University Of Bristol Study Finds That SARS-CoV-2 Induced Platelet Driven Thromboinflammation Contributes To Thrombosis In COVID-19 Patients
A new study by researchers from the University of Bristol – United Kingdom has found that SARS-CoV-2 induced platelet driven thromboinflammation increases the risk of thrombosis in COVID-19 patients.
Numerous past studies have already showed that individuals with COVID-19 are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality.
The study team in their research aimed to characterize the mechanism of altered platelet function in COVID-19 patients.
The platelet proteome, platelet functional responses and platelet-neutrophil aggregates were compared between patients hospitalized with COVID-19 and healthy control subjects using Tandem Mass Tag (TMT) proteomic analysis, Western blotting and flow cytometry.
The study findings showed that COVID-19 patients displayed a different profile of platelet protein expression (858 altered out of 5773 quantified).
Interestingly, levels of COVID-19 plasma markers were enhanced in COVID-19 platelets. Gene ontology (GO) pathway analysis demonstrated that levels of granule secretory proteins were raised, whereas some platelet activation proteins, such as the thrombopoietin receptor and PKCα, were lowered.
Basally, COVID-19 platelets showed enhanced phosphatidylserine (PS) exposure, with unaltered integrin αIIbβ3 activation and P-selectin expression. Agonist-stimulated integrin αIIbβ3 activation and PS exposure, but not P-selectin expression, were significantly decreased in COVID-19 patients.
Furthermore COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This interaction was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction.
The study findings suggest the presence of two platelet populations in patients with COVID-19: one with circulating platelets with an altered proteome and reduced functional responses and another with P-selectin expressing neutrophil-associated platelets.
Corresponding author, Dr Ingeborg Hers from the School of Physiology, Pharmacology and Neuroscience at University of Bristol told Thailand Medical News
, "Our study findings hence concluded that platelet driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.”
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2022.10.12.511145v1
To date, the COVID-19 pandemic has killed over 6.58 million people according to reported data. The disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive sense RNA virus that can lead to various clinical symptoms causing the hospitalization of many people with pneumonitis.
However, studies have also revealed that COVID-19 patients have a high arterial and venous thrombosis rate involving pulmonary embolism, deep-vein thrombosis, ischemic stroke, an
d myocardial infarction.
Furthermore, many autopsy studies have indicated the presence of microthrombi in the heart, lungs, brain, liver, and kidney of COVID-19 patients. This suggests that COVID-19 can cause systemic thrombosis, leading to multi-organ failure.
Interestingly, many clinical case reports have also indicated that COVID-19 patients have a high level of serum coagulation markers such as D-dimer and fibrinogen.
Also, thromboprophylaxis protocols have been observed to improve outcomes in hospitalized COVID-19 patients. However, it did not significantly affect the mortality of patients with severe COVID-19. Therefore, thrombosis remains a prominent COVID-19 feature where high thrombin generation is not the only contributing factor.
It is already known that platelets essential for hemostasis can also lead to thrombosis due to inappropriate activation.
Platelets can become hyperactive, leading to the increased secretion of dense alpha-granules, increased formation of platelet-leukocyte aggregates, and increased aggregation. Platelet transcriptome analysis has shown an overrepresentation of mitochondrial dysfunction and antigen presentation pathways in COVID-19 patients, leading to platelet hyperactivity. However, many studies also reported reduced or impaired platelet function in COVID-19 patients, suggesting that the platelet response is complex.
Despite the fact that the pathogenesis of thrombosis in COVID-19 patients is not well understood, there seem to be a clear indication and several hallmarks of thromboinflammation.
Already it has been demonstrated that SARS-CoV-2 can activate endothelial cells with the help of the angiotensin-converting enzyme 2 (ACE2) receptor, which leads to vascular dysfunction.
Such endothelium damage can activate the innate immune system through neutrophil recruitment, tissue factor (TF) expression, pro-inflammatory cytokines, and complement.
This subsequently leads to higher expression and/or release of prothrombotic factors and upregulation of adhesion molecules which can activate platelets, causing thrombus formation, aggregation, and further activation of the innate immune system.
The platelets can then increase the generation of thrombin through the expression of phosphatidyl serine and tissue factor (TF).
Such a network of interactions between the innate immune system, platelets, coagulation, and damaged endothelium can contribute to thrombosis in COVID-19 patients.
This is the first study to analyze the impact of COVID-19 on platelet proteome and relate the functional responses of platelets and the formation of platelet-neutrophil aggregate in hospitalized COVID-19 patients.
The research involved 22 hospitalized adult COVID-19 patients and 19 healthy controls recruited between October 2020 and February 2021. Information on comorbidities, demographics, medications, admission results and treatment records were collected from all the patients.
Detailed full blood counts were measured for both healthy controls and COVID-19 patients. Platelets were isolated from venous blood, washed, and prepared for tandem mass tag (TMT) proteomic analysis.
The study team then compared the platelet proteome between healthy controls and COVID-19 patients. In addition, Gene ontology pathways were used to analyze COVID-19 elated changes to platelet proteins that affect their function. Finally, western blotting and flow cytometry analysis was carried out.
The mean age of the COVID-19 cohort was 59 years, and the control cohort was 39 years. The mean body mass index (BMI) of COVID-19 patients was reported to be 30.6 kg/m2, while that of the control was 22.7 kg/m2. In addition, 88% of the patients required oxygen therapy and additional medications, including rivaroxaban, dexamethasone, and heparin. Additionally, COVID-19 patients were observed to comprise a higher neutrophil level and lower lymphocyte count.
The study findings showed that out of the 5,773 platelet proteins, 858 were elevated in COVID-19 patients compared to healthy controls. These mainly included the C-reactive protein and the 40S ribosomal proteins.
However, many platelet activations signaling proteins decreased in COVID-19 patients compared to controls. Platelet lysates obtained from COVID-19 patients were reported to show a reduction in thrombopoietin (TPO) receptor cMpl, interferon-induced transmembrane membrane protein 3 (IFITM3), and protein kinase C α (PKCα) expression.
Also, CD147 (basigin), a receptor associated with SAS-CoV-2 interaction, was reported to be present in both control and COVID-19 patient samples.
The study findings also showed that out of the 38 serum proteins associated with COVID-19, 12 were altered in the platelet of COVID-19 patients. Out of them, four proteins were observed to show more than a 4-fold expression that included serum amyloid A (SAA1), galectin 3-binding protein (G3BP), lipopolysaccharide-binding protein (LBP), and C-reactive protein (CRP).
Meanwhile, out of 18 granule secretion proteins associated with COVID-19, 11 were observed to be increased, and seven decreased in platelets from COVID-19 patients. Moreover, out of the 15 platelet activation proteins, 12 were reduced in COVID-19 patients' platelets, including Ser/Thr kinases and tyrosine kinases.
Interestingly however, levels of interleukin-6 receptor subunit, Apolipoprotein E, and cathepsin G were observed to be increased.
The study findings also showed that the activation of integrin αIIbβ3, which is the receptor causing platelet aggregation and alpha-granule marker P-selectin that is expressed on the secretion of alpha-granule secretion, was similar for both controls and COVID-19 patients under basal conditions. However, agonist-induced integrin αIIbβ3 activation was reported to be impaired in platelets of COVID-19 patients, while agonist-stimulated P-selectin expression was reported to be unchanged.
Additionally, under unstimulated conditions, platelets from COVID-19 patients reported a slight increase in phosphatidyl serine exposure which was reduced upon stimulation.
The study team also reported that the platelet-neutrophil interactions were observed to be increased in COVID-19 patients even under unstimulated conditions. P-selectin CD62P blocking antibody was observed to reduce under stimulated basal platelet neutrophil interactions but had no impact on stimulated platelet-neutrophil interactions.
The study findings demonstrate the presence of two different platelet populations in COVID-19 patients. The first is circulating platelets with an altered proteome, and the second is P-selectin expressing neutrophil-associated platelets.
The findings also indicate that platelet-driven thromboinflammation is one of the critical factors that can increase the risk of thrombosis in COVID-19 patients.
The study team said that further research needs to be done to understand the mechanism of this effect.
The team also stressed that the study had certain limitations for instance heparin and dexamethasone may contribute to the platelet phenotype and also the healthy controls were younger with lower BMI and the healthy controls self-reported being SARS-CoV-2 negative, which might be subjected to bias.
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