Giovanni Ciccarelli

Role of Tissue Factor in the Coagulation Network.

It is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of the flowing blood to cell-surface protein, such as tissue factor (TF). Vascular injury and/or atherosclerotic plaque complication is responsible for this exposure, leading to clinical manifestations such as acute coronary syndromes. For many years, TF has been considered one of the major determinants of plaque-related thrombosis. However, the discoveries of different pools of TF that circulates in the blood as cell-associated TF, microparticles bound, and as soluble form have changed this dogma. Recent evidence suggests that an increased circulating TF activity may potentiate the initial thrombogenic stimulus related to vessel wall-associated TF, leading to the formation of larger and/or more stable thrombus and thus more severe clinical manifestations. Different pathological conditions, such as inflammatory status, enhance TF expression and activity. Conversely, TF upregulation may facilitate inflammation by formation of proinflammatory fibrin fragments and coagulation proteases generation, including FVIIa, FXa, and thrombin. Furthermore, the biology of TF has become more complex by the demonstration that, apart from its known effects on blood coagulation, it is involved in intracellular signaling, proliferation, angiogenesis, and tumor metastasis.

Angiography versus Hemodynamics to Predict the Natural History of Coronary Stenoses: A FAME 2-Substudy

Background: Among patients with documented stable coronary artery disease and in whom no revascularization was performed, we compared the respective values of angiographic diameter stenosis (DS) and fractional flow reserve (FFR) in predicting natural history. Methods: The present analysis included the 607 patients from the FAME 2 trial (Fractional Flow Reserve Versus Angiography in Multivessel Evaluation 2) in whom no revascularization was performed. FFR varied from 0.20 to 1.00 (average 0.74±0.16), and DS (by quantitative coronary analysis) varied from 8% to 98% (average 53±15). The primary end point, defined as vessel-oriented clinical end point (VOCE) at 2 years, was a composite of prospectively adjudicated cardiac death, vessel-related myocardial infarction, vessel-related urgent, and not urgent revascularization. The stenoses were divided into 4 groups according to FFR and %DS values: positive concordance (FFR⩽0.80; DS≥50%), negative concordance (FFR>0.80; DS<50%), positive mismatch (FFR⩽0.80; DS<50%), and negative mismatch (FFR>0.80; DS≥50%). Results: The rate of VOCE was highest in the positive concordance group (log rank: X2=80.96; P=0.001) and lowest in the negative concordance group. The rate of VOCE was higher in the positive mismatch group than in the negative mismatch group (hazard ratio, 0.38; 95% confidence interval, 0.21–0.67; P=0.001). There was no significant difference in VOCE between the positive concordance and positive mismatch groups (FFR⩽0.80; hazard ratio, 0.77; 95% confidence interval, 0.57–1.09; P=0.149) and no significant difference in rate of VOCE between the negative mismatch and negative concordance groups (FFR>0.80; hazard ratio, 1.89; 95% confidence interval, 0.96–3.74; P=0.067). Conclusions: In patients with stable coronary disease, physiology (FFR) is a more important determinant of the natural history of coronary stenoses than anatomy (DS). Clinical Trial Registration: URL: https://clinicaltrials.gov. Unique identifier: NCT01132495.Background —Among patients with documented stable coronary artery disease (CAD) and in whom no revascularization was performed, we compared the respective values of angiographic diameter stenosis (DS) and of fractional flow reserve (FFR) in predicting natural history. Methods —The present analysis included the 607 patients from the Fractional flow reserve versus angiography in multivessel evaluation 2 (FAME 2) trial in whom no revascularization was performed. FFR varied from 0.20 to 1.00 (average 0.74 ± 0.16) and DS (by QCA) varied from 8% to 98% (average 53 ± 15). The primary end point, defined as VOCE (Vessel oriented clinical endpoint) at 2 years, was a composite of prospectively adjudicated cardiac death, vessel-related myocardial infarction, vessel-related urgent and not urgent revascularization. The stenoses were divided into 4 groups according to FFR and %DS values: Positive Concordance (PC: FFR≤0.80; DS≥50%); Negative Concordance (NC: FFR>0.80; DS 0.80; DS≥50%). Results —The rate of VOCE was highest in the PC group (Log Rank: X 2 =80.96; p=0.001), and lowest in the NC group. The rate of VOCE was higher in the PM group than in the NM group (H.R. 0.38, 95% C.I. 0.21 - 0.67; p=0.001). There was no significant difference in VOCE between the PC and the PM (both groups with FFR≤0.80, H.R. 0.77, 95% C.I. 0.57 - 1.09; p=0.149) and no significant difference in rate of VOCE between the NM and NC (both groups with FFR>0.80, H.R. 1.89, 95% C.I. 0.96 - 3.74; p=0.067). Conclusions —In patients with stable coronary disease, physiology (FFR) is a more important determinant of the natural history of coronary stenoses than anatomy (DS). Clinical Trial Registration —URL: https://clinicaltrials.gov Unique Identifier: NCT01132495.

Expression of functional tissue factor in activated T-lymphocytes in vitro and in vivo: A possible contribution of immunity to thrombosis?

OBJECTIVE T-lymphocyte activation plays an important role in the pathophysiology of acute coronary syndromes (ACS). Plaques from ACS patients show a selective oligoclonal expansion of T-cells, indicating a specific, antigen-driven recruitment of T-lymphocytes within the unstable lesions. At present, however, it is not known whether T-cells may contribute directly to thrombosis by expressing functional tissue factor (TF). Accordingly, the aim of the present study was to investigate whether T-cells are able to express functional TF in their activated status. METHODS In vitro, CD3(+)-cells, isolated from buffy coats, were stimulated with anti-CD3/CD28 beads, IL-6, TNF-α, IL-17, INF-γ or PMA/ionomycin. Following stimulation, TF expression on cell-surface, at gene and protein levels, as well as its procoagulant activity in whole cells and microparticles was measured. In vivo, TF expression was evaluated in CD3(+)-cells isolated from the aorta and the coronary sinus of ACS-NSTEMI and stable coronary artery disease (SCAD) patients. The presence of CD3(+)-TF(+)cells was also evaluated by immunohistochemistry in thrombi aspirated from ACS-STEMI patients. RESULTS PMA/ionomycin and IL-17 plus INF-γ stimulation resulted in a significant TF increase at gene and protein levels as well as at cell-surface expression. This was accompanied by a parallel increase in FXa generation, both in whole cells and in microparticles, indicating that the induced membrane-bound TF was active. Furthermore, transcardiac TF gradient was significantly higher in CD3(+)-cells obtained from ACS-patients compared to SCAD-patients. Interestingly, thrombi from ACS-STEMI patients resulted enriched in CD3(+)-cells, most of them expressing TF. CONCLUSIONS Our data demonstrate that activated T-lymphocytes in vitro express functional TF on their membranes, suggesting a direct pathophysiological role of these cells in the thrombotic process; this hypothesis is further supported by the observations in vivo that CD3(+)-cells from coronary circulation of ACS-NSTEMI patients show increased TF levels and that coronary thrombi from ACS-STEMI patients are enriched in CD3(+)-cells expressing TF.

Catheter-based measurements of absolute coronary blood flow and microvascular resistance feasibility, safety, and reproducibility in humans

Background— The principle of continuous thermodilution can be used to calculate absolute coronary blood flow and microvascular resistance (R). The aim of the study is to explore the safety, feasibility, and reproducibility of coronary blood flow and R measurements as measured by continuous thermodilution in humans. Methods and Results— Absolute coronary flow and R can be calculated by thermodilution by infusing saline at room temperature through a dedicated monorail catheter. The temperature of saline as it enters the vessel, the temperature of blood and saline mixed in the distal part of the vessel, and the distal coronary pressure were measured by a pressure/temperature sensor-tipped guidewire. The feasibility and safety of the method were tested in 135 patients who were referred for coronary angiography. No significant adverse events were observed; in 11 (8.1%) patients, bradycardia and concomitant atrioventricular block appeared transiently and were reversed immediately on interruption of the infusion. The reproducibility of measurements was tested in a subgroup of 80 patients (129 arteries). Duplicate measurements had a strong correlation both for coronary blood flow (&rgr;=0.841, P<0.001; intraclass correlation coefficient=0.89, P<0.001) and R (&rgr;=0.780, P<0.001; intraclass correlation coefficient=0.89, P<0.001). In Bland–Altman plots, there was no significant bias or asymmetry. Conclusions— Absolute coronary blood flow (in L/min) and R (in mm Hg/L/min or Wood units) can be safely and reproducibly measured with continuous thermodilution. This approach constitutes a new opportunity for the study of the coronary microcirculation.

Lipid Target in Very High-Risk Cardiovascular Patients: Lesson from PCSK9 Monoclonal Antibodies

The role of low-density lipoproteins (LDLs) as a major risk factor for cardiovascular disease has been demonstrated by several epidemiological studies. The molecular basis for LDLs in atherosclerotic plaque formation and progression is not completely unraveled yet. Pharmacological modulation of plasma LDL-C concentrations and randomized clinical trials addressing the impact of lipid-lowering interventions on cardiovascular outcome have clearly shown that reducing plasma LDL-C concentrations results in a significant decrease in major cardiovascular events. For many years, statins have represented the most powerful pharmacological agents available to lower plasma LDL-C concentrations. In clinical trials, it has been shown that the greater the reduction in plasma LDL-C concentrations, the lower the rate of major cardiovascular events, especially in high-risk patients, because of multiple risk factors and recurrent events. However, in a substantial number of patients, the recommended LDL target is difficult to achieve because of different factors: genetic background (familial hypercholesterolemia), side effects (statin intolerance), or high baseline plasma LDL-C concentrations. In the last decade, our understanding of the molecular mechanisms involved in LDL metabolism has progressed significantly and the key role of proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged. This protein is an enzyme able to bind the LDL receptors (LDL-R) on hepatocytes, favoring their degradation. Blocking PCSK9 represents an intriguing new therapeutic approach to decrease plasma LDL-C concentrations, which in recent studies has been demonstrated to also result in a significant reduction in major cardiovascular events.

Upregulation of TH/IL-17 Pathway-Related Genes in Human Coronary Endothelial Cells Stimulated with Serum of Patients with Acute Coronary Syndromes

Background Inflammation plays an essential role in the development and complications of atherosclerosis plaques, including acute coronary syndromes (ACS). Indeed, previous reports have shown that within the coronary circulation of ACS patients, several soluble mediators are released. Moreover, it has been demonstrated that endothelial dysfunction might play an important role in atherosclerosis as well as ACS pathophysiology. However, the mechanisms by which these soluble mediators might affect endothelial functions are still largely unknown. We have evaluated whether soluble mediators contained in serum from coronary circulation of ACS patients might promote changes of gene profile in human coronary endothelial cells (HCAECs). Methods HCAECs were stimulated in vitro for 12 h with serum obtained from the coronary sinus (CS) and the aorta (Ao) of ACS patients; stable angina (SA) patients served as controls. Gene expression profiles of stimulated cells were evaluated by microarray and real-time PCR. Results HCAECs stimulated with serum from CS of ACS patients showed a significant change (upregulation and downregulation) in gene expression profile as compared with cells stimulated with serum from CS of SA patients. Moreover, ad hoc sub analysis indicated the upregulation of Th-17/IL-17 pathway-related genes. Conclusion This study demonstrates that, in ACS patients, the chemical mediators released in the coronary circulation might be able to perturb coronary endothelial cells (ECs) modifying their gene profile. These modified ECs, through downregulation of protective gene and, mainly, through upregulation of gene able to modulate the Th-17/IL-17 pathway, might play a key role in progression of coronary atherosclerosis and in developing future acute events.

Visual and Quantitative Assessment of Coronary Stenoses at Angiography Versus Fractional Flow Reserve

Background— The correlation between angiographic assessment of coronary stenoses and fractional flow reserve (FFR) is weak. Whether and how risk factors impact the diagnostic accuracy of angiography is unknown. We sought to evaluate the diagnostic accuracy of angiography by visual estimate and by quantitative coronary angiography when compared with FFR and evaluate the influence of risk factors (RF) on this accuracy. Methods and Results— In 1382 coronary stenoses (1104 patients), percent diameter stenosis by visual estimation (DSVE) and by quantitative coronary angiography (DSQCA) was compared with FFR. Patients were divided into 4 subgroups, according to the presence of RFs, and the relationship between DSVE, DSQCA, and FFR was analyzed. Overall, DSVE was significantly higher than DSQCA (P<0.0001); nonetheless, when examined by strata of DS, DSVE was significantly smaller than DSQCA in mild stenoses, although the reverse held true for severe stenoses. Compared with FFR, a large scatter was observed for both DSVE and DSQCA. When using a dichotomous FFR value of 0.80, C statistic was significantly higher for DSVE than for DSQCA (0.712 versus 0.640, respectively; P<0.001). C statistics for DSVE decreased progressively as RFs accumulated (0.776 for ⩽1 RF, 0.750 for 2 RFs, 0.713 for 3 RFs and 0.627 for ≥4 RFs; P=0.0053). In addition, in diabetics, the relationship between FFR and angiographic indices was particularly weak (C statistics: 0.524 for DSVE and 0.511 for DSQCA). Conclusions— Overall, DSVE has a better diagnostic accuracy than DSQCA to predict the functional significance of coronary stenosis. The predictive accuracy of angiography is moderate in patients with ⩽1 RFs, but weakens as RFs accumulate, especially in diabetics.

COLCHICINE EXERTS ANTITHROMBOTIC PROPERTIES IN OXLDL-TREATED ENDOTHELIAL CELLS VIA INHIBITION OF TISSUE FACTOR EXPRESSION AND ACTIVITY

Background: Tissue factor (TF) exposure is the key trigger of thrombus formation. Oxidized low-density lipoproteins (oxLDL) induce TF in endothelial cells (ECs), suggesting that oxLDL may act locally promoting thrombosis in atherosclerotic lesions. Colchicine (COL) is an anti-inflammatory agent that

Safety of Percutaneous Left Atrial Appendage Occlusion in Patients with Atrial Fibrillation after Acute Coronary Syndrome: A Single Center Experience

Aims: In patients with atrial fibrillation (AF) and indication to Dual Antiplatelet Therapy (DAPT) for Acute Coronary Syndrome (ACS), triple therapy is recommended. However, this issue is still largely debated. Percutaneous Left Atrial Appendage (LAA) occlusion may represent an alternative therapeutic approach in this subset of patients, as it could lead to a reduction in the incidence of bleeding. Nevertheless, due to lack of clinical evidence, this approach does not represent the gold standard treatment for these patients. The objective of this study is to evaluate the safety of percutaneous LAA occlusion in patients with Atrial Fibrillation and indication of concomitant DAPT for ACS. Methods and Results: Fifteen AF-patients with indications to anticoagulant and DAPT after ACS underwent LAA occlusion with Amplatzer Cardiac Plug (ACP) or Amulet ACP (St. Jude) from October 2014 to May 2016. Procedural success was achieved in 14/15 patients, while in 1 patient a clinically non-relevant pericardial effusion was registered before the trans-septal puncture, without implantation of the device. No peri-procedural death, major bleeding and dislocation or thrombi of the device were recorded. During a median follow-up of 182 days (interquartile range [IQR]: 169 to 183 days), one peri-procedural minor bleeding and two non cardiovascular death were described. Conclusions: LAA occlusion represents a relatively safe procedure for patients with atrial fibrillation and the need of DAPT due to ACS. Randomized trials should be designed to evaluate the efficacy and safety of this procedure in this clinical setting.