Coronavirus News: Canadian Study Reveals That Platelet-Activating Immune Complexes Responsible For COVID-19 Associated Coagulopathy In Critical Cases
: Researchers from McMaster University, Ontario-Canada have revealed that platelet activating immune complexes are the contributing causes to coagulation disorders associated with COVID-19 seen in severe or critically ill COVID-19 patients.
Thrombosis is a prominent feature of COVID-19 and often occurs in patients who are critically ill; however, the mechanism is unclear. This COVID-19 associated coagulopathy (CAC) shares features with heparin-induced thrombocytopenia (HIT), including mild thrombocytopenia and thrombosis. Te study team tested 10 CAC patients for anti-PF4/heparin antibodies and functional platelet activation. HIT was excluded in all samples based on anti-PF4/heparin antibody and serotonin release assay results. Of note, 6 CAC patients demonstrated platelet activation by the serotonin release assay that was inhibited by FcγRIIA receptor blockade, confirming an IgG-specific immune complex (IC)-mediated reaction. Platelet activation was independent of heparin, but inhibitable by both therapeutic and high dose heparin. All 6 samples were positive for IgG-specific antibodies targeting the receptor binding domain (RBD) or the spike protein of the SARS-CoV-2 virus. These samples were additionally characterized by significant endothelial activation, shown by increased von Willebrand factor antigen and activity. ADAMTS13 activity was not severely reduced, and ADAMTS13 inhibitors were not present, ruling out thrombotic thrombocytopenic purpura. The study thus identifies platelet-activating ICs as a mechanism that contributes to CAC thrombosis.
The study findings were published on a preprint server and are currently being peer-reviewed. https://www.medrxiv.org/content/10.1101/2020.11.04.20226076v1
So far, the SARS-CoV-2 coronavirus that causes COVID-19 is known to be associated with coagulation disorders. Thrombosis is commonly seen in patients critically ill with COVID-19, and this has been termed as COVID-19 associated coagulopathy (CAC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197634/
The main features of CAC are similar to heparin-induced thrombocytopenia (HIT), showing features of mild thrombocytopenia (low platelet counts) and diffuse arterial and venous thrombosis. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416717/
Heparin-induced thrombocytopenia or HIT is a condition where prominent features of thrombocytopenia are seen; these are associated with heparin treatment and lead to thrombosis. It is said to be caused by IgG-specific antibodies that target platelet-factor 4 (PF4). These two form complexes called immune complexes (IC). These immune complexes, in turn, can activate the platelets mediated by FcγRIIA receptor. https://pubmed.ncbi.nlm.nih.gov/2818941/
However the immune complexes are not a result of IgG-specific anti-PF4/heparin antibodies in COVID-19, but are caused by endothelial cell activation.
Newer studies have shown that these immune complexes are also associated with severe COVID-19. This study was undertaken to see the association between platelet-activating ICs in CAC patients. https://pubmed.ncbi.nlm.nih.gov/32479986/
In this study, the study team included blood samples from ten critically ill patients with COVID-19. The samples were analyzed at the McMaster Platelet Immunology Laboratory (MPIL). To compare against the critically ill patients, samples from eight convalescent COVID-19 positive patients were also taken. Of these, five belonged to HIT patients prior to the pandemic (HIT group). Seven of the samples were from health controls before the pandemic (HC group).
The study team tested these samples for HIT, and collated other details from the patients to aid in their assessment. These included the gender of the patient, platelet counts (minimum), use of heparin, events related to thrombosis or abnormal coagulation, diagnosis at admission and outcome.
Utilizing anti-PF4/heparin enzymatic immunoassay, the anti-PF4/heparin antibodies were detected in all samples. IgG, IgM, and IgA PF4-heparin antibodies were quantified. IgG-specific anti-PF4/heparin EIA was performed for positive patients. Functional platelet activation was tested using serotonin release assay (SRA) with heparin (0.1, 0.3, and 100 U/mL). IgG-, IgA- and IgM specific antibodies against the RBD (receptor-binding domain) and spike protein of SARS-CoV-2 virus were also tested.
CAC patients with COVID-19 antibodies contain ICs that are capable of activating platelets in the SRA in a manner that is unique from HIT ICs. (A) CAC (n = 10) patient sera compared to (B) HIT patient (n = 5) sera, serving as a control, in the SRA. 14C-serotonin release was measured in the presence of increasing heparin doses or with addition of IV.3 (Fc?RIIA inhibitor). 14Cserotonin release >20% is positive in the SRA (horizontal dashed line). Most CAC patient sera (n = 6, solid line) demonstrate heparin-independent platelet activation, as opposed to classic HIT controls. Platelet activation was inhibited with IV.3 in both groups. (C) IgG, IgA, and IgM COVID-19 antibodies in platelet-activating CAC patient sera (n = 6). Antibodies were measured in the SARS-CoV-2 ELISA and include RBD and spike protein specificity. Values are shown as a ratio of observed optical density to the determined assay cut-off optical density. Values above 1 ratio are considered positive in the SARS-CoV-2 ELISA.
In order to measure endothelial activation, von Willebrand Factor (vWF) antigen levels were assessed using chemiluminescent immunoassay. The team assessed the VWF activity using a special assay. They measured the ADAMTS13 metalloproteinase enzyme and also measured the anti-ADAMTS13 antibody in all patients. This was done to determine the changes in the vWF antigen levels associated with ADAMTS13 activity.
The key findings of the study were:
- Platelet activation was seen using the serotonin release assay among the 6 CAC patients,
-Importantly this platelet activation was inhibited when the FcγRIIA receptor was blocked, indicating an IgG-specific immune complex (IC)-mediated reaction.
-It was found that this platelet activation was independent of heparin; when heparin was administered at therapeutic and high doses, the activation, however, was inhibited
-These 6 CAC samples also were positive for IgG-specific antibodies against the receptor-binding domain (RBD) or the spike protein of the SARS-CoV-2 virus
-Importantly, there was also significant endothelial cell activation in these samples, indicated by increased vWF antigen and activity. Moreover, patients with COVID-19 thrombosis also had raised vWF antigen and its activity. This is not associated, however, with inhibition or block of ADAMTS13.
-There was no indication of thrombotic thrombocytopenic purpura (TTP).
-None of the controls had anti-SARS-CoV-2 antibodies
-Convalescent plasma from non-critically ill COVID-19 patients did not activate platelets in the SRA, despite having high titers of anti-SARS-CoV-2 antibodies. This indicated that the antibodies alone are not capable of platelet activation.
-Endothelial cell activation was found to contribute to coagulopathy
The results also confirm that endothelial cell activation contributes to coagulopathy, as previously shown. This is not secondary to severe ADAMTS13 activity reduction (or the presence of anti-ADAMTS13 antibody, as described in thrombotic thrombocytopenic purpura (TTP). https://www.thelancet.com/journals/lanhae/article/PIIS2352-3026(20)30216-7/fulltext
This pattern is more consistent with a secondary thrombotic microangiopathy and is likely compounded by the presence of platelet-activating ICs. https://pubmed.ncbi.nlm.nih.gov/27754896/
The research findings study provides evidence that immune complexes associated with COVID-19 were different from other severe coagulation disorders, including HIT and TTP.
Hence COVID-19 patients with severe disease who develop thrombosis have immune complexes that activate platelets through FcγRIIA signaling. Patients with COVID-19 thrombosis also had raised vWF antigen levels and activity.
The study team however did not associate this with inhibition or a block of ADAMTS13.
The study team wrote, “These ICs can produce a highly prothrombotic state resembling HIT but with unique platelet activating properties
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