Even as the evidence of the central role played by immune dysregulation in severe COVID-19 pours in, the coagulation system is also being seen to be significantly disrupted in this condition. A new study by University of Tuebingen researchers published on the preprint server medRxiv* in September 2020 shows the strong association between platelet apoptosis and the thrombotic manifestations that may underlie critical COVID-19 illness.
Procoagulant Activity in COVID-19
It is now established that coagulation activity is high in COVID-19 patients who require intensive care. However, it does not seem to fit the definition of disseminated intravascular coagulation (DIC), nor is it the kind of perturbed clotting seen in severe infections. DIC was identified only in severe or critical COVID-19.
However, scientists also noticed that patients with severe COVID-19 in the ICU also had acute clotting manifestations in the lung (pulmonary embolism, PE), ischemic stroke, deep vein thrombosis (DVT), myocardial infarctions, and clots forming in the systemic arteries, in half the patients. Thus, thromboembolism and infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are closely related, and both cellular factors and the plasma components of the coagulation and innate immune system are interwoven into this response.
This scanning electron microscope image shows SARS-CoV-2 (round gold objects) emerging from the surface of cells cultured in the lab. SARS-CoV-2, also known as 2019-nCoV, is the virus that causes COVID-19. The virus shown was isolated from a patient in the U.S. Image captured and colorized at NIAID's Rocky Mountain Laboratories (RML) in Hamilton, Montana. Credit: NIAID
Platelet Activation in COVID-19
Most of the clinical studies on COVID-19 have examined increased plasma coagulation factors, especially D-dimer elevations, which are related to worse clinical features. Other abnormalities have been reported, such as prothrombin time, activated partial thromboplastin time, and a severe fall in the platelet count.
Platelets are not only components of the coagulation pathway, but also release cytokines that activate the inflammatory pathways, as well as detecting pathogen-receptor interactions and immune cytokines. Viral intruders trigger a systemic inflammatory response, which in turn causes platelet activation as a result of antigen recognition and interaction with white blood cells.
Activated platelets are at the heart of procoagulant activity in viral infections, as has been seen in patients with HIV and dengue, where they enter the pathway of apoptosis via the intrinsic or mitochondrial route.
Platelet Apoptosis and Coagulation Activity
The researchers set out to examine whether abnormalities in coagulation are always linked to prior platelet apoptosis.
Platelet apoptosis by the intrinsic or mitochondrial pathway is mediated by the process called the mitochondrial outer membrane permeabilization (MOMP), in which Bcl2 proteins either facilitate or suppress apoptosis. The collapse of the mitochondrial inner membrane potential (ΔΨm) causes cytochrome c into the cytoplasm to form a structure called the apoptosome. This, in turn, activates caspase 9 and leads to the activation of the caspase cascade. In the late stage of apoptosis, phosphatidylserine (PS) externalization occurs on the extracellular membrane.
The phospholipid PS is a lipid component of many cell membranes that may serve to trigger the activation of several steps in the coagulation cascade. But this is probably not the only factor, since damaged endothelium and leukocytes display pathogen- and damage-associated molecular patterns that initiate uncontrolled coagulation.
Clinical Characteristics and Platelet Parameters
The study was based on blood samples taken from 21 successive hospitalized patients with severe COVID-19 symptoms, namely, acute respiratory distress symptoms (ARDS), requiring intensive care, and 4 patients without ARDS. Most patients were male, and 15 had risk factors such as high blood pressure, obesity, diabetes, and heart disease. The mean age was 60 years.
All the patients in the ICU required controlled respiratory support, and six of them also had venous-venous extracorporeal membrane oxygenation (vv-ECMO). The patients received prophylactic or therapeutic doses in 12 and 9 cases, respectively, while 8 patients had aspirin as well.
Over the next 30 days, one-third of the patients died, while two-thirds experienced a drop in platelet counts. Almost 60% of them developed thromboembolism of the kidney, spleen or liver, heart or brain, clotting in the ECMO, or pulmonary embolism.
Platelets from ICU COVID-19 patients showed markedly increased ΔΨm depolarization of the mitochondria, unlike either healthy or non-ICU COVID-19 patients, but identical to that in other seriously ill ICU patients without COVID-19. They also had a higher cytosolic calcium ion concentration
Next, we determined the cytosolic Ca2+ concentration in platelets from ICU COVID-19 patients, almost thrice that in healthy donors, and non-ICU COVID-19 patients, and twice that of other ICU patients.
The researchers looked for externalization of PS in platelets that had undergone apoptosis. They found it was much higher on platelets derived from ICU COVID-19 patients, four times as high as in healthy donors, and twice that in non- ICU COVID-19 patients and other ICU patients. This confirms that platelet apoptosis might be a powerful influence on the clinical severity of the disease in these patients.
Other cytokines that take part in the apoptotic pathway are also higher in these patients. The higher the externalized PS concentration and the cytosol ionic calcium, the lower was the platelet count, and the higher the D-dimer concentration, as well as the incidence of thromboembolism. Cytosolic ionic calcium concentrations were also higher in those who failed to survive relative to other ICU COVID-19 patients.
Thus, the study shows that in severe COVID-19 patients requiring intensive care, platelets express elevated apoptotic markers.
Serum-Mediated Platelet Apoptosis
Both the serum and the IgG antibodies from the COVID-19 patients trigger apoptosis in platelets derived from healthy individuals. The impact of patient serum on platelets could indicate a direct IgG-viral binding or an indirect non-specific platelet-IgG interaction. The latter is likely, mediated by crosslinking of platelets by IgG antibodies through FcγRIIA receptors.
These receptors could be blocked by specific tyrosine kinase inhibitors, for instance, as suggested as a treatment for thrombocytopenia in response to heparin therapy.
Platelet Apoptosis as Therapeutic Target
An important difference between platelet apoptosis in ICU COVID-19 patients and other ICU septic patients was that despite the increased ΔΨm depolarization in both, neither cytosolic ionic calcium or PS externalization was increased in the latter, indicating that apoptosis was occurring by other pathways than by the intrinsic pathway. This could indicate that platelet apoptosis is a valid therapeutic target for COVID-19.
The researchers also found that platelet apoptosis is associated with a higher risk of thromboembolism as well as death in patients with severe COVID-19. Further research is necessary to establish whether thromboembolism in ICU COVID-19 patients is due solely to apoptosis or if activated platelets are also necessary, in the absence of marked thrombocytopenia. In this case, the caspase-mediated mitochondrial pathway of apoptosis is a likely suspect, supported by the finding of higher cleaved-caspase 9 in these patients.
More research is also required to uncover the causal link if any between these events since this was an observational study. However, the researchers say, “Data presented in this study may build a basis for future studies to dissect platelet-mediated pathological mechanisms involved in the progression of COVID-19.”
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.