Multiplate Research Articles

Assessment of hemostatic profile in patients with mild to advanced liver cirrhosis.

BACKGROUNDHemostasis of patients suffering from liver cirrhosis is challenging due to both, pro- and anticoagulatory disorders leading to hemostatic alterations with distinct abnormalities of coagulation. Pathological changes in conventional coagulation analysis and platelet count are common manifestations of decreased liver synthesis of coagulation factors and reduced platelet count in these patients. However, conventional coagulation analysis and platelet count do not reflect in-vivo coagulation status or platelet function. The purpose of this present observational study was therefore to assess the haemostatic profile including plasmatic coagulation using thrombelastometry and impedance aggregometry for platelet function in patients suffering from liver cirrhosis.AIMTo assess the hemostatic profile of cirrhotic patients according to model for end-stage liver disease (MELD) score.METHODSOur study included both in- and outpatients suffering from liver cirrhosis attending the out- and inpatient care of the department of hepatology. Demographic and biochemical data as well as medical history including cause of liver cirrhosis, end stage kidney failure and medication with anticoagulants were recorded. To assess the hemostatic profile, platelet function was analyzed by multiple electrode aggregometry (MEA) using Multiplate® (ADP-, ASPI- and TRAP-test) and thrombelastometry using ROTEM® (EXTEM, INTEM, FIBTEM). Data were compared using Mann-Whitney U- or χ2-test. Spearman correlation was performed to analyze the association between MELD Score and results of thrombelastometry and MEA.RESULTSA total of 68 patients attending the out- and inpatient care suffering from liver cirrhosis were screened. Of these, 50 patients were included and assigned to groups according to MELD score 6 to 11 (n = 25) or ≥ 17 (n = 25). Baseline patient characteristics revealed significant differences for MELD score (8 vs 22, P < 0.0001) and underlying laboratory parameters (international normalized ratio, bilirubine, creatinine) as well as fibrinogen level (275 mg/dL vs 209 mg/dL, P = 0.006) and aPTT (30 s vs 35 s, P = 0.047). MEA showed a moderately impaired platelet function (medians: AUCADP = 43U, AUCASPI = 71U, AUCTRAP = 92U) but no significant differences between both groups. Thrombelastometry using ROTEM® (EXTEM, INTEM, FIBTEM) revealed values within normal range in both groups. No significant correlation was observed between MELD score and results of MEA/thrombelastometry.CONCLUSIONOur data demonstrate a partially impaired hemostatic profile in liver cirrhosis patients unrelated to MELD score. An individual assessment of a potential coagulopathy should therefore be considered.

Changes in Red Blood Cell Properties and Platelet Function during Extracorporeal Membrane Oxygenation.

Extracorporeal membrane oxygenation (ECMO) is associated with frequent hemorrhagic and thromboembolic complications. The multiple effects of ECMO include inflammatory response on contact with the circuit; hemolysis acquired von Willebrand syndrome likely affects the function of red blood cells (RBC) and platelets. The aim of this prospective observational study was to analyze RBC aggregation and elongation (deformability) and platelet aggregation in the first week of ECMO. Sixteen patients were included. Blood samples were taken prior to initiation of ECMO and on days 1, 2, 3, 5, and 7. RBC aggregation and elongation were analyzed using the laser-assisted optical rotational red cell analyzer (Lorrca). Upstroke, top, and amplitude as indices of aggregation showed significant time effects. RBC elongation was not affected at low shear stress. At high shear stress there was an increase in the elongation index at day 2 (p = 0.004), followed by a decrease. Platelet function was analyzed using multiple electrode aggregometry (Multiplate®). In pairwise comparison in the days 1–7 to the value prior to ECMO there was no significant difference in platelet aggregation by any of the three agonists (ADP p = 0.61; TRAP p = 0.77; Ristocetin p = 0.25). This implies that the rheology of RBCs seemed to be more affected by ECMO than platelets. Especially the red blood cell deformability continues to decline at higher shear stress.

Evaluation of functional assays for the diagnosis of heparin induced thrombocytopenia using 5B9, a monoclonal IgG that mimics human antibodies

BackgroundSerotonin release assay (SRA) is considered as the “gold standard” for detecting pathogenic heparin‐induced thrombocytopenia (HIT) antibodies. However, this method is time consuming, expensive, and uses radioelements. Heparin‐induced multiple electrode aggregometry (HIMEA), light transmission aggregometry (LTA) with platelet rich plasma (PRP) or washed platelets (WP), adenosine triphosphate (ATP) release, and flow cytometry (FC) are available alternatives. ObjectivesTo evaluate the performance of these assays, comparatively with SRA, for detecting HIT antibodies, using 5B9, a monoclonal IgG fully mimicking human HIT antibodies. Patients/MethodsHeparin‐dependent platelet activation induced by 5B9 (50/20/10 µg/mL) was evaluated by all assays performed on the same day using platelets from 20 healthy donors. The three methods exhibiting the highest sensitivity to 5B9 were then assessed by testing samples from patients with either likely (n = 10), or indeterminate/unlikely HIT (n = 10). ResultsAll methods exhibited good sensitivity for detecting 5B9 50 µg/mL, but only SRA and HIMEA were positive with 100% of donors using 5B9 20 µg/mL, followed by FC (83%). SRA detected 5B9 10 μg/mL with 90% of donors, while HIMEA and FC were positive in 45% and 44% of cases, respectively. Whereas SRA was positive with 9/10 samples from likely HIT, HIMEA and FC were positive with 6 and 7 of them, respectively. Neither SRA nor HIMEA was positive with indeterminate/unlikely HIT samples, while FC was positive or doubtful in three cases. ConclusionsSerotonin release assay likely remains the most sensitive and specific assay for detecting platelet activating HIT antibodies, but HIMEA or FC are potential alternatives, despite being less performant.

Gender Differences in Platelet Reactivity in Diabetic Patients Receiving Dual Antiplatelet Therapy

BackgroundIncreased comorbidities and a perceived high-bleeding risk often prevent the use of dual antiplatelet therapy (DAPT) in female patients. However, more aggressive antiplatelet treatment would certainly offer additional outcome benefits in coronary artery disease, especially among diabetic patients. The aim of the present study was to evaluate the gender differences in high-residual on treatment platelet reactivity (HRPR) among diabetic patients treated with DAPT. MethodsOur population is represented by a consecutive cohort of diabetic patients treated with DAPT (ASA + clopidogrel, ticagrelor or dose-adjusted prasugrel) for an acute coronary syndrome or elective PCI, undergoing platelet reactivity assessment at 30–90 days post-discharge. Aggregation was assessed by multiple-electrode aggregometry and in diabetic patients naïve to antiplatelet therapy, by light transmission aggregometry, surface expression of P-selectin and plasma concentration of Thromboxane B2. ResultsWe included 472 patients, 113 (23.9%) women. Female gender was associated with more advanced age, and increased comorbidities. Mean platelet reactivity did not differ according to gender. The rate of HRPR was similar in women as compared to men (for ASA: adjusted OR[95%CI] = 0.59[0.27–1.33], p = 0.21, for ADP-antagonists: adjusted OR[95%CI] = 1.24[0.25–1.80], p = 0.27), however, the benefits of the new ADP-antagonists on platelet reactivity were lower in women than in men (p interaction = 0.01).No impact of gender on platelet reactivity was confirmed among 50 diabetic patients naïve to antiplatelet therapy. ConclusionsAmong diabetic patients receiving dual antiplatelet therapy gender does not affect platelet reactivity or high-on treatment platelet reactivity. However, the enhanced platelet inhibition provided by the new-ADP antagonists of new-ADP antagonists could be mitigated in women.

Reduction of Preoperative Waiting Time before Urgent Surgery for Patients on P2Y12 Inhibitors Using Multiple Electrode Aggregometry: A Retrospective Study.

P2Y12 inhibitor discontinuation is essential before most surgical interventions to limit bleeding complications. Based on pharmacodynamic data, fixed discontinuation durations have been recommended. However, as platelet function recovery is highly variable among patients, a more individualized approach based on platelet function testing (PFT) has been proposed. The aim of this retrospective single-centre study was to determine whether PFT using whole blood adenosine diphosphate–multiple electrode aggregometry (ADP–MEA) was associated with a safe reduction of preoperative waiting time. Preoperative ADP–MEA was performed for 29 patients on P2Y12 inhibitors. Among those, 17 patients underwent a coronary artery bypass graft. Twenty one were operated with an ADP–MEA ≥ 19 U (quantification of the area under the aggregation curve), and the waiting time was shorter by 1.6 days (median 1.8 days, IQR 0.5–2.9), by comparison with the current recommendations (five days for clopidogrel and ticagrelor, seven days for prasugrel). Platelet function recovery was indeed highly variable among individuals. With the 19 U threshold, high residual platelet inhibition was associated with perioperative platelet transfusion. These results suggest that preoperative PFT with ADP–MEA could help reduce waiting time before urgent surgery for patients on P2Y12 inhibitors.

Ticagrelor Inhibits Toll-Like and Protease-Activated Receptor Mediated Platelet Activation in Acute Coronary Syndromes

PurposeSince ticagrelor inhibits the cellular uptake of adenosine, thereby increasing extracellular adenosine concentration and biological activity, we hypothesized that ticagrelor has adenosine-dependent antiplatelet properties. In the current study, we compared the effects of ticagrelor and prasugrel on platelet activation in acute coronary syndrome (ACS).MethodsPlatelet surface expression of P-selectin and activated glycoprotein (GP) IIb/IIIa in response to adenosine diphosphate (ADP), the toll-like receptor (TLR)-1/2 agonist Pam3CSK4, the TLR-4 agonist lipopolysaccharide (LPS), the protease-activated receptor (PAR)-1 agonist SFLLRN, and the PAR-4 agonist AYPGKF were measured by flow cytometry in blood from 80 ticagrelor- and 80 prasugrel-treated ACS patients on day 3 after percutaneous coronary intervention. Residual platelet aggregation to arachidonic acid (AA) and ADP were assessed by multiple electrode aggregometry and light transmission aggregometry.ResultsADP-induced platelet activation and aggregation, and AA-induced platelet aggregation were similar in patients on ticagrelor and prasugrel, respectively (all p ≥ 0.3). Further, LPS-induced platelet surface expression of P-selectin and activated GPIIb/IIIa did not differ significantly between ticagrelor- and prasugrel-treated patients (both p > 0.4). In contrast, Pam3CSK4-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly lower in ticagrelor-treated patients (both p ≤ 0.005). Moreover, SFLLRN-induced platelet surface expression of P-selectin and activated GPIIb/IIIa were significantly less pronounced in patients on ticagrelor therapy compared to prasugrel-treated patients (both p < 0.03). Finally, PAR-4 mediated platelet activation as assessed by platelet surface expression of activated GPIIb/IIIa following stimulation with AYPGKF was significantly lower in patients receiving ticagrelor (p = 0.02).ConclusionTicagrelor inhibits TLR-1/2 and PAR mediated platelet activation in ACS patients more strongly than prasugrel.

Heparin Dose and Point-of-Care Measurements of Hemostasis in Cardiac Surgery—Results of a Randomized Controlled Trial

High heparin doses during cardiopulmonary bypass (CPB) have been suggested to reduce thrombin activation and consumption coagulopathy and consequently bleeding complications. The authors investigated the effect of a high heparin dose during CPB on point-of-care measurements of coagulation. The authors hypothesized that during CPB a high heparin dose compared with a lower heparin dose would reduce thrombin generation and platelet activation and tested whether this would be reflected in the results of rotational thromboelastometry (TEM) and platelet aggregation, measured with multiple electrode aggregometry (MEA). Prospective, randomized, controlled, open single-center study. University teaching hospital. Sixty-three consecutive patients undergoing elective coronary artery bypass grafting with CPB were enrolled. Patients were randomly assigned to receive either a high (600 IU/kg, n = 32) or a low (300 IU/kg, n = 31) initial dose of heparin. Target levels of activated clotting time during CPB were >600 seconds in the high heparin dose group and >400 seconds in the low heparin dose group. Blood samples were collected (1) preoperatively after induction of anesthesia, (2) 10 minutes after aortic declamping, (3) 30 minutes after protamine administration, and (4) 3 hours after protamine administration. TEM and MEA were then measured. There was no difference in blood loss up to 18 hours postoperatively (median 735 mL for high dose v 610 mL for low dose; p < 0.056) or transfusions between the groups. Total median heparin dose (54,300 IU v 27,000 IU; p = 0.001) and median antifactor Xa levels during CPB (9.38 U/mL v 5.04 U/mL; p = 0.001) were greater in the high than in the low heparin dose group. However, neither TEM nor MEA results differed significantly between the groups. Compared with a lower dose of heparin during CPB, a high dose of heparin had little effect on the point-of-care measurements of hemostasis, TEM, and MEA. Based on the similarity of platelet and coagulation activity assessments, the higher heparin dose does not appear to offer benefit during CPB.

Offset of ticagrelor prior to coronary artery bypass graft surgery for acute coronary syndromes: effects on platelet function and cellular adenosine uptake

Ticagrelor is an antagonist of both platelet adenosine diphosphate (ADP) receptor P2Y12 and equilibrative nucleoside transporter-1. Optimal timing of ticagrelor cessation prior to coronary artery bypass grafting (CABG) remains unclear. We characterized the offset of ticagrelor’s effects on platelets and cellular adenosine uptake in ticagrelor-treated patients (n = 13) awaiting CABG. Blood was drawn prior to CABG at multiple timepoints 2 to 120 (h) after the last dose of ticagrelor. Platelet function (n = 13) was assessed with multiple electrode aggregometry (MEA), expressed as arbitrary units (U) derived from area-under-the-curve (AUC) in response to ADP, and inhibition of adenosine uptake by high-performance liquid chromatography (n = 7). Mean±SD AUC was 20.3 ± 8.2 U (2 h post-ticagrelor), 33.0 ± 18.3U (24 h), 56.6 ± 30.6U (48 h), 61.4 ± 20.2U (72 h), 82.8 ± 24.2U (96 h) and 96.0 ± 15.3U (120 h). There was a significant difference between 72 h and 120 h (p = .007), but not between 96 h and 120 h (p > .99). By 96 h, all patients had AUC >31U, an accepted cutoff below which surgical bleeding risk is increased. Adenosine uptake showed no significant differences between the timepoints. These data suggest it takes 4 days for platelet reactivity to recover sufficiently after cessation of ticagrelor to avoid the excess risk of CABG-related bleeding. Discontinuing ticagrelor had no measurable effect on cellular adenosine uptake.

Reduced Activity of von Willebrand Factor after Flow-Diverting Stent Implantation for Intracranial Aneurysms: A Link to Acquired von Willebrand Disease?

Vascular devices generating high shear stress can cause type 2A acquired von Willebrand disease, which is characterized by low von Willebrand factor activity accompanied by hemorrhagic complications. The braided mesh structure of flow-diverting stents with a relatively small strut size can create abnormally high shear stress while arterial blood flows through the stent struts into the aneurysm, and flow-diverting stent may be associated with reduced von Willebrand factor activity. Aneurysmal morphologic parameters and patient data were examined retrospectively among patients who had an unruptured intracranial aneurysm treated with a flow-diverting stent. The RISTOtest (test for whole blood ristocetin-induced platelet aggregation) for von Willebrand factor activity, as well as tests for aspirin and clopidogrel/prasugrel effectiveness, were performed immediately before the endovascular procedure and 24 hours later by multiple electrode aggregometry. A total of 39 patients with 56 aneurysms were recruited, and statistical analyses were performed in 32 patents with 49 aneurysms. Compared with the baseline values, von Willebrand factor activity was reduced in 16 patients but increased in 23 patients. Aneurysmal variables (eg, neck area, volume, volume-to-neck area ratio, size ratio, and morphologic index) clearly distinguished patients with reduced von Willebrand factor activity from those with nonreduced von Willebrand factor activity. The receiver operating characteristic curve showed that the morphologic index and volume had the highest discriminative power, with an area under the curve of 0.99. In high-volume/large-neck aneurysms, flow-diverting stent implantation can cause reduced von Willebrand factor activity, which may be linked causally to acquired von Willebrand disease.

Intragastric Application of Aspirin, Clopidogrel, Cilostazol, and BPC 157 in Rats: Platelet Aggregation and Blood Clot.

We suggest that the stable gastric pentadecapeptide BPC 157 may rescue thrombocyte function. We focused on the antithrombotic agent aspirin, clopidogrel, and cilostazol application in rats; arachidonic acid, ADP, collagen, and arachidonic acid/PGE1 platelet aggregation (aggregometry) and blood clot viscoelastic properties (thromboelastometry); and the pentadecapeptide BPC 157. Rats received intragastrically for three days once daily treatment with antithrombotic agents—aspirin (10 mg/kg) or clopidogrel (10 mg/kg) or cilostazol (10 mg/kg). Medication (BPC 157 (10 μg/kg) or an equal volume of saline (5 ml/kg)) was given intragastrically, immediately after each antithrombotic agent application. For multiple electrode aggregometry and modified rotational thromboelastometry studies, blood sampling was at 2 h after last application. Adenosine diphosphate (ADP test 6.5 μM), arachidonic acid (ASPI test 0.5 mM), a combination of arachidonic acid and prostaglandin E1 (ASPI test 0.5 mM and PGE1-test 30 nM), and collagen (COL test 3.2 μg/ml) were used as aggregation agonists. Given with aspirin, clopidogrel, or cilostazol in rats, BPC 157 counteracted their inhibitory effects on aggregation activated by arachidonic acid, ADP, collagen, and arachidonic acid/PGE1. Specifically, this includes recovery of the aggregation induced by arachidonic acid (vs. aspirin, vs. clopidogrel, and vs. cilostazol), arachidonic acid/PGE1 (vs. cilostazol), ADP (vs. clopidogrel), or collagen (vs. clopidogrel). Contrarily, there is no effect on the used tests (extrinsic/intrinsic hemostasis system, the fibrin part of the clot) EXTEM, INTEM, and FIBTEM; clotting time; clot formation time; alpha-angle; maximum clot firmness; lysis index after 30 minutes; and maximum lysis. In conclusion, we revealed that BPC 157 largely rescues thrombocyte function.

Platelet reactivity expressed as a novel platelet reactivity score is associated with higher inflammatory state after coronary artery bypass grafting.

Despite therapy, patients operated using a cardiopulmonary bypass demonstrate increased platelet aggregation, which rebounds to above preoperative levels. The aim of the study was to test the interaction between platelet reactivity/activation and selected inflammatory markers in the post-operative period. In total, 103 patients with non-ST elevation acute coronary syndrome (NSTE-ACS) who were not eligible for percutaneous coronary interventions (PCI), and required urgent revascularization, were included. Platelet reactivity was measured using the PFA-100 platelet analyser, multiple electrode aggregometry, and was expressed as a novel platelet reactivity score (PRS). Patients were divided using their PRS scores into high platelet relativity or low platelet reactivity subgroups (HPR or LPR). Platelet basal activation was measured using immunoassays for soluble P-selectin and soluble CD40L. We measured high-sensitivity C-reactive protein (CRP), and used immunoassays for tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6) as inflammation markers. Significant differences between HPR and LPR groups were found for CRP (mg/l): 81.5 vs. 44.6, p < 0.02; and TNF-α (pg/l): 3.51 vs. 2.37, p < 0.02. A significant association was found between CRP, TNF-α, IL-6 and platelet reactivity (platelet reactivity score). Cohen's k showed: CRP = 0.49, p < 0.0001, TNF-α = 0.37, p < 0.002. Perioperative myocardial infarction and rhythm disturbances occurred more frequently in the high platelet reactivity group: 7 (16.3%) vs. 2 (3.3%), p < 0.04, and 9 (20.9%) vs. 4 (6.7%), p < 0.04, respectively. Inflammatory parameters CRP and TNF-α are strongly associated with platelet reactivity (expressed as PRS) in cardiopulmonary bypass graft patients. Platelet hyperreactivity in the early post-operative period combined with a systemic inflammatory state correlates with a higher risk of post-operative rhythm disturbances and myocardial infarction.

High On-treatment Platelet Reactivity and Low Platelet Response to Aspirin in Patients with Peripheral Arterial Disease Undergoing Percutaneous Transluminal Angioplasty of the Arteries of the Lower Limbs

Introduction - Patients with peripheral arterial disease (PAD) following peripheral percutaneous transluminal angioplasty (PTA) are prone to complications following the procedure, including stent thrombosis and arterial thrombosis, despite treatment with aspirin. High on-treatment platelet reactivity to aspirin is associated with increased risk of ischemic events and other vascular complications in patients following coronary interventions or PTA. We sought to assess platelet responsiveness to aspirin in patients with PAD following PTA of the arteries of lower limbs. Methods - 140 patents with PAD undergoing PTA of the arteries of the lower limbs were included in the study. 29 patients (group 1) were not treated with aspirin therapy prior to the admission and all 29 of them started aspirin treatment (75mg per day) on the day of admission. 111 patients (group 2) were treated with aspirin therapy prior to the admission and the treatment was continued during the study period. Two blood samples were collected from each of the patient in both groups (1 and 2) included in the study: one sample on the day of admission to the hospital, and the second sample one week (7 days) after the first blood collection respectively. We assessed platelet activation pathways involving the arachidonic acid receptor (ASPI). ASPI-tests using multiple electrode aggregometry (MEA) were performed in all blood samples collected in the study. 'Low responders' were patients with an ASPI-test value greater than or equal to a pre-established normal range. Results - ASPI levels in blood samples obtained on the day of admission were between 61 and 1837 (mean 771). Among the patients, who started aspirin treatment on the day of admission (group 1), ASPI levels were from 244 to 1357 (mean 1002) on the day of admission. After a week on aspirin treatment, ASPI levels among them were from 33 to 1057 (mean 368). Therefore, significant (p<0,05) decrease of ASPI levels after a week from the beginning of aspirin treatment was observed. (mean ASPI decrease from 1002 to 368 (= of 634)). There were 3 'low responders' among the patients in group 1 - ASPI levels on the 7th day from admission: 828, 856 and 1057. ASPI levels on the day of admission among them were: 1267, 1306 and 1188, and there were one of the highest ASPI levels obtained in the whole study. Among the patients in group 2 (patients treated with aspirin therapy prior to the admission), changes in ASPI levels were not statistically significant (however, both increases and decreases in ASPI levels were observed between 1st and 7th day of the study). Moreover, the assessment of ASPI levels prior to the beginning of aspirin intake among patients in group 2, including ‘low responders’, was not possible, while all of the patients in that group were treated with aspirin therapy prior to the first blood sample collection. Conclusion - High on-treatment platelet reactivity and low platelet response to aspirin in patients with peripheral arterial disease (PAD) following peripheral percutaneous transluminal angioplasty (PTA) of the arteries of the lower limbs may be the result of their platelets' high reactivity in general. Greater doses of aspirin or other antiplatelet drugs may be required for these patients to avoid postoperative complications.

Gender-based differences in platelet function and platelet reactivity to P2Y12inhibitors.

BackgroundGender influences platelet biology. Women have a larger platelet count, but gender-based differences in platelet function remain debated. We performed a study addressing gender-based differences in platelet function using point-of-care platelet function tests (PFT).MethodsThe patient population consisted of 760 cardiac surgery patients where preoperative PFT (multiple-electrode aggregometry [MEA]) were available. Platelet count and function at the ADPtest and TRAPtest were compared in the overall population and separately in patients with or without residual effects of P2Y12 inhibitors.ResultsWomen had a significantly (P = 0.001) higher platelet count but a non-significantly higher platelet reactivity to ADP. In clopidogrel-treated patients, the platelets ADP reactivity was significantly (P = 0.031) higher in women, and platelet count was the main determinant of platelet hyper-reactivity. Within patients under full clopidogrel effects, women with a platelet count ≥ 200,000 cells/μL had a significantly (P = 0.023) higher rate of high-on-treatment platelet reactivity (HTPR, 45.5%) with respect to males with a platelet count < 200,000 cells/μL (11.9%), with a relative risk of 6.2 (95% confidence interval 1.4–29).ConclusionsOur findings confirm that women have a larger platelet count than men, and that this is associated to a trend towards a higher platelet reactivity. HTPR is largely represented in women with a high platelet count. This generates the hypothesis that women requiring P2Y12 inhibitors could potentially benefit from larger doses of drug or should be treated with anti-platelet agents with a low rate of HTPR.

255Impact of the type of acute coronary syndrome on the pharmocodynamic response to P2Y12 inhibitors in the acute and maintenance phase of therapy

Abstract Background The presence of an acute coronary syndrome (ACS) is per se a predictor of reduced responsiveness to clopidogrel; in particular, patients with ST-elevation myocardial infarction (STEMI) have impaired clopidogrel-induced platelet inhibition than those with other forms of ACS. However, the impact of the type of ACS on the pharmocodynamic efficacy of more potent P2Y12 antagonists such as prasugrel or ticagrelor has not been fully elucidated to date. Purpose To assess the impact of the type of ACS on platelet inhibition mediated by P2Y12 receptor antagonists in the acute and the maintenance phase of therapy in a contemporary cohort of ACS patients undergoing percutaneous coronary intervention (PCI). Methods Substudy of a prospective, national, multicentre, pharmacodynamic registry conducted in a population of ACS patients undergoing PCI and treated with dual antiplatelet therapy including aspirin and a P2Y12 inhibitor as per clinical indication. Patients were classified according to the ACS diagnosis into groups: a) STEMI, b) non-ST-elevation ACS (NSTEACS), c) unstable angina (UA), and d) other (excluded from the present analysis). Platelet function tests (PFT) were performed at day 1 and day 30 (±5) after PCI and included: 1) VerifyNow P2Y12 assay, expressed as P2Y12 reaction units (PRUs); 2) Vasodilator-stimulated phosphoprotein (VASP) assay; and 3) Multiple electrode aggregometry (MEA). Results A total of 965 patients (372 with STEMI, 395 with NSTEACS and 198 with UA) were included in the present substudy. At day 1, the proportions of patients with each type of ACS according to the P2Y12 inhibitor received were: 1) clopidogrel (n=317): STEMI 35.0%, NSTEACS 34.4% and UA 30.6%; 2) prasugrel (n=192): STEMI 70.3%, NSTEACS 17.7% and UA 12.0%; 3) ticagrelor (n=456): STEMI 27.6%, NSTEACS 55.3% and UA 17.1%. A statistically significant reduced platelet inhibition, measured with the VerifyNow system, was observed in STEMI patients compared with the other forms of ACS in patients receiving clopidogrel (STEMI: 217.3±8.1, NSTEACS: 157.1±7.9 and UA: 164.9±8.6 PRUs; p for STEMI vs. NSTEACS &lt;0.001 and p for STEMI vs. UA &lt;0.001) and ticagrelor (STEMI: 57.7±3.8, NSTEACS: 45.2.1±2.6 and UA: 40.6±4.9 PRUs; p for STEMI vs. NSTEACS 0.008 and p for STEMI vs. UA 0.007), while a numerical trend towards greater platelet reactivity in STEMI compared to UA was observed in subjects receiving prasugrel (Figure). Remarkably, at day 30, no significant differences on platelet inhibition were observed according to the ACS type with any of the P2Y12 inhibitors. Similar results were observed with MEA and VASP assays. PD response according to the ACS type Conclusions Patients presenting with STEMI have impaired platelet inhibition mediated by P2Y12 antagonists compared to other types of ACS during the acute phase of therapy, whereas no difference is observed during the maintenance phase of treatment. Acknowledgement/Funding Funded by Instituto de Salud Carlos III through the project PI13/01012 (co-funded by European Regional Development Fund. ERDF, a way to build Europe)

P328Is there evidence of a rebound increase in platelet aggregation following withdrawal of Aspirin or Ticagrelor in patients who have previously undergone PCI and coronary stenting?

Abstract Introduction In patients treated with coronary stents previous studies have demonstrated an increased risk of acute coronary syndrome in after discontinuation of clopidogrel. In this study, we recruited patients already randomised in the GLOBAL LEADERS study allocated to discontinue aspirin treatment, while remaining on ticagrelor, 1 month after coronary stenting (Ticag MonoRx group) and a control group discontinuing ticagrelor at 6–12 months while remaining on aspirin (ASA MonoRx group). Both groups underwent platelet studies at day 0, prior to discontinuation of aspirin or ticagrelor and then on day 2, 7 and 14 day post cessation with multiple electrode aggregometry. Purpose This study was designed to look for evidence of a rebound increase in platelet aggregation in response to collagen after withdrawal of either aspirin or ticagrelor in patients who have been treated with both drugs after PCI with DES implantation. We needed a sample size of 26 patients in each group for 90% power to detect a mean change in platelet aggregation of 100 AU/min with an alpha of 0.05. The primary outcome measure was change in platelet aggregation in response to collagen between baseline and day 2, day 7 and day 14 following cessation of DAPT. A rebound effect was defined as a &gt;10% increase in collagen induced platelet aggregation on either day 2 or day 7 compared to day 14 post discontinuation of either aspirin or ticagrelor. Methods Patients provided written informed consent and underwent MEA using arachidonic acid (AA), adenosine diphosphate (ADP), thrombin receptor activator peptide (TRAP) and collagen in prespecified concentrations timed at 30 mins post phlebotomy. Results were calculated from the area under the curve and expressed as as whole number aggregation units (AU). Inbuilt QC analysis was used to determine the need for repeat assays. Results Collagen induced platelet aggregation was similar in both groups at day 0 (37 AU vs 34 AU; p=0.687) and at day 2 (55 AU vs 40 AU; p=0.12). By day 7, patients on ticagrelor monotherapy had higher collagen induced platelet aggregation (78 AU vs 37 AU; p=0.0001) and this difference was maintained at 14 days (80 AU vs 43 AU; p=0.0001). In patients, assigned to ticagrelor monotherapy after 1 month of DAPT, AA induced platelet aggregation progressively increased from day 0 to day 14. In the patients discontinuing ticagrelor and continuing on aspirin monotherapy, ADP induced platelet aggregation increased from day 0 to day 14. Rebound was seen in 6/17 (35%) patients in the ticagrelor monotherapy group versus 8/17 (47%) patients in the aspirin monotherapy group (p=0.728) with a mean peak of 21 AU (SD 6) and 10 AU (SD 6) respectively above baseline readings, p=0.003. There was no difference in TRAP induced aggregation at any time point. Figure 1 Conclusions Ticagrelor monotherapy was associated with higher collagen induced platelet aggregation than aspirin monotherapy at both 7 and 14 days post cessation of DAPT. Acknowledgement/Funding British Heart Foundation Project Grant PG/14/97/31263, AstraZeneca UK Limited

Homocysteine levels and platelet reactivity in coronary artery disease patients treated with ticagrelor

Background and aimRecurrent atherothrombotic events have been reported in certain higher risk subsets of patients even with ticagrelor, a potent first-line antiplatelet agent for the management of patients with acute coronary syndrome (ACS). Hyperhomocysteinemia is a known determinant of platelet function abnormalities. Therefore, the aim of our study was to evaluate the impact of homocysteine (Hcy) levels on platelet reactivity in patients receiving Ticagrelor. Methods and resultsPatients with ACS undergoing percutaneous coronary revascularization and on dual antiplatelet therapy with ASA + Ticagrelor (90mg/twice a day) were scheduled for platelet function assessment 30–90 days post-discharge. Aggregation tests were performed by Multiple Electrode Aggregometry (MEA). Suboptimal platelet inhibition HRPR-high residual platelet reactivity was defined if above the lower limit of normality (417 AU*min).We included 432 patients, divided according to Hcy tertiles. Higher Hcy levels were associated with age, renal failure, creatinine levels and use diuretics (p < 0.001).Patients with higher Hcy levels displayed a higher platelet reactivity at COL test (p = 0.002), and ADP test (p = 0.04), with a linear relationship between Hcy and platelet aggregation after stimulation with collagen (r = 0.202, p < 0.001), thrombin receptor peptide (r = 0.104, p = 0.05) and ADP (r = 0.145, p = 0.006).However, Hcy levels did not significantly affect the rate of HRPR with Ticagrelor (9.9% vs 13.7% vs 10.7%, p = 0.89; adjusted OR [95% CI] = [0.616–1.51], p = 0.99). ConclusionsAmong patients with ACS, despite the elevated platelet reactivity associated to hyperhomocysteinemia, DAPT with ticagrelor could overcome such phenomenon, achieving an adequate platelet inhibition in the majority of the patients.

Differential Effects of Ticagrelor With or Without Aspirin on Platelet Reactivity and Coagulation Activation: A Randomized Trial in Healthy Volunteers.

Dual antiplatelet therapy (DAPT) is standard in acute coronary heart disease but confers a bleeding risk. To compare the effects of ticagrelor‐monotherapy with ticagrelor‐based DAPT on hemostatic system activation, we conducted a randomized controlled trial in 44 volunteers using a loading‐dose regimen and measured platelet‐aggregometry triggered by adenosine diphosphate (multiple electrode aggregometry (MEA)‐ADP) and arachidonic acid (MEA‐AA), the vasodilator‐stimulated phosphoprotein (VASP), prothrombin fragment 1.2 (f1.2), and d‐Dimer. Ticagrelor‐based DAPT and ticagrelor‐monotherapy significantly decreased MEA‐ADP (Δmean: −51.4 (−56.9; −45.8) and −46.2 (−51.7; −40.7)) and VASP (Δmean: −70.3 (−76.2; −64.4) and −69.6 (−75.5; −63.7)) at 2 hours and over 24 hours. MEA‐AA was reduced significantly by both treatments (Δmean: −72.9 (−80.6; −65.3) and −25.7 (−33.3; −18.0)) at 2 hours, and stronger by ticagrelor‐based DAPT over 24 hours. Both treatments decreased f1.2 (geometric mean ratio (GMR): 0.92 (0.84; 1.01) and 0.88 (0.80; 0.96)) and d‐Dimer (GMR: 0.89 (0.86; 0.92) and 0.91 (0.88; 0.94)) at 2 hours and d‐Dimer over 24 hours. Ticagrelor‐monotherapy and ticagrelor‐based DAPT comparably affect hemostatic system activation.

Minimally invasive single locking plate combined with multiple planar screw internal fixation for pilon fracture

Objective To investigate the clinical effect of minimally invasive single locking plate combined with multiplanar screw internal fixation on pilon fracture of distal tibia. Methods A retrospective case control study was conducted to analyze the clinical data of 51 patients with pilon fracture involving distal tibial articular surface admitted to 81th Group Military Hospital of the Army from January 2013 to August 2017. Among the patients, 20 patients including 15 males and five females, aged (37.5±9.9)years were treated with closed traction reduction or open joint reduction through minimally invasive incision and single main locking plate combined with multiplanar screw placement (study group). There were 13 patients with type II and seven patients with type III according to Ru edi-Allgwer classification of fracture. In addition, 31 patients were treated with open reduction and multi-plate internal fixation (control group), including 25 males and six females, aged (43.4±11.3) years. There were 20 patients with type II and 11 with type III according to Ru edi-Allgwer classification. The operation time, fracture healing time, postoperative complications were compared between the two groups. Burwell-Charnley imaging evaluation criteria were used to assess the quality of fracture reduction. At the last follow-up, ankle function was assessed by the American Orthopedic Foot and Ankle Society (AOFAS) ankle-hind foot functional score. Results All 51 patients were followed up for 6-24 months, with an average of 16 months. The operation time was (82.5±19.2)minutes in the study group and (127.7±40.8)minutes in the control group (P 0.05). Local skin necrosis (not above the plate) occurred in two patients in the study group and in three patients in the control group, with the wounds being healed within 3 months after dressing change and vacuum sealing drainage (VSD). In the control group, one patient had severe infection and recovered after treatment of steel plate removal, debridement and irrigation, and external fixator fixation. Three patients in the control group had skin necrosis resulting in plate exposure, who received transferred skin flaps to cover the wound. The incidence of postoperative complications were 10% (2/20) and 23% in the study group and the control group, respectively (7/31)(P>0.05), with the incidence of severe complications for 0 and 13% (4/31)(P 0.05). At the last follow-up, AOFAS ankle-hind foot function scores were excellent in 12 patients, good in five patients, fair in two patients and poor in one patient in the study group with excellence rate of 85%, while the scores were excellent in 20 patients, good in six patients, fair in three patients and poor in two patients in the control group, with excellence rate of 84% (P>0.05). Conclusion For pilon fracture of distal tibia, both minimally invasive single locking plate combined with multi-plate screw internal fixation and open reduction combined with multi-plate internal fixation have good reduction effect and satisfactory recovery of ankle function, but the former can significantly shorten the operation time and reduce the incidence of serious complications after operation. Key words: Tibial fractures; Fracture fixation, internal; Pilon fractures

Association among Clopidogrel Cessation, Platelet Function, and Bleeding in Coronary Bypass Surgery: An Observational Trial.

This study sought to determine (1) the association between the length of preoperative clopidogrel discontinuation, blood loss, and transfusion requirements and (2) whether preoperative platelet function testing predicts excessive postoperative bleeding in patients undergoing coronary artery bypass grafting (CABG) surgery. In this retrospective analysis, patients undergoing CABG were divided into three groups with regard to the period between preoperative clopidogrel cessation and surgery: group 1 (n = 94, ≤3 days), group 2 (n = 100, 4-5 days), and group 3 (n = 83, 6-7 days), respectively. Impedance aggregometry (Multiplate) with arachidonic acid (ASPI) test assay (used for platelets stimulation) and adenosine diphosphate (ADP) test (used for platelets stimulation) was performed before the surgery. Primary outcome was 24 hours chest tube output (CTO) and transfusion requirements (red blood cell concentrate [RBCC], platelet concentrate [PC], fibrinogen concentrate [FC], and fresh-frozen plasma [FFP]) were considered as secondary outcomes. CTO during 24 hours was significantly higher in group 1 as compared with groups 2 and 3, respectively (p = 0.003). Considering secondary outcomes, RBCC (p = 0.043), PC (p = 0.001), FC (p = 0.003), and FFP (p = 0.010) were more frequently transfused in group 1 as compared with groups 2 and 3, respectively. Multiple electrode aggregometry ASPI and ADP tests were significantly correlated with the 24-hour CTO (ASPI test-rho = -0.258, p < 0.001; ADP test-rho = -0.164, p = 0.007). A significant correlation was observed between clopidogrel-free interval and 24-hour CTO (rho = -0.200, p < 0.001). Receiver-operating characteristics (ROC) curve analysis revealed cutoff values to delineate bleeding tendency (ASPI test ≤ 25 area under the aggregation curve [AUC], ADP test ≤63 AUC, and clopidogrel-free interval ≤3 days). Excessive postoperative bleeding occurred less frequently if the period between clopidogrel discontinuation and surgery was longer than 3 days, as compared with shorter waiting time. Inadequate recovery of the platelets function following clopidogrel cessation in preoperative period was associated with increased bleeding risk. Platelet function testing was found to be a useful tool for postoperative bleeding management in our hands.

Platelet Function Disturbance During Veno-Venous ECMO in ARDS Patients Assessed by Multiple Electrode Aggregometry-A Prospective, Observational Cohort Study.

Extracorporeal (veno-venous) membrane oxygenation (vvECMO) has been shown to have negative effects on platelet number and function. This study aimed to gain more information about the impact of vvECMO on platelet function assessed by multiple electrode aggregometry (MEA). Twenty patients with the indication for vvECMO were included. Platelet function was analyzed using MEA (Multiplate®) before (T-1), 6 h (T0), one (T1), two (T2), three (T3), and seven (T4) days after the beginning of vvECMO. Median aggregational measurements were already below the normal reference range before vvECMO initiation. Platelet aggregation was significantly reduced 6 h after vvECMO initiation compared to T-1 and spontaneously recovered with a significant increase at T2. Platelet count dropped significantly between T-1 and T0 and continuously decreased between T0 and T4. At T4, ADP-induced platelet aggregation showed an inverse correlation with the paO2 in the oxygenator. Platelet function should be assessed by MEA before the initiation of extracorporeal circulation. Although ECMO therapy led to a further decrease in platelet aggregation after 6 h, all measurements had recovered to baseline on day two. This implies that MEA as a whole blood method might not adequately reflect the changes in platelet function in the later stages of extracorporeal circulation.