ASPI Test Research Articles

Mitochondrial dysfunction in platelets from severe trauma patients - A prospective case-control study

IntroductionTrauma-induced coagulopathy (TIC) refers to an abnormal coagulation process, an imbalance between coagulation and fibrinolysis due to several pathological factors, such as haemorrhage and tissue injury. Platelet activation and subsequent clot formation are associated with mitochondrial activity, suggesting a possible role for mitochondria in TIC. Comprehensive studies of mitochondrial dysfunction in platelets from severe trauma patients have not yet been performed. MethodsIn this prospective case-control study, patients with severe trauma (ISS≥16) had venous blood samples taken at arrival to the Emergency Unit of a Level 1 Trauma Centre. Mitochondrial functional measurements (Oxygraph-2k, Oroboros) were performed to determine oxygen consumption in different respiratory states, the H2O2 production and extramitochondrial Ca2+ movements. In addition, standard laboratory and coagulation tests, viscoelastometry (ClotPro) and aggregometry (Multiplate) were performed. Measurements data were compared with age and sex matched healthy control patients. ResultsSevere trauma patients (n = 113) with a median age of 38 years (IQR, 20–51), a median ISS of 28 (IQR, 20–48) met our inclusion criteria. Oxidative phosphorylation in platelet mitochondria from severe trauma patients significantly decreased compared to controls (34.7 ± 8.8 pmol/s/mL vs. 48.0 ± 19.7 pmol/s/mL). The mitochondrial H2O2 production significantly increased and greater endogenous Ca2+ release was found in the polytrauma group. Consistent with these results, clotting time (CT) increased while maximum clot firmness (MCF) decreased with the EX-test and FIB-test in severe trauma samples. Multiplate aggregometry showed significantly decreased ADP-test (38 ± 12 AUC vs. 112 ± 14 AUC) and ASPI test (78 ± 22 AUC vs. 84 ± 28 AUC) also tended to decrease in mitochondria of polytrauma patients as compared with controls. Significant strong correlation has been demonstrated between mitochondrial OxPhos and MCF while it was negatively correlated with ISS (R2=0.448, P˂0.05), INR, CT and lactate level of patients. ConclusionsThe present study revealed that severe trauma is associated with platelet mitochondrial dysfunction resulting in reduced ATP synthesis and impaired extramitochondrial Ca2+ movement. These factors are required for platelet activation, recruitment and clot stability likely thus, platelet mitochondrial dysfunction contributes to the development of TIC.

The results of the cross-sectional study of platelet aggregative function in children with hemorrhagic syndrome

Background. Early diagnosis of thrombocytopathies in children is a relevant objective of pediatrics. Diagnostics helps prevent the development of bleeding, chronic posthemorrhagic anemia, reduces the risk of thrombosis.The aim of the study is an assessment of the incidence of platelet aggregation disorders in children with manifestations of hemorrhagic syndrome against the background of chronic pathology.Methods. The study was conducted on the basis of the Research Institute of Pediatrics and Children’s Health, Scientific Center No. 2, Petrovsky National Research Centre of Surgery in the period from January — until December 2022. 62 children were included in the study, of which 50 children were selected (21 boys and 29 girls) aged 2 years 3 months to 17 years 11 months. The median age was 9.4 (7.2; 13.4). Aggregometry was performed using an impedance semiautomatic aggregometer in whole blood.Results. Depending on the diagnosis, the children were divided into the following groups: cardiovascular diseases (CVD), lysosomal storage disorders (LSD), monogenic hereditary diseases (MHD), children with dysplastic syndrome (DS), children with pathology of the nervous system (NS). Hypoaggregation with thrombin-activating peptide (TRAP test) was detected in 28% of cases (n = 14), more often in children from the group with MHD — 10% (n = 5) and with the presenceof DS — 10% (n = 5). Hypoaggregation with adenosine diphosphate (ADP test) was detected in 20% of cases (n = 10), with arachidonic acid (ASPI test) was detected in 14% (n = 7). Hyperaggregation with the TRAP test was detected in 12% (n = 6), with the ADP test detected in 8% of cases (n = 4). Hyperaggregation with ASPI test was detected in 18% of cases (n = 9).Conclusion. The analyzed results of laboratory tests of platelet aggregation function, hypoaggregation with inducers was observed in more than half of the children, hyperaggregation was observed was present in almost half of the patients.

Association of Impedance Aggregometry-Measured Platelet Aggregation With Thromboembolic Events in Patients Who Undergo Carotid Endarterectomy: A Pilot Study

ObjectivesThe aim of the current study was to assess the relationship between thrombin receptor activator peptide 6 (TRAP test), adenosine-5′-diphosphate (ADP test) and arachidonic acid (ASPI test) and stroke/TIA, using the multiple electrode aggregometry (Multiplate®) in patients undergoing CEA. DesignA retrospective study. SettingVascular surgery operating rooms of university hospital. ParticipantsOne hundred and thirty-one out of 474 patients undergoing CEA between November 2020 and October 2022. InterventionsNone. Measurements & Main ResultsA pre-operative blood sample of all enrolled patients was analyzed using the multiplate analyzer. Receiver operating characteristics curves were generated to test the ability of TRAP, ADP and ASPI in discriminating perioperative thromboembolic stroke/TIA. A logistic LASSO regression model was used to identify factors independently associated with stroke/TIA. Eight patients suffered of perioperative stroke/TIA. Although all the platelet functional assays showed an excellent predictive performance an ADP value exciding 72 U showed the highest specificity (87%) and sensitivity (68%) in discriminating patients who had perioperative thromboembolic stroke/TIA with a negative predictive value of 99% and a positive predictive value of 15%. After LASSO regression an ADP> 72 U and need of shunt during CEA were the only two variables independently associated with perioperative stroke/TIA. ConclusionSince the ADP test was independently associated with perioperative stroke/TIA, the assessment of platelet reactivity using Multiplate® may offer potential utility in monitoring patients undergoing CEA.

Management of Microvascular Bleeding after On-Pump Cardiac Surgery in a Patient with Perioperative Diagnosis of Impairment of Platelet Responses to Adenosine Diphosphate: A Case Report and a Literature Review.

Impairment of platelet responses to adenosine diphosphate (ADP) is typified by mild to severe bleeding diathesis, easy bruising, excessive mucosal and post-operative bleeding. Patients lack full platelet activation and aggregation in response to ADP. Following research of the literature in Scopus, PubMed/MEDLINE, ScienceDirect, and the Cochrane Library, we report only 18 patients described to date with impaired platelet response to ADP, none of whom in the high bleeding-risk surgical setting or exploring potential therapeutic options. Data regarding population, putative genetic mutations, modes of inheritance, functional defects, and related clinical manifestations were retrieved from case series and case reports. A 40-year-old woman was scheduled for on-pump cardiac surgery. Her past medical history included episodes of spontaneous mucocutaneous hemorrhages of the mild entity since childhood. Multiple electrode aggregometry (MEA, Multiplate® Roche Diagnostics, Rotkreuz, Switzerland) was used to evaluate platelet response to thrombin-activated peptide-6 (TRAP), arachidonic acid (ASPI), and ADP. An inadequate platelet aggregation induced using a high concentration of ADP with normal TRAP and ASPI tests was detected preoperatively. Therefore, intravenous desmopressin (DVVAP) 0.3 μg/kg body weight was administered to manage microvascular bleeding developed after weaning from cardiopulmonary bypass (CPB). Proper management of impaired platelet response to ADP requires a systematic assessment. The Multiplate analyzer is a valuable tool to promptly detect the disorder when a high clinical suspect is present and obtain insights during high bleeding-risk surgical procedures. DVVAP can be beneficial as first-line therapy in bleeding patients to improve platelet function.

The LIPL Study Postprandial lipid profile, inflammation and platelet activity in patients with chronic coronary syndrome

Abstract Funding Acknowledgements Type of funding sources: None. Introduction In general, most people spend their day in a non-fasting state. Hence, it is suggested that the changes in atherosclerosis happen mainly under the influence of non-fasting lipids. Up to date, the studies in the postprandial state were primarily performed on healthy subjects. Aim This exploratory, cross-sectional study investigates the change in lipid profile, in biomarkers of inflammation, as well as markers of platelet activation in patients with different cardiovascular risk profiles in the postprandial state. Methods The studied population consists of 66 patients with different cardiovascular risk: patients with a history of chronic coronary syndrome (CCS) and diabetes mellitus type 2 (DM2) (n=20), CCS without DM2 (n=25), and a healthy control group (n=21). Lipid variables and markers of platelet function were assessed during the fasting state (baseline) and three and five hours after a standardized fat meal using a standardized oral fat tolerance test (OFTT), a milkshake with 90g of fat. Platelet activity was measured with the Multiplate Assay using adenosine diphosphate (ADP), arachidonic acid (ASPI) and thrombin-receptor activating peptide (TRAP) as stimulants. Results Patients with CCS and DM2 were significantly older and had the highest BMI. All patients with CCS were on acetylsalicylic acid, and 95% of CCS patients were on high-dose statins. HDL-c decreased significantly during the OFTT, with a peak after five hours (3.46±0.4 mg/dL, p<0.001). Triglycerides (TG) increased significantly during the OFTT, with a peak after five hours (130.2 ± 14.5 mg/dL, p<0.001) irrespective of the group. All other variables of the lipid profile remained stable during the OFTT. The absolute leucocyte and neutrophile count and the neutrophile to lymphocyte ratio (NLR) increased significantly during the OFTT in the whole study population, whereas the absolute lymphocyte count increased significantly only in the control group. A significant increase in platelet activity during OFTT was shown in the control group by the increased absolute platelet count, increased reticulated thrombocytes, and increased platelet reactivity measured by the ASPI and TRAP tests, respectively. There was no significant change in the markers of platelet activity during the OFTT in patients with CCS with/without DM2. Conclusion This study showed that a standardized fatty meal changes HDL-c and TG concentrations and leads to an increased postprandial inflammation response irrespective of the cardiovascular risk and to increased platelet activity in healthy subjects, while there was no increase in platelet activity in higher-risk CCS patients.

Platelet function testing using the Multiplate analyzer after administration of aspirin in Aachen minipigs.

Knowledge of platelet function in pigs and the effectiveness of antiplatelet therapy is important to ensure proper transferability from animal studies to humans. Our aim was to (1) characterize baseline platelet function of Aachen minipigs using the bedside Multiplate analyzer, (2) compare baseline platelet function with Göttingen minipigs, and (3) characterize platelet inhibition within the first 5 minutes after intravenous administration of acetylsalicylic acid (ASA). We characterized the baseline platelet function and hematological parameters in 9 Aachen minipigs. Historical data of 8 unmedicated Göttingen minipigs were used for comparison of baseline values. Platelet inhibition in Aachen minipigs was tested 1–5 minutes after intravenous administration of 500 mg ASA. Multiplate examinations included the following tests: ASPI test (to assess the effect of ASA), adenosine-diphosphate-test (ADP test) and thrombin receptor activating peptide test (TRAP test). Median values and interquartile range (IQR) of the Multiplate baseline tests in Aachen minipigs were as follows: ASPI: 39 U (IQR = 21–71), ADP: 70 U (IQR = 48–73), and TRAP: 8 U (IQR = 6–9), whereas the values in Göttingen minipigs were as follows: ASPI: 70.5 U (IQR = 60–78), ADP: 51 U (IQR = 45–66), and TRAP: 6.5 U (IQR = 4–8). ASPI values of Göttingen minipigs were significantly higher than those of Aachen minipigs (p = 0.046). Intravenous administration of ASA in Aachen minipigs resulted in significant platelet inhibition after 1 minute, which remained stable over a period of 5 minutes (p≤0.038). Aachen minipigs appeared to have a high variance in arachidonic acid-mediated platelet aggregation. In Aachen minipigs, intravenous ASA administration resulted in immediate platelet inhibition.

Multiplate Platelet Function Testing upon Emergency Room Admission Fails to Provide Useful Information in Major Trauma Patients Not on Platelet Inhibitors

Platelet dysfunction is a suggested driver of trauma-induced coagulopathy. However, there is still a paucity of data regarding the impact of injury pattern on platelet function and the association of platelet dysfunction on transfusion requirements and mortality. In this retrospective cohort study, patients were grouped into those with isolated severe traumatic brain injury (TBI group), those with major trauma without TBI (MT group), and a combination of both major trauma and traumatic brain injury (MT + TBI group). Platelet function was assessed by whole blood impedance aggregometry (Multiplate®, MP). Three different platelet activators were used: adenosine-diphosphate (ADP test), arachidonic acid (ASPI test), and thrombin activated peptide-6 (TRAP test). Blood transfusion requirements within 6 h and 24 h and the association of platelet dysfunction on mortality was investigated. A total of 328 predominantly male patients (75.3%) with a median age of 53 (37–68) years and a median ISS of 29 (22–38) were included. No significant difference between the TBI group, the MT group, and the MT + TBI group was detected for any of the investigated platelet function tests. Unadjusted and adjusted for platelet count, the investigated MP assays revealed no significant group differences upon ER admission and were not able to sufficiently predict massive transfusion, neither within the first 6 h nor for the first 24 h after hospital admission. No association between platelet dysfunction measured by MP upon ER admission and mortality was observed. Conclusion: Injury pattern did not specifically impact platelet function measurable by MP. Platelet dysfunction upon ER admission measurable by MP was not associated with transfusion requirements and mortality. The clinical relevance of platelet function testing by MP in trauma patients not on platelet inhibitors is questionable.

Plasma Fibrinogen Independently Predicts Hypofibrinolysis in Severe COVID-19.

High rates of thrombosis are present in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Deeper insight into the prothrombotic state is essential to provide the best thromboprophylaxis care. Here, we aimed to explore associations among platelet indices, conventional hemostasis parameters, and viscoelastometry data. This pilot study included patients with severe COVID-19 (n = 21) and age-matched controls (n = 21). Each patient received 100 mg aspirin therapy at the time of blood sampling. Total platelet count, high immature platelet fraction (H-IPF), fibrinogen, D-dimer, Activated Partial Thromboplastin Time, von Willebrand factor antigen and von Willebrand factor ristocetin cofactor activity, plasminogen, and alpha2-antiplasmin were measured. To monitor the aspirin therapy, a platelet function test from hirudin anticoagulated whole blood was performed using the ASPI test by Multiplate analyser. High on-aspirin platelet reactivity (n = 8) was defined with an AUC > 40 cut-off value by ASPI tests. In addition, in vitro viscoelastometric tests were carried out using a ClotPro analyser in COVID-associated thromboembolic events (n = 8) (p = 0.071) nor the survival rate (p = 0.854) showed associations with high on-aspirin platelet reactivity status. The platelet count (p = 0.03), all subjects. COVID-19 patients presented with higher levels of inflammatory markers, compared with the controls, along with evidence of hypercoagulability by ClotPro. H-IPF (%) was significantly higher among non-survivors (n = 18) compared to survivors (p = 0.011), and a negative correlation (p = 0.002) was found between H-IPF and plasminogen level in the total population. The platelet count was significantly higher among patients with high on-aspirin platelet reactivity (p = 0.03). Neither the ECA-A10 (p = 0.008), and ECA-MCF (p = 0.016) were significantly higher, while the tPA-CFT (p < 0.001) was significantly lower among patients with high on-aspirin platelet reactivity. However, only fibrinogen proved to be an independent predictor of hypofibrinolysis in severe COVID-19 patients. In conclusion, a faster developing, more solid clot formation was observed in aspirin ‘non-responder’ COVID-19 patients. Therefore, an individually tailored thromboprophylaxis is needed to prevent thrombotic complications, particularly in the hypofibrinolytic cluster.

Why Me? To Be an Ultra-Responder to Antiplatelet Therapy: A Case Report

Background: Platelet function testing is a valid tool to investigate the clinical response to antiplatelet therapy in different clinical settings; in particular, it might supply helpful information in patients with cerebrovascular disease. Oral antiplatelet treatment, such as Aspirin (ASA) and Clopidogrel, is the gold standard in secondary stroke prevention of non-cardiogenic ischemic stroke; conversely, its application as a primary prevention therapy is not routinely recommended in patients with vascular risk factors. Multiple electrode platelet aggregometry (MEA) impedance aggregometer is a validated device to test platelet inhibition induced by ASA or Clopidogrel.Case Report: We report the case of a 78-year-old patient without relevant clinical history, taking ASA as primary prevention strategy, who was admitted for sudden onset of dysarthria and left facial hyposthenia during physical effort. Brain CT revealed two small subcortical bilateral spontaneous intracranial hemorrhages. Platelet aggregometry with MEA performed upon admission revealed a very strong platelet inhibition induced by ASA (result of the ASPI Test was 5 U, consistent with an ultra-responsiveness to ASA, and the cutoff value of correct responsiveness is <40 U). MRI at longitudinal follow-up revealed the presence of two small cavernous angioma underlying hemorrhagic spots.Conclusion: The evaluation of platelet reactivity in stroke patients undergoing antiplatelet therapies, not commonly performed in clinical practice, could be useful to optimize prevention strategies; the verification of the biological effectiveness of ASA or Clopidogrel could be a valid tool in the definition of each patient's risk profile, particularly in patients with cerebrovascular disease known to be at increased risk for both hemorrhagic and thrombotic complications.

Pre-operative platelet reactivity is a strong, independent predictor of bleeding complications after branched endovascular thoracoabdominal aortic aneurysm repair

Endovascular aortic repair (EVAR) an alternative to open surgical repair of thoracoabdominal aortic aneurysm (TAAA). The effect of EVAR on platelet reactivity is unknown. We prospectively determined the effect of branched EVAR (bEVAR) on platelet reactivity in patients with TAAA, and evaluated the predictive value of preoperative platelet reactivity for post-operative bleeding in 50 consecutive patients undergoing elective bEVAR (mean age 70.9 ± 5.7 years, 66% male). Blood samples were collected within 24 hours before bEVAR, after bEVAR and at hospital discharge. Platelet reactivity was assessed with impedance aggregometry using ASPI, ADP and TRAP tests. Platelet reactivity decreased within 24 hours after bEVAR compared to the measurement before bEVAR in all tests (p ≤ 0.04), with a further decrease in hospital discharge in the ADP test (p = .004). Twenty-three patients experienced post-operative bleeding complications (transfusion ≥2 red blood cell [RBC] units). Preoperative platelet reactivity below the cutoff value of 30 AUC units predicted post-operative bleeding with 78% sensitivity and 59% specificity (p = .045). In the multivariable analysis, platelet reactivity was the only independent predictor of postoperative bleeding (OR 6.507, 95% CI 1.227–34.506, p = .028). We conclude that platelet reactivity decreases following bEVAR of TAAA and is a strong and independent predictor for postoperative bleeding complications.

Platelet Receptor Activity for Predicting Survival in Patients with Intracranial Bleeding.

Blood coagulation disorders in patients with intracranial bleeding as a result of head injuries or ruptured aneurysms are a diagnostic and therapeutic problem and appropriate assessments are needed to limit CNS damage and to implement preventive measures. The aim of the study was to monitor changes in platelet aggregation and to assess the importance of platelet dysfunction for predicting survival. Platelet receptor function analysis was performed using the agonists arachidonic acid (ASPI), adenosine diphosphate (ADP), collagen (COL), thrombin receptor activating protein (TRAP), ristocetin (RISTO) upon admission to the ICU and on days 2, 3, and 5. On admission, the ASPI, ADP, COL, TRAP, and RISTO tests indicated there was reduced platelet aggregation, despite there being a normal platelet count. In ‘Non-survivors’, the platelet response to all agonists was suppressed throughout the study period, while in ‘Survivors’ it improved. Measuring platelet function in ICU patients with intracranial bleeding is a strong predictor related to outcome: patients with impaired platelet aggregation had a lower 28-day survival rate compared to patients with normal platelet aggregation (log-rank test p = 0.014). The results indicated that measuring platelet aggregation can be helpful in the early detection, diagnosis, and treatment of bleeding disorders.

Bleeding risk stratification in coronary artery surgery: the should-not-bleed score

BackgroundAn estimated 20% of allogeneic blood transfusions in the United States are associated with cardiac surgery. It is estimated that 11% of red cell resources were used for transfusion support of patients undergoing coronary artery bypass grafting (CABG) with a documented wide variability in transfusion rate (7.8 to 92.8%). To address the issue of unnecessary transfusions within the CABG population, we developed a model to predict which patients are at low risk of bleeding for whom transfusion treatment might be considered unnecessary. Herein we present our “SHOULD-NOT-BLEED-SCORE” application developed for the Windows® software platform which is based on our previous research.MethodsThis study is aimed to develop a user-friendly application that stratifies patients with respect to bleeding risk. The statistical model we used in our previous research was focused on detection of CABG patients at low risk of bleeding. The rationale behind such an approach was to identify a CABG patient subgroup at low risk of bleeding. By identifying patients at low risk of bleeding we can define a subgroup of patients for whom transfusion treatment might be considered unnecessary. We developed a Windows platform application based on risk modelling which we previously calculated for 1426 patients undergoing elective CABG from January 2010 to January 2018.ResultsThe SHOULD-NOT-BLEED-SCORE risk score is developed for the Windows software platform. A mathematical model that is based on multivariate analysis was used for app development. The variables that entered the scoring system were: Age; Body Mass Index; Chronic Renal Failure; Preoperative Clopidogrel Exposure; Preoperative Red Blood Cells Count; Preoperative Fibrinogen Level; Preoperative Multiplate ASPI test area under the curve (AUC) units. The SHOULD-NOT-BLEED-SCORE identifies/predicts patients without a risk for excessive bleeding with strong discriminatory performance (Receiver Operating Curve (ROC) analysis AUC 72.3%, p < 0.001).ConclusionThe SHOULD-NOT-BLEED risk scoring application may be useful in the preoperative risk screening process. The clinical and economic burden associated with unnecessary transfusions may be adequately addressed by a preoperative scoring system detecting patients at low risk of bleeding for whom transfusion treatment might be considered unnecessary.

Relation Between Platelet Reactivity Levels and Diabetic Retinopathy Stage in Patient with Type 2 Diabetes Mellitus by Using Multiplate Whole Blood Aggregometry

ABSTRACT Purpose: To test the hypothesis of a possible association between platelet reactivity and the severity of diabetic retinopathy using Multiplate whole blood aggregometry in type 2 diabetes mellitus patients. Methods: Of 157 patients were divided to three groups based on the severity of diabetic retinopathy (normal, non-proliferative and proliferative [ordinal among group 1-2-3]). Platelet reactivity was measured using arachidonic acid response to the ASPI and ADP platelet test. The association between DR stage and the degree of platelet reactivity (predictor variable) ASPI, ADP, systolic blood pressure, age, hypertension, body mass index (BMI), HbA1c, creatinine, Microalbumin, platelet, triglyceride/HDL and Hscrp variables were evaluated using ordinal logistic regression models (Model 1). The association between DR presence (outcome variable (group 1 vs group 2 and 3)) and the presence of variables was evaluated using binary logistic regression models (Model 2).Results: A comparison of the laboratory parameters of the three groups revealed that the ASPI, ADP, glucose and HbA1c values were significantly higher in Group-3 than Group-1. ASPI (odds-ratio OR: 1.044[1.021–1.09], p < .001], ADP (OR: 1.033[1.010–1.10], p: 0.002] and HbA1c (OR: 2.42(1.22, 4.94), p < .001) were demonstrated to be associated with stage of DR while the other variables were not. In binary logistic regression (model-2) analysis; ASPI (OR: 1.061[1.031–1.1], p < .001], ADP (OR: 1.03(1.01, 1.06), p: 0.045] and HbA1c (OR: 4.37 (1.67, 11.36)], p: 0.002) were associated with DR while the other variables were not. Conclusion: Herewith, we demonstrated that higher platelet reactivity measured by multiplate ASPI and ADP was significantly associated with stages of DR. Therefore, these measurements may be useful to predict the severity of DR in the clinical practice of physicians.

Impact of renin angiotensin system inhibitors on homocysteine levels and platelets reactivity in patients on dual antiplatelet therapy

Background and aimsDual antiplatelet therapy (DAPT) and Renin-angiotensin system inhibitors (RASi) represent the cornerstone in the treatment of patients undergoing percutaneous coronary interventions (PCI), mainly after an acute ischemic event. However, high-on treatment residual platelet reactivity (HRPR), is not infrequent despite optimal medical treatment. Homocysteine (Hcy) is a metabolite of methionine catabolism linked to atherothrombosis. Recently, a potential crosstalk between RAS and Hcy has been suggested, potentially favouring platelet aggregation and cardiovascular disease.Therefore, we aimed to investigate the impact of RASi on Hcy levels and platelet aggregation in patients on DAPT after PCI. Methods and resultsPatients undergoing PCI on DAPT with ASA plus an ADP-antagonist (clopidogrel, ticagrelor or prasugrel), were included. RASi comprised angiotensin converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB). Aggregation tests were performed by Multiple Electrode Aggregometry. We included 1210 patients, of whom 862 (71.2%) were on treatment with RASi. Overall, DAPT composition was ASA+clopidogrel in 566 (46.8%) patients, ASA+ticagrelor in 428 (35.4%) and ASA+prasugrel in 216 (17.9%). Median values of Hcy were higher in RASi patients (p = 0.006), who displayed a higher percentage of Hcy above the median value (52.4% vs. 44.8%, p = 0.019, adjustedOR [95%CI] = 1.40 [1.04–1.88], p = 0.027). No differences in HRPR rate were found according to RASi use for ASPI test (3.6% vs. 3.3%, p = 0.88) and ADP test (25.6% vs. 24.3%,p = 0.62; adjustedOR [95%CI] = 1.23 [0.89–1.70], p = 0.220) and according to ADP-antagonist type. A direct linear relationship was observed between platelet reactivity and Hcy in both patients receiving RASi and untreated ones, with higher values of platelet aggregation being observed in patients with Hcy above the median, independently from RASi administration and DAPT strategy. ConclusionIn patients on DAPT after PCI, RASi treatment did not emerge as an independent predictor of HRPR. However, the levels of Hcy were significantly elevated in patients on RASi and related to higher values of platelet reactivity, independently from the DAPT strategy.

Hypothermia-Associated Coagulopathy: A Comparison of Viscoelastic Monitoring, Platelet Function, and Real Time Live Confocal Microscopy at Low Blood Temperatures, an in vitro Experimental Study.

IntroductionHypothermia has notable effects on platelets, platelet function, fibrinogen, and coagulation factors. Common laboratory techniques cannot identify those effects, because blood samples are usually warmed to 37°C before analysis and do not fully reflect the in vivo situation. Multiple aspects of the pathophysiological changes in humoral and cellular coagulation remain obscure. This in vitro experimental study aimed to compare the measurements of thromboelastometry (TEM), multiple-electrode aggregometry (MEA) and Real Time Live Confocal Imaging for the purpose of identifying and characterizing hypothermia-associated coagulopathy.MethodsBlood samples were drawn from 18 healthy volunteers and incubated for 30 min before being analyzed at the target temperatures (37, 32, 24, 18, and 13.7°C). At each temperature thromboelastometry and multiple-electrode aggregometry were measured. Real Time Live Confocal Imaging was performed at 4, 24, and 37°C. The images obtained by Real Time Live Confocal Imaging were compared with the functional results of thromboelastometry and multiple-electrode aggregometry.ResultsThromboelastometry standard parameters were impaired at temperatures below baseline 37°C (ANOVA overall effect, p < 0.001): clotting time was prolonged by 27% at 13.7°C and by 60% at 18°C (p < 0.044); clot formation time was prolonged by 157% (p < 0.001). A reduction in platelet function with decreasing temperatures was observed (p < 0.001); the area under the curve at 13.7°C was reduced by 96% (ADP test), 92% (ASPI test), and 91% (TRAP test) of the baseline values. Temperature-associated changes in coagulation were visualized with Real Time Live Confocal Imaging. Molecular changes such as the temperature-associated decrease in the fibrin network are paralleled by cellular effects like the lesser activity of the platelets as a result of decreased temperature. The maximum clot firmness (MCF) in TEM only changed slightly within the temperature range tested.ConclusionThe inhibitory effects of temperature on clot formation were visualized with Real Time Live Confocal Microscopy and compared with standard point-of-care testing. Inhibition of clotting factors and impaired platelet function are probably a result of hypothermia-induced impairment of thrombin. Measurement of MCF in TEM does not fully concur with Real Time Live Confocal Microscopy or MEA in hypothermia.

Synergistic platelet inhibition between Omega-3 and acetylsalicylic acid dose titration; an observational study

BackgroundOmega-3 and acetylsalicylic acid (ASA) are two widely used “over-the-counter” drugs. Previous research has shown multiple electrode aggregometry (MEA) can detect ASA and varying Omega-3 platelet inhibiting effects. Synergistic platelet inhibiting effects of ASA and Omega-3 have been found using other methods than MEA. The aim of this study was to investigate the antiplatelet effects of Omega-3, and ASA synergism with MEA.MethodsTen healthy male volunteers ingested Omega-3 (1260 mg/day) for 5 days. MEA was used to analyse platelet function before and after Omega-3 intake. Aggregation was initiated using three different agonists and measured as area under the curve (AUC): adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP) and arachidonic acid (ASPI). Two concentrations of ASA were dose titrated ex vivo to 2 out of 3 ASPI test cells in order to measure synergism between Omega-3 and ASA.ResultsFollowing 5 days Omega-3 intake, ADP, TRAP and ASPI AUC did not change significantly. In vitro ASA before Omega-3 intake, reduced ASPI AUC < 30 U, indicating a strong platelet inhibiting effect. Below this AUC level, the 5 days Omega-3 intake increased ASPI-AUC with the ex vivo added low dose ASA (P = 0.02) and high dose ASA (P = 0.04).ConclusionsNo synergism between ASA and Omega-3 was found using the MEA ASPI test. The surprising increase in ASPI-AUC following Omega-3 intake and ex vivo ASA suggest that there are methodological issuses with the MEA ASPI test.Trial registrationTrial registration ISRCTN78027929. Registered 19 May 2015.

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.

Estimating short- and long-term reference change values and index of individuality for tests of platelet function

BackgroundIn order to manage risks of bleeding and thrombosis after some surgical procedures, platelet function is often measured repeatedly over days or weeks using laboratory tests of platelet function. To interpret test results in the perioperative period, it is necessary to understand analytical, biological and between-person variation. MethodsWe collected three separate blood specimens from 16 healthy volunteers on the first study day, and one additional specimen from each volunteer 1, 2, and 3 months later. Arachidonic acid-induced and adenosine diphosphate (ADP)-induced platelet function were measured in duplicate by whole blood impedance aggregometry using Multiplate (ASPI/ADP tests) and VerifyNow (Aspirin Reaction Units [ARU] and P2Y12 Reaction Units [PRU]). The analytical variation (CVA), within-subject variation (CVI), between-subject variation (CVG), index of individuality (II), and reference change values (RCV) were calculated. ResultsVerifyNow ARU demonstrated the smallest short-term and long-term variability (CVA, CVI, and CVG ~1%), resulting in short- and long-term RCV values <5%. II was also higher (1.92) for VerifyNow ARU than other platelet function tests. Multiplate ASPI and ADP tests had the highest RCV both short-(19.0% and 25.2%, respectively) and long-term (32.1% and 39.6%, respectively) due to increased CVA (>5%) and CVI (3.9–13.1%). VerifyNow PRU had a lower RCV than Multiplate ADP; but was the only test with II <0.6. ConclusionsVerifyNow ARU results can be interpreted relative to a fixed cut-off or population-based reference interval; or relative to small changes in an individual’s previous values. VerifyNow PRU and Multiplate ASPI and ADP tests should only be interpreted based upon relative change; and can only distinguish relatively large (>23%) changes over several weeks.

257Ticagrelor decreases concentrations of prothrombotic extracellular vesicles compared to clopidogrel

Abstract Introduction The platelet P2Y12 antagonist ticagrelor reduces all-cause and cardiovascular mortality in patients after acute myocardial infarction (AMI) compared to clopidogrel, but the underlying mechanism is unknown. Because activated platelets release prothrombotic extracellular vesicles (EVs) exposing P-selectin, fibrinogen and phosphatidylserine (PS), we hypothesized that ticagrelor inhibits the release of EVs compared to clopidogrel. Purpose To compare the effects of ticagrelor and clopidogrel on the concentrations of platelet EVs in relation to platelet reactivity, and on the concentrations of EVs from other P2Y12-exposing cells (leukocytes, endothelial cells). Methods After the percutaneous coronary intervention, patients with first AMI (n=60, age 64.5±10.8 years, 68% male) were randomized in 1:1 ratio to antiplatelet therapy with ticagrelor or clopidogrel. Venous blood was collected from fasting patients at randomisation, 48 hours after randomisation, and 6 months following the index hospitalization with informed consent. Flow cytometry (Apogee A60 Micro) was used to determine plasma concentrations of EVs labelled with antibodies for activated platelets (CD61, CD62p; PEVs), fibrinogen, phosphatidylserine (PS), leukocytes (CD45; LEVs) and endothelial cells (CD146; EEVs). To maximize the reliability, the analysis of the 1,224 flow cytometry data files was performed with software enabling automatic flow rate stabilization, diameter and refractive index determination, size distribution fitting, fluorescent gate determination, and statistics reporting (MATLAB R2018a). To differentiate between EVs and small platelets, only particles &lt;700 nm were included. Platelet reactivity was assessed by impedance aggregometry (Multiplate Analyzer) using the ASPI test (arachidonic acid, 0.5 mM) and the ADP test (adenosine diphosphate, 6.5 μM). Results Concentrations of PEVs exposing P-selectin, fibrinogen and PS were lower after 6 months of therapy with ticagrelor compared to clopidogrel, and decreased over time (p=0.034; p=0.021; p=0.018, respectively). Concentrations of LEVs were lower both after 72 hours and after 6 months of therapy with ticagrelor compared to clopidogrel (p=0.018; p&lt;0.001, respectively). There was a trend towards higher concentrations of EEVs both after 72 hours and after 6 months of therapy with ticagrelor, compared to clopidogrel. There were no correlations between the concentrations of PEVs and platelet reactivity. Conclusions Ticagrelor decreases concentrations of prothrombotic EVs, compared to clopidogrel, suggesting that the clinical benefits of ticagrelor may be at least partly explained by inhibition of EV release from activated platelets. Further research is warranted to link this effect with clinical outcomes. Acknowledgement/Funding Medical University of Warsaw (1WR/PM2/18)

Platelet function testing in pigs using the Multiplate® Analyzer.

For endovascular research pigs are an established animal model. However, experiences regarding analyses of platelet inhibition in pigs using the Multiplate® Analyzer are limited. The aims of the present study were to investigate if (1) the Multiplate® Analyzer is a suitable method for examination of porcine platelet function using manufacturers’ recommendations for human blood, and (2) platelet inhibition can be induced with acetylsalicylic acid (ASA) and clopidogrel in pigs reliably, and if (3) non-responders to one of the drug can be detected. Additionally we examined differences in (4) the effectiveness of ASA between oral administration and intravenous application, and (5) between domestic pigs (German Landrace; GL) and miniature pigs (MP). We investigated platelet function of 36 unmedicated pigs (GL n = 28; MP n = 8). In addition, 32 blood samples taken from medicated pigs (GL n = 15; MP n = 17) were analysed. Platelet inhibition was induced in four different ways: (1) 500 mg ASA intravenously (n = 11), (2) 500 mg ASA intravenously and 450 mg clopidogrel orally (n = 5), (3) 250 mg ASA orally (n = 11), (4) 250 mg ASA orally and 75 mg clopidogrel orally (n = 5). Results of the ASPI and ADP test of the Multiplate® Analyzer subtests in unmedicated and medicated pigs were in a comparable range to results known from humans. Application of ASA decreased the mean values of the ASPI test significantly regardless of the application method. Joined administration of ASA and clopidogrel also decreased the mean values of the ADP test significantly. Both, oral and intravenous administrations of ASA as well as oral administration of clopidogrel effectively inhibited platelet function in pigs. One pig did not respond to clopidogrel. We found no differences between domestic and miniature pigs regarding reference values in unmedicated pigs and the effectiveness of ASA and clopidogrel.