Emilia Antonucci

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

Summary.  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 μmol 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: ≥ 54% and ≥ 66% for LTA induced by 2 and 10 μmol L–1 ADP respectively, and ≥ 264 P2Y12 Reaction Units (PRU) for VerifyNow. The cut‐off for PFA‐100 C/ADP CT was ≥ 68 s. RPR was detected in 25.1% of patients by 2 μmol L–1 ADP‐induced LTA (ADP‐LTA), in 23.2% by 10 μmol 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 (ρ = 0.62, P < 0.001) and significant agreement (k = 0.34, P < 0.001) with LTA induced by 2 μmol L–1 ADP. The correlation was similar but the agreement was better between VerifyNow and 10 μmol L–1 ADP‐LTA (ρ =  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.

Bleeding Risk During Oral Anticoagulation in Atrial Fibrillation Patients Older Than 80 Years

OBJECTIVES We sought to evaluate the rate of bleeding in relation to age (<80 and > or =80 years), the quality of anticoagulation (expressed as time spent in international normalized ratio therapeutic range), and factors associated with bleeding events. BACKGROUND Stroke prevention in patients with atrial fibrillation (AF) is an increasingly crucial public health target, particularly in patients ages > or =80 years. METHODS We conducted a prospective observational study on 783 patients with AF on oral anticoagulant treatment (OAT). RESULTS Patients spent a median 14%, 71%, and 15% of time below, within, and above the intended therapeutic range, respectively. No difference in OAT quality was found between patients age <80 and > or =80 years. During follow-up, 94 patients experienced bleeding complications (rate 3.7 x 100 patient/years), 37 major (rate 1.4 x 100 patient/years), and 57 minor (rate 2.2 x 100 patient/years). Different rates of major hemorrhage were observed between patients age <80 and > or =80 years (0.9 vs. 1.9 x 100 patient/years; p = 0.004). Bleeding risk also was greater in patients with a history of previous cerebral ischemic event (odds ratio [OR]: 2.5; 95% confidence interval: 1.3 to 4.8; p = 0.007). A Cox regression analysis confirmed age > or =80 years associated with bleeding risk (OR: 2.0). CONCLUSIONS These results indicate that the rate of major bleeding complications may be kept acceptably low also in very elderly AF patients on OAT, provided a careful management of anticoagulation is obtained.

Assessment of platelet function on whole blood by multiple electrode aggregometry in high-risk patients with coronary artery disease receiving antiplatelet therapy.

This study sought to compare Multiplate impedance platelet aggregometry (IPA) with light transmission aggregometry (LTA) and the PFA-100 for determining the prevalence of residual platelet reactivity (RPR) by the Multiplate IPA in 297 patients with acute coronary syndrome receiving dual antiplatelet therapy. Aggregations were induced by adenosine-5 diphosphate (ADP), arachidonic acid, and collagen. PFA-100 closure times were measured by collagen and ADP and epinephrine (CEPI) cartridges. Significant correlations were observed between Multiplate IPA and LTA after all stimulations (P < .0001) and between Multiplate IPA (arachidonate and collagen) and PFA-100 CEPI closure time (P < .0001 for both). Cutoff values of Multiplate IPA (for all stimulations) were calculated for the identification of RPR. Between the Multiplate IPA and LTA good agreement was found with all 3 agonists (P < .0001 for all). Multiplate IPA might represent a reliable, handy, rapid tool to monitor antiplatelet therapy in clinical practice and for clinical investigations.

D-dimer to guide the duration of anticoagulation in patients with venous thromboembolism: a management study

The optimal duration of anticoagulation in patients with venous thromboembolism (VTE) is uncertain. We investigated whether persistently negative D-dimers in patients with vein recanalization or stable thrombotic burden can identify subjects at low recurrence risk. Outpatients with a first VTE (unprovoked or associated with weak risk factors) were eligible after at least 3 months (12 in those with residual thrombosis) of anticoagulation. They received serial D-dimer measurements using commercial assays with predefined age/sex-specific cutoffs and were followed for up to 2 years. Of 1010 patients, anticoagulation was stopped in 528 (52.3%) with persistently negative D-dimer who subsequently experienced 25 recurrences (3.0% pt-y; 95% confidence interval [CI], 2.0-4.4%). Of the remaining 482 patients, 373 resumed anticoagulation and 109 refused it. Recurrent VTE developed in 15 patients (8.8% pt-y; 95% CI, 5.0-14.1) of the latter group and in 4 of the former (0.7% pt-y; 95% CI, 0.2-1.7; hazard ratio = 2.92; 95% CI, 1.87-9.72; P = .0006). Major bleeding occurred in 14 patients (2.3% pt-y; 95% CI, 1.3-3.9) who resumed anticoagulation. Serial D-dimer measurement is suitable in clinical practice for the identification of VTE patients in whom anticoagulation can be safely discontinued. This study was registered at clinicaltrials.gov as #NCT00954395.

p-Coumaric acid, a common dietary phenol, inhibits platelet activity in vitro and in vivo.

p-Coumaric acid (3-(4-hydroxyphenyl)-2-propenoic acid; 4CA), is a ubiquitous plant metabolite with antioxidant and anti-inflammatory properties. The antiplatelet activity of this compound was analysed both ex vivo and in vitro. 4-CA, administered to rabbits for 2 weeks at the dose of 5 mg/kg, mixed with food, inhibited ADP-induced platelet aggregation without affecting blood coagulation. This effect was associated with a marked increase in plasma antioxidant activity, measured as ferric reducing ability of plasma, and with the reduction of thromboxane B2 production. The antiplatelet effect was confirmed by in vitro experiments on human blood: 4CA (500 microM and 1 mM) reduced ADP-induced platelet aggregation (55 x 2 (se 4 x 01) % and 35 x 6 (se 2 x 35) % relative to basal level, respectively). 4CA was able to modify platelet function, measured with PFA-100, a shear-inducing device that simulates primary haemostasis. 4CA interfered also with arachidonic acid cascade, reducing thromboxane B2 production and lipopolysaccharide-induced prostaglandin E2 generation (ic50 371 and 126 microM, respectively). The data show that 4CA is an antioxidant compound with good antiplatelet activity at doses that can be obtained with dietary intervention, suggesting possible applications for primary prevention of vascular disease.

Comparison of methods for monitoring residual platelet reactivity after clopidogrel by point-of-care tests on whole blood in high-risk patients.

Cardiovascular events are more frequent in high-risk coronary artery disease (CAD) patients on dual antiplatelet therapy with a residual platelet reactivity (RPR) than in those showing inhibition of ADP-inducible platelet activation. It is known that post-interventional RPR is a clinically important entity confirming it as a risk factor for thrombo-ischaemic events. Multiple electrode platelet aggregometry (MEA) on whole blood has been recently proposed as a rapid tool to evaluate RPR in high-risk CAD patients on clopidogrel therapy. It was the aim of this study to detect RPR in 801 high-risk CAD patients on dual antiplatelet therapy comparing MEA with the VerifyNow P2Y12 assay on whole blood and classical light transmission aggregation (LTA) on platelet-rich plasma. ADP (10 microM) was employed as agonist for MEA and LTA. The prevalence of RPR was 20.6% by MEA, 16.1% by LTA and 30.8% by VerifyNow. MEA showed a significant correlation (rho=0.62, p<0.0001) with VerifyNow and a moderate agreement (k=0.52, p<0.001) with 81.5% of concordant values. A significant correlation was found between MEA and LTA (rho=0.71, p<0.001) with a good agreement (k=0.63, p<0.001) and 88.8% of concordant values. MEA in relation to LTA showed a sensitivity of 80% and a specificity of 91%. MEA might represent a reliable method and valid alternative in comparison with other available platelet function assays. It might help to guide antiplatelet therapy and thus improve clinical outcome of high-risk CAD patients.

Gender differences in stroke risk of atrial fibrillation patients on oral anticoagulant treatment

The efficacy of adjusted-dose oral anticoagulant treatment (OAT) in the prevention of stroke in atrial fibrillation (AF) is well documented. Available data show that AF patients are widely heterogeneous in terms of ischaemic stroke risk. The role of female gender as a predictor of stroke risk is inconsistent, in particular it is unclear if warfarin treatment is able to prevent stroke equally in both sexes. We performed a prospective study on 780 AF patients on OAT, followed by an Anticoagulation Clinic, to evaluate if female gender is a risk factor for stroke among patients on OAT and if the quality of anticoagulation is different between genders. No difference was found in relation to the quality of anticoagulation between genders (p=0.5). During follow-up 33 patients had major bleedings (rate 1.37 x 100 pt/yrs) but no difference was found between genders in bleeding risk. Forty patients had ischaemic events [rate 1.66 x 100 pt/yrs; males rate 1.2 x 100 pt/yrs; females rate 2.43 x 100 pt/yrs; p=0.042; relative risk (RR) of females vs. males 2.0 (95% confidence interval [CI] 1.3-3.1); p= 0.004]. The higher rate of ischaemic events in females with respect to males was confirmed at Cox regression analysis after correction for age (p=0.009). In addition, strokes occurring in females were more disabling, and RR for severe and fatal stroke, defined according to Modified Rankin scale, of females vs. males was 3.1 (95% CI 1.3-6.5; p=0.001). In conclusion, our data show a higher risk of stroke in anticoagulated AF females with respect to males, despite a similar quality of anticoagulation.

The balance between pro- and anti-inflammatory cytokines is associated with platelet aggregability in acute coronary syndrome patients

BACKGROUND Residual platelet reactivity (RPR) on antiplatelet therapy in ischemic heart disease patients is associated with adverse events. Clinical, cellular and pharmacogenetic factors may account for the variable response to antiplatelet treatment. OBJECTIVE We sought to explore the interplay of multiple pro-inflammatory and anti-inflammatory cytokines with platelet function in patients with acute coronary syndrome (ACS) undergoing percutaneous coronary intervention (PCI) on dual antiplatelet therapy. METHODS In 208 ACS patients undergoing PCI on dual antiplatelet therapy we measured platelet function by platelet aggregation with two agonists [1mM arachidonic acid (AA) and 10muM ADP]. IL-1beta, IL-1ra, IL-4, IL-6, IL-8, IL-10, IL-12, IP-10, IFN-gamma, MCP-1, MIP-1alpha, MIP-1beta, TNF-alpha, and VEGF levels were determined by using the Bio-Plex cytokine assay (Bio-Rad Laboratories Inc., Hercules, CA, USA). We defined patients with RPR those with platelet aggregation by AA >or=20% and/or ADP (10micromol) >or=70%. RESULTS We documented a significant association between IP-10, IFN-gamma, IL-4 and RPR by both AA- and ADP-induced platelet aggregation after adjustment for age, sex, cardiovascular risk factors, ejection fraction, BMI, vWF and CRP. Patients with pro-inflammatory cytokines not compensated by anti-inflammatory cytokines had higher risk of RPR by both AA and ADP (AA: OR=3.85, 95% CI 1.52-9.74; ADP: OR=2.49, 95% CI 1.33-4.68) with respect to patients with balanced anti-/pro-inflammatory cytokines. Patients with anti-inflammatory response overwhelming pro-inflammatory response have lower risk of RPR (AA: OR=0.55, 95% CI 0.28-1.06; ADP: OR=0.47, 95% CI 0.26-0.87). CONCLUSION Our study provides new insights into the interplay of anti-/pro-inflammatory cytokines with platelet hyper-reactivity in high-risk patients.

Culprit Factors for the Failure of Well-Conducted Warfarin Therapy to Prevent Ischemic Events in Patients With Atrial Fibrillation The Role of Homocysteine

Background and Purpose— In patients with atrial fibrillation (AF), oral anticoagulant therapy (OAT) is effective in reducing stroke and embolism. However, despite OAT, ischemic events do occur in some patients. Studies specifically addressing the identification of risk factors for ischemic events during well-conducted OAT are not available. In this study, we prospectively investigated the role of classic risk factors and homocysteine levels in the occurrence of ischemic complications in 364 AF patients on OAT. Methods— The quality of anticoagulation levels and the occurrence of bleeding and thrombotic events were recorded. Results— During follow-up (859 patient years) 21 patients had ischemic complications (rate 2.4×100 patient-years). Homocysteine plasma levels were higher in these patients than in patients without ischemic complications during OAT (P<0.01), whereas no difference was observed in relation to the quality of OAT. The presence of a history of previous ischemic events, hypertension, and homocysteine plasma levels over the 90th percentile were all associated with an increased risk of ischemic events during OAT (odds ratio [OR]=7, 4.5, and 4.7, respectively). The coexistence of these risk factors markedly increased the risk (OR=13.1; 95% CI, 3.7 to 45.7; P=0.001). Conclusion— In conclusion, our results indicate that AF patients with multiple risk factors may not be sufficiently protected by OAT, even when this is well conducted.

Comparison between different D-Dimer cutoff values to assess the individual risk of recurrent venous thromboembolism: analysis of results obtained in the DULCIS study

D‐dimer assay, generally evaluated according to cutoff points calibrated for VTE exclusion, is used to estimate the individual risk of recurrence after a first idiopathic event of venous thromboembolism (VTE).