Clinical impact of high platelet reactivity in patients with atrial fibrillation and concomitant percutaneous coronary intervention on dual or triple antithrombotic therapy.

High platelet reactivity (HPR) on clopidogrel is an established thrombotic risk factor after percutaneous coronary intervention (PCI). The introduction of more potent antiplatelet drugs has partially surpassed this issue. However, in the setting of concomitant atrial fibrillation (AF) and PCI clopidogrel is still the most adopted P2Y12 inhibitor. In the present study all consecutive patients with history of AF discharged from our cardiology ward with dual (DAT) or triple (TAT) antithrombotic therapy after a PCI from April 2018 to March 2021 were enrolled in an observational registry. For all subjects, blood serum samples were collected and tested for platelet reactivity by arachidonic acid and ADP (VerifyNow system) and genotyping of the CYP2C19*2 loss-of-function polymorphism. We recorded at 3 and 12-months follow-up: (1) major adverse cardiac and cerebrovascular events (MACCE), (2) major hemorrhagic or clinically relevant non-major bleeding and (3) all-cause mortality. A total of 147 patients were included (91, 62% on TAT). In 93.4% of patients, clopidogrel was chosen as P2Y12 inhibitor. P2Y12 dependent HPR resulted an independent predictor of MACCE both at 3 and 12 months (HR 2.93, 95% C.I. 1.03 to 7.56, p = 0.027 and HR 1.67, 95% C.I. 1.20 to 2.34, p = 0.003, respectively). At 3-months follow-up the presence of CYP2C19*2 polymorphism was independently associated with MACCE (HR 5.21, 95% C.I. 1.03 to 26.28, p = 0.045). In conclusion, in a real-world unselected population on TAT or DAT, the entity of platelet inhibition on P2Y12 inhibitor is a potent predictor of thrombotic risk, suggesting the clinical utility of this laboratory evaluation for a tailored antithrombotic therapy in this high-risk clinical scenario. The present analysis was performed in patients with AF undergoing PCI on dual or triple antithrombotic therapy. At 1 year follow-up MACCE incidence was consistent, and it was not different in different antithrombotic pattern groups. P2Y12 dependent HPR was a potent independent predictor of MACCE both at 3- and 12-months follow-up. In the first 3 months after stenting the carriage of CYP2C19*2 allele was similarly associated with MACCE. Abbreviation: DAT, dual antithrombotic therapy; HPR, high platelet reactivity; MACCE, major adverse cardiac and cerebrovascular events; PRU, P2Y12 reactive unit; TAT, triple antithrombotic therapy. Created with BioRender.com.

Effects of a 3-month dietary intervention with a lacto-ovo-vegetarian diet on vitamin B12 levels in a group of omnivores: results from the CARDIVEG (Cardiovascular Prevention with Vegetarian Diet) study.

Previous studies have suggested that vegetarianism can result in a reduction of vitamin B12 circulating levels. The aim of the present study was to investigate the effects of a 3-month dietary intervention with a lacto-ovo-vegetarian diet (VD) on the levels of circulating vitamin B12 in a group of omnivores. We analysed fifty-four omnivorous subjects who followed a VD as a first dietary intervention within the CARDIVEG (Cardiovascular Prevention with Vegetarian Diet) study, a dietary intervention study. VD resulted in a significant reduction (P<0·001) of 51·2 % of vitamin B12 intake and in a significant reduction (P=0·005) of 6·2 % of the circulating levels of vitamin B12 (-24·5 pg/ml). Changes in vitamin B12 intake were significantly correlated with changes in circulating levels of vitamin B12 (R 0·61, P<0·001). Subgroup analyses showed that reduction in circulating vitamin B12 levels was more evident in participants who were younger, overweight, non-smokers and had hypercholesterolaemia. A logistic regression analysis showed that a reduction in vitamin B12 intake greater than the first quartile of the delta changes obtained in the study population (-28·5 %) conferred a significantly higher risk of experiencing a decrease in circulating vitamin B12 levels (OR 10·1; 95 % CI 1·3, 76·1). In conclusion, a 3-month VD period determined a significant reduction in circulating levels of vitamin B12, being significantly correlated with the reduction in vitamin B12 intake. Although a well-planned VD can provide adequate nutrition across all life stages, special care must be taken to ensure adequate vitamin B12 intake and to help prevent deficiency.

Mediterranean, but not lacto-ovo-vegetarian, diet positively influence circulating progenitor cells for cardiovascular prevention: The CARDIVEG study.

AIM: To evaluate the possible association between dietary habits and progenitor cells using data obtained from a randomized crossover trial using two different diets, lacto-ovo-vegetarian (VD) and Mediterranean (MD), the CARDIVEG study. METHODS AND RESULTS: Eighty clinically healthy subjects with a low-to-moderate cardiovascular risk profile (61 F; 19 M; mean age: 50.7 ± 11.6 years) were randomly assigned to isocaloric VD and MD diets lasting three months each, and then crossed. The two diets showed no effects on endothelial progenitor cells and circulating endothelial cells but opposite effects on circulating progenitor cells. In fact, VD determined significant (p < 0.05) and negative changes on circulating progenitor cells, with an average geometric variation of -130 cells/106 events for CD34+/CD45-/dim, -80 cells/106 events for CD133+/CD45-/dim, and -84 cells/106 events for CD34+/CD133+/CD45-/dim while MD determined significant (p < 0.05) and positive changes for CD34+/CD45-/dim levels, with a geometric mean increase of +54 cells/106 events. No significant correlations were observed between changes in progenitor cells and changes in inflammatory parameters during the VD phase. On the other hand, during the MD phase negative correlations between changes of CD34+/CD45-/dim and interleukin-6 (R = -0.324; p = 0.004) as well as interleukin-8 (R = -0.228; p = 0.04) and monocyte chemotactic protein-1 (R = -0.277; p = 0.01), were observed. These correlations remained significant also after adjustment for confounding factors only for CD34+/CD45-/dim and interleukin-6 (ß = -0.282; p = 0.018) and monocyte chemotactic protein-1 (ß = -0.254; p = 0.031). CONCLUSIONS: MD, but not VD, reported a significant and positive effect on circulating progenitor cells in a group of subjects at low-to-moderate cardiovascular risk, probably acting through the modulation of inflammatory parameters.

Aging process, adherence to Mediterranean diet and nutritional status in a large cohort of nonagenarians: Effects on endothelial progenitor cells.

BACKGROUND AND AIM: Adherence to the Mediterranean Diet (MD) has been associated with a longer and better life. The aim of this study was to examine the effects of adherence to the MD, and of nutritional habits on endothelial progenitor (EPCs) and circulating progenitor (CPCs) cells in a cohort of nonagenarians enrolled within the Mugello Study, an epidemiological study aimed at investigating both clinically relevant geriatric items and various health issues, including those related to nutritional status. METHODS AND RESULTS: Four hundred twenty-one nonagenarians (306 F, 115 M, mean age: 93.1 ± 3.2 years) were evaluated. Adherence to MD was assessed through the Mediterranean Diet Score. Elderly subjects who were in the fourth quartile of the Mediterranean diet score showed significantly higher EPCs than subjects grouped into the other three quartiles. After adjustment for confounders, elderly subjects who were in the highest quartile of adherence to the MD score reported to have EPCs' levels significantly higher than those who reported lower values of adherence to the MD. Furthermore, by analyzing different food categories, it was reported that daily consumption of olive oil and a higher consumption of fruit and vegetables showed higher CPCs CD34+ and EPCs CD34+/KDR+ than subjects with not daily or lower consumption. CONCLUSION: Our results support the hypothesis that the adherence to MD, as well as a daily consumption of olive oil and fruit and vegetables, characteristics of MD, may protect against the development of endothelial dysfunction through increasing EPCs and CPCs in older age.

Cytochrome P450 2C19*2 polymorphism and cardiovascular recurrences in patients taking clopidogrel: a meta-analysis.

Several polymorphisms in genes that encode platelet components (receptors or enzymes), or cytochrome P450 enzyme isoforms, involved in clopidogrel metabolism, have been proposed as possible mechanisms for nonresponsiveness to clopidogrel. Among them, a great deal of attention has been focused on the loss-of-function CYP2C19(*)2 (or 681 G > A) polymorphism. We performed a meta-analysis of all the prospective studies that have been published, which analyze the role of such a polymorphism in recurrent cardiovascular events in patients with coronary artery disease (CAD) being treated with clopidogrel. Studies were searched in MedLine, Embase, Web of Science, The Cochrane Systematic Review Database, Google Scholar and bibliographies of retrieved articles up to January 2010. The principal underlying hypothesis was that the presence of the (*)2 variant allele of the polymorphism would be associated with an increased risk of clinical recurrence. Data were available for a total of 8043 patients from seven cohort prospective studies, who were followed for a period of time ranging from 6 months to 8.3 years. The summary risk ratios (RRs) for the prospective cohort studies included showed a significant association between the CYP2C19(*)2 polymorphism and an increased risk of major adverse cardiovascular events in the follow-up (RR: 1.96 (1.14-3.37); P = 0.02). When studies evaluating stent thrombosis (n = 4) for a total of 4975 patients were considered, the presence of the variant allele was associated with an increased risk of stent thrombosis (RR: 3.82 (2.23-6.54); P = 0.0001). The current meta-analysis, carried out on nearly 8000 patients with CAD undergoing clopidogrel treatment, shows that the CYP2C19(*)2 polymorphism is associated with an increased risk of major adverse cardiovascular events and stent thrombosis.

Platelet aggregability is modulated by eNOS locus in non-type 2 diabetic patients with acute coronary syndrome.

BACKGROUND AND AIM: Platelet nitric oxide (NO) synthesis is compromised in patients with acute coronary syndrome (ACS), and platelet NO availability may be critically relevant in determining the extent of thrombosis in ACS patients. It has been demonstrated that an impaired responsiveness to the antiaggregatory effects of NO may affect platelet dysfunction in diabetic patients with ACS. Since NO availability may be genetically determined, we have investigated the role of endothelial nitric oxide synthase (eNOS) gene in influencing platelet aggregability in relation to the presence (n=247) or absence (n=883) of type 2 diabetes in ACS patients. METHODS AND RESULTS: We have genotyped 1130 consecutive high risk ACS patients on dual antiplatelet therapy, previously investigated in relation to platelet function. eNOS 4a allele frequency was significantly higher in diabetic vs. non-diabetic patients (p=0.02). In non-diabetic patients the eNOS 4a allele significantly modulated platelet aggregability in response to arachidonic acid (AA), but not to collagen and adenosine diphosphate (ADP) stimulus, after Bonferroni correction for multiple testing. After adjustment for age, gender, smoking habit, hypertension and ejection fraction ≤40%, the eNOS 4a allele remained significantly and independently associated with platelet aggregability in response to AA stimulus [ß (SE)=0.17 (0.07), p=0.01]. When platelet aggregation values were considered according to the presence or absence of high residual platelet reactivity (RPR) eNOS 4a, but not -786C and 894T, allele was significantly associated with RPR by AA stimulus. The haplotype reconstruction analysis for eNOS gene showed that the -786C/894G/4a and -786C/894G/4b haplotypes significantly influenced platelet aggregation after AA stimulus. CONCLUSIONS: Our study indicates that eNOS 4a allele, may be a determinant of higher platelet aggregability and residual platelet reactivity in non-diabetic ACS patients.

High sensitivity C-reactive protein predicts the development of new carotid artery plaques in older persons.

BACKGROUND AND AIM: Previous studies have shown that increased levels of C-reactive protein (CRP) predict cardiovascular events, including stroke, myocardial infarction and death from cardiovascular causes. Previous studies have also shown that increased levels of CRP are strong predictors of the progression of pre-existing carotid artery plaques. However, whether CRP is involved in the development of new plaques, that may or may not be associated with clinical events, in subjects with clean carotid arteries has been scarcely investigated. METHODS AND RESULTS: 486 "InCHIANTI" Study participants (200 men and 286 women, 72% aged 65 years and over) free from carotid artery plaques at baseline, also underwent carotid artery scan three years later. We tested the association of baseline characteristics, cardiovascular risk factors and inflammatory markers with the development of new carotid artery plaques. Older participants were significantly more likely to develop new plaques. Independent of age, the relative risks of developing new plaques associated with heavy smoking and family history of atherosclerosis were 1.7 (95%CI 1.5-1.9) and 1.9 (95%CI 1.2-3.1), respectively. Participants with high (>3 µg/mL) and moderate (≥1 and ≤3 µg/mL) CRP levels had a relative risk of 2.2 (95%CI 1.9-2.6) and 1.9 (95%CI 1.6-2.3) respectively, when compared with subjects with low (<1 µg/mL) CRP levels. Surprisingly, risk factors such as hypertension, diabetes, dyslipidemia and overweight/obesity were not significant predictors of the development of new carotid artery plaques. CONCLUSIONS: High CRP levels independently predict the development of new plaques in older persons with carotid arteries free from atherosclerotic lesions.

Effects of a dairy product (pecorino cheese) naturally rich in cis-9, trans-11 conjugated linoleic acid on lipid, inflammatory and haemorheological variables: a dietary intervention study.

BACKGROUND AND AIM: Some studies recently reported a favourable effect for cis-9, trans-11 conjugated linoleic acid (CLA) on plasma lipoprotein profile of healthy subjects. Aim of this crossover intervention study was to evaluate the influence of a short-term dietary intake of a cheese derived from sheep's milk naturally rich in CLA on several atherosclerotic biomarkers, in comparison with a commercially available cheese. METHODS AND RESULTS: Ten subjects (6 F; 4 M) with a median age of 51.5 followed for 10 weeks a diet containing 200 g/week of cheese naturally rich in CLA (intervention period) and for the same period a diet containing a commercially available cheese of the same quantity (placebo period). Consumption of the dairy product naturally rich in cis-9, trans-11 CLA determined a significant (p<0.05) reduction in inflammatory parameters such as interleukin-6 (pre: 8.08+/-1.57 vs. post: 4.58+/-0.94 pg/mL), interleukin-8 (pre: 45.02+/-5.82 vs. post: 28.59+/-2.64 pg/mL), and tumour necrosis factor-alpha (pre: 53.58+/-25.67 vs. post: 32.09+/-17.42 pg/mL) whereas no significant differences in the placebo period were observed. With regard to haemorheological parameters, the test period significantly ameliorated erythrocytes' filtration rate (pre: 7.61+/-0.71% vs. post: 9.12+/-0.97%; p=0.03) with respect to the placebo period. Moreover, a reduction in the extent of platelet aggregation, induced by arachidonic acid [pre: 87.8+/-1.76% vs. post: 77.7+/-3.56%; p=0.04] was observed during the test period in comparison with the placebo period. CONCLUSIONS: Dietary short-term intake of the tested dairy product naturally rich in cis-9, trans-11 CLA appeared to cause favourable biochemical changes of atherosclerotic markers.

Different methodologies for evaluating the effect of clopidogrel on platelet function in high-risk coronary artery disease patients.

BACKGROUND: Two point-of-care (POC) systems have been recently proposed as rapid tools with which to evaluate residual platelet reactivity (RPR) in coronary artery disease (CAD) patients. OBJECTIVES AND METHODS: We compared Platelet Function Analyzer-100 (PFA-100) closure times (CTs) by collagen/adenosine 5'-diphosphate (ADP) (C/ADP CT) cartridge and the VerifyNow P2Y12 Assay (VerifyNow) with light transmission aggregation (LTA) induced by 2 and 10 micromol L(-1) ADP in 1267 CAD patients on dual antiplatelet therapy who underwent percutaneous coronary intervention. We also performed the vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay by cytofluorimetric analysis in a subgroup of 115 patients. RESULTS: Cut-off values for identifying RPR were: > or = 54% and > or = 66% for LTA induced by 2 and 10 micromol L(-1) ADP respectively, and > or = 264 P2Y12 Reaction Units (PRU) for VerifyNow. The cut-off for PFA-100 C/ADP CT was > or = 68 s. RPR was detected in 25.1% of patients by 2 mumol L(-1) ADP-induced LTA (ADP-LTA), in 23.2% by 10 micromol L(-1) ADP-LTA, in 24.4% by PFA-100, and in 24.7% by VerifyNow. PFA-100 results did not parallel those obtained with LTA. VerifyNow showed a significant correlation (rho = 0.62, P < 0.001) and significant agreement (k = 0.34, P < 0.001) with LTA induced by 2 micromol L(-1) ADP. The correlation was similar but the agreement was better between VerifyNow and 10 micromol L(-1) ADP-LTA (rho = 0.64, P < 0.0001; k = 0.43, P < 0.001). Significant relationships were found between VASP platelet reactivity index and both ADP-LTA and VerifyNow. PFA-100 C/ADP CT did not significantly correlate with any of the other assays. CONCLUSIONS: Our results show a significant correlation between LTA and VerifyNow but not the PFA-100 C/ADP assay. Clinical validation studies for POC systems are necessary.

Markers of hypercoagulability and inflammation predict mortality in patients with heart failure.

BACKGROUND AND AIMS: Plasma levels of inflammatory markers are increased in chronic heart failure (HF) and are also subclinical indicators of future HF. Inflammation is strictly correlated with clotting activation, but the association between inflammation, hypercoagulability and prognosis in HF has not been previously reported. METHODS AND RESULTS: Markers of inflammation (interleukin-6; IL-6, and C-reactive protein; CRP) and hypercoagulability (D-dimer; DD, and thrombin-antithrombin III complex; TAT) were prospectively assessed in 214 subjects with New York Heart Association (NYHA) functional class II-IV HF. During a median follow-up of 8.5 months, 32 patients had an event: 13 died and 19 were hospitalized because of worsening of HF. IL-6, DD and TAT levels were all significantly associated with increased risk of death after adjustment for other known HF prognostic factors (age, gender, traditional cardiovascular risk factors, NYHA class, systolic left ventricular function, renal failure, hemoglobin, serum sodium) in a Cox multivariate proportional hazard model (P = 0.003, P = 0.01 and P = 0.02, respectively). When these markers were added simultaneously to the known prognostic factors in a new Cox multivariate model, only DD levels were significant predictors of mortality (hazard ratio [95% confidence interval; CI]: 11 [2.7-45.1], P = 0.001). The Kaplan-Meier curve revealed a significantly better outcome in patients with DD below 450 ng mL(-1). NT-pro-BNP was the only significant predictor of rehospitalization (HR [95% CI]: 5.3 [2.0-13.8], P < 0.001). CONCLUSION: Hypercoagulability and inflammation, as assessed by DD, TAT and IL-6 levels, are associated with an increased mortality risk in HF.

Erythrocyte oxidative stress is associated with cell deformability in patients with retinal vein occlusion.

Essentials Retinal vein occlusion (RVO), characterized by blood hyperviscosity, has an unclear pathogenesis. We aimed to find out if hemorheological profile is altered by oxidative stress in RVO patients. Red blood cell (RBC) oxidative stress is associated to whole blood viscosity and RBC deformability. Reactive oxygen species alter RBC membrane rigidity, playing a key role in RVO pathogenesis. SUMMARY: Background Retinal vein occlusion (RVO) is characterized by vision loss resulting from hypoperfusion and hypoxia of the retina. RVO pathogenesis is not yet fully understood, although blood hyperviscosity has been observed. Erythrocyte deformability plays a key role in determining blood viscosity, and it is critical to microvascular perfusion and oxygen delivery. It has been shown that oxidative stress-induced erythrocyte membrane fluidity alterations are linked to the progression of cardiovascular diseases. Objectives To determine whether erythrocytes from RVO patients show signs of oxidative stress, and whether this condition can modify the hemorheologic profile in these patients. Patients and Methods We analyzed the entire hemorheologic profile and erythrocyte oxidative stress - reactive oxygen species (ROS) production and membrane lipid peroxidation - in 128 RVO patients and 128 healthy subjects, matched for age and sex. Fluorescence anisotropy was used to evaluate the fluidity of erythrocyte membranes. Results In RVO patients, erythrocyte oxidative stress was present and positively correlated with whole blood viscosity and erythrocyte deformability. Multivariate linear regression analysis after adjustment for age, cardiovascular risk factors, medications, leukocyte number and mean corpuscular volume indicated that erythrocyte-derived ROS and erythrocyte lipid peroxidation were significantly and positively correlated with erythrocyte membrane viscosity and deformability. Moreover, in vitro experiments demonstrated that ROS have a key role in erythrocyte membrane fluidity. Conclusions Our findings indicate that erythrocyte oxidative stress plays a key role in the pathogenesis of RVO, and pave the way to new therapeutic interventions.

Dietary habits, lifestyle and cardiovascular risk factors in a clinically healthy Italian population: the 'Florence' diet is not Mediterranean.

OBJECTIVE: To evaluate dietary habits and lifestyle of Italian subjects, to provide current data on adequacy of the nutritional guidelines and recommendations especially in relation to primary prevention of cardiovascular diseases and to assess the influence of dietary habits on lipid profile and homocysteine levels. DESIGN: Cross-sectional. SETTING: Population-based study. SUBJECTS: A sample of 520 clinically healthy subjects (211 males, 309 females) with a mean age of 46 y, living in Florence area, Italy. INTERVENTIONS: Dietary pattern was assessed by trained dietitians through a semiquantitative food questionnaire. Fasting blood samples were drawn for assessment of lipid profile, homocysteine and circulating vitamins. RESULTS: Contribution from total fats was over 30% in about 70% of subjects and intake of saturated fatty acids (SFA) was above the recommended values in at least 40% of the study population. Furthermore, almost the whole (99.6%) population reported low intake of polyunsaturated fatty acids (PUFA). High levels of total cholesterol were present in over 40% of the study population, whereas abnormal values of LDL-cholesterol were observed in about 30%. High levels of homocysteine were found in 11.7% of the study population. An extremely high percentage of subjects reported low intake of vitamins, especially with regard to folic acid (89%), vitamin B(6) (70.1%) and vitamin E (99.6%). In a multiple linear regression model, circulating levels of vitamin B(12) and folic acid, and intake of alcohol and vitamin C resulted in being independently associated with homocysteine plasma levels. CONCLUSIONS: In a typical Mediterranean country, general outlines of Mediterranean diet are not completely followed, especially concerning total fats, SFA, PUFA and vitamins' intake. SPONSORSHIP: Ministero della Salute (Italy) - 'Progetto per la Salute e la Prevenzione di Malattia' 2001-2003.

Cytokine gene expression and production by human LPS-stimulated mononuclear cells are inhibited by sulfated heparin-like semi-synthetic derivatives.

BACKGROUND: The K5 polysaccharide obtained from Escherichia coli strain 010:K5:H4 is a polymer of the disaccharidic unit formed by D-glucuronic acid and N-acetylglucosamine. This structure is akin to N-acetylheparosan, the precursory polymer of heparin and of heparan sulfate. This structural affinity with N-acetylated heparin and with de-sulfated heparin makes the K5 polysaccharide extremely useful for the preparation of sulfated heparin-like semi-synthetic derivatives. It has been demonstrated that heparins are able to inhibit tissue factor and cytokine production and expression by human monocytes. OBJECTIVE: The aim of this study was to evaluate the effects of four different heparin-like semi-synthetic derivatives on inflammatory cytokine production and expression by human mononuclear cells. RESULTS: The simultaneous addition of lipopolysaccharide (LPS; 0.2 and 10 micro g mL(-1)) and the K5 polysaccharide did not inhibit interleukin (IL)-1beta, IL-6 or tumor necrosis factor (TNF)-alpha production by stimulated mononuclear cells. IL-1beta, IL-6 and TNF-alpha concentrations in supernatants of LPS-stimulated mononuclear cells were not influenced by the addition of N,O-sulfated K5 polysaccharide (K5-N, OS) and epimerized N-sulfated K5 polysaccharide (K5 NS epi) at 5 and 10 microg mL(-1), whereas the addition of epimerized N,O-sulfated K5 polysaccharide (K5-N, OS epi) (5 and 10 microg mL(-1)) and O-sulfated K5 polysaccharide (K5-OS) (5 and 10 microg mL(-1)) to LPS-stimulated cells caused a significant dose-dependent inhibition of IL-1beta, IL-6 and TNF-alpha. All sulfated heparin-like semi-synthetic derivatives did not influence the IL-10 production by LPS-stimulated mononuclear cells. In LPS-stimulated cells (0.2 and 10 microg mL(-1)), K5-OS or K5-N, OS epi at 5 and 10 microg mL(-1) markedly decreased TNF-alpha mRNA expression. CONCLUSIONS: These results indicate that the sulfated heparin-like semi-synthetic derivatives K5-OS and K5-N, OS epi are able to inhibit both expression and production of inflammatory cytokines, whereas they do not influence the anti-inflammatory cytokine IL-10, suggesting a potential role for these products as modulators of inflammatory reactions.

Activation of the immune system and coronary artery disease: the role of anti-endothelial cell antibodies.

On the basis of the role of immuno-mediated inflammation in atherosclerosis we investigated, (1) the prevalence of anti-endothelial cell antibodies (AECA) in ischaemic heart disease (IHD); (2) if beta2-glycoprotein I (beta2-GPI) was the target antigen of AECA; (3) the relationship between AECA, tissue factor (TF) and tissue factor pathway inhibitor (TFPI). In 93 consecutive IHD patients undergoing percutaneous transluminal coronary angioplasty (PTCA) and 105 controls AECA were detected by ELISA on human umbilical vein endothelial cells (HUVEC). AECA positive sera were evaluated for anti-beta2-GPI antibodies by ELISA. TF and TFPI plasma levels were assessed by ELISA. Twelve of 93 (12.9%) IHD patients and only one of 105 controls (0.95%) were AECA positive. The prevalence of AECA was higher in unstable angina (UA) than in effort angina (EA) (P=0.01). Three of 12 AECA positive sera resulted positive for anti-beta2-GPI and showed a marked decrease in EC-binding when tested on HUVEC cultured in serum-free medium. The binding was restored by the addition of beta2-GPI. TF and TFPI levels were similar in AECA positive and AECA negative patients. The rate of angiographically documented clinical recurrences was 66.7% in the AECA positive and 14.8% in the AECA negative group (P=0.0004) with a significant relationship between restenosis and AECA (P<0.0001), unchanged by the inclusion of cardiovascular risk factors in the regression model. Our results suggest a 'role' for AECA in the immune-mediated inflammation in UA beta2-GPI is not the only AECA target antigen. AECA are not responsible for high TF and TFPI levels. The high rate of clinical recurrences after PTCA, confirmed by angiography, in AECA positive patients is in line with such a role and suggests further large-scale 'ad hoc' studies.

Prevalence of factor V Leiden mutation in non-European populations.

A difference in the prevalence of venous thromboembolism (TE) in major human groups has been described and an uneven distribution of FV Leiden mutation over the world has recently been reported. We investigated FV Leiden mutation in 584 apparently healthy subjects mostly from populations different from those previously investigated: 170 Europeans (Spanish, Italians), 101 sub-saharan Africans (Fon, Bariba, Berba, Dendi), 115 Asians (Indonesians, Chinese, Tharus), 57 Amerindians (Cayapa), 84 Afroamericans (Rio Cayapa, Viche), and 57 Ethiopians (Amhara, Oromo). The mutation was detected in only 1/115 Asian (Tharu) and in 5/170 Europeans (4 Italians, 1 Spanish). These data confirm that in non-Europeans the prevalence of FV mutation is at least 7 times lower than in Europeans and provide indirect evidence of a low prevalence not only of the FV Leiden gene but also of other genes leading to more severe thrombophilia. Finally, findings from the literature together with those pertaining to this study clearly show a marked heterogeneity among Europeans.

Procedure-dependence and tissue factor-independence of hypercoagulability during orthopaedic surgery.

The increased risk for deep vein thrombosis (DVT) after orthopaedic surgery has been well documented as well as hypercoagulable state during both total hip arthroplasty (THA) and total knee replacement (TKR). To investigate the influence of the surgical procedure [posterolateral (PL) or lateral (L) approach for THA, use of tourniquet (TQ) or not use of TQ for TKR] on the hypercoagulability and the role of extrinsic pathway activation and endothelial stimulation during orthopaedic surgery we have examined 40 patients (20 patients undergoing primary THA--10 with PL approach and 10 with L approach--and 20 patients undergoing TKR--10 with TQ application and 10 without TQ). Thrombin-antithrombin complexes (TAT), tissue factor (TF), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM) and von Willebrand factor antigen (vWF:Ag) were analyzed before and during the orthopaedic surgery. During THA, TAT plasma levels increased more markedly in patients assigned to the L than PL approach (p <0.05); during TKR an elevation of TAT of higher degree (p <0.05) was observed when TQ was not applicated. Blood clotting activation was significantly (p <0.001) more relevant during THA than TKR. No changes in TF and vWF:Ag plasma levels were observed in all patients undergoing THA and TKR. TFPI plasma levels significantly (p <0.05) decreased 1 h after the end of the THA in group PL and group L, whereas they remained unaffected in the two groups of patients undergoing TKR. Similarly TM plasma levels significantly decreased during THA, but not during TKR. In conclusion, these results show that: 1) the site of surgical procedures and the type of approach affect the degree of hypercoagulability, 2) the blood clotting activation takes place in the early phases of orthopaedic surgery, without signs of extrinsic pathway and endothelial activation.

Cytokine gene expression in human LPS- and IFNgamma-stimulated mononuclear cells is inhibited by heparin.

In addition to its well-understood anticoagulant activity, heparin is known to modulate a variety of biological functions including immunologic responses. In order to investigate whether heparin influences the humoral immunity by interacting with cellular elements and affecting gene expression in blood circulating cells. we studied the effect of heparin on IL-1beta, IL-6 and TNFalpha mRNAs in human lipopolysaccharide-(LPS)- or interferon-gamma(IFN-gamma)-stimulated mononuclear cells. The study of mRNA was carried out by an initial PCR screening followed by a Northern blot quantitative analysis. Heparin (0.5 U/ml) turned out to inhibit all three cytokine gene expressions. The mRNA decrease was 37 +/- 6% for IL-1beta, 53 +/- 3% for IL-6 and 47 +/- 4% for TNFalpha with LPS stimulus. No differences could be observed in the inhibitory effect of heparin on IFNgamma-stimulated cells. This effect of heparin was confirmed in a subset of experiments performed on purified monocytes. These results suggest an important immunosuppressive effect of heparin on cell-mediated immune responses.

Tissue factor and homocysteine levels in ischemic heart disease are associated with angiographically documented clinical recurrences after coronary angioplasty.

BACKGROUND: In ischemic heart disease (IHD) patients high plasma levels of Tissue Factor (TF), the trigger of coagulation cascade, are present. Homocysteine (Hcy) is a risk factor for coronary artery disease, and several different pathophysiological mechanisms by which Hcy may play a role in thrombus formation have been postulated in "in vitro" studies. We investigated the "in vivo" role of Hcy in affecting plasma levels of TF, its inhibitor Tissue Factor Pathway Inhibitor (TFPI) and hypercoagulability. METHODS AND RESULTS: We investigated 119 IHD patients who underwent PTCA and compared them with 103 healthy subjects. TF, TFPI, Thrombin-Antithrombin complexes (TAT) and Hcy levels were significantly higher in the patients than in the controls. A positive correlation was found between Hcy and TF (r = 0.54; p < 0.0001), Hcy and TFPI (r = 0.26; p <0.05) as well as Hcy and TAT (r = 0.33; p <0.0001) levels. An inverse correlation existed between folate intake and Hcy levels (r = -0.28; p = 0.001). Hcy levels within the first quartile and in the highest quartile were associated with a lower (p < 0.001) and higher (p <0.0001) rate of clinical recurrences, respectively. Patients with TF values in the first quartile had a lower rate of angiographically documented clinical recurrences as compared to those in the fourth quartile (p <0.01); those in the highest quartile of TF showed a higher rate of recurrences (p = 0.001). Multivariate analysis confirmed these results (first quartile of Hcy: OR 0.02, C1 0.002-0.27; fourth quartile of Hcy: OR 36.5, C1 3.6-365/first quartile of TF: OR 0.006, C1 0.001-0.44; fourth quartile of TF: OR 16.4, C1 3.0 - 90.0), also after adjustment for risk factors and Hcy and TF respectively. CONCLUSIONS: In this study we show that TF, TFPI and TAT levels are correlated with Hcy plasma levels in IHD patients, providing evidence of an "in vivo" pathophysiological mechanism of hyperhomocysteinemia. The observed association between angiographically documented clinical recurrences and TF and Hcy values awaits confirmation in studies designated to evaluate this issue on a larger number of patients.

Tissue factor reduction and tissue factor pathway inhibitor release after heparin administration.

Elevated plasma levels of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) and large amounts of monocyte procoagulant activity (PCA) have been documented in unstable angina (UA) patients. In in vitro experiments heparin is able to blunt monocyte TF production by inhibiting TF and cytokine gene expression by stimulated cells and after in vivo administration it reduces adverse ischemic outcomes in UA patients. TF and TFPI plasma levels and monocyte PCA have been investigated in 28 refractory UA patients before and during anticoagulant subcutaneous heparin administration (thrice daily weight- and PTT-adjusted for 3 days) followed by 5000 IU X 3 for 5 days. After 2-day treatment, immediately prior to the heparin injection, TF and TFPI plasma levels [(median and range): 239 pg/ml, 130-385 pg/ ml and 120 ng/ml, 80-287 ng/ml] were lower in comparison to baseline samples (254.5 pg/ml, 134.6-380 pg/ml and 135.5 ng/ml, 74-306 ng/ml). Four h after the heparin injection TF furtherly decreased (176.5 pg/ml, 87.5-321 pg/ml; -32.5%. p<0.001) and TFPI increased (240.5 ng/ml, 140-450 ng/ml; +67%, p<0.0001). After 7-day treatment, before the injection of heparin, TF and TFPI plasma levels (200 pg/ml, 128-325 pg/ml and 115 ng/ml, 70-252 ng/ml) significantly decreased (p<0.05) in comparison to the pre-treatment values. On the morning of the 8th day, 4 h after the injection of heparin TF plasma levels and monocytes PCA significantly decreased (156.5 pg/ml, 74-259 pg/ml and from 180 U/105 monocytes, 109-582 U/10(5) monocytes to 86.1 U/10(5) monocytes, 28-320 U/10(5) monocytes; - 38% and -55% respectively) and TFPI increased (235.6 ng/ml, 152-423 ng/ ml; +70%, p<0.001). In conclusion, heparin treatment is associated with a decrease of high TF plasma levels and monocyte procoagulant activity in UA patients. These actions of heparin may play a role in determining the antithrombotic and antiinflammatory properties of this drug.

Elevated tissue factor and tissue factor pathway inhibitor circulating levels in ischaemic heart disease patients.

Several studies have shown that thrombosis and inflammation play an important role in the pathogenesis of Ischaemic Heart Disease (IHD). In particular, Tissue Factor (TF) is responsible for the thrombogenicity of the atherosclerotic plaque and plays a key role in triggering thrombin generation. The aim of this study was to evaluate the TF/Tissue Factor Pathway Inhibitor (TFPI) system in patients with IHD. We have studied 55 patients with IHD and not on heparin [18 with unstable angina (UA), 24 with effort angina (EA) and 13 with previous myocardial infarction (MI)] and 48 sex- and age-matched healthy volunteers, by measuring plasma levels of TF, TFPI, Prothrombin Fragment 1-2 (F1+2), and Thrombin Antithrombin Complexes (TAT). TF plasma levels in IHD patients (median 215.4 pg/ml; range 72.6 to 834.3 pg/ml) were significantly (p<0.001) higher than those found in control subjects (median 142.5 pg/ml; range 28.0-255.3 pg/ml). Similarly, TFPI plasma levels in IHD patients were significantly higher (median 129.0 ng/ml; range 30.3-316.8 ng/ml; p<0.001) than those found in control subjects (median 60.4 ng/ml; range 20.8-151.3 ng/ml). UA patients showed higher amounts of TF and TFPI plasma levels (TF median 255.6 pg/ml; range 148.8-834.3 pg/ml; TFPI median 137.7 ng/ml; range 38.3-316.8 ng/ml) than patients with EA (TF median 182.0 pg/ml; range 72.6-380.0 pg/ml; TFPI median 115.2 ng/ml; range 47.0-196.8 ng/ml) and MI (TF median 213.9 pg/ml; range 125.0 to 341.9 pg/ml; TFPI median 130.5 ng/ml; range 94.0-207.8 ng/ml). Similar levels of TF and TFPI were found in patients with mono- or bivasal coronary lesions. A positive correlation was observed between TF and TFPI plasma levels (r = 0.57, p<0.001). Excess thrombin formation in patients with IHD was documented by TAT (median 5.2 microg/l; range 1.7-21.0 microg/l) and F1+2 levels (median 1.4 nmol/l; range 0.6 to 6.2 nmol/l) both significantly higher (p<0.001) than those found in control subjects (TAT median 2.3 microg/l; range 1.4-4.2 microg/l; F1+2 median 0.7 nmol/l; range 0.3-1.3 nmol/l). As in other conditions associated with cell-mediated clotting activation (cancer and DIC), also in IHD high levels of circulating TF are present. Endothelial cells and monocytes are the possible common source of TF and TFPI. The blood clotting activation observed in these patients may be related to elevated TF circulating levels not sufficiently inhibited by the elevated TFPI plasma levels present.