Exosite 2-Directed Ligands Attenuate Protein C Activation by the Thrombin-Thrombomodulin Complex.

Thrombin activity, inhibition, and localization are regulated by two exosites that flank the active site. Substrates, cofactors, and inhibitors bind to exosite 1 to promote active site access, whereas exosite 2 interactions hold thrombin on cells, platelets, and proteins. The exosites also serve allosteric roles, whereby ligand binding alters thrombin activity. Previously, we showed that ligands that bind exosite 2 attenuate the exosite 1-mediated interaction of thrombin with fibrin, demonstrating allosteric connection between the exosites. To determine the functional consequences of these inter-exosite interactions, we examined the effect of exosite 2 ligands on thrombin's interaction with thrombomodulin, a key cofactor that binds exosite 1 and redirects thrombin activity to the anticoagulant protein C pathway. Exosite 2-directed ligands, which included the HD22 aptamer, glycoprotein 1bα-derived peptide, and fibrinogen γ'-chain peptide, reduced the level of exosite 1-mediated thrombin binding to the thrombomodulin peptide consisting of the fourth, fifth, and sixth epidermal-like growth factor-like domains, decreasing affinity by >10-fold, and attenuated thrombomodulin-dependent activation of protein C by 60-80%. The ligands had similar effects on thrombin-mediated protein C activation with intact soluble thrombomodulin and with thrombomodulin on the surface of cultured endothelial cells. Their activity was exosite 2-specific because it was attenuated when RA-thrombin, a variant lacking exosite 2, was used in place of thrombin. These results indicate that additional reactions mediated by exosite 1 are amenable to regulation by exosite 2 ligation, providing further evidence of inter-exosite allosteric regulation of thrombin activity.

Evolving use of new oral anticoagulants for treatment of venous thromboembolism.

The new oral anticoagulants (NOACs), which include dabigatran, rivaroxaban, apixaban, and edoxaban, are poised to replace warfarin for treatment of the majority of patients with venous thromboembolism (VTE). With a rapid onset of action and the capacity to be administered in fixed doses without routine coagulation monitoring, NOACs streamline VTE treatment. In phase 3 trials in patients with acute symptomatic VTE, NOACs have been shown to be noninferior to conventional anticoagulant therapy for prevention of recurrence and are associated with less bleeding. Rivaroxaban and dabigatran are already licensed for VTE treatment in the United States, and apixaban and edoxaban are under regulatory consideration for this indication. As the number of approved drugs increases, clinicians will need to choose the right anticoagulant for the right VTE patient. To help with this decision, this review (1) compares the pharmacologic profiles of the NOACs, (2) outlines the unique design features of the phase 3 trials that evaluated the NOACs for VTE treatment, (3) reviews the results of these trials highlighting similarities and differences in the findings, (4) provides perspective about which VTE patients should receive conventional treatment or are candidates for NOACs, and (5) offers suggestions about how to choose among the NOACs.

Overview of the new oral anticoagulants: opportunities and challenges.

The non-vitamin K antagonist oral anticoagulants (NOACs) are replacing warfarin for many indications. These agents include dabigatran, which inhibits thrombin, and rivaroxaban, apixaban, and edoxaban, which inhibit factor Xa. All 4 agents are licensed in the United States for stroke prevention in atrial fibrillation and for treatment of venous thromboembolism and rivaroxaban and apixaban are approved for thromboprophylaxis after elective hip or knee arthroplasty. The NOACs are at least as effective as warfarin, but are not only more convenient to administer because they can be given in fixed doses without routine coagulation monitoring but also are safer because they are associated with less intracranial bleeding. As part of a theme series on the NOACs, this article (1) compares the pharmacological profiles of the NOACs with that of warfarin, (2) identifies the doses of the NOACs for each approved indication, (3) provides an overview of the completed phase III trials with the NOACs, (4) briefly discusses the ongoing studies with the NOACs for new indications, (5) reviews the emerging real-world data with the NOACs, and (6) highlights the potential opportunities for the NOACs and identifies the remaining challenges.

Oral direct factor Xa inhibitors.

Vitamin K antagonists, such as warfarin, have been the mainstay of oral anticoagulation for many decades. Although effective, warfarin has numerous limitations, including a variable dose requirement from patient to patient because of differences in dietary vitamin K intake, common genetic polymorphisms, and multiple drug interactions that affect its pharmacodynamics and metabolism. Consequently, warfarin requires frequent monitoring to ensure that a therapeutic anticoagulant effect has been achieved because excessive anticoagulation can lead to bleeding, and because insufficient anticoagulation can result in thrombosis. Such monitoring is burdensome for patients and physicians and is costly for the health care system. These limitations have prompted the development of new oral anticoagulants that target either factor Xa or thrombin. Although the path to the development of these drugs has been long, the new drugs are at least as effective and safe as warfarin, but they streamline clinical care because they can be administered in fixed doses without routine coagulation monitoring. This article focuses on rivaroxaban, apixaban, and edoxaban, the oral factor Xa inhibitors in the most advanced stages of development. After 20 years of discovery research, these agents are already licensed for several indications. Thus, the long path to finding replacements for warfarin has finally reached fruition. Therefore, development of the oral factor Xa inhibitors represents a translational science success story.

Cross-immunity against SARS-COV-2 variants of concern in naturally infected critically ill COVID-19 patients.

Critically ill patients infected with SARS-CoV-2 display adaptive immunity, but it is unknown if they develop cross-reactivity to variants of concern (VOCs). We profiled cross-immunity against SARS-CoV-2 VOCs in naturally infected, non-vaccinated, critically ill COVID-19 patients. Wave-1 patients (wild-type infection) were similar in demographics to Wave-3 patients (wild-type/alpha infection), but Wave-3 patients had higher illness severity. Wave-1 patients developed increasing neutralizing antibodies to all variants, as did patients during Wave-3. Wave-3 patients, when compared to Wave-1, developed more robust antibody responses, particularly for wild-type, alpha, beta and delta variants. Within Wave-3, neutralizing antibodies were significantly less to beta and gamma VOCs, as compared to wild-type, alpha and delta. Patients previously diagnosed with cancer or chronic obstructive pulmonary disease had significantly fewer neutralizing antibodies. Naturally infected ICU patients developed adaptive responses to all VOCs, with greater responses in those patients more likely to be infected with the alpha variant, versus wild-type.

The association between anticoagulation and adverse outcomes after a positive SARS-CoV-2 test among older outpatients: A population-based cohort study.

INTRODUCTION: Anticoagulation may improve outcomes in patients with COVID-19 when started early in the course of illness. MATERIALS AND METHODS: This was a population-based cohort study using linked administrative datasets of outpatients aged ≥65 years old testing positive for SARS-CoV-2 between January 1 and December 31, 2020 in Ontario, Canada. The key exposure was anticoagulation with warfarin or direct oral anticoagulants before COVID-19 diagnosis. We calculated propensity scores and used matching weights (MWs) to reduce baseline differences between anticoagulated and non-anticoagulated patients. The primary outcome was a composite of death or hospitalization within 60 days of a positive SARS-CoV-2 test. We used the Kaplan-Meier method and cumulative incidence functions to estimate risk of the primary and component outcomes at 60 days. RESULTS: We studied 23,159 outpatients (mean age 78.5 years; 13,474 [58.2%] female), among whom 3200 (13.8%) deaths and 3183 (13.7%) hospitalizations occurred within 60 days of the SARS-CoV-2 test. After application of MWs, the 60-day risk of death or hospitalization was 29.2% (95% CI 27.4%-31.2%) for anticoagulated individuals and 32.1% (95% CI 30.7%-33.5%) without anticoagulation (absolute risk difference [ARD], -2.9%; p = 0.005). Anticoagulation was also associated with a lower risk of death: 18.6% (95% CI 17.0%-20.2%) with anticoagulation and 20.9% (95% CI 19.7%-22.2%) in non-anticoagulated patients (ARD -2.3%; p = 0.005). CONCLUSIONS: Among outpatients aged ≥65 years, oral anticoagulation at the time of a positive SARS-CoV-2 test was associated with a lower risk of a composite of death or hospitalization within 60 days.

Immunothrombosis Biomarkers for Distinguishing Coronavirus Disease 2019 Patients From Noncoronavirus Disease Septic Patients With Pneumonia and for Predicting ICU Mortality.

IMPORTANCE: Coronavirus disease 2019 patients have an increased risk of thrombotic complications that may reflect immunothrombosis, a process characterized by blood clotting, endothelial dysfunction, and the release of neutrophil extracellular traps. To date, few studies have investigated longitudinal changes in immunothrombosis biomarkers in these patients. Furthermore, how these longitudinal changes differ between coronavirus disease 2019 patients and noncoronavirus disease septic patients with pneumonia are unknown. OBJECTIVES: In this pilot observational study, we investigated the utility of immunothrombosis biomarkers for distinguishing between coronavirus disease 2019 patients and noncoronavirus disease septic patients with pneumonia. We also evaluated the utility of the biomarkers for predicting ICU mortality in these patients. DESIGN SETTING AND PARTICIPANTS: The participants were ICU patients with coronavirus disease 2019 (n = 14), noncoronavirus disease septic patients with pneumonia (n = 19), and healthy age-matched controls (n = 14). MAIN OUTCOMES AND MEASURES: Nine biomarkers were measured from plasma samples (on days 1, 2, 4, 7, 10, and/or 14). Analysis was based on binomial logit models and receiver operating characteristic analyses. RESULTS: Cell-free DNA, d-dimer, soluble endothelial protein C receptor, protein C, soluble thrombomodulin, fibrinogen, citrullinated histones, and thrombin-antithrombin complexes have significant powers for distinguishing coronavirus disease 2019 patients from healthy individuals. In comparison, fibrinogen, soluble endothelial protein C receptor, antithrombin, and cell-free DNA have significant powers for distinguishing coronavirus disease 2019 from pneumonia patients. The predictors of ICU mortality differ between the two patient groups: soluble thrombomodulin and citrullinated histones for coronavirus disease 2019 patients, and protein C and cell-free DNA or fibrinogen for pneumonia patients. In both patient groups, the most recent biomarker values have stronger prognostic value than their ICU day 1 values. CONCLUSIONS AND RELEVANCE: Fibrinogen, soluble endothelial protein C receptor, antithrombin, and cell-free DNA have utility for distinguishing coronavirus disease 2019 patients from noncoronavirus disease septic patients with pneumonia. The most important predictors of ICU mortality are soluble thrombomodulin/citrullinated histones for coronavirus disease 2019 patients, and protein C/cell-free DNA for noncoronavirus disease pneumonia patients. This hypothesis-generating study suggests that the pathophysiology of immunothrombosis differs between the two patient groups.

Biomarkers of coagulation, endothelial function, and fibrinolysis in critically ill patients with COVID-19: A single-center prospective longitudinal study.

BACKGROUND: Immunothrombosis and coagulopathy in the lung microvasculature may lead to lung injury and disease progression in coronavirus disease 2019 (COVID-19). We aim to identify biomarkers of coagulation, endothelial function, and fibrinolysis that are associated with disease severity and may have prognostic potential. METHODS: We performed a single-center prospective study of 14 adult COVID-19(+) intensive care unit patients who were age- and sex-matched to 14 COVID-19(-) intensive care unit patients, and healthy controls. Daily blood draws, clinical data, and patient characteristics were collected. Baseline values for 10 biomarkers of interest were compared between the three groups, and visualized using Fisher's linear discriminant function. Linear repeated-measures mixed models were used to screen biomarkers for associations with mortality. Selected biomarkers were further explored and entered into an unsupervised longitudinal clustering machine learning algorithm to identify trends and targets that may be used for future predictive modelling efforts. RESULTS: Elevated D-dimer was the strongest contributor in distinguishing COVID-19 status; however, D-dimer was not associated with survival. Variable selection identified clot lysis time, and antigen levels of soluble thrombomodulin (sTM), plasminogen activator inhibitor-1 (PAI-1), and plasminogen as biomarkers associated with death. Longitudinal multivariate k-means clustering on these biomarkers alone identified two clusters of COVID-19(+) patients: low (30%) and high (100%) mortality groups. Biomarker trajectories that characterized the high mortality cluster were higher clot lysis times (inhibited fibrinolysis), higher sTM and PAI-1 levels, and lower plasminogen levels. CONCLUSIONS: Longitudinal trajectories of clot lysis time, sTM, PAI-1, and plasminogen may have predictive ability for mortality in COVID-19.

A regional massive hemorrhage protocol developed through a modified Delphi technique.

BACKGROUND: A massive hemorrhage protocol (MHP) enables rapid delivery of blood components in a patient who is exsanguinating pending definitive hemorrhage control, but there is variability in MHP implementation rates, content and compliance owing to challenges presented by infrequent activation, variable team performance and patient acuity. The goal of this project was to identify the key evidence-based principles and quality indicators required to develop a standardized regional MHP. METHODS: A modified Delphi consensus technique was performed in the spring and summer of 2018. Panellists used survey links to independently review and rate (on a 7-point Likert scale) 43 statements and 8 quality indicators drafted by a steering committee composed of transfusion medicine specialists and technologists, and trauma physicians. External stakeholder input from all hospitals in Ontario was sought. RESULTS: Three rounds were held with 36 experts from diverse clinical backgrounds. Consensus was reached for 42 statements and 8 quality indicators. Additional modifications from external stakeholders were incorporated to form the foundation for the proposed MHP. INTERPRETATION: This MHP template will provide the basis for the design of an MHP toolkit, including specific recommendations for pediatric and obstetrical patients, and for hospitals with limited availability of blood components or means to achieve definitive hemorrhage control. We believe that harmonization of MHPs in our region will simplify training, increase uptake of evidence-based interventions, enhance communication, improve patient comfort and safety, and, ultimately, improve patient outcomes.

The impact of social media promotion with infographics and podcasts on research dissemination and readership.

OBJECTIVE: In 2015 and 2016, the Canadian Journal of Emergency Medicine (CJEM) Social Media (SoMe) Team collaborated with established medical websites to promote CJEM articles using podcasts and infographics while tracking dissemination and readership. METHODS: CJEM publications in the "Original Research" and "State of the Art" sections were selected by the SoMe Team for podcast and infographic promotion based on their perceived interest to emergency physicians. A control group was composed retrospectively of articles from the 2015 and 2016 issues with the highest Altmetric score that received standard Facebook and Twitter promotions. Studies on SoMe topics were excluded. Dissemination was quantified by January 1, 2017 Altmetric scores. Readership was measured by abstract and full-text views over a 3-month period. The number needed to view (NNV) was calculated by dividing abstract views by full-text views. RESULTS: Twenty-nine of 88 articles that met inclusion were included in the podcast (6), infographic (11), and control (12) groups. Descriptive statistics (mean, 95% confidence interval) were calculated for podcast (Altmetric: 61, 42-80; Abstract: 1795, 1135-2455; Full-text: 431, 0-1031), infographic (Altmetric: 31.5, 19-43; Abstract: 590, 361-819; Full-text: 65, 33-98), and control (Altmetric: 12, 8-15; Abstract: 257, 159-354; Full-Text: 73, 38-109) articles. The NNV was 4.2 for podcast, 9.0 for infographic, and 3.5 for control articles. Discussion Limitations included selection bias, the influence of SoMe promotion on the Altmetric scores, and a lack of generalizability to other journals. CONCLUSION: Collaboration with established SoMe websites using podcasts and infographics was associated with increased Altmetric scores and abstract views but not full-text article views.

The effect of an infographic promotion on research dissemination and readership: A randomized controlled trial.

OBJECTIVE: Journals use social media to increase the awareness of their publications. Infographics show research findings in a concise and visually appealing manner, well suited for dissemination on social media platforms. We hypothesized that infographic abstracts promoted on social media would increase the dissemination and online readership of the parent research articles. METHODS: Twenty-four articles were chosen from the six issues of CJEM published between July 2016 and June 2017 and randomized to infographic or control groups. All articles were disseminated through the journal's social media accounts (Twitter and Facebook). Control articles were promoted using a screen capture image of each article's abstract on the journal's social media accounts. Infographic articles were promoted similarly using a visual infographic. Infographics were also published and promoted on the CanadiEM.org's website and social media channels. Abstract views, full-text views, and the change in Altmetric score were compared between groups using unpaired two-tailed t-tests. RESULTS: There were no significant differences in the groups at baseline. Abstract views (mean, 95% CI) were higher in the infographics (379, 287-471) than the control group (176, 136-215, p<0.001). Mean change in Altmetric scores was higher in the infographics (26, 18-34) than in the control group (3, 2-4, p<0.0001). There was no difference in full-text views between the infographics (50, 0-101) and control groups (25, 18-32). CONCLUSION: The promotion of CJEM articles using infographics on social media and the CanadiEM.org website increased Altmetric scores and abstract views. Infographics may have a role in increasing awareness of medical literature.

Mechanistic Basis for the Differential Effects of Rivaroxaban and Apixaban on Global Tests of Coagulation.

Rivaroxaban and apixaban are both small molecules that reversibly inhibit factor Xa. Compared with rivaroxaban, apixaban has minimal effects on the prothrombin time and activated partial thromboplastin time. To investigate this phenomenon, we used a factor Xa-directed substrate in a buffer system. Although rivaroxaban and apixaban inhibited factor Xa with similar K i values at equilibrium, kinetic measurements revealed that rivaroxaban inhibited factor Xa up to 4-fold faster than apixaban ( p < 0.001). Using a discontinuous chromogenic assay to monitor thrombin production by prothrombinase in a purified system, rivaroxaban was 4-fold more potent than apixaban (K i values of 0.7 ± 0.3 and 2.9 ± 0.5 nM, respectively; p = 0.02). Likewise, in thrombin generation assays in plasma, rivaroxaban prolonged the lag time and suppressed endogenous thrombin potential to a greater extent than apixaban. To characterize how the two inhibitors differ in recognizing factor Xa, inhibition of prothrombinase was monitored in real-time using a fluorescent probe for thrombin. The data were fit using a mixed-inhibition model and the individual association and dissociation rate constants were determined. The association rates for the binding of rivaroxaban to either free factor Xa or factor Xa incorporated into the prothrombinase complex were 10- and 1,193-fold faster than those for apixaban, respectively, whereas dissociation rates were about 3-fold faster. Collectively, these findings suggest that rivaroxaban and apixaban differ in their capacity to inhibit factor Xa and provide a plausible explanation for the observation that rivaroxaban has a greater effect on global tests of coagulation than apixaban.

Dabigatran and Argatroban Diametrically Modulate Thrombin Exosite Function.

Thrombin is a highly plastic molecule whose activity and specificity are regulated by exosites 1 and 2, positively-charged domains that flank the active site. Exosite binding by substrates and cofactors regulates thrombin activity by localizing thrombin, guiding substrates, and by inducing allosteric changes at the active site. Although inter-exosite and exosite-to-active-site allostery have been demonstrated, the impact of active site ligation on exosite function has not been examined. To address this gap, we used surface plasmon resonance to determine the effects of dabigatran and argatroban, active site-directed inhibitors, on thrombin binding to immobilized γA/γA-fibrin or glycoprotein Ibα peptide via exosite 1 and 2, respectively, and thrombin binding to γA/γ'-fibrin or factor Va, which is mediated by both exosites. Whereas dabigatran attenuated binding, argatroban increased thrombin binding to γA/γA- and γA/γ'-fibrin and to factor Va. The results with immobilized fibrin were confirmed by examining the binding of radiolabeled thrombin to fibrin clots. Thus, dabigatran modestly accelerated the dissociation of thrombin from γA/γA-fibrin clots, whereas argatroban attenuated dissociation. Dabigatran had no effect on thrombin binding to glycoprotein Ibα peptide, whereas argatroban promoted binding. These findings not only highlight functional effects of thrombin allostery, but also suggest that individual active site-directed thrombin inhibitors uniquely modulate exosite function, thereby identifying potential novel mechanisms of action.