Platelet Activation Markers Research Articles

Preanalytical conditions for multiparameter platelet flow cytometry

BackgroundFlow cytometry is an important technique for understanding multiple aspects of blood platelet biology. Despite the widespread use of the platform for assessing platelet function, the optimization and careful consideration of preanalytical conditions, sample processing techniques, and data analysis strategies should be regularly assessed. When set up and designed with optimal conditions, it can ensure the acquisition of robust and reproducible flow cytometry data. However, these parameters are rarely described despite their importance. ObjectivesWe aimed to characterize the effects of several preanalytical variables on the analysis of blood platelets by multiparameter fluorescent flow cytometry. MethodsWe assessed anticoagulant choice, sample material, sample processing, and storage times on 4 distinct and commonly used markers of platelet activation, including fibrinogen binding, expression of CD62P and CD42b, and phosphatidylserine exposure. ResultsThe use of suboptimal conditions led to increases in basal platelet activity and reduced sensitivities to stimulation; however, the use of optimal conditions protected the platelets from artifactual stimulation and preserved basal activity and sensitivity to activation. ConclusionThe optimal preanalytical conditions identified here for the measurement of platelet phenotype by flow cytometry suggest a framework for future development of multiparameter platelet assays for high-quality data sets and advanced analysis.

Platelets and the Atherosclerotic Process: An Overview of New Markers of Platelet Activation and Reactivity, and Their Implications in Primary and Secondary Prevention.

The key role played by platelets in the atherosclerosis physiopathology, especially in the acute setting, is ascertained: they are the main actors during thrombus formation and, thus, one of the major investigated elements related to atherothrombotic process involving coronary arteries. Platelets have been studied from different points of view, according with the technology advances and the improvement in the hemostasis knowledge achieved in the last years. Morphology and reactivity constitute the first aspects investigated related to platelets with a significant body of evidence published linking a number of their values and markers to coronary artery disease and cardiovascular events. Recently, the impact of genetics on platelet activation has been explored with promising findings as additional instrument for patient risk stratification; however, this deserves further confirmations. Moreover, the interplay between immune system and platelets has been partially elucidated in the last years, providing intriguing elements that will be basic components for future research to better understand platelet regulation and improve cardiovascular outcome of patients.

Associations between ambient PM2.5 and markers of platelet activity in pregnant women in Puerto Rico

Associations between ambient PM2.5 and markers of platelet activity in pregnant women in Puerto Rico

Complement inhibition can decrease the haemostatic response in a microvascular bleeding model at multiple levels.

Haemostasis is a crucial process by which the body stops bleeding. It is achieved by the formation of a platelet plug, which is strengthened by formation of a fibrin mesh mediated by the coagulation cascade. In proinflammatory and prothrombotic conditions, multiple interactions of the complement system and the coagulation cascade are known to aggravate thromboinflammatory processes and increase the risk of arterial and venous thrombosis. Whether those interactions also play a relevant role during the physiological process of haemostasis is not yet completely understood. The aim of this study was to investigate the potential role of complement components and activation during the haemostatic response to mechanical vessel injury. We used a microvascular bleeding model that simulates a blood vessel, featuring human endothelial cells, perfusion with fresh human whole blood, and an inducible mechanical injury to the vessel. We studied the effects of complement inhibitors against components of the lectin (MASP-1, MASP-2), classical (C1s), alternative (FD) and common pathways (C3, C5), as well as a novel triple fusion inhibitor of all three complement pathways (TriFu). Effects on clot formation were analysed by recording of fibrin deposition and the platelet activation marker CD62P at the injury site in real time using a confocal microscope. With the inhibitors targeting MASP-2 or C1s, no significant reduction of fibrin formation was observed, while platelet activation was significantly reduced in the presence of the FD inhibitor. Both common pathway inhibitors targeting C3 or C5, respectively, were associated with a substantial reduction of fibrin formation, and platelet activation was also reduced in the presence of the C3 inhibitor. Triple inhibition of all three activation pathways at the C3-convertase level by TriFu reduced both fibrin formation and platelet activation. When several complement inhibitors were directly compared in two individual donors, TriFu and the inhibitors of MASP-1 and C3 had the strongest effects on clot formation. The observed impact of complement inhibition on reducing fibrin clot formation and platelet activation suggests a role of the complement system in haemostasis, with modulators of complement initiation, amplification or effector functions showing distinct profiles. While the interactions between complement and coagulation might have evolved to support haemostasis and protect against bleeding in case of vessel injury, they can turn harmful in pathological conditions when aggravating thromboinflammation and promoting thrombosis.

Evaluation of a rail logistics transmission system for the transportation of blood components within a medical centre.

Rail logistics transmission systems (RLTSs) are commonly used for the transportation of blood samples, pathological specimens and other medical materials in many hospitals, as they are rapid, secure, cost-effective and intelligent. However, few studies have evaluated blood component transportation from blood banks to the patient care areas of hospitals using RLTS. In this study, we evaluate the RLTS used for the transportation of blood components within a medical centre. The dispatch of blood components, including packed red blood cells (pRBCs), fresh frozen plasma (FFP), cryoprecipitate and platelet units, from a blood bank to critical care areas or general wards was done using RLTS. Parameters such as the delivery time, temperature, physical integrity and blood component quality were evaluated via analytical testing using specimens obtained before and after transportation by RLTS. The turnaround time and temperature of all tested blood units via RLTS transportation were able to meet the clinical demands of blood component delivery (median time: 323 s [118-668 s]; temperature variation: 4.5-8.9°C for pRBCs and FFP and 21.5-23.5°C for cryoprecipitate and platelet units). Furthermore, parameters of pRBC quality, including the haemolysis index and potassium and lactate dehydrogenase levels in plasma, were not significantly different before and after transportation through RLTS. Similarly, RLTS transportation affected neither the basic coagulation test results in FFP and cryoprecipitate specimens nor platelet aggregation and activation markers in apheresis platelet specimens. Hospital-wide delivery of blood components via RLTS seems to be safe, reliable and cost-effective and does not have any negative impact on blood quality. Therefore, the establishment of standard criteria, protocols and guidelines based on further studies is needed.

Soluble C-type lectin-like receptor 2 in stroke (CLECSTRO) study: protocol of a multicentre, prospective cohort of a novel platelet activation marker in acute ischaemic stroke and transient ischaemic attack

IntroductionSoluble C-type lectin-like receptor 2 (sCLEC-2) is a new biomarker for platelet activation, which can be easily measured by usual blood collection. We conducted the CLECSTRO, a prospective, observational cohort...

VEGF-A plasma levels are associated with impaired DLCO and radiological sequelae in long COVID patients.

Long COVID, also known as post-acute sequelae of COVID-19 (PASC), is characterized by persistent clinical symptoms following COVID-19. To correlate biomarkers of endothelial dysfunction with persistent clinical symptoms and pulmonary function defects at distance from COVID-19. Consecutive patients with long COVID-19 suspicion were enrolled. A panel of endothelial biomarkers was measured in each patient during clinical evaluation and pulmonary function test (PFT). The study included 137 PASC patients, mostly male (68%), with a median age of 55years. A total of 194 PFTs were performed between months 3 and 24 after an episode of SARS-CoV-2 infection. We compared biomarkers evaluated in PASC patients with 20 healthy volunteers (HVs) and acute hospitalized COVID-19 patients (n = 88). The study found that angiogenesis-related biomarkers and von Willebrand factor (VWF) levels were increased in PASC patients compared to HVs without increased inflammatory or platelet activation markers. Moreover, VEGF-A and VWF were associated with persistent lung CT scan lesions and impaired diffusing capacity of the lungs for carbon monoxide (DLCO) measurement. By employing a Cox proportional hazards model adjusted for age, sex, and body mass index, we further confirmed the accuracy of VEGF-A and VWF. Following adjustment, VEGF-A emerged as the most significant predictive factor associated with persistent lung CT scan lesions and impaired DLCO measurement. VEGF-A is a relevant predictive factor for DLCO impairment and radiological sequelae in PASC. Beyond being a biomarker, we hypothesize that the persistence of angiogenic disorders may contribute to long COVID symptoms.

Effect of proprotein convertase subtilisin/kexin type 9 on platelet activation associated with sepsis

To investigate the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on platelet activation in sepsis. (1) Clinical trial: a prospective study was conducted. Patients with sepsis and septic shock aged ≥ 18 years old who met the diagnostic criteria of Sepsis-3 admitted to the department of intensive care medicine of the Affiliated Hospital of Binzhou Medical College from January to October in 2021 were selected as subjects. Healthy subjects in the same period were taken as healthy control group. Platelet count (PLT) in the first routine blood test after admission was recorded. Venous blood was taken 1 day after diagnosis, and serum PCSK9 level was determined by enzyme-linked immunosorbent assay (ELISA). The differences of PCSK9 level and PLT between the two groups were compared, and subgroup analysis was conducted based on PLT for patients with sepsis. The correlation between PCSK9 level and PLT in septic patients was analyzed by Pearson correlation method. (2) Animal experiment: 80 male C57BL/6 mice were randomly divided into control group, sepsis model group [lipopolysaccharide (LPS) group], PCSK9 inhibitor pretreatment group (PCSK9 inhibitor+LPS group) and PCSK9 inhibitor control group (PCSK9 inhibitor group), with 20 mice in each group. The mouse model of sepsis was reproduced by intraperitoneal injection of LPS 12 mg/kg, and the control group and PCSK9 inhibitor group were intraperitoneally injected with the same amount of sterile normal saline. PCSK9 inhibitor+LPS group and PCSK9 inhibitor group were pretreated with PCSK9 inhibitor 5 mg/kg intraperitoneal injection for 7 days before injection of LPS or normal saline, respectively, and the control group and LPS group were injected with an equal amount of sterile normal saline. The lung tissues were taken for pathological and immunohistochemical observation 24 hours after modeling. Blood was taken from the heart for determining PLT. Platelet activation was detected by flow cytometry. The expression level of platelet-activation marker CD40L was detected by Western blotting. (1) Clinical trial: there were 57 cases in the sepsis group and 27 cases in the healthy control group. Serum PCSK9 level in the sepsis group was significantly higher than that in the healthy control group (μg/L: 232.25±72.21 vs. 191.72±54.92, P < 0.05), and PLT was significantly lower than that in the healthy control group [×109/L: 146.00 (75.50, 204.50) vs. 224.00 (194.00, 247.00), P < 0.01]. Subgroup analysis showed that the serum PCSK9 level in the thrombocytopenia patients (n = 20) was significantly higher than that in the non-thrombocytopenia patients (n = 37; μg/L: 264.04±60.40 vs. 215.06±72.95, P < 0.01). Correlation analysis showed a significant negative correlation between serum PCSK9 levels and PLT in septic patients (r = -0.340, P = 0.010). (2) Animal experiment: there were no significant pathological changes in lung tissue in the control group and PCSK9 inhibitor group under light microscope, and no significant differences in PLT, platelet activation and plasma CD40L protein expression was found between the two groups. In the LPS group, a large number of inflammatory cells were infiltrated in the pulmonary interstitium, the alveolar structure was damaged obviously, the alveolar septum was widened, the alveolar cavity was extensively bleeding, the capillary dilatation with bleeding and platelet aggregation were found, the PLT was significantly decreased, the platelet activation and the expression level of CD40L protein in plasma were significantly increased. The infiltration of inflammatory cells in lung tissue of mice in the PCSK9 inhibitor+LPS group was reduced to a certain extent, the thickening of alveolar septa was reduced, the platelet aggregation in lung tissue was decreased as compared with the LPS group, the PLT was significantly increased (×109/L: 515.83±46.60 vs. 324.83±46.31, P < 0.05), the platelet activation and the expression level of CD40L protein in plasma were significantly decreased [positive expression rate of platelet activation dependent granule surface facial mask protein CD62P: (12.15±1.39)% vs. (18.33±2.74)%, CD40L protein (CD40L/β-actin): 0.77±0.08 vs. 1.18±0.10, both P < 0.05]. PCSK9 level has a certain effect on promoting platelet activation in sepsis, and inhibition of PCSK9 level may have potential research value in improving adverse outcomes caused by sepsis thrombocytopenia.

Characterization of extracellular vesicles in COVID-19 infection during pregnancy.

Introduction: SARS-CoV-2 infection may cause a severe inflammatory response, inflicting severe morbidity and mortality. This risk is modestly increased in pregnant patients. Despite the hypercoagulability and immunosuppression associated with pregnancy, most pregnant women experience a mild COVID-19 infection. Maternal extracellular vesicles (EVs) may interact with endothelial and immune components to facilitate a favorable disease course. This pilot study aimed to explore the characteristics of EVs released during COVID-19 infection occurring during the third trimester of pregnancy. Methods: In this prospective study, blood samples were obtained from 16 healthy non-pregnant (NP), 18 healthy-pregnant (HP), and 22 COVID-19 positive pregnant subjects (CoV-P). Disease course and pregnancy outcomes were assessed and EVs were characterized. Of note, limited volumes of sample acquired from the subjects made it necessary to use smaller and different subsets of samples for each analysis. Results: The majority (91%) of the COVID-19-pregnant subjects (18 mild and 2 moderate disease) experienced good pregnancy-related outcomes. EV concentrations were higher in healthy-pregnant subjects compared to non-pregnant subjects (p = 0.0041) and lower in COVID-19-pregnant subjects compared to healthy-pregnant subjects (p = 0.0150). CD63 exosome marker expression was higher in EVs of healthy-pregnant subjects and COVID-19-pregnant subjects compared to EVs of non-pregnant subjects (p = 0.0149, p = 0.0028, respectively). Similar levels of SARS-CoV-2 entry proteins (ACE-2 and TMPRSS2) were found in all three groups. Cytokine content increased in healthy-pregnant subject-EVs compared to non-pregnant EVs, while IL-2 and IL-6 levels were decreased in COVID-19-pregnant subject-EVs compared to healthy-pregnant subject-EVs (p = 0.043, p = 0.0390, respectively). CD8+, cytotoxic T-cell marker, was lower in non-pregnant EVs compared to healthy-pregnant subject-EVs and to COVID-19-pregnant subjects (p = 0.0108, p < 0.0001, respectively). COVID-19- pregnant subject-EVs demonstrated higher levels of platelet activation marker (CD62P) than non-pregnant (p = 0.0327) and healthy-pregnant subjects (p = 0.0365). Endothelial marker EV-CD144+ was lower in healthy-pregnant subjects versus non-pregnant subjects (p = 0.0093), but similar in COVID-19-pregnant and non-pregnant subjects. Other EVs' coagulation markers/activity, D-Dimer and fibrinogen levels were similar in healthy-pregnant subjects and COVID-19 positive pregnant subjects. Conclusion: COVID-19 positive pregnant subjects' EVs demonstrated an attenuated inflammatory response, with no additional activation of the coagulation system.

Decreased Platelet Aggregation in Patients with Decompensated Liver Cirrhosis and TIPS Implantation.

Transjugular intrahepatic portosystemic shunt (TIPS) implantation is an effective treatment of portal hypertension in patients with decompensated liver cirrhosis. However, some patients develop TIPS thrombosis with recurrence of portal hypertension. The role of platelets in TIPS thrombosis and the necessity of antiplatelet therapy is unclear. Therefore, we aimed to study platelet function in patients with liver cirrhosis prior to and after TIPS implantation. Platelet aggregation was tested in peripheral and portal-vein blood patient samples on the day (D) of TIPS implantation (D0), D4 and D30 following the procedure (platelet count above 100 × 103/µL, aspirin starting on D5) using whole-blood impedance aggregometry (WBIA) and light transmission aggregometry (LTA). In addition, surface platelet activation markers (P-selectin, activated GPIIb/IIIa) and platelet-neutrophil complexes (PNCs) were assessed by flow cytometry. Thrombin receptor activating peptide 6 (TRAP-6), adenosine diphosphate (ADP) and arachidonic acid (AA) were used as agonists. Healthy subjects were included as controls. Agonist-induced platelet aggregation was reduced (WBIA: TRAP-6 p < 0.01, ADP p < 0.01, AA p < 0.001; LTA: TRAP-6 p = 0.13, ADP p = 0.05, AA p < 0.01) in patients (D0, n = 13) compared with healthy subjects (n = 9). While surface activation markers at baseline were negligibly low, the percentage of PNCs was higher in patients than in controls (p < 0.05). ADP-induced P-selectin expression was increased (p < 0.001), whereas TRAP-6-induced GPIIb/IIIa activation was impaired (p < 0.001) in patients versus controls. PNC formation in response to agonists was not different between groups. Results did not differ between peripheral and portal-vein blood of patients (D0, n = 11) and did not change over time (D0, D4, D30) following TIPS implantation (n = 9). In summary, patients with decompensated liver cirrhosis display in vitro platelet aggregation defects in response to various agonists. Defective aggregation persists upon TIPS implantation. Therefore, we conclude that antiplatelet treatment to prevent TIPS thrombosis is questionable.

Peripheral artery disease and markers of endothelial dysfunction and platelet activation in people with HIV.

People with HIV (PWH) have increased risk of peripheral artery disease (PAD), but the pathogenesis is unknown. We aimed to determine associations between markers of endothelial dysfunction and platelet activation and both PAD at baseline and de novo PAD in PWH. 1012 PWH from the longitudinal Copenhagen Comorbidity in HIV Infection (COCOMO) study and 57 age- and sex-matched population controls were included. Plasma samples were collected at baseline and analyzed for soluble thrombomodulin, syndecan-1 and CD40 ligand (sCD40L). Ankle-brachial index was measured at baseline and two-year follow-up in PWH. Logistic and Poisson regression models were used to test associations. PWH had higher concentrations of soluble thrombomodulin (P=0.03) and syndecan-1 (P<0.001), and lower concentrations of sCD40L (P<0.001) compared to controls. High concentration of soluble thrombomodulin, but not syndecan-1 or sCD40L, was associated with lower odds of PAD in PWH at baseline after adjustments (adjusted odds ratio: 0.50 [0.28, 0.90], P=0.02). None of the markers were associated with de novo PAD. PWH had higher concentrations of soluble thrombomodulin and syndecan-1 and lower concentration of sCD40L compared to controls. Soluble thrombomodulin was associated with lower odds of PAD at baseline. Further studies are needed to elucidate the pathogenesis of PAD in people with HIV.

Venous Thromboembolism Risk Assessment among Beta-thalassemia Patients

BACKGROUND: Thromboembolic (TE) disorders are among the most common complications of beta-thalassemia. We designed this cross-sectional study to investigate the state of hypercoagulability and platelet activation in patients with beta-thalassemia. PATIENTS AND METHODS: Seventy-five patients diagnosed with beta-thalassemia by hemoglobin electrophoresis were divided into three groups of 25 patients each: Group I (thalassemia trait), Group II (thalassemia intermedia [TI]), and Group III (thalassemia major). In addition, 50 healthy individuals were included as controls. Both patients and control groups were subjected to clinical and laboratory assessment, which included measurement of protein C, anti-thrombin III, D-dimer, CD41, CD42, CD61, and CD62P, and detection of beta-chain mutations. RESULTS: Levels of the platelet activation marker CD62P were significantly higher in beta-thalassemia patients (26.28 ± 18.01%) than in controls (4.78 ± 2.27%) (P &lt; 0.001). The D-dimer level was significantly higher in beta-thalassemia patients (348.41 ± 571.01 ng/mL) than in controls (71.6 ± 39.61 ng/mL) (P &lt; 0.001). Protein-C and AT-III levels were significantly lower in beta-thalassemia patients (71.45 ± 13.26%, 78.38 ± 15.32%) in comparison with controls (94.9 ± 13.03%, 96.52 ± 11.01%) (P &lt; 0.001 and P &lt; 0.001, respectively). TE disorders were found in 7/25 (9%) beta-thalassemia patients, especially in older and postsplenectomy patients. TE was most commonly found in beta-TI. Beta-chain mutations were found in all patients with TE disorders, especially compound heterozygous intervening sequence (IVS) (IVS1.6 [T &gt; C]/IVS1.110 [G &gt; A]). CONCLUSION: Postsplenectomy teenagers and adults with beta-thalassemia with lower levels of natural anticoagulant in the blood, an increased level of D-dimer, and platelet activator factor had a significantly higher risk for TE than those with childhood beta-thalassemia and the control group. In comparison with other beta-thalassemia patients, TI with beta-chain mutations is more likely to develop TE.

Multimodal analysis of granulocytes, monocytes, and platelets in patients with cystic fibrosis before and after Elexacaftor-Tezacaftor-Ivacaftor treatment.

Cystic fibrosis (CF) is a monogenetic disease caused by an impairment of the cystic fibrosis transmembrane conductance regulator (CFTR). CF affects multiple organs and is associated with acute and chronic inflammation. In 2020, Elexacaftor-Tezacaftor-Ivacaftor (ETI) was approved to enhance and restore the remaining CFTR functionality. This study investigates cellular innate immunity, with a focus on neutrophil activation and phenotype, comparing healthy volunteers with patients with CF before (T1, n = 13) and after six months (T2, n = 11) of ETI treatment. ETI treatment reduced sweat chloride (T1: 95 mmol/l (83|108) vs. T2: 32 mmol/l (25|62), p < 0.01, median, first|third quartile) and significantly improved pulmonal function (FEV1 T1: 2.66 l (1.92|3.04) vs. T2: 3.69 l (3.00|4.03), p < 0.01). Moreover, there was a significant decrease in the biomarker human epididymis protein 4 (T1: 6.2 ng/ml (4.6|6.3) vs. T2: 3.0 ng/ml (2.2|3.7), p < 0.01) and a small but significant decrease in matrix metallopeptidase 9 (T1: 45.5 ng/ml (32.5|140.1) vs. T2: 28.2 ng/ml (18.2|33.6), p < 0.05). Neutrophil phenotype (CD10, CD11b, CD62L, and CD66b) and function (radical oxygen species generation, chemotactic and phagocytic activity) remained largely unaffected by ETI treatment. Likewise, monocyte phenotype and markers of platelet activation were similar at T1 and T2. In summary, the present study confirmed a positive impact on patients with CF after ETI treatment. However, neither beneficial nor harmful effects of ETI treatment on cellular innate immunity could be detected, possibly due to the study population consisting of patients with well-controlled CF.

Citrate Phosphate Dextrose Alters Coagulation Dynamics Ex Vivo

IntroductionCitrate-phosphate-dextrose (CPD) is the most common anticoagulant for blood product storage in the United States. It was developed to prolong shelf life, though there is little research regarding its impact on function following transfusion. We used flow cytometry (FC), thromboelastography (TEG), and a clot contraction assay called the zFlex platform to measure platelet activation and global clot formation in blood samples anticoagulated with either CPD or in a standard blue top citrate (BTC) tube. MethodsSamples were obtained through venipuncture of the antecubital fossa from healthy donors who had not recently taken antiplatelet medication. Samples for FC analysis were spun to obtain platelet-rich plasma, while TEG and zFlex utilized recalcified whole blood. ResultsMean fluorescence intensity for CD62p (P-selectin, marker of platelet activation) in baseline samples was equal, while mean fluorescence intensity in samples activated with thrombin receptor activating peptide was higher in CPD than BTC (65,814 ± 4445 versus 52,483 ± 5435, P = 0.007). TEG results demonstrated similar maximum amplitude for CPD (62.7 ± 1.8 mm versus 61 ± 1 mm) (P = 0.33), though reaction time and kinetics time were significantly longer in CPD versus BTC. CPD R-time: 7.9 ± 0.4 min versus BTC: 3.8 ± 0.4 (P < 0.001). CPD K-time: 2.2 ± 0.2 min versus BTC: 1.6 ± 0.1 min (P < 0.001). Clot contraction strength was not different between the two groups on zFlex: CPD 4353 ± 6 = 517 μN versus BTC 4901 ± 390 μN (P = 0.39). ConclusionsOur findings suggest that CPD does not affect platelet function (minimal difference on FC and no difference in ultimate clot strength, which is ∼80% due to platelet function) but may alter clot dynamics by attenuating thrombin generation.

Rivaroxaban Combined with Atorvastatin Inhibits Acute Pulmonary Embolism by Promoting the Expression of NRF2/NQO1.

Atorvastatin and direct oral factor Xa inhibitors (for instance, rivaroxaban) are co-administrated in patients with atrial fibrillation. However, no studies have been conducted on the function of these two agents in acute pulmonary embolism (APE). Therefore, we investigated the effects of rivaroxaban + atorvastatin in rats with APE and explored the underlying mechanisms. Patients with APE were enrolled, and rats with APE were generated for different regimens. The mean pulmonary arterial pressure (mPAP), heart rate, and PaO2 of APE patients and rats were measured. The plasma levels of oxidative stress- and inflammation-related factors were measured, and the expression of platelet activation markers (CD63 and CD62P) was detected. The proteins targeted by rivaroxaban and atorvastatin, the targets associated with APE, and the genes aberrantly expressed in rats with APE were intersected to obtain candidate factors. Rivaroxaban + atorvastatin reduced mPAP and increased PaO2 in patients and rats with APE. Rivaroxaban + atorvastatin repressed oxidative stress, inflammatory levels, and platelet activation during APE. NRF2 and NQO1 were increased in the lung of rats treated with rivaroxaban + atorvastatin. The therapeutic effect of the combination on APE rats was suppressed after NRF2 downregulation. NRF2 promoted the NQO1 transcription. NQO1 eliminated the inhibitory effect of sh-NRF2 on the combined therapy. The alleviating effect of rivaroxaban + atorvastatin administration against APE correlates with NRF2/NQO1 expression.

#2668 SUPERIOR BIOCOMPATIBILITY OF REGIONAL CITRATE ANTICOAGULATION COMPARED TO HEPARIN DURING EXPANDED HEMODIALYSIS WITH THERANOVA® MEMBRANE

Abstract Background and Aims Regional citrate anticoagulation (RCA) is known to improve the biocompatibility of hemodialysis (HD) by reducing complement, leukocyte and platelet activation. As continuous exposure to bio-incompatible HD may accelerate cardiovascular disease and increase mortality, improved biocompatibility may be important for clinical outcomes, including long-term survival of chronic HD patients. We found no reports of RCA during expanded hemodialysis (HDx), except our recent pilot study [1]. The aim of our trial was to compare RCA and full dose unfractionated heparin (UFH) during HDx with respect to biocompatibility parameters. Methods We performed a randomized, cross-over trial in 32 patients on chronic HD. Each had 2 HDx procedures with Theranova® membrane: one with UFH and one with RCA. RCA was achieved with 8% trisodium citrate at a flow rate of 150 mL/min. Anticoagulation with UFH was achieved by a bolus of 30 I.U. per kg of body weight, followed by an infusion of 18 I.U. per kg of body weight per hour. Anticoagulation was monitored by post-filter ionized calcium (RCA) or activated partial thromboplastin time (UFH). The primary outcomes were serum concentrations of C3a (marker of complement activation) and myeloperoxidase (MPO; marker of leukocyte activation) and plasma concentrations of platelet factor 4 (PF4; marker of platelet activation). Blood was drawn prior to, after 15 minutes and just before the end of HDx. The area under the biocompatibility marker concentration-time curve (AUC) was calculated for an individual biocompatibility marker from all three concentrations using the linear trapezoidal rule. As a secondary outcome, adhesion of blood cells and activation of clotting on inner surfaces of dialyzer fibers were visualized using scanning electron microscopy (SEM) in a subset of patients (n = 5). Biocompatibility parameters were compared between the groups using paired t-test or Wilcoxon signed-rank test. Results The trial included 22 males and 10 females. Patients were of mean age 65 ± 12 years with median dialysis vintage of 31 (IQR 17-89) months. Mean duration of HDx was 4.4 ± 0.4 hours (always the same for an individual patient). All patients had arteriovenous fistulas. Analysis of PF4 included only 8 patients, while the rest had concentrations of PF4 above the measuring range, probably due to unspecific platelet degranulation because of preanalytical factors. The concentrations of C3a, MPO and PF4 rose significantly after 15 minutes of HDx with UFH, while we observed no significant rises during HDx with RCA (Table 1). The AUC for biocompatibility markers were significantly smaller during HDx with RCA in comparison to UFH: 1163.0 ± 306.8 vs. 1444.1 ± 385.9 µgh/L for C3a (p&amp;lt;0.001); 78.2 (IQR 39.0-131.2) vs. 275.1 (IQR 136.9-492.9) µgh/L for MPO (p&amp;lt;0.001) and 100.0 (IQR 51.8-196.6) vs. 379.4 (IQR 295.0-553.0) IUh/mL for PF4 (p = 0.04). SEM showed almost no deposition on the membrane with comparable clotting score: 2 (IQR 1-3) for RCA vs. 2 (IQR 1-2.25) for UFH (p = 0.40). Conclusion Given the higher activation of complement, leukocytes and platelets during HDx with UFH, we conclude that RCA offers superior biocompatibility compared to UFH during HDx. Whether improved biocompatibility has clinical relevance remains to be explored.

#3944 COMPARISON OF CLINICAL PERFORMANCE AND HEMOCOMPATIBILITY OF DIALYZERS APPLIED DURING POST-DILUTION ONLINE HEMODIAFILTRATION (HDF) – EMPORA III STUDY

Abstract Background and Aims Performance and hemocompatibility are the two main functions of a hemodialyzer. Synthetic dialysis membranes made from polysulfone (PSU) or polyethersulfone (PES) are mainly used for dialysis treatments and are blended with the hydrophilic agent polyvinylpyrrolidone (PVP), to improve hemocompatibility during dialysis treatments. A novel PSU-based dialyzer (FX CorAL) with an increased and stabilized PVP content has been developed and has shown strong performance and a favourable hemocompatibility profile within previous short term clinical studies. In the present clinical study, we now investigated the performance and hemocompatibility of the FX CorAL vs. two comparators over a longer follow-up time and applied an extended panel of hemocompatibility biomarkers. This allowed us to analyse treatment-specific performance as well as an extensive intra- and interdialytic hemocompatibility profile. Method eMPORA III was a prospective, open, controlled, multicentre crossover trial with randomized treatment sequences conducted in DE, CZ and HU. It randomized stable patients receiving regular post-dilution online HDF to FX CorAL 600, FX CorDiax 600 (both Fresenius Medical Care) and xevonta Hi 15 (B. Braun), each for 4 weeks. The primary outcome was β2-m removal rate (RR) during 4 hrs HDF. Non-inferiority (margin: 5%) and superiority of FX CorAL 600 versus comparators were tested at α = 2.5% (one-sided), with adjustment for multiple tests. Secondary endpoints were RR and/or clearance of β2-m and other molecules, as well as intra- and interdialytic changes of markers of complement activation (C3a, sC5b-9), cell activation / inflammation (white blood cells (WBC), PMN elastase, IL-6, IL-8, LTB-4, sICAM-1, hsCRP), platelet activation (platelet count, β-TG, TxB-2), and oxidative stress (MDA, GSH-Px). Intradialytic hemocompatibility markers were analysed descriptively as differences vs. the values determined at the start of the HDF session (LS means). Results Eighty-two patients were included and analysed in the safety population, with n = 76 presenting data for the primary outcome (ITT population). FX CorAL 600 showed the highest β2-m RR (LS mean: 76.31%), followed by FX CorDiax 600 (75.71%) and xevonta Hi 15 (74.49%). Non-inferiority to its comparators was statistically significant (p&amp;lt;0.0001 each). Superiority was shown vs. xevonta Hi 15 (p&amp;lt;0.0001), but not vs. FX CorDiax 600 (p = 0.0606). The secondary endpoints β2-m clearance as well as myoglobin clearance and RR affirmed these results; small molecule clearances and/or RR were similar between dialyzers. Analyses of hemocompatibility markers within one HDF session, found the following significant differences: Complement activation • C3a 15 min: FX CorAL: 31%; FX CorDiax: 59% (p = 0.003 vs. FX CorAL); xevonta: 41% (p = 0.029 vs. FX CorAL) • sC5b-9 60 min: FX CorAL: 24%; FX CorDiax: 25% (p = 0.337); xevonta: 41% (p&amp;lt;0.0001) Cell activation/ Inflammation • WBC 15 min: FX CorAL: -7.7%; FX CorDiax: -11.1% (p = 0.015); xevonta: -10.1% (p = 0.246) • Monocytes 15 min: FX CorAL: -30.9%; FX CorDiax: -30.3% (p = 0.533); xevonta: -38.2% (p = 0.012) • Neutrophils 15 min: FX CorAL: -3.0%; FX CorDiax: -7.5% (p = 0.019); xevonta: -6.1% (p = 0.258) • PMN elastase 60 min: FX CorAL: 18%; FX CorDiax: 42% (p = 0.003); xevonta: 52% (p = 0.0003) • LTB-4 15 min: FX CorAL: 170%; FX CorDiax: 217% (p = 0.047); xevonta: 186% (p = 0.539) Platelet activation • β-TG 60 min: FX CorAL: -15%; FX CorDiax: 4% (p&amp;lt;0.0001); xevonta: 18% (p&amp;lt;0.0001) Hemocompatibility markers showed no interdialytic changes, i.e., no significant changes within treatment periods. Nine serious adverse events occurred in this study, none of which was dialyser related. Conclusion FX CorAL 600 efficiently removed middle and small molecules and was non-inferior to both comparators and significantly superior to xevonta Hi 15 in β2-m RR. The typical drop in WBC, monocyte, and neutrophil count during dialysis as well as the rise of complement (C3a, sC5b-9) and cell / platelet activation markers (PMN elastase, LTB-4 and β-TG) were lower or comparable during treatment with FX CorAL vs. both comparators, indicating superior hemocompatibility properties.

Procoagulant effect of extracellular vesicles in patients after transcatheter aortic valve replacement or transcatheter aortic valve replacement with percutaneous coronary intervention.

Patients with severe aortic stenosis (AS) after replacement of the transcatheter aortic valve (TAVR) are more likely to develop thrombotic complications such as cerebral embolism and artificial valve thrombosis. However, the mechanism is not yet well defined. We aimed to explore the plasma extracellular vesicles (EVs) levels and their role in the induction of procoagulant activity (PCA) in patients receiving TAVR alone or TAVR with percutaneous coronary intervention (PCI). EVs were analyzed with flow cytometer. Markers of platelet and endothelial cell activation were quantified using selective enzyme-linked immunosorbent assay (ELISA) kits. Procoagulant activity (PCA) was assessed by clotting time, purified clotting complex assays, and fibrin production assays. Our results confirmed that EVs with positive phosphatedylserin (PS+EV), platelet EVs (PEVs) and positive tissue factor EVs (TF+EVs) were higher in patients following TAVR than before TAVR, particularly in TAVR with PCI. Furthermore, endothelial-derived EVs (EEVs) were also higher in patients after TAVR with PCI than pre-TAVR, however, the EEVs levels in TAVR alone patients were gradually reduce than pre-TAVR. In addition, we further proved that total EVs contributed to dramatically shortened coagulation time, increased intrinsic/extrinsic factor Xa and thrombin generation in patients after TAVR, especially in TAVR with PCI. The PCA was markedly attenuated by approximately 80% with lactucin. Our study reveals a previously unrecognized link between plasma EV levels and hypercoagulability in patients after TAVR, especially TAVR with PCI. Blockade of PS+EVs may improve the hypercoagulable state and prognosis of patients.

Gender differences in serum markers of inflammation and platelet activation in patients with non-valvular atrial fibrillation

The prevalence of atrial fibrillation is high and comparable in both sexes. Such factors as differently expressed blood biomarkers in women and men may play a role in the occurrence of atrial fibrillation and the development of thrombotic complications. To study markers of inflammation and platelet activation in patients with atrial fibrillation of non-valvular origin, receiving anticoagulant therapy and having a history of thrombotic complications and patients with atrial fibrillation without thrombotic complications, depending on the gender of the patients. The study included 22 healthy volunteers and 60 patients diagnosed with atrial fibrillation receiving anticoagulant therapy, of which 21 patients developed thrombotic complications. Serum levels of α2- macroglobulin, hsC-reactive protein, fetuin A, α-1-acid glycoprotein, L-selectin, serum amyloid P, adipsin, and platelet factor 4 were studied on FLEXMAP 3D using Acute Phase diagnostic test systems Panel 3. A comparative study of the content of biomarkers demonstrated an increased concentration of C-reactive protein in men and women in both groups of patients with atrial fibrillation; decrease in fetuin A and L-selectin in the group of women with thrombosis compared with women without thrombotic complications and compared with healthy women. There were no gender differences in the concentration of fetuin A and L-selectin in the group of patients with atrial fibrillation without thrombotic complications and in healthy volunteers. The level of adipsin had no gender differences in the group of patients with atrial fibrillation with thrombosis and in healthy volunteers, however, it was significantly increased in women without thrombosis. The content of platelet factor 4 in women in both groups of patients exceeded the value of this indicator in healthy women; no gender differences were found in the groups of patients with atrial fibrillation. Low levels of fetuin A and L-selectin, with a simultaneous increase in C-reactive protein and platelet factor 4, lead to an increase of prothrombogenic potential and to a change in the balance of pro- and antiinflammatory mediators towards increased inflammation in female patients with atrial fibrillation.

Evidence that platelets from transfusion-dependent β-thalassemia patients induce T cell activation

A hypercoagulable state leading to increased risk for thrombotic events represents one of the most common complications observed in transfusion-dependent β-thalassemia (TDT) patients. TDT patients have increased frequencies of circulating activated platelets. However, there is no information so far if platelets from TDT patients can activate T cells. In the present study we showed that T cells treated with platelets from TDT patients showed significant increased surface expression of CD69 compared to the T cells treated with platelets from healthy individuals. Patients with splenectomy showed increased T cell activation compared to patients with intact spleen. No T cell activation was observed following incubation with plasma alone, nor with platelets from healthy subjects. The percentages of regulatory T cells (Tregs) were also examined. TDT patients showed statistically significant increased percentages of Tregs compared to healthy controls. Additionally, we observed a positive statistically significant correlation between the percentages of Tregs and the platelet-induced activated T cells in patients who were not treated with aspirin. TDT patients showed increased levels of sP-selectin, suPAR and GDF-15, molecules implicated in platelet activation. We show that platelets from TDT patients can activate T cells in vitro. This activation correlates with markers of platelet activation and increased numbers of Tregs, perhaps in an effort to eliminate immune dysregulation, conceivably secondary to platelet activation.