intake for seven days. The total consumption of milk was obtained from these records. Details of all deaths and vascular events were collected during the following 20 years. Incident ischaemic strokes and heart disease events were diagnosed by standard criteria. SETTING: The Caerphilly cohort, a representative population sample of men in South Wales, aged 45-59 when first seen in 1979-83. PARTICIPANTS: A representative 3:10 subsample of the men in the cohort. MAIN RESULTS: 665 men (87% of those approached) returned satisfactory seven day diet diaries. After adjustment, the relative odds of an event in the men whose milk consumption was the median or higher, relative to those with lower intakes of milk, were 0.52 (0.27 to 0.99) for an ischaemic stroke and 0.88 (0.56 to 1.40) for an ischaemic heart disease event. Deaths from all causes were similar in the two milk consumption groups (relative odds 1.08; 0.74 to 1.58). CONCLUSIONS: These results give no convincing evidence of an increased risk of vascular disease from milk drinking. Rather, the subjects who drank more than the median amount of milk had a reduced risk of an ischaemic stroke, and possibly a reduced risk of an ischaemic heart disease event. These conclusions are in agreement with the results of a previously reported overview of 10 large, long term cohort studies based on food frequency intake records.

Do total and high density lipoprotein cholesterol and triglycerides act independently in the prediction of ischemic heart disease? Ten-year follow-up of Caerphilly and Speedwell Cohorts.

Several studies have suggested that men with raised plasma triglycerides (TGs) in combination with adverse levels of other lipids may be at special risk of subsequent ischemic heart disease (IHD). We examined the independent and combined effects of plasma lipids at 10 years of follow-up. We measured fasting TGs, total cholesterol (TC), and high density lipoprotein cholesterol (HDLC) in 4362 men (aged 45 to 63 years) from 2 study populations and reexamined them at intervals during a 10-year follow-up. Major IHD events (death from IHD, clinical myocardial infarction, or ECG-defined myocardial infarction) were recorded. Five hundred thirty-three major IHD events occurred. All 3 lipids were strongly and independently predictive of IHD after 10 years of follow-up. Subjects were then divided into 27 groups (ie, 3(3)) by the tertiles of TGs, TC, and HDLC. The number of events observed in each group was compared with that predicted by a logistic regression model, which included terms for the 3 lipids (without interactions) and potential confounding variables. The incidence of IHD was 22.6% in the group with the lipid risk factor combination with the highest expected risk (high TGs, high TC, and low HDLC) and 4.7% in the group with the lowest expected risk (P<0.01). A comparison of the predicted number of events in the 27 groups with the number of events observed showed that a logistic regression provided an adequate fit without the need to incorporate interactions between lipids in the model. Conclusions are as follows: (1) Serum TGs, TC, and HDLC are independently predictive of IHD at 10 years of follow-up. (2) Combinations of adverse levels of the 3 major lipid risk factors have no greater impact on IHD than that expected from their individual contributions in a logistic regression model. There was no evidence that men with low HDL/raised TGs were at significantly greater risk than that predicted from the independent effects of the 2 lipids considered individually.

Plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality: an individual participant meta-analysis.

CONTEXT: Plasma fibrinogen levels may be associated with the risk of coronary heart disease (CHD) and stroke. OBJECTIVE: To assess the relationships of fibrinogen levels with risk of major vascular and with risk of nonvascular outcomes based on individual participant data. DATA SOURCES: Relevant studies were identified by computer-assisted searches, hand searches of reference lists, and personal communication with relevant investigators. STUDY SELECTION: All identified prospective studies were included with information available on baseline fibrinogen levels and details of subsequent major vascular morbidity and/or cause-specific mortality during at least 1 year of follow-up. Studies were excluded if they recruited participants on the basis of having had a previous history of cardiovascular disease; participants with known preexisting CHD or stroke were excluded. DATA EXTRACTION: Individual records were provided on each of 154,211 participants in 31 prospective studies. During 1.38 million person-years of follow-up, there were 6944 first nonfatal myocardial infarctions or stroke events and 13,210 deaths. Cause-specific mortality was generally available. Analyses involved proportional hazards modeling with adjustment for confounding by known cardiovascular risk factors and for regression dilution bias. DATA SYNTHESIS: Within each age group considered (40-59, 60-69, and > or =70 years), there was an approximately log-linear association with usual fibrinogen level for the risk of any CHD, any stroke, other vascular (eg, non-CHD, nonstroke) mortality, and nonvascular mortality. There was no evidence of a threshold within the range of usual fibrinogen level studied at any age. The age- and sex- adjusted hazard ratio per 1-g/L increase in usual fibrinogen level for CHD was 2.42 (95% confidence interval [CI], 2.24-2.60); stroke, 2.06 (95% CI, 1.83-2.33); other vascular mortality, 2.76 (95% CI, 2.28-3.35); and nonvascular mortality, 2.03 (95% CI, 1.90-2.18). The hazard ratios for CHD and stroke were reduced to about 1.8 after further adjustment for measured values of several established vascular risk factors. In a subset of 7011 participants with available C-reactive protein values, the findings for CHD were essentially unchanged following additional adjustment for C-reactive protein. The associations of fibrinogen level with CHD or stroke did not differ substantially according to sex, smoking, blood pressure, blood lipid levels, or several features of study design. CONCLUSIONS: In this large individual participant meta-analysis, moderately strong associations were found between usual plasma fibrinogen level and the risks of CHD, stroke, other vascular mortality, and nonvascular mortality in a wide range of circumstances in healthy middle-aged adults. Assessment of any causal relevance of elevated fibrinogen levels to disease requires additional research.

Exon 3 of the alpha folate receptor gene contains a 5' splice site which confers enhanced ovarian carcinoma specific expression.

The human folate receptor (FR) is overexpressed in ovarian carcinoma. FR transcripts are heterogeneous due to the use of two promoters, P1 and P4, and alternative splicing of exon 3. RNase protection assay and RT-PCR revealed higher levels of the transcripts that include exon 3 in lines and specimens from ovarian carcinoma. A P1-chloramphenicol acetyltransferase (CAT) construct containing exon 3 demonstrated efficient reporter expression only in ovarian carcinoma. 5' and 3' deleted variants of the P1-CAT construct were analyzed by RT-PCR of the exogenous transcripts and reporter activity. A 5' splice site and 35 bp downstream intronic region of exon 3 appeared to regulate enhanced FR expression in ovarian carcinoma.

Antioxidant flavonols and ischemic heart disease in a Welsh population of men: the Caerphilly Study.

Antioxidant flavonols and their major food source, black tea, have been associated with a lower risk of ischemic heart disease (IHD) and stroke in Dutch men. We investigated whether flavonol intake predicted a lower rate of IHD in 1900 Welsh men aged 45-59 y, who were followed up for 14 y. Flavonol intake, mainly from tea to which milk is customarily added, was not related to IHD incidence [relative risk (RR), highest compared with lowest quartile: 1.0; 95% CI: 0.6, 1.6; P for trend = 0.996; n = 186] but was weakly positively related to IHD mortality (RR: 1.6; 95% CI: 0.9, 2.9; P = 0.119; n = 131) and cancer mortality (RR: 1.3; 95% CI: 0.7, 2.3; P = 0.150; n = 104) and strongly related to total mortality (RR: 1.4; 95% CI: 1.0, 2.0; P = 0.014; n = 334). Men with the highest consumption of tea (> 1.2 L, or > 8 cups/d) had an RR of 2.4 (95% CI: 1.5, 3.9) of dying in the follow-up period compared with men consuming < 300 mL/d (< 2 cups/d). We conclude that intake of antioxidant flavonols is not inversely associated with IHD risk in the United Kingdom. Possibly, flavonols from tea to which milk is added are not absorbed; experimental evidence suggests that adding milk to tea abolishes the plasma antioxidant-raising capacity of tea. The apparent association between tea consumption and increased mortality in this population merits further investigation.

Factors affecting bone density in young adults.

A 14-y follow-up of 581 children who took part in a randomized controlled trial of the effect of a milk supplement on growth of children was conducted to investigate the supplement's effect on adult bone mineral content (BMC) and density (BMD). BMC and BMD of the nondominant forearm were measured by single-photon absorptiometry in 371 subjects (64%) aged 20-23 y, at a proximal site (shaft of radius and ulna) and at a distal site near the wrist. BMCs and BMDs tended to be higher in the intervention group (NS). Cross-sectionally, BMD was positively associated with body weight (P less than 0.01) in both sexes; inversely associated with alcohol consumption (P less than 0.05), and positively with manual occupation (NS) in men; positively associated with current intakes of calcium (P less than 0.05), vitamin D (P less than 0.01), and sports activity during adolescence (P less than 0.01), and inversely with parity (NS) in women. In multiple linear-regression analysis body weight and sports activity during adolescence were stronger determinants of female BMD than was diet.

Alcohol and platelet aggregation: the Caerphilly Prospective Heart Disease Study.

Intakes of alcohol and saturated fatty acids were determined through a dietary questionnaire from 1600 men (aged 49-66 y) in the Caerphilly Prospective Heart Disease Study. Platelet aggregation induced by thrombin adenosine disphosphate (ADP), and collagen was studied in subjects who had fasted and had not recently taken drugs affecting platelets. In subjects who drank alcohol, the odds ratio of a high response to aggregation was significantly reduced (primary ADP, P less than 0.05; secondary ADP, P less than 0.001; collagen, P less than 0.02). The significance was enhanced by adjusting for smoking and by including only the subjects with a high intake of saturated fatty acids or a low intake of polyunsaturated fatty acids. By contrast, the responsiveness to thrombin was slightly increased at all levels of alcohol consumption. We therefore suggest that part of the effects of alcohol on coronary heart disease may be mediated by a dose-dependent effect on certain platelet functions, modulated by the intake of dietary fat.

Fruit and vegetable consumption and cancer mortality in the Caerphilly Study.

We investigated whether the consumption of fruit and vegetables lowered cancer mortality in a cohort of 2112 Welsh men ages 45-69 years (The Caerphilly Study), which was followed-up for 13.8 years. At baseline (between 1979 and 1983), participants completed a 56-item food frequency questionnaire from which the consumption of fruit and vegetables was calculated. Relative risks (RR) were estimated with Cox proportional hazard analysis, with death from various types of cancer as a dependent variable, and fruit, vegetables, vitamin C, beta-carotene, dietary fiber, and potential confounders as independent variables. Mean consumption of vegetables and fruit at baseline was 118 g/day and 83 g/day, respectively. During follow-up 114 men died from cancer, including 51 men who died from respiratory tract cancer and 45 men who died from digestive tract cancer. Fruit consumption and the intake of dietary fiber were inversely related to respiratory tract cancer, but after adjustment for potential confounders including age, smoking, and social class, the association with fruit consumption became nonsignificant. Vegetable and fruit consumption was, independently from other risk factors, inversely related to mortality from cancer of the digestive tract (P for trend = 0.021), mainly due to an inverse association with fruit consumption (RR for the highest quartile versus the lowest was 0.3; 95% CI, 0.1-0.8). Vitamin C, beta-carotene, and dietary fiber were not significantly associated with cancers of the digestive tract. Vegetable and fruit consumption was also inversely related to all-cause cancer mortality, and the strongest association was observed for fruit consumption (RR in the highest versus lowest quartile was 0.5; 95% CI, 0.3-1.0). Consumption of vegetables and particularly the consumption of fruit could considerably lower the risk of dying from cancer in middle-aged men.

Homocysteine and ischaemic heart disease in the Caerphilly cohort.

Elevated circulating total homocyst(e)ine concentrations are associated with a higher prevalence of ischaemic heart disease (IHD). We utilized data from the Caerphilly Prospective Cohort Study to assess the predictive power of the serum total homocyst(e)ine concentration for future IHD. Serum total homocyst(e)ine concentrations were measured in 2290 men in the Caerphilly cohort, a representative population sample of men aged 50-64 years. During a 5-year follow-up period, 56 men suffered fatal IHD, 77 had a non-fatal myocardial infarction, while 21 were found to have ECG evidence of myocardial infarction (MI) when examined at follow-up. The mean serum total homocyst(e)ine concentration in the total of 154 men who experienced an incident IHD event was 12.4 micromol/l, whereas the 2136 men who experienced no such event had a mean level of 11.7 micromol/l. The difference between these means, examined by logistic regression and standardising for the effects of differences in age, social class, smoking, BMI, diabetes, HDL-cholesterol and prevalent IHD is 0.47 micromol/l (95% CI = -0.13 to 1.11 micromol/l). The mean difference for the 56 men who died, and whose death was attributed to IHD, is 0.81 micromol/l (95% CI= -0.17 to 1.88 micromol/l) after correction for confounding factors. Vitamin nutritional status and alcohol intake were significant negative determinants of serum total homocyst(e)ine concentrations; the effect of alcohol is explained by the folic acid content of beer, which is the preferred alcoholic beverage in Caerphilly. It is concluded that the serum total homocyst(e)ine concentration is weakly predictive of IHD events, though in the present data adjustments for other factors attenuated the relationship and it became not statistically significant (P > 0.05).

Triglyceride as a risk factor for ischaemic heart disease in British men: effect of adjusting for measurement error.

OBJECTIVE: To assess the influence of differential precision in the measurement of the correlated variables total cholesterol and high density lipoprotein (HDL) cholesterol on the estimates of the risk of ischaemic heart disease (IHD) associated with plasma triglyceride levels. DESIGN, SETTING AND PARTICIPANTS: The Caerphilly Heart Disease Study (CHDS), a prospective cohort study of 2512 middle-aged men living in the town of Caerphilly, south Wales, UK. The results from two sub-studies were used to estimate the degree of measurement imprecision (laboratory error and within-person variation) in triglycerides, total cholesterol and HDL cholesterol. MAIN OUTCOME MEASURES: Multivariable risk estimates for major IHD calculated from logistic regression analysis, adjusted and not adjusted for measurement imprecision. Major IHD events were defined as death from IHD, clinical non-fatal myocardial infarction or electrocardiographic myocardial infarction. RESULTS: There were 261 men with major IHD events during follow-up. In age-adjusted analyses, taking measurement imprecision into account strengthened associations with IHD for all lipid factors. The odds ratio (OR) for one S.D. increase in triglycerides, ignoring measurement imprecision, was 1.36 (95% confidence interval [95% CI] 1.20-1.55) but 1.57 (95% CI 1.30-1.89) when taking imprecision into account. The standardised odds ratio for triglycerides adjusted for measurement imprecision and the two other lipid factors was 1.35 (95% CI 1.09-1.69). In this model, the triglyceride level showed a stronger association than total cholesterol (OR 1.28; 95% CI 1.05-1.56) and HDL cholesterol (OR for one S.D. decrease 1.20; 95% CI 0.97-1.49). When adding fasting blood glucose and diastolic blood pressure, however, the effect of triglycerides was reduced and ceased to be statistically significant (OR 1.19; 95% CI 0.95-1.49). This was further attenuated upon inclusion of body mass index, smoking status and history of pre-existing IHD. Total cholesterol remained a statistically significant (P < 0.05) risk factor in all models. CONCLUSIONS: In contrast to other cohort studies, triglyceride concentration in the CHDS shows an association with the risk of IHD which is independent of total and HDL cholesterol. This effect was pronounced after adjustment for measurement imprecision. It was reduced, however, when adjusted for other factors. While hypertriglyceridaemia may exert an influence independent of other lipid factors, insulin resistance is probably the underlying metabolic disturbance.

British studies of aspirin and myocardial infarction.

The MRC Epidemiology Unit in Cardiff, Wales conducted three randomized, controlled trials of aspirin use in patients who had had myocardial infarction. Follow-up study for one to two years was conducted in 1,239 men after they experienced myocardial infarction and another was based on 1,682 patients followed for one year after infarction. Although the results are not statistically significant in either trial, they are consistent with a reduction of about 26 and 17 percent in the mortality rate of patients during the year after infarction. The third trial, which was concerned with a very early mortality rate, was based on a sample of 2,530 patients. This gave no evidence of benefit from aspirin use during the acute phase of infarction.

Blood lead and platelet aggregation--evidence for a causal association.

Adenosine diphosphate (ADP) and thrombin are agonists of platelet aggregation in which intracellular calcium plays a significant role as a signal transducer. Lead is a well established toxicological agent affecting intracellular mechanisms controlling free ionized calcium concentration in a number of cell lines. This epidemiological study is the first demonstration of a significant relationship between the extent of primary ADP-induced platelet aggregation measured by optical densitometry in platelet rich plasma (PRP) and blood lead concentration. High blood lead levels are associated with decreased aggregation in this population of 2,150 men aged 49-65 years. This is reflected by a negative regression relationship of -0.19% of maximal extent (PRP vs platelet poor plasma) of aggregation per microgram of Pb/dl (T = -3.82, p less than 0.001). In contrast, no relationship is noted between thrombin-induced aggregation and blood lead concentration. Smoking behaviour represents a potential confounder which may be the explanation for the observed relationship. However, because smoking status is simultaneously related to both ADP- and thrombin-induced aggregation, but is simultaneously related to ADP-induced aggregation and blood lead concentration in a different way, the observed relation is likely to be causal. The mechanisms by which ADP and thrombin effect intracellular calcium transduction signals appear to be distinctively different. The findings in this population-based study are not inconsistent with this difference.

The association of platelet and red cell count with platelet impedance changes in whole blood and light-scattering changes in platelet rich plasma: evidence from the Caerphilly Collaborative Heart Disease Study.

This epidemiological study was undertaken to explore possible relationships among various haematological indices, prevalent ischaemic heart disease and platelet "function" as measured by two rather different methods. ADP-induced platelet impedance changes in whole blood were strongly associated with prevalent ischaemic heart disease in a general population of 49-66 year men at increased risk. Adenosine diphosphate (ADP) induced platelet aggregation in platelet rich plasma (PRP) at a constant platelet count and also the whole blood platelet count and red cell (RBC) count were strongly and independently related to ADP-induced platelet impedance changes. Both platelet count and platelet aggregation in PRP assessed by changes in optical density were directly related to increasing platelet "sensitivity" as measured by impedance changes in whole blood but RBC count was inversely related. Positive independent relationships between platelet impedance changes and plasma viscosity and fibrinogen were markedly attenuated when platelet count was taken into account, but this finding does not discount a role for these factors in platelet aggregation. No relationship was noted between white blood cell (WBC) count and platelet impedance changes; however, a significant inverse relationship was noted with platelet aggregation in PRP. These findings indicate that laboratory-based experimental findings can be observed in population based studies, and that these haematological factors may be important indicators of ischaemic disease in the population.

Cigarette smoking sensitizes and desensitizes impedance-measured ADP-induced platelet aggregation in whole blood.

The effect of smoking on platelet aggregation appears to produce conflicting results, with some studies indicating an enhancement and others a decrease of aggregation. This epidemiological study of 120 male smokers, a subset of the Caerphilly Heart Disease Study, examined the relationship of two dimensions of smoking (time proximity of last cigarette before venepuncture and serum nicotine concentration) with threshold dose of adenosine diphosphate (ADP) necessary to induce platelet aggregation in whole blood. Means (range) of ADP threshold dose and nicotine concentration were 1.66 (0.5-2.5, censored) microM and 12.2 (0-35.2) ng/ml. Men smoking within 30 min of venepuncture demonstrated lower ADP threshold doses (-0.48 microM lower [95% C.I.: -0.95, -0.02])--reflecting increased sensitivity. Men with higher nicotine concentration had higher ADP threshold doses (Regression Coefficient: +0.032 microM per ng/ml [95% C.I.: 0.003, 0.062])--reflecting decreased sensitivity. Men smoking 30 min or more before venepuncture who also had high nicotine concentration (25-30 ng/ml) demonstrated the highest ADP threshold doses compared to never smokers and to men smoking the previous day (approximately 2.20 vs 1.86 and 1.81 microM). Relations involving nicotine concentration do not necessarily reflect a pharmacological effect although the potential for a short term nicotine mediated tolerance effect cannot be dismissed. These observations support an hypothesis suggesting a temporal sequence of platelet sensitization and desensitization during smoking.

Fibrin D-dimer, tissue plasminogen activator, plasminogen activator inhibitor, and the risk of major ischaemic heart disease in the Caerphilly Study.

Plasma levels of fibrin D-dimer, tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI) have been associated with ischaemic heart disease (IHD). However their associations with incident IHD in samples of the general population are not established. D-dimer antigen, tPA antigen and PAI activity were measured in stored, fasting plasma samples from 1,998 men aged 45-65 examined between 1984 and 1988, during the first re-examination of the Caerphilly Study cohort. These variables were related to major IHD events (myocardial infarction or IHD death) which occurred in 129 men during a follow-up period which averaged 61 months. Mean fibrin D-dimer was higher in men who developed IHD events (90 vs. 71 ng/ml; age-adjusted logarithmic mean difference 0.21; 95% CI 0.11, 0.30; p <0.0001). This association remained after adjusting for baseline IHD and for other risk factors including fibrinogen: the adjusted relative odds of IHD in the highest fifth of D-dimer were 3.5 (95% CI 1.8, 6.9; p = 0.0003). Mean tPA antigen was also higher in men who developed IHD (12.6 vs. 11.6 ng/ml; mean difference 0.9; 95% CI 0.2, 1.7; p = 0.02); however this difference largely disappeared after adjusting for other risk factors. PAI activity was not associated with risk of IHD.