Background: Thrombogenicity is a known complication of COVID-19, resulting from SARS-CoV-2 infection, with significant effects on morbidity and mortality. Objective: We aimed to better understand the effects of COVID-19 on fibrinogen and the resulting effects on clot structure, formation, and degradation. Methods: Fibrinogen isolated from COVID-19 patients and uninfected subjects was used to form uniformly concentrated clots (2 mg/ml), which were characterized using confocal microscopy, scanning electron microscopy, atomic force microscopy, and endogenous and exogenous fibrinolysis assays. Neuraminidase digestion and subsequent NANA assay were used to quantify sialic acid residue presence; clots made from the desialylated fibrinogen were then assayed similarly to the original fibrinogen clots. Results: Clots made from purified fibrinogen from COVID-19 patients were shown to be significantly stiffer and denser than clots made using fibrinogen from noninfected subjects. Endogenous and exogenous fibrinolysis assays demonstrated that clot polymerization and degradation dynamics were different for purified fibrinogen from COVID-19 patients compared with fibrinogen from noninfected subjects. Quantification of sialic acid residues via the NANA assay demonstrated that SARS-CoV-2-positive fibrinogen samples contained significantly more sialic acid. Desialylation via neuraminidase digestion resolved differences in clot density. Desialylation did not normalize differences in polymerization, but did affect rate of exogenous fibrinolysis. Discussion: These differences noted in purified SARS-CoV-2-positive clots demonstrate that structural differences in fibrinogen, and not just differences in gross fibrinogen concentration, contribute to clinical differences in thrombotic features associated with COVID-19. These structural differences are at least in part mediated by differential sialylation.

Objective: To evaluate the feasibility of recruitment, preliminary efficacy, and acceptability of auricular percutaneous electrical nerve field stimulation (PENFS) for the treatment of fibromyalgia in veterans, using neuroimaging as an outcome measure and a biomarker of treatment response. Design: Randomized, controlled, single-blind. Setting: Government hospital. Subjects: Twenty-one veterans with fibromyalgia were randomized to standard therapy (ST) control or ST with auricular PENFS treatment. Methods: Participants received weekly visits with a pain practitioner over 4 weeks. The PENFS group received reapplication of PENFS at each weekly visit. Resting-state functional connectivity magnetic resonance imaging (rs-fcMRI) data were collected within 2 weeks prior to initiating treatment and 2 weeks following the final treatment. Analysis of rs-fcMRI used a right posterior insula seed. Pain and function were assessed at baseline and at 2, 6, and 12 weeks post-treatment. Results: At 12 weeks post-treatment, there was a nonsignificant trend toward improved pain scores and significant improvements in pain interference with sleep among the PENFS treatment group as compared with the ST controls. Neuroimaging data displayed increased connectivity to areas of the cerebellum and executive control networks in the PENFS group as compared with the ST control group following treatment. Conclusions: There was a trend toward improved pain and function among veterans with fibromyalgia in the ST + PENFS group as compared with the ST control group. Pain and functional outcomes correlated with altered rs-fcMRI network connectivity. Neuroimaging results differed between groups, suggesting an alternative underlying mechanism for PENFS analgesia.

Orthologous proteins contain sequence disparity guided by natural selection. In certain cases, species-specific protein functionality predicts pharmacological enhancement, such as greater specific activity or stability. However, immunological barriers generally preclude use of nonhuman proteins as therapeutics, and difficulty exists in the identification of individual sequence determinants among the overall sequence disparity. Ancestral sequence reconstruction (ASR) represents a platform for the prediction and resurrection of ancient gene and protein sequences. Recently, we demonstrated that ASR can be used as a platform to facilitate the identification of therapeutic protein variants with enhanced properties. Specifically, we identified coagulation factor VIII (FVIII) variants with improved specific activity, biosynthesis, stability, and resistance to anti-human FVIII antibody–based inhibition. In the current study, we resurrected a panel of ancient mammalian coagulation factor IX (FIX) variants with the goal of identifying improved pharmaceutical candidates. One variant (An96) demonstrated 12-fold greater FIX activity production than human FIX. Addition of the R338L Padua substitution further increased An96 activity, suggesting independent but additive mechanisms. after adeno-associated virus 2 (AAV2)/8-FIX gene therapy, 10-fold greater plasma FIX activity was observed in hemophilia B mice administered AAV2/8-An96–Padua as compared with AAV2/8-human FIX–Padua. Furthermore, phenotypic correction conferred by the ancestral variant was confirmed using a saphenous vein bleeding challenge and thromboelastography. Collectively, these findings validate the ASR drug discovery platform as well as identify an ancient FIX candidate for pharmaceutical development.

Background: Platelet function testing to monitor antiplatelet therapy is important for reducing thromboembolic complications, yet variability across testing methods remains challenging. Here we evaluated the agreement of four different testing platforms used to monitor antiplatelet effects of aspirin (ASA) or P2Y12 inhibitors (P2Y12-I). Methods: Blood and urine specimens from 20 patients receiving dual antiplatelet therapy were analyzed by light transmission aggregometry (LTA), whole blood aggregometry (WBA), VerifyNow PRUTest and AspirinWorks. Result interpretation based on pre-defined cutoff values was used to calculate raw agreement indices, and Pearson's correlation coefficient determined using individual units of measure. Results: Agreement between LTA and WBA for P2Y12-I-response was 60% (r = 0.65, high-dose ADP; r = 0.75, low-dose ADP). VerifyNow agreed with LTA in 75% (r = 0.86, high-dose ADP; r = 0.75, low-dose ADP) and WBA in 55% (r = 0.57) of cases. Agreement between LTA and WBA for ASA-response was 45% (r = 0.09, high-dose collagen WBA; r = 0.19, low-dose collagen WBA). AspirinWorks agreed with LTA in 60% (r = 0.32) and WBA in 35% (r = 0.02, high-dose collagen WBA; r = 0.08, low-dose collagen WBA) of cases. Conclusions: Overall agreement varied from 35 to 75%. LTA and VerifyNow demonstrated the highest agreement for P2Y12-I-response, followed by moderate agreement between LTA and WBA. LTA and AspirinWorks showed moderate agreement for aspirin response, while WBA showed the weakest agreement with both LTA and AspirinWorks. The results from this study support the continued use of LTA for monitoring dual antiplatelet therapy, with VerifyNow as an appropriate alternative for P2Y12-I-response. Integration of results obtained from these varied testing platforms with patient outcomes remains paramount for future studies.

BACKGROUND: Retrospective studies suggested that storage age of RBCs is associated with inflammation and thromboembolism. The Red Cell Storage Duration Study (RECESS) trial randomized subjects undergoing complex cardiac surgery to receive RBCs stored for shorter versus longer periods, and no difference was seen in the primary outcome of change in multiple organ dysfunction score. STUDY DESIGN AND METHODS: In the current study, 90 subjects from the RECESS trial were studied intensively using a range of hemostasis, immunologic, and nitric oxide parameters. Samples were collected before transfusion and on Days 2, 6, 28, and 180 after transfusion. RESULTS: Of 71 parameters tested, only 4 showed a significant difference after transfusion between study arms: CD8+ T-cell interferon-γ secretion and the concentration of extracellular vesicles bearing the B-cell marker CD19 were higher, and plasma endothelial growth factor levels were lower in recipients of fresh versus aged RBCs. Plasma interleukin-6 was higher at Day 2 and lower at Days 6 and 28 in recipients of fresh versus aged RBCs. Multiple parameters showed significant modulation after surgery and transfusion. Most analytes that changed after surgery did not differ based on transfusion status. Several extracellular vesicle markers, including two associated with platelets (CD41a and CD62P), decreased in transfused patients more than in those who underwent surgery without transfusion. CONCLUSIONS: Transfusion of fresh versus aged RBCs does not result in substantial changes in hemostasis, immune, or nitric oxide parameters. It is possible that transfusion modulates the level of platelet-derived extracellular vesicles, which will require study of patients randomly assigned to receipt of transfusion to define.

ABSTRACT: The coronavirus disease (COVID-19) pandemic has threatened millions of lives worldwide with severe systemic inflammation, organ dysfunction, and thromboembolic disease. Within our institution, many critically ill COVID-19-positive patients suffered major thrombotic events, prompting our clinicians to evaluate hypercoagulability outside of traditional coagulation testing.We determined the prevalence of fibrinolysis shutdown via rotational thromboelastometry (ROTEM, Instrumentation Laboratories, Bedford, Mass) in patients admitted to the intensive care unit over a period of 3 weeks. In 25 patients who had a ROTEM test, we found that 11 (44%) met criteria for fibrinolysis shutdown. Eight of 9 (73%) of the VTE patients met criteria for fibrinolysis shutdown.Given the high rate of fibrinolysis shutdown in these patients, our data support using viscoelastic testing to evaluate for the presence of impaired fibrinolysis. This may help identify patient subsets who might benefit from the administration of fibrinolytics.

Patients critically ill with COVID-19 are at risk for thrombotic events despite prophylactic anticoagulation. Impaired fibrinolysis has been proposed as an underlying mechanism. Our objective was to determine if fibrinolysis stimulated by tissue plasminogen activator (tPA) differed between COVID patients and controls. Plasma from 14 COVID patients on prophylactic heparin therapy was obtained and compared with heparinized plasma from 14 different healthy donors to act as controls. Kaolin activated thromboelastography with heparinase was utilized to obtain baseline measurements and then repeated with the addition of 4 nM tPA. Baseline fibrinogen levels were higher in COVID plasma as measured by maximum clot amplitude (43.6 ± 6.9 mm vs. 23.2 ± 5.5 mm, p < 0.0001) and Clauss assay (595 ± 135 mg/dL vs. 278 ± 44 mg/dL, p < 0.0001). With the addition of tPA, fibrinolysis at 30 min after MA (LY30%) was lower (37.9 ± 16.5% vs. 58.9 ± 18.3%, p = 0.0035) and time to 50% lysis was longer (48.8 ± 16.3 vs. 30.5 ± 15.4 min, p = 0.0053) in the COVID-19 samples. Clotting times and rate of fibrin polymerization (‘R’ or ‘α’ parameters) were largely the same in both groups. Clot from COVID patients contains a higher fibrin content compared to standard controls and shows resistance to fibrinolysis induced by tPA. These findings suggest the clinical efficacy of thrombolytics may be reduced in COVID-19 patients.

Study Objective: The objective of our study was to determine safety and pharmacology (pharmacokinetics and preliminary efficacy) of intranasal (IN) ketamine for uncontrolled cancer-related pain. Design: Dose escalation clinical trial. Setting: Outpatient. Patients: Ten adult patients with uncontrolled cancer-related pain. Intervention: Each patient received escalating doses of ketamine over four visits, each 2–5 days apart: 10 mg IN at visit 1, 10 mg intravenous (IV) at visit 2, 30 mg IN at visit 3, and 50 mg IN at visit 4. Measurements: Pain was measured before and after drug administration for up to 4 h using the 11 point (0–10) Numerical Pain Rating Scale (NPRS). Main Results: All subjects had advanced cancer, with intractable pain, despite being on moderate dosage of opioids. There was a statistically significant reduction in median NPRS by 1.5 (1–4), 3 (2–3), and 4 (3–5) points at 60 min after receiving the medication and remained decreased by 1.5 (1–2), 2 (1–2) and 1 (1–4) points at the end of the study visit (240 min) with the 10 mg, 30 mg and 50 mg IN dosage, respectively. The median percentage of maximal pain relief being 22.5 (16.6–71.5), 65.5 (40–100), and 69.25 (50–100) for 10 mg, 30 mg and 50 mg IN dosage, respectively and 100 (75–100) with 10 mg IV dose. All side effects (nausea and feeling of unreality) resolved by the end of each study visit. No severe adverse events occurred. Conclusion: In this single-institution study, all dosages of IN ketamine administered in the study (10, 30, and 50 mg) provided significant pain relief for intractable cancer-related pain and were well tolerated. The 50 mg dose provided maximal pain relief without major side effects. Further study focused on repeated administration efficacy and safety for cancer-related pain is warranted.

BACKGROUND: The purpose of this multicenter pilot study was to: (1) determine the frequency of regional cerebral oxygen saturation (rSco 2) desaturations during cardiac surgery involving cardiopulmonary bypass (CPB); (2) evaluate the accuracy of clinician-identified rSco 2 desaturations compared with those recorded continuously during surgery by the near-infrared spectroscopy (NIRS) monitor; and (3) assess the effectiveness of an intervention algorithm for reversing rSco 2 desaturations. METHODS: Two hundred thirty-five patients undergoing coronary artery bypass graft and/or valvular surgery were enrolled at 8 US centers in this prospective observational study. NIRS (Invos™ 5100C; Covidien) was used to monitor rSco 2 during surgery. The frequency and magnitude of rSco 2 decrements >20% from preanesthesia baseline were documented, and the efficacy of a standard treatment algorithm for correcting rSco 2 was determined. The data from the NIRS monitor were downloaded at the conclusion of surgery and sent to the coordinating center where the number of clinician-identified rSco 2 desaturation events was compared with the number detected by the NIRS monitor. RESULTS: The average rSco 2 obtained at baseline (mean ± SD, 61% ± 11%; 99% confidence interval, 57%-65%) and during CPB (62% ± 14%; 57%-67%) was not different. However, rSco 2 after separation from CPB (56% ± 11%; 53%-60%) was lower than measurements at baseline and during CPB (P < 0.001). During CPB, rSco 2 desaturations occurred in 61% (99% confidence interval, 50%-75%) of patients. The area under the curve for product of magnitude and duration of the rSco 2 was (mean ± SD, 145.2; 384.8% × min). Clinicians identified all patients with an rSco 2 desaturation but identified only 340 (89.5%) of the 380 total desaturation events. Of the 340 clinician-identified rSco 2 desaturation events, 115 resolved with usual clinical care before implementation of the treatment algorithm. For the remaining 225 events, the treatment algorithm resulted in resolution of the rSco 2 desaturation in all but 18 patients. CONCLUSIONS: This multicenter pilot study found that 50% to 75% of patients undergoing cardiac surgery experience one or more rSco 2 desaturations during CPB. Nearly 10% of desaturation events were not identified by clinicians, suggesting that appropriate alarming systems should be adopted to alert clinicians of such events. The intervention algorithm was effective in reversing clinically identified rSco 2 desaturations in the majority of events.

Fibromyalgia is a chronic pain state that includes widespread musculoskeletal pain, fatigue, psychiatric symptoms, cognitive and sleep disturbances, and multiple somatic symptoms. Current therapies are often insufficient or come with significant risks, and while there is an increasing demand for non-pharmacologic and especially non-opioid pain management such as that offered through complementary and alternative medicine therapies, there is currently insufficient evidence to recommend these therapies. Percutaneous electrical neural stimulation (PENS) is an evidence-based treatment option for pain conditions that involves electrical current stimulation through needles inserted into the skin. Percutaneous electrical neural field stimulation (PENFS) of the auricle is similar to PENS, but instead of targeting a single neurovascular bundle, PENFS stimulates the entire ear, covering all auricular branches of the cranial nerves, including the vagus nerve. The neural mechanisms of PENFS for fibromyalgia symptom relief are unknown. Objective: We hypothesize that PENFS treatment will decrease functional brain connectivity between the default mode network (DMN) and right posterior insula in fibromyalgia patients. We expect that the decrease in functional connectivity between the DMN and insula will correlate with patient-reported analgesic improvements as indicated by the Defense and Veterans Pain Rating Scale (DVPRS) and will be anti-correlated with patient-reported analgesic medication consumption. Exploratory analyses will be performed for further hypothesis generation. Methods: A total of 20 adults from the Atlanta Veterans Affairs Medical Center diagnosed with fibromyalgia will be randomized into 2 groups: 10 subjects to a control (standard therapy) group and 10 subjects to a PENFS treatment group. The pragmatic, standard therapy group will include pharmacologic treatments such as anticonvulsants, non-steroidal anti-inflammatory drugs, topical agents and physical therapy individualized to patient comorbidities and preferences, prescribed by a pain management practitioner. The PENFS group will include the above therapies in addition to the PENFS treatments. The PENFS subject group will have the Neuro-Stim System placed on the ear for 5 days then removed and replaced once per week for 4 weeks. The primary outcome will be resting functional magnetic resonance imaging connectivity between DMN and insula, which will also be correlated with pain relief and functional improvements. This connectivity will be analyzed utilizing functional connectivity magnetic resonance imaging (fcMRI) and will be compared with patient-reported analgesic improvements as indicated by the DVPRS and patient-reported analgesic medication consumption. Pain and function will be further evaluated using Patient-Reported Outcomes Measurement Information System measures and measures describing a person�s functional status from Activity and Participation section of the International Classification of Functioning Disability and Health. Results: This trial has been funded by the Veterans Health Administration Program Office. This study attained approval by the Emory University/Veterans Affairs (VA) institutional review board and VA Research & Development committee. Institutional review board expedited approval was granted on 2/7/17 (IRB00092224). The study start date is 6/1/17 and estimated completion date is 5/31/20. The recruitment started in June 2017. Conclusions: This is a feasibility study that is meant to demonstrate the practicality of using fcMRI to study the neural correlates of PENFS outcomes and provide information regarding power calculations in order to design and execute a larger randomized controlled clinical trial to determine the efficacy of PENFS for improving pain and function.