A Strategy for Gene Knockdown in Dinoflagellates.

Dinoflagellates are unicellular protists that display unusual nuclear features such as large genomes, condensed chromosomes and multiple gene copies organized as tandem gene arrays. Genetic regulation is believed to be controlled at the translational rather than transcriptional level. An important player in this process is initiation factor eIF4E which binds the 7-methylguanosine cap structure (m7G) at the 5'-end of mRNA. Transcriptome analysis of eleven dinoflagellate species has established that each species encodes between eight to fifteen eIF4E family members. Determining the role of eIF4E family members in gene expression requires a method of knocking down their expression. In other eukaryotes this can be accomplished using translational blocking morpholinos that bind to complementary strands of RNA, therefore inhibiting the mRNA processing. Previously, unmodified morpholinos lacked the ability to pass through cell membranes, however peptide-based reagents have been used to deliver substances into the cytosol of cells by an endocytosis-mediated process without damaging the cell membrane. We have successfully delivered fluorescently-tagged morpholinos to the cytosol of Amphidinium carterae by using a specific cell penetrating peptide with the goal to target an eIF4e-1a sequence to inhibit translation. Specific eIF4e knockdown success (up to 42%) has been characterized via microscopy and western blot analysis.

Tissue Factor Pathway Inhibitor Levels During Veno-Arterial Extracorporeal Membrane Oxygenation in Adults.

Tissue factor pathway inhibitor (TFPI) has multiple anticoagulant properties. To our knowledge, no studies have measured TFPI levels in adult veno-arterial (VA) extracorporeal membrane oxygenation patients. We hypothesized that adult VA ECMO patients would have increased TFPI levels and slowed tissue factor triggered thrombin generation. Twenty VA ECMO patients had TFPI levels and thrombin generation lag time measured on ECMO day 1 or 2, day 3, and day 5. TFPI levels and thrombin generation lag time were compared against healthy control plasma samples. Mean TFPI levels were significantly higher in ECMO patients on ECMO day 1 or 2 = 81,877 ± 19,481 pg/mL, day 3 = 73,907 ± 26,690 pg/mL, and day 5 = 77,812 ± 23,484 pg/mL compared with control plasma = 38,958 ± 9,225 pg/mL (P < 0.001 for all comparisons). Median thrombin generation lag time was significantly longer in ECMO patients on ECMO day 1 or 2 = 10.0 minutes [7.5, 13.8], day 3 = 9.0 minutes [6.8, 12.1], and day 5 = 10.7 minutes [8.3, 15.2] compared with control plasma = 3.6 minutes [2.9, 4.2] (P < 0.001 for all comparisons). TFPI is increased in VA ECMO patients and tissue factor triggered thrombin generation is slowed. Increased TFPI levels could contribute to the multifactorial coagulopathy that occurs during ECMO.

Hematologic evaluation of intraoperative autologous blood collection and allogeneic transfusion in cardiac surgery.

BACKGROUND: Acute normovolemic hemodilution is recommended as a technique to reduce allogeneic red blood cell (RBC) transfusions in cardiac surgery, but its efficacy to reduce non-RBC transfusion has not been consistently demonstrated. We hypothesized that intraoperative large-volume autologous whole blood (AWB) collection and reinfusion improves viscoelastic coagulation parameters. STUDY DESIGN AND METHODS: Prospective observational study of cardiac surgery patients at the University of Maryland Medical Center between December 2017 and August 2019. Rotational thromboelastometry parameters were compared between AWB and control groups (n = 25 in each group) at three time points: T1, baseline; T2, on cardiopulmonary bypass (CPB) after the cross-clamp removal; and T3, 30-60 minutes after protamine administration. The study's primary outcomes were whole blood viscoelastic coagulation parameters that included EXTEM clotting time (CT), FIBTEM amplitude at 10 minutes, and EXTEM amplitude at 10 minutes (EXTEM-A10 ). Chest tube drainage and allogeneic transfusion were secondary outcomes. RESULTS: Reinfusion of AWB after CPB resulted in a significantly shorter EXTEM CT; mean difference, -11.4 seconds (-21.4 to -1.4; P = .03). It also resulted in a greater percentage increase in EXTEM A10 from T2 to T3; mean difference, 7.8% (95% CI, 1.1%-14.5%; P = .02). Statistical significance was not found in 24-hour chest tube drainage. CONCLUSION: Large-volume AWB collection and reinfusion are feasible in selected cardiac surgical patients, and may be associated with prohemostatic effects according to thromboelastometry, warranting further investigation with a prospective randomized study.

A Pharmacokinetic and Pharmacodynamic Investigation of an ε-Aminocaproic Acid Regimen Designed for Cardiac Surgery With Cardiopulmonary Bypass.

OBJECTIVE: To investigate the pharmacokinetics and pharmacodynamics of an ε-aminocaproic acid (EACA) regimen designed for cardiac surgery with cardiopulmonary bypass (CPB). DESIGN: Prospective observational study requiring blood sampling to measure EACA concentrations and fibrinolysis markers (fibrinogen, D-dimer, α2-antiplasmin, and tissue plasminogen activator-plasminogen activator inhibitor [tPA-PAI-1] complex). SETTING: Single-center, tertiary medical center. PARTICIPANTS: Patients who underwent cardiac surgery with CPB between 2018 and 2019 for aortic or mitral valve replacement/repair or coronary artery bypass grafting. Previous sternotomy patients were included. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: The pharmacokinetics of EACA, during CPB, were described by a 3-compartment disposition model. EACA concentrations were greater than 130 mg/L in all patients after CPB and in most patients during CPB. The D-dimer level trended up and reached a peak median level of 1.35 mg/L of fibrinogen equivalence units (FEU) at 15 minutes after protamine administration. The median change in D-dimer (ΔD-dimer) from baseline to 15 minutes after protamine was 0.34 (-0.48 to 3.81) mg/L FEU. ΔD-dimer did not correlate with EACA concentration intraoperatively, urine output, body weight, glomerular filtration rate, cell salvage volume, and ultrafiltration volume. The median 24-hour chest tube output was 445 (180-1,011) mL. CONCLUSION: This regimen provided maximum EACA concentrations near the time of protamine administration, with a total perioperative dose of 15 g. Most patients had EACA concentrations greater than the target during CPB. ΔD-dimer did not correlate with EACA concentration. The median 24-hour chest tube output compared well to similar studies that used higher doses of EACA.

Clotting Time Results Are Not Interchangeable Between EXTEM and FIBTEM on Rotational Thromboelastometry.

OBJECTIVE: To explore how cytochalasin D (CyD) affects clot initiation and to compare clotting times (CTs) of EXTEM and FIBTEM on rotational thromboelastometry in cardiac surgical patients undergoing cardiopulmonary bypass (CPB). DESIGN: Retrospective cohort study with translational in vitro coagulation experiments. SETTING: Single-center, tertiary, academic medical center. PARTICIPANTS: Patients who underwent cardiac surgery with CPB between November 2015 and August 2017. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: The study's primary measurements were CTEXTEM and CTFIBTEM before and after CPB. Additionally, the authors performed translational in vitro coagulation experiments using commercial plasma. In these experiments, the impact of CyD on in vitro thrombin generation (TG) was assessed using 10 platelet-rich plasma (PRP) samples and calibrated automated thrombogram. The impact of CyD on ROTEM-CT also was evaluated in vitro using the same 10 PRP samples. One hundred fifty-three patients had clinical CTEXTEM and CTFIBTEM measurements. CTFIBTEM was shorter than CTEXTEM before and after CPB by 6.8% (95% confidence interval [CI], 5.5-8.1) and 8.9% (95% CI, 4.7-13.0), respectively. These results correlated with in vitro experiments, where TG lag time was shortened by CyD and CTFIBTEM was shorter than CTEXTEM. CONCLUSION: CyD shortens the onset of TG and clot formation, resulting in shorter CTFIBTEM than CTEXTEM. The authors' data suggest that CTEXTEM and CTFIBTEM are not interchangeable. Additional clinical studies are warranted to assess if CTFIBTEM can be used to optimize the indication for plasma transfusion.

Coagulation Factor Levels and Underlying Thrombin Generation Patterns in Adult Extracorporeal Membrane Oxygenation Patients.

BACKGROUND: There is a paucity of data on the underlying procoagulant-anticoagulant balance during extracorporeal membrane oxygenation (ECMO). We hypothesized that adult ECMO patients would have an imbalance between procoagulant and anticoagulant factors, leading to an abnormal underlying thrombin generation (TG) pattern. METHODS: Twenty adult venoarterial (VA) ECMO patients had procoagulant and anticoagulant factor levels measured temporally on ECMO day 1 or 2, day 3, and day 5. In heparin-neutralized plasma, underlying TG patterns, and sensitivity to activated protein C were assessed using calibrated automated thrombogram. TG parameters including lag time, peak TG, and endogenous thrombin potential (ETP) were compared against 5 normal plasma controls (3 males and 2 females) obtained from a commercial supplier. Thrombomodulin (TM) was added to some samples to evaluate for activated protein C resistance. RESULTS: Procoagulant factors (factor [F] II, FV, and FX) were mostly in normal reference ranges and gradually increased during the first 5 ECMO days (P = .022, <.001, <.001). FVIII levels were elevated at all time points and did not change (P = .766). In contrast, FXI was in the low-normal range but did not increase during ECMO (P = .093). Antithrombin (AT) and protein C levels were below normal but increased during the first 5 ECMO days (P = .002 and P = .014). Heparinase-treated samples showed prolonged lag time, increased peak TG, and increased ETP compared to controls; mean difference in lag time on ECMO day 1 or 2 = 6.0 minutes (99% confidence interval [CI], 2.8-9.2), peak TG = 193.4 (99% CI, 122.5-264.3), and ETP = 1170.4 (99% CI, 723.2-1617.6). After in vitro TM treatment, differences in TG parameters were accentuated and ECMO samples appeared insensitive to TM treatment; mean difference in lag time on ECMO day 1 or 2 = 9.3 minutes (99% CI, 6.2-12.4), peak TG = 233.0 (99% CI, 140.9-325.1), and ETP = 1322.5 (99% CI, 764.8-1880.2). Similar differences in TG parameters were observed on ECMO days 3 and 5. CONCLUSIONS: Contact activation occurs during ECMO, but procoagulant factor levels are generally preserved. Although heparin-neutralized TG is delayed, peak TG and ETP are supranormal in the setting of high FVIII and low AT and protein C levels. Resistance to TM is also apparent. These changes demonstrate a possible mechanism for hypercoagulability during adult VA ECMO.