As extracellular vesicles (EVs) have become a prominent topic in life sciences, a growing number of studies are published on a regular basis addressing their biological relevance and possible applications. Nevertheless, the fundamental question of the true vesicular nature as well as possible influences on the EV secretion behavior have often been not adequately addressed. Furthermore, research regarding endothelial cell-derived EVs (EndoEVs) often focused on the large vesicular fractions comprising of microvesicles (MV) and apoptotic bodies. In this study we aimed to further extend the current knowledge of the influence of pre-isolation conditions, such as cell density and conditioning time, on EndoEV release from human umbilical vein endothelial cells (HUVECs). We combined fluorescence nanoparticle tracking analysis (NTA) and the established fluorescence-triggered flow cytometry (FT-FC) protocol to allow vesicle-specific detection and characterization of size and surface markers. We found significant effects of cell density and conditioning time on both abundance and size distribution of EndoEVs. Additionally, we present detailed information regarding the surface marker display on EVs from different fractions and size ranges. Our data provide crucial relevance for future projects aiming to elucidate EV secretion behavior of endothelial cells. Moreover, we show that the influence of different conditioning parameters on the nature of EndoEVs has to be considered.

Specific antagonists have been developed for the reversal of direct oral anticoagulants (DOAC). We investigated the impact of these reversal agents on the plasma concentration and visco-elastic test results of dabigatran and factor Xa inhibitors. After baseline measurements of dabigatran, the plasma concentration, and the visco-elastic ClotPro^® ecarin clotting time (ECA-CT), we added the reversal agent Idarucizumab in vitro and these two analyses were repeated. Likewise, the baseline plasma concentration of apixaban, edoxaban, and rivaroxaban as well as ClotPro^® Russell’s viper venom test clotting time (RVV-CT) were measured and reanalyzed following Andexanet alfa spiking. We analyzed fifty blood samples from 37 patients and 10 healthy volunteers. Idarucizumab decreased the measured dabigatran plasma concentration from 323.9 ± 185.4 ng/mL to 5.9 ± 2.3 ng/mL and ECA-CT from 706.2 ± 344.6 s to 70.6 ± 20.2 s, (all, p < 0.001). Andexanet alfa decreased the apixaban concentration from 165.1 ± 65.5 ng/mL to 9.8 ± 8.1 ng/mL, edoxaban from 152.4 ± 79.0 ng/mL to 36.4 ± 19.2 ng/mL, and rivaroxaban from 153.2 ± 111.8 ng/mL to 18.1 ± 9.1 ng/mL (all p < 0.001). Andexanet alfa shortened the RVV-CT of patients with apixaban from 239.2 ± 71.7 s to 151.1 ± 30.2 s, edoxaban from 288.2 ± 65.0 s to 122.7 ± 37.1 s, and rivaroxaban from 225.9 ± 49.3 s to 103.7 ± 12.1 s (all p < 0.001). In vitro spiking of dabigatran-containing blood with Idarucizumab substantially reduced the plasma concentration and ecarin-test clotting time. Andexanet alfa lowered the concentration of the investigated factor Xa-inhibitors but did not normalize the RVV-CT. In healthy volunteers’ blood, Idarucizumab spiking had no impact on ECA-CT. Andexanet alfa spiking of non-anticoagulated blood prolonged RVV-CT (p = 0.001), potentially as a consequence of a competitive antagonism with human factor Xa.

Severe bleeding remains a prominent cause of early in-hospital mortality in major trauma patients. Thus, prompt prediction of patients at risk of massive transfusion (MT) is crucial. We investigated the ability of the inflammatory marker interleukin (IL)-6 to forecast MT in severely injured trauma patients. IL-6 plasma levels were measured upon admission. Receiver operating characteristic curves (ROCs) were calculated, and sensitivity and specificity were determined. In this retrospective study, a total of 468 predominantly male (77.8%) patients, with a median injury severity score (ISS) of 25 (17–34), were included. The Youden index for the prediction of MT within 6 and 24 h was 351 pg/mL. Patients were dichotomized into two groups: (i) low-IL-6 < 350 pg/mL and (ii) high-IL-6 ≥ 350 pg/mL. IL-6 ≥ 350 pg/mL was associated with a lower prothrombin time index, a higher activated partial thromboplastin time, and a lower fibrinogen concentration compared with IL-6 < 350 pg/mL (p <0.0001 for all). Thromboelastometric parameters were significantly different between groups (p <0.03 in all). More patients in the high-IL-6 group received MT (p <0.0001). The ROCs revealed an area under the curve of 0.76 vs. 0.82 for the high-IL-6 group for receiving MT in the first 6 and 24 h. IL-6 ≥ 350 pg/mL predicted MT within 6 and 24 h with a sensitivity of 45% and 58%, respectively, and a specificity of 89%. IL-6 ≥ 350 pg/mL appears to be a reasonable early predictor for coagulopathy and MT within the first 6 and 24 h intervals. Large-scale prospective studies are warranted to confirm these findings.

Bleeding guidelines currently recommend use of viscoelastic testing (VET) to direct haemostatic resuscitation in severe haemorrhage. However, VET-derived parameters of clot initiation, such as clotting time (CT) and activated clotting time (ACT), might not adequately reflect a clinically relevant interaction of procoagulant and anticoagulant activity, as revealed by thrombin generation assays. The aim of this study was to evaluate the ability of CT and ACT to indicate thrombin generation activity. Citrated whole blood obtained from 13 healthy volunteers underwent a 50% crystalloid dilution (DL-50%), followed by spiking with four-factor prothrombin complex concentrate (DL-50% + 4F-PCC). Changes in thrombin generation activity were compared with the VET parameters CT and ACT derived from four commercially available viscoelastic devices (ROTEM® Delta, ClotPro®, TEG®6s, and Quantra®) and standard coagulation tests. Dilution of whole blood resulted in a marked increase in velocity index, peak height, and endogenous thrombin potential (all P<0.01), with a further substantial increase after spiking with 4F-PCC (all P<0.001). In contrast, CT and ACT were significantly prolonged in response to DL-50% on all devices (all P<0.05). Subsequent spiking of diluted blood with 4F-PCC had no impact on CT and ACT derived from VET analysers, but it restored standard coagulation tests without reaching baseline values (all P<0.01). Upregulated thrombin generation parameters after PCC spiking were not displayed by CT, ACT, or standard tests. Our results do not support treatment algorithms using prolonged CT or ACT as a trigger for administration of PCC to augment thrombin generation.

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.