Paolo Ferraresi

Prospective evaluation of on-clopidogrel platelet reactivity over time in patients treated with percutaneous coronary intervention relationship with gene polymorphisms and clinical outcome.

OBJECTIVES This study sought to investigate the evolving pattern over time of on-clopidogrel platelet reactivity (PR) and its relationship with genotype and clinical outcomes after percutaneous coronary intervention. BACKGROUND Whether on-clopidogrel PR and role of genotype differ over time is unknown. METHODS On-clopidogrel PR before percutaneous coronary intervention, and 1 and 6 months thereafter via VerifyNow P2Y12 (Accumetrics Inc., San Diego, California), CYP2C19*2, *17, CYP3A5*3, and ABCB1 polymorphisms were evaluated in 300 patients. Death, stroke, myocardial infarction, and bleedings were assessed up to 1 year. RESULTS On-clopidogrel PR varied significantly over time, being higher at baseline than at 1 and 6 months after. From baseline to 1 month, 83 of 300 patients varied their response status. This was mainly due to baseline poor responders becoming full responders (75 of 83). Genotype justifies roughly 18% of this trend. CYP2C19*2 and *17 influence on PR was consistent over time, whereas that of ABCB1 appeared of greater impact at baseline. On-clopidogrel PR at 1 month independently best predicts ischemic and bleeding events. We found a therapeutic window (86 to 238 P2Y₁₂ reactivity units) with a lower incidence of both ischemic and bleeding complications. A risk score was created by combining genotype (ABCB1 and CYP2C19*2), baseline PR, and creatinine clearance to predict 1-month poor responsiveness and 1-year poor prognosis. CONCLUSIONS In patients at steady state for clopidogrel undergoing percutaneous coronary intervention, PR decreases from baseline to 1 month. Genotype influences ≈18% of this trend. On-clopidogrel PR at 1 month is the strongest predictor of adverse outcomes, and this can be predicted by combining genotype to baseline phenotype and clinical variables.

Polymorphisms in the factor VII gene and the risk of myocardial infarction in patients with coronary artery disease

BACKGROUND High plasma levels of coagulation factor VII have been suggested to be predictors of death due to coronary artery disease. Since polymorphisms in the factor VII gene contribute to variations in factor VII levels, such polymorphisms may be associated with the risk of myocardial infarction, which is precipitated by thrombosis. METHODS We studied a total of 444 patients, 311 of whom had severe, angiographically documented coronary atherosclerosis. Of these 311 patients, 175 had documentation of a previous myocardial infarction. As a control group, 133 patients with normal coronary arteriograms were also included. We measured the levels of activated factor VII and assessed three polymorphisms in the factor VII gene, one involving the promoter (A1 and A2 alleles), one involving the catalytic region (R353Q), and one involving intron 7. RESULTS Each of the polymorphisms influenced factor VII levels. Patients with the A2A2 and QQ genotypes had the lowest levels of activated factor VII (66 percent and 72 percent lower, respectively, than the levels in patients with the wild-type genotypes). The frequencies of the various genotypes in the patients free of coronary artery disease were similar to those in the entire population of patients with coronary artery disease. In the latter group, there were significantly more heterozygotes and homozygotes for the A2 and Q alleles among those who had not had a myocardial infarction than among those who had had an infarction (P=0.008 for the presence of the promoter polymorphism and P=0.01 for the presence of the R353Q polymorphism by chi-square analysis). The adjusted odds ratio for myocardial infarction among the patients with the A1A2 or RQ genotype was 0.47 (95 percent confidence interval, 0.27 to 0.81). CONCLUSIONS Our findings suggest that certain factor VII genotypes have a role in protection against myocardial infarction. This may explain why some patients do not have myocardial infarction despite the presence of severe coronary atherosclerosis.

Tissue Factor and Coagulation Factor VII Levels During Acute Myocardial Infarction: Association With Genotype and Adverse Events

Objective—We investigated in patients with ongoing myocardial infarction (MI) whether coagulation factor VII (FVII) and tissue factor (TF) levels are affected at admission by genetic components and whether they may predict subsequent cardiovascular events. Methods and Results—256 patients admitted for MI were evaluated for FVII and TF antigen levels before any treatment at entry, and were genotyped for FVII and TF polymorphisms. FVII gene insertions at −323, 11293 and the −402G/A change predicted FVII levels and explained 14% of variance. The −603 TF gene polymorphism failed to affect significantly TF levels (P=0.07). These variables were correlated with the incidence of death (36 patients) and reinfarction (9 patients) after a median follow-up of 397 days. Events were independently predicted by FVII (HR 2.1, 95% CI 1.2 to 5.7) and TF (HR 4.1, 95% CI 2 to 11) levels. Composite end point was significantly worse when both parameters were above the receiver-operating characteristics (ROC) values (HR 8.3, 95% CI 5 to 18, compared with FVII and TF below), and above the ROC value of TF (>630 pg/mL) it differed among FVII genotype groups. Conclusions—Admission FVII and TF antigen levels, partially predicted by polymorphisms, are independent predictors of mortality and reinfarction in patients with acute MI.

Relationship between paraoxonase Q192R gene polymorphism and on-clopidogrel platelet reactivity over time in patients treated with percutaneous coronary intervention.

Recently, paraoxonase-1 (PON1) has been identified as the crucial enzyme for clopidogrel bioactivation, with its common Q192R polymorphism determining the rate of active metabolite formation [1]. In a first report, PON1 QQ homozygous individuals showed considerably lower platelet inhibition by clopidogrel and a higher risk of stent thrombosis than RR homozygous individuals [1]. This finding was not confirmed in a subsequent study [2]. In the current analysis, we investigated the relationship between PON1 Q192R polymorphism and on-clopidogrel platelet reactivity (PR) at different time points. We also compared its incidence with that of other common gene polymorphisms [3], and we discuss how it is related to clinical outcome. For this purpose, 300 patients undergoing percutaneous coronary intervention (PCI) for ischemic heart disease (with the exception of ST-segment elevation myocardial infarction) were recruited. This study population has been previously investigated [3]. The main aim of the study was to evaluate whether clopidogrel response varied over time [3]. Also, the relationship between on-clopidogrel PR variation, genotype and clinical outcome was investigated. Briefly, all patients were treated with aspirin and clopidogrel (600 mg as a loading dose at least 12 h before PCI). Blood samples were drawn just before PCI, and at 1 and 6 months after PCI. To evaluate on-clopidogrel PR, we used VerifyNow P2Y12 (Accumetrics, San Diego, CA, USA). The results were expressed in P2Y12 reaction units (PRUs). A poor clopidogrel response was defined as a PRU value ‡ 235. The occurrence of death, myocardial infarction, stroke, stent thrombosis (definite and probable) and bleeds (Thrombolysis in Myocardial Infarction [TIMI] criteria) was assessed. In the present substudy, we integrated the previous data with that of PON1 Q192R gene polymorphism (rs662, genotyped with the TaqMan allelic discrimination assay; Applied Biosystems, Foster City, CA, USA). Our present aim was to assess the relationship of on-clopidogrel PR at different time points with PON1 Q192R gene polymorphism, as compared with other common gene polymorphisms. This study was approved by the local Ethics Committee, and all patients gave written informed consent. Continuous data are presented as mean ± standard deviation and were compared by t-test and one-way ANOVA .I t is of note that PRU values are normally distributed (P =0 .7 with the Kolmogorov–Smirnov test). A linear mixed model was used to quantify changes in on-clopidogrel PR over time while integrating the roles of baseline, genetic and procedural characteristics. Also, a multivariable linear regression model was used to assess clinical, procedural and genetic determinants of on-clopidogrel PR. Categorical variables were summarized in terms of numbers and percentages, and were compared by the use of two-sided Fishers exact test. A two-sided P-value of < 0.05 was considered to be significant. All analyses were performed with STATISTICA 8 (Statsoft, Tulsa, Okla, USA) and

Idiopathic central retinal vein occlusion in a thrombophilic patient with the heterozygous 20210 G/A prothrombin genotype

PURPOSE To report the occurrence of monolateral central retinal vein occlusion in a patient with heterozygous 20210 G/A prothrombin genotype, known to be associated with high thrombophilic risk. METHODS A monolateral central retinal vein occlusion was diagnosed in a 71-year-old woman, who had suffered from a deep vein thrombosis in her left leg at the age of 36 years. Mutations of the genes involved in the coagulation process were investigated by DNA polymerase chain reaction. RESULT DNA analysis showed the patient to be heterozygous for the prothrombin 20210 G/A genetic variation. CONCLUSION The 20210 G/A prothrombin gene mutation may be associated with central retinal vein occlusion.

The heterozygous 20210 G/A genotype prevalence in patients affected by central and branch retinal vein occlusion: a pilot study

Abstract. Background: Several inherited conditions have been associated with an increased or decreased incidence of retinal vein occlusion (RVO). The A allele in the 20210 G/A prothrombin gene has been found to be associated with systemic venous thrombosis. The aim of this study has been to verify the prevalence of this mutation in patients affected by central RVO (CRVO) or branch RVO (BRVO). Methods: A retrospective study was carried out on 100 consecutive patients suffering from RVO, more than 50 years old, unaffected by systemic diseases known to be associated with markedly increased RVO occurrence. We determined the frequency of this mutation by performing mutagenised amplification of exon 14 followed by restriction analysis of the amplified DNA fragment. Results: The overall frequency of prothrombin 20210A allele in RVO patients was 6.0%. All heterozygous patients had suffered from CRVO. In this study subgroup, the frequency of the 20210 G/A prothrombin heterozygosis was 12.0%. The difference in the frequency of this the genetic variant between the CRVO and BRVO groups was statistically significant. None of the conventional RVO risk factors were statistically related to the occurrence of the disease in either the CRVO or the BRVO subgroup. Conclusion: The prevalence of the prothrombin 20210A mutation observed in CRVO patients is significantly higher than in the normal Italian population. Moreover, the prevalence is significantly greater in CRVO than in BRVO patients. These results raise the possibility that the prothrombin 20210A variant may be considered as a risk factor for CRVO.

Increased factor VIII coagulant activity levels in male carriers of the factor V R2 polymorphism.

A common factor V gene haplotype, the FVR2 haplotype (FVHR2), has been associated with a reduced cofactor activity in activated protein C-mediated activated factor VIII inactivation. Our aim was to investigate the role of FVHR2 as a possible determinant of factor VIII levels in a population study. A total of 516 individuals (401 men, 115 women; mean age 58.4 ± 10.8 years) were enrolled within the frame of a regional cardiovascular survey, characterized for factor VIII coagulant activity (FVIII:c) and factor V coagulant activity (FV:c) levels, and genotyped for factor V polymorphisms. In men without signs of overt inflammation, FVHR2 carriers had higher levels of FVIII:c than noncarriers (154 IU/dl, 95% confidence interval = 143–166 versus 142 IU/dl, 95% confidence interval = 138–147; P = 0.045) and were more represented in individuals with high (≥ 150 IU/dl) FVIII:c levels (21.2 versus 10.8%; odds ratio = 2.27, 95% confidence interval = 1.17–4.39 after adjustment for age, blood group and high-sensitivity C-reactive protein levels). In conclusion, this clinical report suggests the common FVHR2 as a possible independent determinant of FVIII:c levels. The report concomitantly addresses the relationship between factor V and factor VIII levels and supports the hypothesis of a mild prothrombotic role of FVHR2 by means of increased factor VIII levels.

Asymptomatic carriership of factor V Leiden and genotypes of the fibrinogen gene cluster

Summary. We investigated the role of frequent fibrinogen polymorphisms in venous thromboembolic disease in conjunction with inherited thrombophilia. Two hundred unrelated subjects, all carriers of the factor V R506Q mutation (FV Leiden), were genotyped at the fibrinogen gene cluster. Among these subjects, 100 had experienced previous venous thromboembolism (VTE) and 100 were still asymptomatic for VTE. Significant differences were observed between the groups for the BclI polymorphism (P = 0·004). Scanning, by sequencing the DNA regions flanking the BclI marker, revealed new polymorphisms, a C to T transition and a G to T transversion at 1520 and 3369 base pairs 3′ to the β gene stop codon respectively. These markers showed less association with the clinical phenotype than BclI itself. A combined genotype including 10 markers was more frequent among the asymptomatic subjects (17%) than among patients (3%), and was associated with a reduction in fibrinogen antigen level (2·42 ± 0·35 vs 2·69 ± 0·41 g/l, P = 0·028) among the asymptomatic subjects. Our data suggest that, in the presence of inherited thrombophilia, frequent fibrinogen polymorphisms may interact to modulate the risk of venous thromboembolism.

Temporal and Genotype-Driven Variation of Factor VII Levels in Patients With Acute Myocardial Infarction:

defined as the elevation of creatine kinase MB and/or troponin I above the upper reference limit (5 and 0.1 ng/mL, respectively) in 2 or more consecutives samples. At entry, all patients were submitted to primary percutaneous coronary interventions (PCI) and received adjunctive therapy with aspirin (250 mg intravenously followed by 100 mg/d), clopidogrel (loading dose 300 mg followed by 75 mg/d), 50 U/Kg of unfractionated heparin, and glycoprotein IIb/IIIa inhibitors. According to current guidelines, patients received standard medical therapy with β-blockers, angiotensin-converting enzyme inhibitors, statins, and nitrates. The local Ethics Committee approved the study. All patients gave written informed consent.

Angiotensin-converting enzyme insertion/deletion polymorphism and risk of restenosis after directional coronary atherectomy followed by stent implantation

The D allele of the insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene is associated with higher plasma and tissue ACE levels, which enhance the stimulus for neo-intimal hyperplasia. Plaque debulking before stenting reduces the plaque-related determinants of in-stent restenosis and provides an ideal clinical model for studying neointimal hyperplasia. We prospectively studied 113 consecutive patients undergoing elective DCA followed by stent implantation. The presence of I/D in ACE genome DNA was analysed by means of polymerase chain reaction. Follow-up coronary angiography was performed 6-12 months after DCA, and all of the angiograms were quantitatively analysed. The baseline clinical and angiographic characteristics of the patients with a D/D (33%), I/D (52%) and I/I (15%) genotype were well balanced. There were no significant differences in minimal lumen diameter before and after the procedure or at follow-up, and no significant differences in acute gain, late loss or the loss index. Our results indicate that ACE I/D polymorphism does not influence the risk of developing angiographic restenosis in patients undergoing DCA followed by stent implantation.