Evaluation of the protein C pathway in critically ill patients with severe COVID-19 as compared to bacterial sepsis

Introduction Endothelial dysfunction has been shown to play a role in severe COVID-19, the pathophysiology of which may be attributed to a myriad of factors including unmitigated immune and inflammatory response, viral-induced injury to the endothelium, end-stage organ failure, and coagulopathy. In addition, severe COVID-19 is most often seen in patients with multiple comorbidities, which themselves are often associated with endothelial dysfunction (such as myocardial and renal failure, as well as thrombotic disorders). However, the literature is still emerging on this topic and there appears to be no consensus on the extent to which endothelial dysfunction plays a role in severe COVID-19. Method The aim of this study was to assess the functionality of the endothelial protein C pathway in hospitalized patients > 18 years of age with severe COVID-19 as compared to those hospitalized with bacterial sepsis. COVID-19 (n = 31) and sepsis (n = 47) patients who were admitted to the ICU were assessed for rates of thrombin and activated protein C (APC) generation. Indirect markers of thrombin formation, including thrombin-antithrombin (TAT) complex, prothrombin fragment 1 + 2 (F1 + 2), as well as D-dimer, and protein C (PC) were measured additionally. Statistical analysis was performed via the Mann-Whitney test and a p value of < 0.05 was considered statistically significant. Results Plasma levels of free thrombin in COVID-19 and sepsis patients did not differ significantly, with (median, IQR) 0.59 (0.46-1.21) vs 0.57 (0.46-1.10) pmol/L, respectively. TAT was also increased at similar extent in both cohorts (192;111-325 pmol/L in COVID-19 patients, 148;73-213 pmol/L in sepsis patients), whereas F1 + 2 was higher in COVID-19 than in sepsis patients, with 850 (440-1940) vs 380 (130-620) pmol/L (p = 1.3 x 10-5). Interestingly, rates of APC formation did not significantly differ between the two groups, with 7.47 (1.99- 19.14) vs 9.87 (2.08-16.87) pmol/L ( Fig. 1). D-dimer and protein C were significantly higher in the COVID-19 patients than in those with sepsis (14.3 vs 8.1 mg/L, p = 0.01, and 92.9 % vs 58.5 %, p = 3x10-8, respectively). Conclusion We hypothesized that APC formation rates in response to thrombin formation would be significantly lower in patients with severe COVID-19 as compared to those with bacterial sepsis due to the well-known association between severe COVID-19 disease burden and endothelial dysfunction due to the downregulation of thrombomodulin expression. However, our results indicate that this may not be universally true in this patient population, as our observations suggest a largely intact functionality of the protein C pathway. Further studies are warranted to investigate the pathophysiology of severe COVID-19. (Figure Presented).

Haemostatic differences between SARS-CoV- 2 PCR-positive and -negative patients at the time of hospital admission

Background: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) is associated with a prothrombotic phenotype with an increased risk for thrombosis. Aim(s): To investigate whether COVID-19 is associated with changes in coagulation parameters upon presentation at the emergency department and whether these changes are associated with the development of thrombotic complications in patients with SARS-CoV- 2 infection. Method(s): A single centre, cross-sectional cohort study: The MArkers in COVID-19 And Relations to Outcomes in the Netherlands (MACARON) study was conducted. All patients suspected of SARS-CoV- 2 infection referred to the emergency department of the Meander Medical Center between March-May 2020 were included. 519 patients (26% PCR positive, median age 66 (range 19-97 years), 52.2% male) were included from whom an oro-and nasopharyngeal swab was obtained for detection of SARS-CoV- 2 by polymerase chain reaction (PCR). Blood samples for laboratory analysis were obtained from all patients. Thrombosis was defined as a clinical diagnosis of venous thromboembolism or atherothrombotic event based upon radiology and laboratory results. Result(s): SARS-CoV- 2 PCR positive patients had increased fibrinogen levels (5.41 g/L vs. 4.21 g/L, p < 0.001) and decreased levels of protein C (85.1% vs. 96.1%, p < 0.001) and alpha2-macroglobulin (4.41 muM vs. 5.11 muM, p < 0.001) compared to the PCR negative patients. In addition, we found more acquired activated protein C resistance in PCR positive patients. Furthermore, we found that elevated levels of factor VIII (208% vs. 162%, p = 0.028) and von Willebrand Factor (208% vs. 186%, p = 0.038) and decreased ADAMTS-13 levels (597 ng/ml vs. 691 ng/ml, p < 0.001) were associated with increased occurrence of thrombosis in PCR positive patients (thrombosis vs. non-thrombosis). Conclusion(s): We found that PCR positive patients had a more pronounced prothrombotic phenotype with endothelial activation upon hospital admission showing that coagulation tests may be considered useful to discriminate severe cases of COVID-19 at risk for thrombosis.

Building a better NET: Neutrophil extracellular trap targeted therapeutics in the treatment of infectious and inflammatory disorders

Infectious and inflammatory stimuli induce the release of neutrophil extracellular traps (NETs), webs of cell-free (cf) DNA complexed with histones and antimicrobial proteins, that capture and kill pathogens. Despite their protective role in the initial stages of sepsis, excessive NET release accompanied by NET degradation, leads to the release of NET degradation products (NDPs), including cfDNA, histones, and myeloperoxidase that injure the microvasculature. Murine studies have shown that clearance or neutralization of NDPs improves outcomes, demonstrating that NETs have a causal link to disease and are not merely biomarkers. Recently, elevated NDPs have been associated with disease severity in sepsis and coronavirus disease 2019, raising further interest in targeting NETs. Many propose eliminating NETs, either by preventing their release, or by degrading them. However, NET inhibition may impede the innate immune response and is difficult to achieve in rapid-onset conditions such as sepsis. On the other hand, approaches that accelerate NET degradation have met with mixed results in murine studies, raising the concern that this strategy may liberate NET-captured pathogens while increasing circulating levels of harmful NDPs. Alternative NET-directed strategies include therapies that neutralize, sequester, or remove NDPs from the circulation. Others propose modifying released NETs to decrease their capacity to induce collateral tissue damage while enhancing their ability to capture microorganisms. Synthetic NETs have also been designed to combat antibiotic-resistant organisms. Although it is still in its infancy, the field of NET-targeted therapeutics is advancing rapidly and may soon find application in the treatment of sepsis and other inflammatory disorders.

Supplementary research on K150del variant of activated protein C.

Activated protein C (APC) is an anticoagulant with potent cytoprotective and anti-inflammatory effects. K150del, a natural variant of APC, is associated with reduced anticoagulant activity. We performed a comprehensive study to analyze the functional alterations of the K150del mutant. Transcriptome analysis of HEK 293T cells treated with wild and mutant APC revealed differentially expressed genes enriched in inflammatory, apoptotic, and virus defense-related signaling pathways. Both wild and mutant APC displayed concentration-dependent cytoprotective effects. Low concentrations of K150del mutant resulted in decreased anti-inflammatory and anti-apoptotic activities, whereas its higher concentrations restored these effects. Expression of virus defense-related genes improved in mouse lung tissues after repeated administration of the APC variant. These results suggest that the APC K150del mutant could help clinicians to accurately predict disease risks and serve as a potential auxiliary therapeutic in viral infections, including 2019 coronavirus disease (COVID-19).

Old drug, new Trick? The rationale for the treatment of COVID-19 with activated protein C.

As the COVID-19 pandemic continues, researchers seek to identify efficacious treatments. Current approaches to COVID-19 therapeutics focus on antiviral agents, convalescent plasma, monoclonal antibodies, immunomodulators and more traditional therapies such as steroids [1-6]. Reversing disturbances in coagulation has also been identified as a priority area for candidate therapies, such as through the Accelerating COVID-19 Therapeutic Interventions and Vaccines 4 adaptive clinical trial (ACTIV-4) which is currently evaluating aspirin, heparins and apixaban [7]. Since there is a clear relationship between mechanisms of coagulation and the immune response, it is possible that reversing disturbances in coagulation may diminish the dysregulated immune response observed in COVID-19. The basis for this hypothesis is described below and is followed by discussion of a proposed candidate therapy - activated protein C. By treating COVID-19 patients using a novel approach, which does not focus on immune-based or antiviral treatments, but instead which addresses both the anti-thrombotic and inflammatory consequences of infection, the hope is that new therapeutic targets can be considered and new candidate therapies, such as activated protein C, may be evaluated.

COVID-19 hypothesis: Activated protein C for therapy of virus-induced pathologic thromboinflammation.

Seriously ill patients with coronavirus disease 2019 (COVID-19) at risk for death exhibit elevated cytokine and chemokine levels and D-dimer, and they often have comorbidities related to vascular dysfunctions. In preclinical studies, activated protein C (APC) provides negative feedback downregulation of excessive inflammation and thrombin generation, attenuates damage caused by ischemia-reperfusion in many organs including lungs, and reduces death caused by bacterial pneumonia. APC exerts both anticoagulant activities and direct cell-signaling activities. Preclinical studies show that its direct cell-signaling actions mediate anti-inflammatory and anti-apoptotic actions, mortality reduction for pneumonia, and beneficial actions for ischemia-reperfusion injury. The APC mutant 3K3A-APC, which was engineered to have diminished anticoagulant activity while retaining cell-signaling actions, was safe in phase 1 and phase 2 human trials. Because of its broad spectrum of homeostatic effects in preclinical studies, we speculate that 3K3A-APC merits consideration for clinical trial studies in appropriately chosen, seriously ill patients with COVID-19.