BRD4 degraders may effectively counteract therapeutic resistance of leukemic stem cells in AML and ALL.

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are life-threatening hematopoietic malignancies characterized by clonal expansion of leukemic blasts in the bone marrow and peripheral blood. The epigenetic reader BRD4 and its downstream effector MYC have recently been identified as potential drug targets in human AML and ALL. We compared anti-leukemic efficacies of the small-molecule BET inhibitor JQ1 and the recently developed BRD4 degraders dBET1 and dBET6 in AML and ALL cells. JQ1, dBET1, and dBET6 were found to suppress growth and viability in all AML and ALL cell lines examined as well as in primary patient-derived AML and ALL cells, including CD34+/CD38- and CD34+/CD38+ leukemic stem and progenitor cells, independent of the type (variant) of leukemia or molecular driver expressed in leukemic cells. Moreover, we found that dBET6 overcomes osteoblast-induced drug resistance in AML and ALL cells, regardless of the type of leukemia or the drug applied. Most promising cooperative or even synergistic drug combination effects were seen with dBET6 and the FLT3 ITD blocker gilteritinib in FLT3 ITD-mutated AML cells, and with dBET6 and the multi-kinase blocker ponatinib in BCR::ABL1+ ALL cells. Finally, all BRD4-targeting drugs suppressed interferon-gamma- and tumor necrosis factor-alpha-induced expression of the resistance-related checkpoint antigen PD-L1 in AML and ALL cells, including LSC. In all assays examined, the BRD4 degrader dBET6 was a superior anti-leukemic drug compared with dBET1 and JQ1. Together, BRD4 degraders may provide enhanced inhibition of multiple mechanisms of therapy resistance in AML and ALL.

Management of Immune Thrombotic Thrombocytopenic Purpura without Therapeutic Plasma Exchange.

Immune thrombotic thrombocytopenic purpura (iTTP) is a rare, life-threatening autoimmune disorder caused by ADAMTS13 deficiency. Caplacizumab, an anti-VWF nanobody, is approved for iTTP treatment, reducing the need for therapeutic plasma exchange (TPE) and improving platelet count recovery and survival. We conducted a retrospective study on 42 acute iTTP cases in Austria and Germany, treated with a modified regimen aimed at avoiding TPE if platelet count increased after the first caplacizumab dose. Baseline characteristics and patient outcomes were compared with a control group of 59 patients with iTTP, receiving frontline treatment with TPE, caplacizumab, and immunosuppression. The main outcome was the time to platelet count normalization. Secondary outcomes included clinical response, exacerbation, refractory iTTP, iTTP-related deaths, and the time to platelet count doubling. The median time to platelet count normalization was similar between the two cohorts (3 and 4 days; P = 0.31). There were no significant differences in clinical response, exacerbations, refractoriness, iTTP-related deaths, or time to platelet count doubling reflecting the short-term treatment response. Four patients did not respond to the first caplacizumab dose and TPE was subsequently initiated. Cytomegalovirus infection, HIV/hepatitis B co-infection, an ovarian teratoma with associated anti-platelet antibodies, and multiple platelet transfusion before the correct diagnosis may have impeded immediate treatment response in these patients. In conclusion, caplacizumab and immunosuppression alone, without TPE, rapidly controlled thrombotic microangiopathy and achieved a sustained clinical response in iTTP. Our study provides a basis for TPE-free iTTP management in experienced centers via shared decision-making between patients and treating physicians.

The plasma miRNome and venous thromboembolism in high-grade glioma: miRNA Sequencing of a nested case-control cohort.

Patients with high-grade gliomas are at high risk of venous thromboembolism (VTE). MicroRNAs (miRNAs) are small non-coding RNAs with multiple roles in tumour biology, haemostasis and platelet function. Their association with VTE risk in high-grade glioma has not been comprehensively mapped so far. We thus conducted a nested case-control study within 152 patients with WHO grade IV glioma that had been part of a prospective cohort study on VTE risk factors. At inclusion a single blood draw was taken, and patients were thereafter followed for a maximum of 2 years. During that time, 24 patients (16%) developed VTE. Of the other 128 patients, we randomly selected 24 age- and sex-matched controls. After quality control, the final group size was 21 patients with VTE during follow-up and 23 without VTE. Small RNA next-generation sequencing of plasma was performed. We observed that hsa-miR-451a was globally the most abundant miRNA. Notably, 51% of all miRNAs showed a correlation with platelet count. The analysis of miRNAs differentially regulated in VTE patients-with and without platelet adjustment-identified potential VTE biomarker candidates such as has-miR-221-3p. Therewith, we here provide one of the largest and deepest peripheral blood miRNA datasets of high-grade glioma patients so far, in which we identified first VTE biomarker candidates that can serve as the starting point for future research.

Tissue factor pathway inhibitor is associated with risk of venous thromboembolism and all-cause mortality in patients with cancer.

Venous thromboembolism (VTE) is a common complication in patients with cancer. Data on the role of natural inhibitors of coagulation for occurrence of cancer-associated VTE are limited, thus, we investigated the association of tissue factor pathway inhibitor (TFPI) with risk of VTE and all-cause mortality in patients with cancer. Total TFPI antigen levels were measured with a commercially available enzyme-linked immunosorbant assay in patients included in the Vienna Cancer and Thrombosis Study, a prospective observational cohort study with the primary outcome VTE. Competing risk analysis and Cox regression analysis were performed to explore the association of TFPI levels with VTE and all-cause mortality. TFPI was analyzed in 898 patients (median age 62 years; interquartile range [IQR], 53-68; 407 (45%) women). Sixty-seven patients developed VTE and 387 died (24-month cumulative risk 7.5% and 42.1%, respectively). Patients had median TFPI levels at study inclusion of 56.4 ng/mL (IQR, 45.7-70.0), with highest levels in tumor types known to have a high risk of VTE (gastroesophageal, pancreatic and brain cancer: 62.0 ng/mL; IQR, 52.0-75.0). In multivariable analysis adjusting for age, sex, cancer type and stage, TFPI levels were associated with VTE risk (subdistribution hazard ratio per doubling =1.63, 95% confidence interval [CI]: 1.03-2.57). When patients with high and intermediate/low VTE risk were analyzed separately, the association remained independently associated in the high risk group only (subdistribution hazard ratio =2.63, 95% CI: 1.40-4.94). TFPI levels were independently associated with all-cause mortality (hazard ratio =2.36, 95% CI: 1.85-3.00). In cancer patients increased TFPI levels are associated with VTE risk, specifically in patients with high-risk tumor types, and with all-cause mortality.

Plasma Clot Properties in Patients with Pancreatic Cancer.

Pancreatic cancer is one of the most prothrombotic malignancies. Plasma clot properties may be altered in patients with pancreatic cancer, and circulating tissue factor (TF) may play an important role. We applied a modified plasma clot formation assay (only CaCl2 and phospholipids were added to initiate clotting) and a standard clotting assay (lipidated TF was also added) to investigate whether plasma clot properties are altered in pancreatic cancer patients (n = 40, 23 female) compared to sex-matched healthy controls. The modified assay was also performed in the presence of a TF blocking antibody. With this modified assay, we detected an increased plasma clot formation rate (Vmax) and an increased delta absorbance (ΔAbs, indicating fibrin fiber thickness) in patients compared to controls. These differences were not detected with the standard clotting assay. Following addition of a TF blocking antibody in in our modified assay, Vmax decreased significantly in patients only, ΔAbs significantly decreased in patients and in healthy controls, the lag phase did not change, and the time to peak fibrin generation increased in patients only. Taken together, these findings indicate the presence of a prothrombotic state in pancreatic cancer patients, which depends on TF and is detectable with our modified assay but not with a standard clotting assay.

Consensus report on markers to distinguish procoagulant platelets from apoptotic platelets: communication from the Scientific and Standardization Committee of the ISTH.

BACKGROUND: Procoagulant platelets are a subpopulation of highly activated platelets that promote coagulation through surface-exposed, negatively charged phospholipids, especially phosphatidylserine. Procoagulant platelets are important for clot stabilization during hemostasis, and an increased number of these platelets is associated with thrombotic risk. There is a need for harmonization in this area since many of the markers and methods used to assess procoagulant platelets are not specific when used in isolation but are also associated with platelet apoptosis. OBJECTIVES: We initiated this project to identify a minimum set of markers and/or methods that can detect and distinguish procoagulant platelets from apoptotic platelets. METHODS: The study design involved a primary panel with 27 international experts who participated in an online survey and moderated virtual focus group meetings. Primary and secondary panel members were then invited to provide input on themes and statements generated from the focus groups. RESULTS: This led to a recommendation to use flow cytometry and a combination of the following 3 surface markers to differentiate procoagulant platelets from apoptotic platelets: P-selectin (CD62P), phosphatidylserine (recognized by annexin V), and the platelet-specific receptor GPIX (CD42a) or αIIb integrin (CD41, GPIIb). CONCLUSION: Procoagulant platelets are expected to be positive for all 3 markers, while apoptotic platelets are positive for annexin V and the platelet-specific surface receptor(s) but negative for P-selectin.

Extracellular vesicles from amniotic fluid, milk, saliva, and urine expose complexes of tissue factor and activated factor VII.

BACKGROUND: Tissue factor (TF) is expressed in the adventitia of the vessel wall and on extracellular vesicles (EVs) in body fluids. TF and activated coagulation factor (F) VII(a) together form the so-called extrinsic tenase complex, which initiates coagulation. AIM: We investigated whether EVs in amniotic fluid, milk, saliva, and urine expose functional extrinsic tenase complexes that can trigger coagulation. METHODS: Milk, saliva, and urine were collected from healthy breastfeeding women (n = 6), and amniotic fluid was collected from healthy women undergoing routine amniocentesis (n = 7). EVs were isolated from body fluids by size exclusion chromatography (SEC) and clotting experiments were performed in the presence and absence of antibodies against TF and FVIIa in normal plasma and in FVII-deficient plasma. The ability of body fluids to generate FXa also was determined. RESULTS: Amniotic fluid, milk, saliva, and urine triggered clotting of normal plasma and of FVII-deficient plasma, which was almost completely inhibited by an anti-FVII antibody and to a lesser extent by an anti-TF antibody. Fractionation of body fluids by SEC showed that only the fractions containing EVs triggered clotting in normal plasma and FVII-deficient plasma and generated FXa, which again was almost completely inhibited by an anti-FVII antibody and partially by an anti-TF antibody. CONCLUSION: Here we show that EVs from amniotic fluid, milk, saliva, and urine expose complexes of TF and FVIIa (i.e., extrinsic tenase complexes) that directly activate FX. Based on our present findings we propose that these EVs from normal body fluids provide hemostatic protection.

Expression of CD98hc in Pancreatic Cancer and Its Role in Cancer Cell Behavior.

Background: Cluster of differentiation 98 heavy chain (CD98hc) is a transmembrane protein, which functions both as a coreceptor of ß-integrins, enhancing intracellular integrin-dependent downstream signaling, and as a transporter of branched-chain and aromatic amino acids. As such, it is pivotal in cell cycle regulation and protection of oxidative, nutritional and DNA replication stress. Overexpression of CD98hc occurs widely in cancer cells and is associated with poor clinical prognosis. The role of CD98hc in pancreatic cancer remains to be elucidated. The aim of this study was to determine the expression of CD98hc in pancreatic ductal adenocarcinoma and to define its potential functional role in cancer cell biology. Methods: Immunohistochemical staining for CD98hc was performed on 222 tissue samples of patients with pancreatic ductal adenocarcinoma. The pancreatic cancer cell lines PANC-1 and BxPC-3 were used to determine the effect of CD98hc expression on cancer cell behavior using cell adhesion, cell trans-migration and cell spreading assays. Flow cytometry was performed to study the rate of apoptosis after detachment or serum starvation. shRNA-lentiviral constructs were used to knock down or reconstitute full length or mutated CD98hc. Results: Up to 20% of pancreatic ductal adenocarcinomas express CD98hc in the acinar cells (13%) and islet cells (20%) embedded in tumor tissue. Although expression of CD98hc in tumor tissue was not associated with a particular tumor stage or grade, our data show a trend towards longer overall survival of pancreatic cancer patients without CD98hc expression as compared to those with immunohistochemical positivity. In vitro downregulation of CD98hc in the pancreatic cancer cell lines PANC-1 and BxPC-3 significantly inhibits cell proliferation (p<0.05), self-renewal (p<0.05) and anchorage-independent growth (p<0.05). Conclusion: CD98hc is expressed in a remarkable percentage of pancreatic ductal adenocarcinomas. Due to its important role in cell behavior and malignant cell transformation, it may be a promising molecular target for potential new therapeutic approaches in pancreatic cancer in the future.

Intraperitoneal Activation of Coagulation and Fibrinolysis in Patients with Cirrhosis and Ascites.

Development of ascites is the most common form of decompensation of cirrhosis. We aimed to investigate the coagulation system in ascitic fluid and plasma of patients with cirrhosis. We determined coagulation parameters and performed clotting and fibrinolysis experiments in ascitic fluid and plasma of thoroughly characterized patients with cirrhosis and ascites (n = 25) and in plasma of patients with cirrhosis but without ascites (n = 25), matched for severity of portal hypertension. We also investigated plasma D-dimer levels in an independent cohort of patients (n = 317) with clinically significant portal hypertension (HVPG ≥ 10 mmHg), grouped according to ascites severity. Ascitic fluid was procoagulant in a clotting assay. The procoagulant potential of ascitic fluid was abolished by depletion of extracellular vesicles from ascitic fluid by filtration or by addition of a tissue factor-neutralizing antibody. Compared with plasma, extracellular vesicle-associated tissue factor activity was high in ascitic fluid, while activities of other coagulation factors were low. The extracellular vesicle-depleted fraction of ascitic fluid induced fibrinolysis, which was prevented by aprotinin, indicating the presence of plasmin in ascitic fluid. Plasma peak thrombin generation and parameters reflecting fibrinolysis were independently associated with the presence of ascites. Finally, plasma D-dimer levels were independently linked to ascites severity in our second cohort comprising 317 patients. In conclusion, coagulation and fibrinolysis become activated in ascites of patients with cirrhosis. While tissue factor-exposing extracellular vesicles in ascitic fluid seem unable to pass the peritoneal membrane, fibrinolytic enzymes get activated in ascitic fluid and may re-enter the systemic circulation and induce systemic fibrinolysis.

Extracellular Vesicles in Human Milk.

Milk supports the growth and development of infants. An increasing number of mostly recent studies have demonstrated that milk contains a hitherto undescribed component called extracellular vesicles (EVs). This presents questions regarding why milk contains EVs and what their function is. Recently, we showed that EVs in human milk expose tissue factor, the protein that triggers coagulation or blood clotting, and that milk-derived EVs promote coagulation. Because bovine milk, which also contains EVs, completely lacks this coagulant activity, important differences are present in the biological functions of human milk-derived EVs between species. In this review, we will summarize the current knowledge regarding the presence and biochemical composition of milk EVs, their function(s) and potential clinical applications such as in probiotics, and the unique problems that milk EVs encounter in vivo, including survival of the gastrointestinal conditions encountered in the newborn. The main focus of this review will be human milk-derived EVs, but when available, we will also include information regarding non-human milk for comparison.

Long-term follow-up after successful treatment of vaccine-induced prothrombotic immune thrombocytopenia.

BACKGROUND: Cases of ChAdOx1 nCoV-19 (AstraZeneca) vaccinated patients with thrombocytopenia, elevated D-dimer, and elevated platelet factor 4 (PF4) antibody levels with- and without thrombosis have been reported. No recommendations regarding the duration of anticoagulation have been made, because data on the long-term course beyond the first weeks is lacking. OBJECTIVE: To report on the treatment, medical course, and longitudinal follow-up of laboratory parameters in patients with vaccine-induced prothrombotic immune thrombocytopenia (VIPIT). PATIENTS: We followed VIPIT patients with- (n = 3) and without (n = 3) venous thromboembolism fulfilling the aforementioned laboratory criteria. RESULTS: Elevated D-dimer (median: 35.10 µg/ml, range: 17.80-52.70), thrombocytopenia (42 G/l, 20-101), and strong positivity in the platelet factor 4 (PF4)/heparin-enzyme-immunoassay (2.42 optical density [OD], 2.06-3.13; reference range < 0.50) were present in all patients after vaccination (10 days, 7-17). Routine laboratory parameters rapidly improved upon initiation of treatment (comprising therapeutic non-heparin anticoagulation in all patients and high dose immunoglobulins ± corticosteroids in 5 patients). PF4 antibody levels slowly decreased over several weeks. Patients were discharged in good physical health (8 days, 5-13). VIPIT did not recur during follow-up (12 weeks, 8-17). Five of 6 patients fully recovered (in 2 patients thrombosis had resolved, in 1 patient exertional dyspnea persisted). CONCLUSIONS: Remissions without sequelae can be achieved upon rapid initiation of treatment in patients with VIPIT. Platelet factor 4 antibody levels slowly decreased over several weeks but VIPIT did not recur in any of our patients. Continuation of anticoagulation in VIPIT patients at least until PF4 antibody negativity is reached seems reasonable.

Hemostatic Biomarkers and Venous Thromboembolism Are Associated With Mortality and Response to Chemotherapy in Patients With Pancreatic Cancer.

Objective: Pancreatic cancer activates coagulation and increases risk of venous thromboembolism (VTE). We aimed at characterizing the association of hemostatic biomarkers and VTE with mortality and chemotherapy response. Approach and Results: Pancreatic cancer patients (N=145) were included in a prospective, observational cohort study (CATS [Vienna Cancer and Thrombosis Study]). Hemostatic biomarkers (D-dimer, extracellular vesicle-tissue factor activity, prothrombin fragment 1+2, fibrinogen, factor VIII, PAI-1 [plasminogen activator inhibitor 1], sP-selectin [soluble P-selectin], thrombin generation assay) were measured at inclusion. The impact of VTE on overall survival/progression-free survival (OS/PFS) was evaluated by multistate modeling. The association of biomarkers with OS was analyzed by Cox-regression and with PFS and disease control rate in patients initiating palliative chemotherapy (n=95) by Cox-regression and logistic regression. Multivariable analysis included stage, grade, sex, age, performance status, VTE (time-dependent), vascular infiltration/compression, and tumor marker levels (carbohydrate-antigen 19-9, carcinoembryonic antigen). VTE occurrence was associated with shorter OS (transition hazard ratio, 3.40 [95% CI, 2.05-5.64]) and shorter PFS (transition hazard ratio, 2.10 [1.16-3.79]). Median post-VTE OS/PFS in months was 5.5 [2.2-6.5] and 3.0 [1.5-3.9], compared with 13.4 [9.7-16.6] and 7.5 [5.9-9.8] in patients without VTE (both P<0.001). D-dimer, extracellular vesicle-tissue factor activity, PAI-1, and sP-selectin were associated with increased mortality (hazard ratio per doubling, 1.27 [1.00-1.61]; 1.63 [1.14-2.36]; 1.25 [1.06-1.47]; 1.52 [1.05-2.20]). In patients initiating palliative chemotherapy, higher D-dimer predicted shorter PFS (hazard ratio per doubling, 1.27 [1.01-1.60]) and lower disease control rate (odds ratio per doubling, 0.59 [0.36-0.98]). Conclusions: VTE diagnosis is associated with shorter OS and PFS. Higher baseline levels of D-dimer, extracellular vesicle-tissue factor activity, PAI-1, and sP-selectin were independently prognostic for increased mortality, and D-dimer predicted response to palliative chemotherapy.

Extracellular Vesicle-Associated Tissue Factor Activity in Prostate Cancer Patients with Disseminated Intravascular Coagulation.

Patients with advanced prostate cancer may develop fulminant disseminated intravascular coagulation (DIC). Circulating extracellular vesicles (EVs)-exposing tissue factor (TF), the initiator of the coagulation cascade, may play an important role. We included 7 prostate cancer patients with DIC, 10 age- and stage-matched cancer controls without DIC, and 10 age-matched healthy male individuals. EV-TF activity was highly elevated in prostate cancer patients with DIC (11.40 pg/mL; range: 4.34-27.06) compared with prostate cancer patients without DIC (0.09 pg/mL; range: 0.00-0.30, p = 0.001) and healthy controls (0.18 pg/mL; range: 0.09-0.54; p = 0.001). Only EVs from patients with DIC reduced fibrin clot formation time of pooled plasma in a TF-dependent manner. Next, we performed in vitro co-culture experiments including EVs derived from a prostate cancer cell line with high (DU145) and low (LNCaP) TF expression, peripheral blood mononuclear cells (PBMCs), and platelets. Co-incubation of DU145 EVs with PBMCs and platelets significantly increased EV-TF activity in conditioned medium and induced TF activity on monocytes. No such effects were seen in co-culture experiments with LNCaP EVs. In conclusion, the findings indicate that elevated EV-TF activity plays a role in the development of prostate-cancer-related DIC and may result from interactions between tumor-derived EVs, monocytes, and platelets.

Successful treatment of vaccine-induced prothrombotic immune thrombocytopenia (VIPIT).

Cases of unusual thrombosis and thrombocytopenia after administration of the ChAdOx1 nCoV-19 vaccine (AstraZeneca) have been reported. The term vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) was coined to reflect this new phenomenon. In vitro experiments with VIPIT patient sera indicated that high-dose intravenous immunoglobulins (IVIG) competitively inhibit the platelet-activating properties of ChAdOx1 nCoV-19 vaccine induced antibodies. Here, we report a case of a 62-year-old woman who had received this vaccine and developed VIPIT. She visited the emergency ward because of petechiae and hematomas. In the laboratory work-up, thrombocytopenia, low fibrinogen, elevated D-dimer, and positivity in the platelet factor 4/heparin-enzyme-immunoassay were present. Signs and symptoms of thrombosis were absent. Upon immediate therapy with non-heparin anticoagulation, high-dose IVIG, and prednisolone, laboratory parameters steadily improved and the patient was discharged from hospital without thrombotic complications. We conclude that early initiation of VIPIT treatment results in a swift response without thrombotic complications.

Emicizumab for the treatment of acquired hemophilia A.

Acquired hemophilia A (AHA) is a severe bleeding disorder caused by inhibiting autoantibodies to coagulation factor VIII (FVIII). For hemostatic treatment, bypassing agents and human or porcine FVIII are currently standard of care. Emicizumab is a bispecific, FVIII-mimetic therapeutic antibody that reduced the annualized bleeding rates in congenital hemophiliacs. Here, we report on 6 male and 6 female patients with AHA treated with emicizumab (all data medians and interquartile range), age 74 (64-80) years, initial FVIII <1%; inhibitor titer 22.3 Bethesda units (BU)/mL (range, 3-2000). Eight patients had severe bleeding. Emicizumab was started, 3 mg/kg subcutaneously, weekly for 2 to 3 doses, followed by 1.5 mg/kg every 3 weeks to keep the lowest effective FVIII levels. For FVIII monitoring, chromogenic assays with human and bovine reagents were used. All patients received immunosuppression with steroids and/or rituximab. After the first dose of emicizumab, activated partial thromboplastin time normalized in 1 to 3 days, FVIII (human reagents) exceeded 10% after 11 (7.5-12) days. Hemostatic efficacy was obtained and bypassing therapy stopped after 1.5 (1-4) days. FVIII (bovine reagents) exceeded 50%, indicating complete remission after 115 (67-185) days, and emicizumab was stopped after 31 (15-79) days. A median of 5 injections (range, 3-9) were given. No patient died of bleeding or thromboembolism, and no breakthrough bleeding was observed after the first dose of emicizumab. In conclusion, emicizumab seems to be an effective hemostatic therapy for AHA, with the advantages of subcutaneous therapy, good hemostatic efficacy, early discharge, and reduction of immunosuppression and adverse events.

Human milk triggers coagulation via tissue factor-exposing extracellular vesicles.

Almost a century ago, it was discovered that human milk activates the coagulation system, but the milk component that triggers coagulation had until now been unidentified. In the present study, we identify this component and demonstrate that extracellular vesicles (EVs) present in normal human milk expose coagulant tissue factor (TF). This coagulant activity withstands digestive conditions, mimicking those of breastfed infants, but is sensitive to pasteurization of pooled donor milk, which is routinely used in neonatal intensive care units. In contrast to human milk, bovine milk, the basis of most infant formulas, lacks coagulant activity. Currently, the physiological function of TF-exposing vesicles in human milk is unknown, but we speculate that these vesicles may be protective for infants. Another explanation could be nipple skin damage, which occurs in most breastfeeding women. Milk-derived TF-exposing EVs may seal the wound and thereby reduce bleeding and breast inflammation.

Influence of hemoadsorption during cardiopulmonary bypass on blood vesicle count and function.

BACKGROUND: Extracorporeal circulation during major cardiac surgery triggers a systemic inflammatory response affecting the clinical course and outcome. Recently, extracellular vesicle (EV) research has shed light onto a novel cellular communication network during inflammation. Hemoadsorption (HA) systems have shown divergent results in modulating the systemic inflammatory response during cardiopulmonary bypass (CPB) surgery. To date, the effect of HA on circulating microvesicles (MVs) in patients undergoing CPB surgery is unknown. METHODS: Count and function of MVs, as part of the extracellular vesicle fraction, were assessed in a subcohort of a single-center, blinded, controlled study investigating the effect of the CytoSorb device during CPB. A total of 18 patients undergoing elective CPB surgery with (n = 9) and without (n = 9) HA device were included in the study. MV phenotyping and counting was conducted via flow cytometry and procoagulatory potential was measured by tissue factor-dependent MV assays. RESULTS: Both study groups exhibited comparable counts and post-operative kinetics in MV subsets. Tissue factor-dependent procoagulatory potential was not detectable in plasma at any timepoint. Post-operative course and laboratory parameters showed no correlation with MV counts in patients undergoing CPB surgery. CONCLUSION: Additional artificial surfaces to the CPB-circuit introduced by the use of the HA device showed no effect on circulating MV count and function in these patients. Larger studies are needed to assess and clarify the effect of HA on circulating vesicle counts and function. Trial registration ClinicalTrials.Gov Identifier: NCT01879176; registration date: June 17, 2013; https://clinicaltrials.gov/ct2/show/NCT01879176.