Fibrinolysis can be abnormally activated in several critical care settings but it's often misdiagnosed by standard laboratory tests. Although rotational thromboelastometry can assess the whole coagulative process, its ability to detect fibrinolysis has been questioned. Aim of this study was to investigate the ability of thromboelastometry in detecting induced fibrinolysis in an in-vitro model. Whole blood samples were taken from 18 healthy volunteers. Each sample was split and added with increasing urokinase concentrations till to reach 0, 50, 75 and 100 IU/ml. Thromboelastometry tests, extem and aptem, were performed on the obtained samples. If significant lysis at 50 IU/ml was recorded, also 10, 25 and 35 IU/ml drug concentrations were tested. No lytic effects were detected in 10 IU/ml samples. Lysis variables were the most sensitive in detecting fibrinolysis even at 25 IU/ml (P < 0.05). Clot firmness parameters were also affected by urokinase, but only at the two highest drug concentrations (P < 0.05). Extem/aptem ratio enhanced the sensitivity of these parameters only if lysis was more marked. Analysing groups of different lysis severity, the time to achieve maximum clot firmness could anticipate an ongoing fulminant or intermediate lysis with 100% sensitivity and 100% specificity (P < 0.05) when lower than 1341.5 s. Rotational thromboelastometry could detect fibrinolysis when it was induced in vitro by 25 IU/ml urokinase or more. Apart from the parameters specific for lysis, time to achieve maximum clot firmness appeared as the earliest indicator of fibrinolysis with high sensitivity and specificity especially if a more intense lysis was going on.

BACKGROUND:Hormonal fertility treatments are associated with increased coagulation factors inducing procoagulant milieu and possibly thrombotic risk.OBJECTIVE:To assess coagulation by ROTEM and coagulation tests in apparently healthy infertile women before oocyte donation procedure.METHODS:We enrolled 51 women (Assisted Reproductive Technology Centre, Florence). ROTEM and coagulation parameters were assessed before the start of infertility treatment.RESULTS:We divided women in 3 groups according to the number of cardiovascular risk factors: Group A (n = 10), Group B (n = 16), and Group C (n = 25). By considering ROTEM Extem test, a significantly increased of MCF, TPI, and G were observed in groups B (p = 0.005, p = 0.03, and p = 0.007) and C (p = 0.01, p = 0.05, and p = 0.005) in comparison to group A. As regards ROTEM Intem test, the TPI and G values were significantly higher in groups B and C in comparison to group A (p < 0.01). MCF by Fibtem test significantly increased in groups B and C than in group A (p = 0.004 and p = 0.002, respectively). FVIII, vWF:Ag and D-dimer values significantly increase according to the presence of≥2 risk factors.CONCLUSIONS:Data from coagulative assessment permit to sensitively identify women with potentially procoagulable state, which represents a risk factor of thromboembolic event during hormone treatment.

Background Coronary artery disease (CAD) has been recognized as a serious and potentially life‐threatening complication of Hepatitis C Virus (HCV) infection. High on‐treatment platelet reactivity has been associated with high risk of ischemic events in patients with CAD, but data regarding the association with HCV infection are still lacking. This post hoc analysis aims to assess high on‐treatment platelet reactivity, severity of CAD, and long‐term outcomes of patients with acute coronary syndrome (ACS) who were infected with HCV. Methods and Results Patients with ACS who were infected with HCV (n=47) were matched to patients with ACS and without HCV (n=137) for age, sex, diabetes mellitus, hypertension, and renal function. HCV‐infected patients with ACS had higher levels of platelet reactivity (ADP10–light transmittance aggregometry, 56±18% versus 44±22% [P=0.002]; arachidonic acid–light transmittance aggregometry, 25±21% versus 16±15% [P=0.011]) and higher rates of high on‐treatment platelet reactivity on clopidogrel and aspirin compared with patients without HCV. Moreover, HCV‐infected patients with ACS had higher rates of multivessel disease (53% versus 30%; P=0.004) and 3‐vessel disease (32% versus 7%; P<0.001) compared with patients without HCV. At long‐term follow‐up, estimated rates of major adverse cardiovascular events (cardiac death, nonfatal myocardial infarction, and ischemia‐driven revascularization) were 57% versus 34% (P=0.005) in HCV‐ and non–HCV‐infected patients with ACS, respectively. In addition, thrombolysis In Myocardial Infarction (TIMI) major bleeding rates were higher in HCV‐infected patients (11% versus 3%; P=0.043) compared with noninfected patients. Multivariable analysis demonstrated that HCV infection was an independent predictor of high on‐treatment platelet reactivity, severity of CAD, and long‐term outcome. Conclusions In this hypothesis‐generating study, patients with ACS and HCV infection showed increased on‐treatment platelet reactivity, more severe CAD, and worse prognosis compared with patients without HCV.

Background Clopidogrel nonresponsiveness is a prognostic marker after percutaneous coronary intervention. Prasugrel and ticagrelor provide a better platelet inhibition and represent the first‐line antiplatelet treatment in acute coronary syndrome. We sought to assess the prognostic impact of high platelet reactivity (HPR) and the potential clinical benefit of a “tailored” escalated or changed antiplatelet therapy in patients with chronic total occlusion. Methods and Results From Florence CTO‐PCI (chronic total occlusion‐percutaneous coronary intervention) registry, platelet function assessed by light transmission aggregometry, was available for 1101 patients. HPR was defined by adenosine diphosphate test ≥70% and optimal platelet reactivity by adenosine diphosphate test <70%. The endpoint of the study was long‐term cardiac survival. Patients were stratified according to light transmission aggregometry results: optimal platelet reactivity (82%) and HPR (18%). Means for the adenosine diphosphate test were 44±16% versus 77±6%, respectively. Three‐year survival was significantly higher in the optimal platelet reactivity group compared with HPR patients (95.3±0.8% versus 86.2±2.8%; P <0.001). With the availability of new P2Y 12 inhibitors, a deeper platelet inhibition (46±17%) and similar survival to the optimal platelet reactivity group were achieved in patients with HPR on clopidogrel therapy after escalation. Conversely, HPR on clopidogrel therapy “not switched” was associated with cardiac mortality (hazard ratio 2.37; P =0.003) after multivariable adjustment. Conclusions HPR on treatment could be a modifiable prognostic marker by new antiaggregants providing a deeper platelet inhibition associated with clinical outcome improvement in complex chronic total occlusion patients. A “tailored” antiplatelet therapy, also driven by the entity of platelet inhibition, could be useful in these high risk setting patients.

BACKGROUND:Patients undergoing cardiac surgery are subject to severe alterations of the coagulation system. The four cardiac surgery centers in Tuscany (Italy) structured and shared an algorithm (Granducato Patient Blood Management algorithm, G-PBMa) with predefined interventions for patient blood management. The aim of the study is to analyze the impact of that algorithm on the transfusion needs and bleeding-related outcomes in a large patient population. METHODS:Multicenter retrospective observational study on 3839 patients undergoing cardiac surgery at the four cardiac centers in Tuscany. The G-PBMa was released at the end of 2015 and it was structured in three parts: pre-, intra-, and post-operative. The year 2014, before the G-PBMa (1955 patients) and the year 2016 (1884 patients) after the G-PBMa in place were compared. Logistic regression analyses were used. RESULTS:The main changes introduced were the routine application of viscoelastic tests in bleeding patients (+72%) and the use of fibrinogen and prothrombin complex concentrate (+67%). The G-PBMa resulted in a significant reduction in the overall transfusion rate and in the transfusion rate of the separate blood products (relative risk for transfusions: 0.75, 95% confidence interval 0.65-0.85, P = 0.001). For preoperative hemoglobin values of between 8 and 10 g/dL, the absolute difference in RBC transfusion rate before and after the G-PBMa introduction ranged around 15%-17%. The G-PBMa introduction determined lower (P = 0.02) chest drain blood loss, lower (P = 0.001) postoperative acute kidney injury and shorter (P = 0.001) hospital stay. CONCLUSIONS:The G-PBMa was effective in reducing blood loss, transfusion requirements, and resulted in a better outcome.

OBJECTIVES:Few data are available on the association between a different entity of platelet inhibition on antiplatelet treatment and clinical outcomes in patients with peripheral artery disease (PAD). The aim of this study was to evaluate the degree of on-treatment platelet reactivity, and its association with ischaemic and haemorrhagic adverse events at follow up in PAD patients undergoing percutaneous transluminal angioplasty (PTA). METHODS:In this observational, prospective, single centre study, 177 consecutive patients with PAD undergoing PTA were enrolled, and treated with dual antiplatelet therapy with aspirin and a P2Y12 inhibitor. Platelet function was assessed on blood samples obtained within 24 h from PTA by light transmission aggregometry (LTA) using arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists of platelet aggregation. High on-treatment platelet reactivity (HPR) was defined by LTA ≥ 20% if induced by AA, and LTA ≥ 70% if induced by ADP. Follow up was performed to record outcomes (death, major amputation, target vessel re-intervention, acute myocardial infarction and/or myocardial revascularisation, stroke/TIA, and bleeding). RESULTS:HPR by AA and HPR by ADP were found in 45% and 32% of patients, respectively. During follow up (median duration 23 months) 23 deaths (13%) were recorded; 27 patients (17.5%) underwent target limb revascularisation (TLR), two (1.3%) amputation, and six (3.9%) myocardial revascularisation. Twenty-four patients (15.6%) experienced minor bleeding. On multivariable analysis, HPR by AA and HPR by ADP were independent predictors of death [HR 3.8 (1.2-11.7), p = .023 and HR 4.8 (1.6-14.5), p = .006, respectively]. The median value of LTA by ADP was significantly lower in patients with bleeding complications than in those without [26.5% (22-39.2) vs. 62% (44.5-74), p < .001). LTA by ADP ≤ 41% was independently associated with bleeding HR 14.6 (2.6-24.0), p = .001] on multivariable analysis. CONCLUSIONS:In this study a high prevalence of on-clopidogrel and aspirin high platelet reactivity was found, which was significantly associated with the risk of death. Conversely, a low on-clopidogrel platelet reactivity was associated with a higher risk of bleeding. These results document that the entity of platelet inhibition is associated with both thrombotic and bleeding complications in PAD patients.

IntroductionSeveral specific assays are commercially available to determine dabigatran anticoagulant activity. Aims of this multicenter and multiplatform study were to compare five methods for dabigatran measurement and investigate their performances in the low concentration range. MethodsDabigatran levels were analyzed in 295 plasma samples from patients enrolled in the START-Laboratory Register by the following methods using dedicated calibrators and controls: STA-ECA II (Diagnostica Stago), standard and low range Hemoclot Thrombin Inhibitors (Hyphen BioMed), Direct Thrombin Inhibitor Assay (Instrumentation Laboratory), Direct Thrombin Inhibitor Assay (Siemens), Technoclot DTI (Technoclone). ResultsMethods showed variable agreement with the Hemoclot Thrombin Inhibitors assay used as reference test, with modest under- or overestimations (Bland-Altman bias from -17.3 to 4.0ng/mL). Limits of detection and quantification varied depending on the assay (4-52 and 7-82ng/mL, respectively). Between-run precision and accuracy were good for all methods for both quality control levels. Assay's repeatability assessed at very low dabigatran concentrations (from 10 to 60ng/mL) was also acceptable, variability generally increased at lower drug levels. ConclusionThe five dabigatran-specific assays evaluated in this study provided reliable assessment of dabigatran plasma levels, although showing different performances.

Backgroup: High residual platelet reactivity (HRPR) in patients receiving clopidogrel and aspirin has been associated with high risk of ischemic events after percutaneous coronary intervention (PCI). Evidence existson difference in pathophysiological mechanisms of acute coronary syndrome in relation to gender. Methods: The RECLOSE 2-ACS study was a prospective, observational, single-center cohort study of 1789 consecutive patients with ACS undergoing an invasive procedure in whom platelet reactivity after PCI was prospectively assessed. The study enrolled consecutive patients undergoing coronary stent implantation for ACS from April 2005 to April 2009 at the Division of Cardiology of Careggi Hospital, Florence, Italy. Platelet reactivity was assessed by light transmittance aggregometry (LTA), using adenosine diphosphate (ADP) and arachidonic acid (AA) as agonists, on blood samples obtained 12-18 hours from PCI. High residual platelet reactivity by ADP was defined as platelet aggregation of 70% or greater. HaPR by AA was defined as platelet aggregation of 20% or greater. Results: Platelet reactivity by ADP or by AA were significantly higher in women than in men [ADP: 49(31-64) % vs 45 (26-30)%, p<0.01; AA: 13 (8-19) % vs 12 (7-17)%, p<0.01]. According to gender, in the high residual platelet reactivity by ADP group the primary end point event rate in women was 36.2 % and 18.9 % in men; stent thrombosis rate was 8.8% in women and 6.2% in men. In the high residual platelet reactivity by AA group the primary end point event rate in women was 21.4 % and 17.1 % in men; stent thrombosis rate was 8.3 % in women and 7.5 % in men. By multivariable Cox regression analysis, high residual platelet reactivity by ADP was independently associated with the primary end point in men but not in women [hazard ratio [HR], Men:1.6; 95% CI, 1.1-2.5; P =0.027; Women HR:1.1 (0.5-2.5), p=0.520]. High residual platelet reactivity by AA was independently associated with the primary end point in men but not in women [HR, Men:1.4; 95% CI, 1.1-2.4; p=0.049; Women HR:1.6 (0.9-3.2), p=0.142]. and with cardiac mortality (HR, 1.81; 95% CI, 1.18- 2.76; p=.006). Similarly, high residual platelet reactivity by ADP was independently associated with stent thrombosis in men but not in women [HR, Men:1.9; 95% CI, 1.1-3.8; p=0.049; Women HR:2.2 (0.8-7.5), p=0.111]. High residual platelet reactivity by AA was independently associated with stent thrombosi in men but not in women [HR, Men:2.0; 95% CI, 1.1-3.6; P =0.031; Women HR:2.8 (0.9-9.2), p=0.850]. Conclusions: Despite the higher prevalence of adverse cardiovascular events in women than in men, high on treatment platelet reactivity by ADP or by AA represents an indepent predictor of major adverse cardiovascular events or stent thrombosis only in male patients.

Introduction: Direct oral anticoagulant (DOAC) intra-and inter-individual variability was previously reported, but its magnitude is still considered negligible for patient management.Objective: To evaluate inter-and intra-individual variability in real-world atrial fibrillation patients on dabigatran, rivaroxaban or apixaban in four Italian anticoagulation clinics and to assess the correlation between DOAC plasma concentration and creatinine-clearance (CrCl).Materials and Methods: A total of 330 consecutive patients were enrolled, of which 160 were on dabigatran (70 and 90 taking 150 mg or 110 mg twice-daily, respectively), 71 on rivaroxaban (37 and 34 taking 20 mg or 15 mg once-daily) and 99 on apixaban (73 and 26 taking 5 mg or 2.5 mg twice-daily). Blood was taken at trough and peak within the first month (15-25 days) of treatment. Diluted-thrombin-time (dTT) calibrated for dabigatran and anti-FXa calibrated for rivaroxaban or apixaban was performed.Results: Mean inter-individual variability expressed as overall CV values for all drugs was lower at peak (CV = 46%) than at trough (CV = 63%). Mean CV% intra-individual variability was 36.6% at trough and 34.0% at peak. Correlation with CrCl was poor for all drugs and only dabigatran at trough showed a significant correlation.Conclusion: This multicenter study confirms high DOAC inter-individual variability that cannot be explained by the rate of renal clearance to which the three DOAC were subjected since the correlation with CrCl was relatively poor. This poor correlation suggests caution in using CrCl as the sole laboratory parameter to indirectly evaluate residual circulating DOAC. (C) 2015 Elsevier Ltd. All rights reserved.

Introduction: Thrombin generation (TG) is a central step of the coagulation system involved in hemostatic and thrombotic roles. Scarce data evaluating in the acute phase the association between TG and the risk of cardiovascular death of acute coronary syndrome (ACS) patients are available, in the era of percutaneous coronary intervention (PCI) and stenting with the use of dual antiplatelet treatment.Materials and methods: We investigated TG in 292 ACS patients undergoing PCI with stent implantation on dual antiplatelet treatment. Venous samples were obtained 12-24 h after PCI. TG was assessed using the Calibrated Automated Thrombogram (CAT).Results: At two years of follow-up, 57 out of 292 patients (19.5%) died from cardiovascular causes. Higher values of endogenous thrombin potential (ETP) [1115.9 (705-1441.3) vs 940.2 (666.0-1253.1), p = 0.049], peak [176.1 (80.5-259.4) vs 107.3 (59.9-181.1), p = 0.002] and velocity index [61.75 (21.03-97.88) vs 25.64 (11.95-50.90), p < 0.001] were observed in relation to survival patients. At the multivariate model adjusted for the Global Registry of Acute Coronary Events risk score, the association between TG and cardiovascular death remained significant for ETP [OR (95% CI): 2.58 (1.10-6.03), p = 0.029], peak [OR (95% CI): 3.27 (1.35-7.92), p = 0.009] and velocity index [OR (95% CI): 3.06 (1.27-7.39), p = 0.013]. This result was confirmed after adjustment for high on-treatment platelet reactivity [ETP: OR (95% CI) 2.35 (1.11-5.00), p = 0.027; peak: OR (95% CI) 2.42 (1.13-5.15), p = 0.022; velocity index: OR (95% CI) 2.43 (1.14-5.20), p = 0.022].Conclusions: ACS patients with a residual TG after PCI and stent implantation have a significantly higher risk of long-term cardiovascular death. These results might be useful in improving risk stratification for ACS patients and support the need of a tailored antithrombotic therapy. (C) 2016 Published by Elsevier Ltd.

Essentials Prothrombin and partial thromboplastin time (PT/PTT) measure direct oral anticoagulants (DOACs). PT, PTT and specific tests for DOACs were performed on patients treated for atrial fibrillation. Normal PT/PTT don't exclude DOAC activity and their prolongation doesn't confirm DOAC action. The use of PT or PTT to evaluate DOAC activity could cause dangerous misinterpretations.

Objective: To evaluate the possible role of high on-aspirin platelet reactivity (HaPR) in a prospective cohort of acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI). Background: Several studies documented that high on-clopidogrel platelet reactivity (HcPR) is associated with increased risk of ischemic events in ACS patients undergoing PCI. On the contrary, conflicting data are available on HaPR and clinical outcome.Methods: Platelet reactivity was assessed by light transmittance aggregometry using arachidonic acid as an agonist in 1789 ACS patients undergoing PCI.Results: HaPR was found in 20.3% of patients. These patients were significantly older, with a higher prevalence of hypertension, diabetes and reduced ejection fraction. Patients with non ST-segment elevation ACS, 3-vessel disease and multivessel PCI had a significantly higher prevalence of HaPR. In addition, stent number and length, and use of drug-eluting stents were significantly higher in HaPR patients. At 24-month follow-up the prevalence of cardiac death was 9.7% in HaPR and 3.8% in non-HaPR [HR2.63(1.72-4.02), p < 0.0001], that of stent thrombosis 6.1% in HaPR and 2.6% in non-HaPR [HR2.4(1.42-4.07), p < 0.001], with no significant differences in other clinical end-points. At multivariate analysis, HaPR was confirmed as an independent risk factor for cardiac death [HR1.88(1.21-2.93), p = 0.005] and stent thrombosis [HR1.91(1.12-3.28), p = 0.018]. The addition of HaPR to a model including clinical and procedural risk factors and HcPR led to a significant improvement in the prediction of cardiac death (NRI 39 +/- 10%, p = 0.0003) and stent thrombosis (NRI 34.7 +/- 13.2%, p = 0.009).Conclusion: HaPR was found to be an independent risk factor for cardiac death and stent thrombosis in ACS patients undergoing PCI. (C) 2015 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

INTRODUCTION:Limited data are available on the natural history of high on treatment platelet reactivity (HPR) by arachidonic acid and ADP - markers of unfavorable prognosis in acute coronary syndrome patients -.MATERIAL AND METHODS:In a cohort of acute coronary syndrome male patients (n=101), we evaluated the time-course of HPR by ADP (platelet aggregation by 10μM ADP≥70%) and arachidonic acid (platelet aggregation by 1mmol arachidonic acid≥20%) measuring platelet function in the acute phase (T0), at 6months (T1) and 1year (T2).RESULTS:We identified persistent (HPR at T0,T1 and T2), acute non persistent (HPR only at T0), and late (HPR only at T1 or T2). Patients with persistent HPR by ADP were more frequently with higher values of BMI. Patients carrying CYP2C19*2 variant were more prevalent in the group of persistent HPR (33%). Significant higher values of immature platelet fraction and high immature platelet fraction at 6 and 12months and mean platelet volume were present in patients with late HPR. Immature platelet fraction was the only variable significantly associated with late HPR by ADP at multivariate analysis (OR=1.6 (1.08-2.3), p=0.016). Patients with persistent HPR by arachidonic acid were more frequently diabetics. Immature platelet fraction at 6months and high immature platelet fraction at 6 and 12months were the parameters associated with late HPR by AA (OR=1.4 (1.0-1.9), p=0.036; OR=1.5 (1.08-2.4), p=0.05; OR=4.9 (1.3-18.8), p=0.018, respectively).CONCLUSIONS:About 25% of 101 patients has persistent HPR; they are more frequently diabetics, overweight or carriers of CYP2C19*2. The occurrence of an inflammatory state, indicated by the increase of immature platelet fraction, is associated with the occurrence of late HPR.