Wael Sumaya

Hirudin anticoagulation allows more rapid determination of P2Y₁₂ inhibition by the VerifyNow P2Y12 assay

VerifyNow (VN) P2Y12 is a point-of-care assay used to assess response to P2Y12 inhibitors. Sodium citrate (citrate) is the standard anticoagulant used for this assay but requires a pre-incubation period. Hirudin is an alternative anticoagulant for platelet function studies that maintains physiological divalent cation levels. We investigated whether hirudin anticoagulation might allow more rapid testing of P2Y₁₂ inhibition at the time of percutaneous coronary intervention (PCI). Blood was collected from the arterial sheath of aspirin-treated patients undergoing elective, urgent or emergency coronary angiography±PCI and aliquots were anticoagulated with either citrate or hirudin. For each anticoagulant, VN P2Y12 was performed both immediately and after 20 minutes. A total of 98 patients were included in this study following pre-treatment with clopidogrel (n=88), prasugrel (n=6) or no P2Y₁₂ inhibitor (n=4). PRU with hirudin immediately (PRU_H_Imm) and PRU with citrate 20 minutes post sampling (PRU_C_20) were very strongly correlated (R=0.95) though PRU_H_Imm tended to be lower than PRU_C_20 so that optimal correlation was estimated by the equation PRU_H_Imm=0.95xPRU_C_20 (p<0.001). Bland-Altman plots showed good agreement between PRU_H_Imm and (0.95xPRU_C_20). Platelet reactivity was more stable over the studied time course with hirudin as compared to citrate. We therefore conclude that VN P2Y12 with hirudin anticoagulation can be performed more rapidly and results are strongly correlated with delayed citrate measurements. Further studies are warranted to assess the utility of this method for improving clinical outcomes in patients undergoing PCI.

Fibrin clot properties independently predict adverse clinical outcome following acute coronary syndrome: a PLATO substudy

Abstract Aims To determine whether fibrin clot properties are associated with clinical outcomes following acute coronary syndrome (ACS). Methods and results Plasma samples were collected at hospital discharge from 4354 ACS patients randomized to clopidogrel or ticagrelor in the PLATelet inhibition and patient Outcomes (PLATO) trial. A validated turbidimetric assay was employed to study plasma clot lysis time and maximum turbidity (a measure of clot density). One-year rates of cardiovascular (CV) death, spontaneous myocardial infarction (MI) and PLATO-defined major bleeding events were assessed after sample collection. Hazard ratios (HRs) were estimated using Cox proportional hazards models. After adjusting for CV risk factors, each 50% increase in lysis time was associated with CV death/spontaneous MI [HR 1.17, 95% confidence interval (CI) 1.05–1.31; P < 0.01] and CV death alone (HR 1.36, 95% CI 1.17–1.59; P < 0.001). Similarly, each 50% increase in maximum turbidity was associated with increased risk of CV death (HR 1.24, 95% CI 1.03–1.50; P = 0.024). After adjustment for other prognostic biomarkers (leukocyte count, high-sensitivity C-reactive protein, high-sensitivity troponin T, cystatin C, N-terminal pro B-type natriuretic peptide, and growth differentiation factor-15), the association with CV death remained significant for lysis time (HR 1.2, 95% CI 1.01–1.42; P = 0.042) but not for maximum turbidity. These associations were consistent regardless of randomized antiplatelet treatment (all interaction P > 0.05). Neither lysis time nor maximum turbidity was associated with major bleeding events. Conclusion Fibrin clots that are resistant to lysis independently predict adverse outcome in ACS patients. Novel therapies targeting fibrin clot properties might be a new avenue for improving prognosis in patients with ACS.

Stability of VerifyNow P2Y12 assay results with citrate anticoagulation as compared to hirudin anticoagulation over 20-min period

Antiplatelet therapy plays a central role in the management of ischaemic heart disease [1]. While poor inhibition of P2Y12 receptor has been linked to higher incidence of ischaemic events, enhanced inhibition has been associated with increased risk of bleeding events [2]. Platelet function testing has been suggested as a way to individualise therapy in order to minimize the risk of both bleeding and ischaemic events [3]. The VerifyNow analyser (VN) (Accumetrics, San Diego, CA) is a point-of-care device that provides rapid assessment of P2Y12 inhibition and results obtained with VN are reported as P2Y12 reaction units (PRU) [4]. Traditionally, trisodium citrate (‘‘citrate’’) has been used with the VN and measurements with citrate require an incubation period of at least 10 minutes and can be performed up to 4 hours post sampling, as per manufacturer’s instruction. Recently, we have demonstrated the possibility for the test to be performed immediately post sampling with hirudin anticoagulation and showed that measurements with hirudin were stable over 20 minutes whereas PRU results obtained with citrate immediately were significantly lower than those obtained after 20 minutes incubation [5]. In this study, we assessed the stability of PRU results obtained with citrate between the recommended minimum incubation time of 10 minutes compared to 20 minutes post sampling as well as compared to measurements with hirudin performed immediately or 20 minutes post sampling. Ten patients with stable coronary artery disease undergoing elective percutaneous angioplasty provided informed consent according to a protocol approved by the local ethics committee. All patients had been pretreated with aspirin and either 600 mg clopidogrel at least 2 hours pre-sampling (n1⁄4 6) or 75 mg daily for at least 5 days (n1⁄4 4). All blood samples for VN testing were obtained by venipuncture using a syringe and a needle. The first 2 mls were discarded and blood was added gently, avoiding turbulence, to uncapped anticoagulant tubes without the use of vacuum. Blood tubes were then capped and inverted gently to mix the blood with the anticoagulant. We used two tubes containing 3.2% citrate (Vacuette, Greiner BioOne, Kremsmünster, Austria) and two tubes containing415 mg/ml hirudin (Double-wall hirudin blood tube, Verum Diagnostica, Munich, Germany) for every patient. The VN P2Y12 test was performed at two time points for each anticoagulant: immediately and at 20 min post sampling with hirudin and at 10 minutes and 20 minutes post sampling with citrate. All statistical analyses were performed using PASW statistic version 19 (IBM SPSS Inc., New York, NY). Data are presented as mean SD and analysis was carried out using paired samples t-test for continuous variables. Measurements with hirudin provided more consistent results over the studied period as compared to measurements with citrate where PRU values tended to be significantly lower at 10 minutes (187 40) versus at 20 minutes (205 38; p1⁄4 0.009) (Table I, Figure 1). Measurements with hirudin as compared to measurements with citrate at 20 min further confirmed our previous study where optimum correlation between both measurements was estimated by the equation PRU_H_Imm1⁄4 0.95 X PRU_C_20. Base PRU (BASE) results representing platelet aggregation in response to thrombin receptor activating peptide (TRAP) were stable over the studied time with each anticoagulant, however measurements with hirudin were significantly different to measurements with citrate. These results are consistent with the results shown in our previous study [5]. The consistency in PRU results obtained with hirudin over the studied time might be explained by the mechanism of action of hirudin, which directly inhibits thrombin without affecting divalent cation levels [6] and this might help provide a stable medium for homeostasis over a 20-min period. Other studies assessing the use of hirudin with Multiple Electrode Aggregometry (MEA) demonstrated stability in results obtained with hirudin up to 12 hours post sampling [7, 8]. Citrate, on the other hand, appears to transiently blunt platelet reactivity, possibly due to the sudden shift in extracellular ionized cations affecting receptor activity and/or intracellular signalling. The limitations of this study are small sample size (although the results are consistent with our previous larger study) and assessment of only a 20-minute window since our primary focus related to rapid testing of P2Y12 inhibition in the clinical setting. We did not assess other assays, such as the VN aspirin assay since this is not currently recommended in clinical practice [9], and Keywords

The challenges of antithrombotic therapy in patients with left ventricular thrombosis

Platelets assume the driving seat when it comes to arterial thrombosis. Subsequent to plaque rupture, they adhere to exposed subendothelial agonists, such as collagen and von Willebrand factor (vWF), in order to produce an initial platelet plug. The subendothelial ligands activate platelets, which change in shape and secrete thromboxane A2 (TXA2) and ADP. TXA2 activates platelets further and ADP amplifies and sustains activation through platelet P2Y12 receptors, leading to further expansion of the platelet-rich thrombus (Take home figure). Thrombin is also generated at the site of arterial injury, with activated platelets as well as tissue factor playing dominant roles in this, but pre-clinical data suggest that thrombin may play a greater role in stabilization of the core of the arterial thrombus rather than its expansion. As a result, preventative therapy following acute coronary syndrome (ACS) consists of dual antiplatelet therapy (DAPT) with aspirin and a P2Y12 inhibitor. 3,4

Contemporary management of ST-segment elevation myocardial infarction.

Abstract Coronary heart disease is the leading cause of death worldwide. In the United States, approximately 1 of every 6 deaths in 2007 was caused by coronary heart disease. Clinical presentation in the acute setting is mostly due to atherosclerotic plaque rupture leading to flow limitation in the affected vessel, and myocardial ischemia and infarction. ST-segment elevation myocardial infarction is usually associated with complete occlusion of the coronary artery and carries the worst prognosis in terms of in-hospital mortality. Despite various advances in treatment options, including percutaneous coronary intervention, ischemic heart disease still carries a significant morbidity and mortality. In this article, we aim to provide a summary of a few key advances in the management of ST-segment elevation myocardial infarction.

Pharmacodynamic Effects of a 6-Hour Regimen of Enoxaparin in Patients Undergoing Primary Percutaneous Coronary Intervention (PENNY PCI Study)

Delayed onset of action of oral P2Y 12 inhibitors in ST-elevation myocardial infarction (STEMI) patients may increase the risk of acute stent thrombosis. Available parenteral anti-thrombotic strategies, to deal with this issue, are limited by added cost and increased risk of bleeding. We investigated the pharmacodynamic effects of a novel regimen of enoxaparin in STEMI patients undergoing primary percutaneous coronary intervention (PPCI). Twenty patients were recruited to receive 0.75 mg/kg bolus of enoxaparin (pre-PPCI) followed by infusion of enoxaparin 0.75 mg/kg/6 h. At four time points (pre-anti-coagulation, end of PPCI, 2–3 hours into infusion and at the end of infusion), anti-Xa levels were determined using chromogenic assays, fibrin clots were assessed by turbidimetric analysis and platelet P2Y 12 inhibition was determined by VerifyNow P2Y12 assay. Clinical outcomes were determined 14 hours after enoxaparin initiation. Nineteen of 20 patients completed the enoxaparin regimen; one patient, who developed no-reflow phenomenon, was switched to tirofiban after the enoxaparin bolus. All received ticagrelor 180 mg before angiography. Mean (± standard error of the mean) anti-Xa levels were sustained during enoxaparin infusion (1.17 ± 0.06 IU/mL at the end of PPCI and 1.003 ± 0.06 IU/mL at 6 hours), resulting in prolonged fibrin clot lag time and increased lysis potential. Onset of platelet P2Y 12 inhibition was delayed in opiate-treated patients. No patients had thrombotic or bleeding complications. In conclusion, enoxaparin 0.75 mg/kg bolus followed by 0.75 mg/kg/6 h provides sustained anti-Xa levels in PPCI patients. This may protect from acute stent thrombosis in opiate-treated PPCI patients who frequently have delayed onset of oral P2Y 12 inhibition.

ULTRA-LOW-DOSE TWICE-DAILY ASPIRIN IMPROVES HEMOSTASIS AND MAINTAINS PLATELET INHIBITION IN ACUTE CORONARY SYNDROME PATIENTS RECEIVING TICAGRELOR

The PLATO study suggested that higher aspirin doses may be inferior in ticagrelor treated patients with acute coronary syndromes (ACS). Reducing bleeding risk whilst maintaining antithrombotic benefits would improve outcomes. We characterised a novel regimen of ultra-low-dose aspirin given twice

Study of Two Dose Regimens of Ticagrelor Compared with Clopidogrel in Patients Undergoing Percutaneous Coronary Intervention for Stable Coronary Artery Disease (STEEL-PCI)

Background: Ticagrelor has superior efficacy to clopidogrel in the management of acute coronary syndromes but has not been assessed in patients undergoing percutaneous coronary intervention for stable coronary artery disease. We compared the pharmacodynamic effects of ticagrelor and clopidogrel in this stable population. Methods: One hundred eighty aspirin-treated stable coronary artery disease patients, who were planned to undergo elective percutaneous coronary intervention in a single center, were randomized 1:1:1 to either a standard clopidogrel regimen or 1 of 2 regimens of ticagrelor, either 90 mg (T90) or 60 mg twice daily (T60), both with a 180 mg loading dose. Cellular adenosine uptake was assessed, at the time of the procedure and pre- and postdose at 1 month, by adding adenosine 1 µmol/L to aliquots of anticoagulated whole blood and mixing with a stop solution at 0, 15, 30, and 60 seconds, then measuring residual plasma adenosine concentration by high-performance liquid chromatography. Systemic plasma adenosine concentration and platelet reactivity were assessed at the same timepoints. High-sensitivity troponin T was measured pre- and 18 to 24 hours postpercutaneous coronary intervention. Results: One hundred seventy-four patients underwent an invasive procedure, of whom 162 received percutaneous coronary intervention (mean age 65 years, 18% female, 21% with diabetes mellitus). No effect on in vitro adenosine uptake was seen postdose at 1 month for either ticagrelor dose compared with clopidogrel (residual adenosine at 15 seconds, mean±SD: clopidogrel 0.274±0.101 µmol/L; T90 0.278±0.134 µmol/L; T60 0.288±0.149 µmol/L; P=0.37). Similarly, no effect of ticagrelor on in vitro adenosine uptake was seen at other timepoints, nor was plasma adenosine concentration affected (all P>0.1). Both maintenance doses of ticagrelor achieved more potent and consistent platelet inhibition than clopidogrel (VerifyNow P2Y12 reaction units, 1 month, mean±SD: predose, T60: 62±47, T90: 40±38, clopidogrel 181±44; postdose, T60: 34±30, T90: 24±21, clopidogrel 159±57; all P<0.0001 for ticagrelor versus clopidogrel). High platelet reactivity was markedly less with both T60 and T90 compared with clopidogrel (VerifyNow P2Y12 reaction units>208, 1 month postdose: 0%, 0%, and 21%, respectively). Median (interquartile range) high-sensitivity troponin T increased 16.9 (6.5–46.9) ng/L for clopidogrel, 22.4 (5.5–53.8) ng/L for T60, and 17.7 (8.1–43.5) ng/L for T90 (P=0.95). There was a trend toward less dyspnea with T60 versus T90 (7.1% versus 19.0%; P=0.09). Conclusions: Maintenance therapy with T60 or T90 had no detectable effect on cellular adenosine uptake at 1 month, nor was there any effect on systemic plasma adenosine levels. Both regimens of ticagrelor achieved greater and more consistent platelet inhibition than clopidogrel but did not appear to affect troponin release after percutaneous coronary intervention. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02327624.