Workshop on COVID-19-Associated Coagulopathy (CAC)

The National Heart, Lung, and Blood Institute’s (NHLBI) Division of Blood Diseases and Resources (DBDR) convened a workshop “COVID-19-Associated Coagulopathy (CAC)” on May 6-7, 2021. The workshop brought together both national and international experts to discuss the most recent science, clinical outcomes, and future directions for research and treatment. The purpose of the workshop was to: (1) identify gaps in the current understanding of CAC; (2) better understand the molecular mechanisms of vascular endothelial cell dysfunction; (3) examine the hyperinflammatory response that contributes to CAC; and (4) identify risk factors predisposing patients to CAC in order to provide more effective early treatment for high-risk patients.

Background:

COVID-19-Associated Coagulopathy (CAC) has played a major role in the morbidity and mortality of the SARS-CoV-2 pandemic and is postulated to result from the dysregulated interactions between inflammatory, immune, and coagulation systems. Elevated plasma D-dimer levels, a biomarker of active thrombosis, have been reported in approximately 25% of COVID patients for up to 4 months following infection. Understanding the triggering events and downstream effects of this pathologic prothrombotic state is key to improving survival and decreasing post- COVID-19 effects that have impacted millions of survivors.

Patients who develop clinical symptoms associated with COVID-19 have clotting events with higher frequency and severity than has been observed in patients with similar viral respiratory infections. These thromboembolic events, including pulmonary thrombosis, pulmonary embolism, microthrombosis, venous thromboembolism (VTE) and arterial thrombosis, are believed to stem from dysfunctional interactions between an overactive inflammatory response and coagulation pathways. These patients often present with abnormal coagulation profiles, notable for elevated C-reactive protein, D-dimer, P-selectin and fibrin(ogen) levels. They frequently exhibit a heavily prothrombotic state, but paradoxically, with little change in partial thromboplastin time, or prothrombin time, and in which platelets are rarely depleted to a level that results in bleeding. This distinguishes CAC from classical disseminated intravascular coagulopathy (DIC). Dysregulation of the finely balanced procoagulant/fibrinolytic state may contribute to the pathophysiology of CAC; however details of the underlying mechanisms have yet to be delineated.

Discussion:

SARS-CoV-2 infection can evoke hyperinflammation via stimulation of both the adaptive and innate immune systems. The virus may also perturb the vascular endothelium of the lung, which can lead to further activation of the coagulation and fibrinolytic systems. Currently, the mechanisms of COVID-19 thrombosis linking vascular endothelial cell injury with the host immune responses and the coagulation and fibrinolysis systems have yet to be elucidated, especially at the molecular and cellular levels.

This workshop was held to identify scientific gaps and opportunities in research areas of critical importance. Improved knowledge and understanding of the prothrombotic state in CAC will help to develop early diagnostics and to provide more effective therapeutics to mitigate or eliminate CAC complications.

The following topics, which focused on the basic mechanistic aspects of CAC, were identified as high research priorities:

1. Dysfunction of Vascular Endothelial Cells
   
   Cumulative observations of thrombotic complications in patients with SARS-CoV-2 infection have highlighted the role that the vascular endothelium plays in maintaining normal hemostasis. The entry of SARS-CoV-2 into human lung epithelial cells is mediated by binding to angiotensin converting enzyme 2 (ACE2), in synergy with transmembrane protease serine 2 (TMPRSS2). While initial stages of COVID-19 may be driven by SARS-CoV- 2 infection of the alveolar epithelium in the lung, close junctions with capillary beds may lead to vascular endothelial cell damage and subsequent entry of the virus into the systemic circulation. Endothelial glycocalyx degradation has been linked to vascular leakage via activation of bradykinin receptors type 1 (B1) and dysregulation of the kallikrein-kinin system (KKS). These effects, together with high levels of D-dimer and P-selectin, likely reflect early triggering events in the coagulation cascade, for which detailed mechanisms still remain unclear. Additionally, tissue factor (TF) and extracellular vesicles (EVs) are released from multiple cell types (including endothelial cells), which are associated with increased disease severity.
   
   One distinguishing feature of COVID-19 is the broad range of neurological symptoms including ischemic stroke and delirium in severe cases. Notable symptoms of post-acute sequelae of SARS- CoV-2 infection (PASC), which includes “long COVID” or “long-haul COVID”, are frequent neurological events including brain fog, fatigue, combined with other symptoms such as concentration/memory problems, taste/smell alteration, and sleep disorders. SARS-CoV-2 can directly infect human and mouse neurons; however, the dependency of viral entry via  ACE2 at the blood brain barrier is incompletely understood. This process is likely to promote vascular leakage, cytokine release, and cell adhesion leading to microthrombi formation, possibly resulting in multifocal micro-vascular brain injury.
   
   Key questions:
   • Do all vascular endothelial cells have the same physiologic properties and susceptibility to COVID-19 injury regardless of location, or are there differences due to organ specificity?
   • What roles do TF and EVs play in COVID-19? What is the source of these agents and what is their function?
   • Is TF a biomarker that can be utilized to identify patients at risk of VTE?
   • Is vascular leakage in the lung alveolar space a mechanism to allow viral entry into the blood system?
   • What role does CAC play in multi-organ failure?
   • How does CAC affect the central nervous system, particularly in the brain?

2. COVID-19-Associated Hypercoagulability
   
   Increased levels of inflammatory cytokines, associated fatty acids and signaling molecules (e.g., IL-6, triglycerides, prostaglandin) as well as circulating von Willebrand factor (vWF) and factor VIII suggest that COVID-19 induces an acute phase response and endothelial activation, which leads to prothrombotic complications. More importantly, elevated levels of D-dimer and extravascular fibrin deposition are observed among severely ill COVID-19 patients, indicating dysregulation of the balance in thrombin generation (fibrin forming) and plasmin generation (fibrin dissolving) pathways. Thrombin, fibrin, and plasmin are not only key players in thrombosis, but also effectors in host responses during infection. Therefore, these observations suggest that changes in the balance of procoagulant and fibrinolytic activity may contribute in multiple ways to COVID-19 pathophysiology and severity. However, the underlying mechanisms controlling these events have not been identified. In particular, the initial triggering events leading to COVID-associated coagulopathies are poorly understood. In addition, increased cellular interactions, including activated platelets, macrophages, and neutrophils during CAC require additional evaluation. Thus, further research into the basic triggering mechanisms involved in CAC is clearly needed to better identify high-risk individuals and to provide more effective therapeutic options and improved clinical outcomes.
   
   Genome-wide association studies (GWAS) and country-wide meta-regression analyses    reveal that the chromosome 3p21 locus and blood group O are highly associated with developing VTE, cerebrovascular ischemic events, and myocardial infarction. Among many other genes, the gene encoding interferon-induced transmembrane protein 3 (IFITM3), is highly expressed in platelets and megakaryocytes during SARS-CoV-2 infection.  It seems that IFITM3 regulates fibrinogen endocytosis in megakaryocytes and platelets and further contributes to platelet hyperreactivity in COVID-19. However, whether platelets express ACE2 and whether inhibiting platelet activation improves outcomes in COVID-19 remains unknown.
   
   Key questions:
   • Why does plasma from patients with COVID-19 have prothrombotic/pro-inflammatory activity in vitro?
   • What are the relative contributions of leukocytes, endothelial cells, and platelets in the pathophysiology of CAC?
   • Does CAC play a role in PASC?
   • What biomarkers can be used to identify patients at high risk for CAC?
   • Is early therapy effective in preventing CAC?
   • Do platelets express ACE2? Does SARS-CoV-2 directly infect the platelets?
   • Does inhibiting platelet activation reduce COVID-19 severity?
   • What are the relevant coagulation-related or thrombosis-related genetic variants? How do these relate to outcomes?
   • What are the causal mechanisms linking patient-specific genetic loci to COVID-19? Could existing GWAS databases, transcriptomic and proteomic studies be leveraged to identify novel drug targets?
   • What role does protein glycosylation play during SARS-CoV-2 infections?

3. COVID-19-Associated Hyperimmune Response
   
   Entry of SARS-CoV-2 into the vasculature may trigger an inflammatory response and further activate the coagulation system leading to intravascular thrombosis. In severe COVID-19, a dysregulated immune response may include autoantibody production and further contribute to the COVID prothrombotic state. For instance, neutrophil extracellular traps (NETs) are elevated among COVID-19 patients, which in turn may drive thrombo- inflammation, including the formation of platelet-neutrophil aggregates. Elevated circulating markers of complement activation are seen, suggesting that activation of the innate immune response also plays a role in developing macro- and microvascular thrombosis.
   
   Various autoantibodies including anti-NET and antiphospholipid antibodies are also observed in patients hospitalized for COVID-related pneumonia, suggesting that dysregulated antibody responses are associated with disease severity. Triggering events that initiate the production of autoantibodies and their downstream effects in CAC need to be further determined.
   
   Key questions:
   • What role do NETs play in CAC?
   • What drives autoantibody production in acute illness and what role does it play in disease progression?
   • What are the key features of anti-NET antibodies and what role do they play in CAC?
   • What are the triggering events in COVID-related autoantibody production?
   • What role does complement activation and deposition play in CAC?
   • What role do JAK-STAT inhibitors play in CAC?

Additional questions for future research:

• What are the distinguishing features of COVID-19 coagulopathies compared to other procoagulant conditions? How do we define CAC?
• What are the mechanisms responsible for vascular endothelial cell injury in COVID-19 patients and what role does the innate or adaptive immune response play in CAC?
• The delicate balance between pro- and anti-coagulation factors sustains hemostasis in normal individuals. In patients infected with SARS-CoV-2, what are the factors or conditions that disturb this hemostatic balance and trigger CAC?
• What are the short-term and long-term complications of CAC? In particular, is Long-COVID or PASC linked to CAC?
• COVID-19 is known to cause severe lung injury and respiratory failure; however COVID-19 has also been implicated in causing multi-organ failure including kidneys, liver, heart and central nervous system. What role does CAC play in causing extra-pulmonary organ failure?
• What genetic traits predispose patients infected with SARS-CoV-2 to hypercoagulability?
• What other risk factors (e.g., age, gender, pregnancy, obesity, diabetes, etc.) predispose patients with CAC to increased morbidity and mortality?
• Can we develop more effective therapies to prevent or mitigate the vascular injury during COVID-19?

In summary, CAC involves different inflammatory and coagulation pathways often times leading to complex, multi-organ dysfunction and a range of sequalae causing significant morbidity and mortality. Our mechanistic understanding of the pathophysiology of CAC remains limited, particularly with respect to the different stages of SARS-CoV-2 infection and disease manifestation. Early prospective trials using conventional treatment for other coagulopathies (e.g., DIC or sepsis-induced coagulopathy) including anticoagulation with heparin, have shown no significant benefit or improvement in thrombosis, need for ECMO support, or mortality for patients admitted to ICU. In light of this, focused research aimed at elucidating the mechanism of CAC, at the molecular and cellular levels, is crucial to developing and identifying effective therapeutics for high-risk patients with CAC. A longer-term research goal is to apply insights from understanding CAC to the increased thrombotic risk associated with other viral respiratory diseases.

Publication Plans:

Workshop organizers plan to develop a white paper that will expand upon the deliberations and detailed research opportunities.

NHLBI Contact:

Kyung Moon, Ph.D., DBDR, NHLBI, NIH
Email: Kyung.moon@nih.gov