Canadian Study Finds That SARS-CoV-2 Infected Cells Trigger Host Tissue Factors That Leads To Platelet Activation And Coagulation.

New study findings by Canadian researchers from Université Laval-Quebec, Canada CHU de Quebec, Canada University of Ottawa, Ottawa and National Research Council Canada are helping to explain the mechanisms promoting platelet activation by the SARS-CoV-2 resulting in thrombosis and coagulation…hallmarks of COVID-19. The study that was also supported by scientist from the University of North Carolina at Chapel Hil-USA, UMR-CBMN CNRS-University Bordeaux-France. Abulcasis University-Morocco and the Mohammed VI University of Health Sciences-Morocco found that SARS-CoV-2 infected cells trigger host tissue factors (TFs) that leads to platelet activation and coagulation.


 
Earlier, deciphering the details of this mechanism was made not possible due to inherent challenges at discriminating the contribution of viral versus host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles, as they are concomitantly released during infection and share biophysical properties.
 
In order to study this, the study team evaluated whether SARS-CoV-2 itself, or components derived from SARS-CoV-2-infected human lung epithelial cells, could activate isolated platelets from healthy donors.
 
Platelet activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbβ3, degranulation, aggregation under flow conditions and the release of extracellular vesicles.
 
The study findings showed that neither SARS-CoV-2 nor purified Spike activate platelets.
 
However, interestingly, TF (tissue factor) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets.
 
All virions and extracellular vesicles were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with virus or extracellular vesicles, which were indistinguishable. Increased TF mRNA expression and activity were also found in lungs in a murine model of COVID-19 and in plasma of severe COVID-19 patients, respectively.
 
Corresponding author, Dr Eric Boilard from CHU de Quebec told Thailand Medical News, “The study findings confirms that TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Importantly blockade of molecules in this pathway may interfere in platelet activation and coagulation that is characteristic of COVID-19.”
 
The study findings were published in the peer reviewed journal: Blood Advances. https://ashpublications.org/bloodadvances/article/doi/10.1182/bloodadvances.2022007444/485001/Platelet-activation-by-SARS-CoV-2-implicates-the
 
The study examined the impact of SARS-CoV-2 or components derived from infected cells in platelet activation.
 
The study team collected venous blood samples from healthy donors. SARS-CoV-2 was also obtained and propagated in human lung epithelial A549 cells that overexpressed angiotensin-converting enzyme 2 (ACE2). Culture supernatant (C.Med) obtained from non-infected cells was used as a negative control.
 
The actual potential of SARS-CoV-2 in platelet activation was assessed based on the frequency of platelets that expressed the active forms of αIIbβ3 integrin (αIIbβ3*) and CD62P (αIIbβ3*CD62P), the release of EVs (CD41+EVs), and the expression density of αIIbβ3*.
 
The study team also obtained plasma preparations from non-immune donors who had no SARS-CoV-2 antibodies.
 
Actual platelet activation was further assessed by incubating the recombinant full-trimeric SARS-CoV-2 spike (S) protein, a recombinant SARS-CoV-2 receptor binding protein (RBD), and RBD-contained S1 subunit in plasma samples. The plasma deficient factors from coagulation factors like FI, FII, FV, FVII, FVIII, FX, FIX, FXI, FXII, FXIII, vWF, protein S, and protein C were incubated with SARS-CoV-2 or C.Med and were subsequently added to platelets for the assessment of activation.
 
The study team furthermore assessed the role of thrombin in the plasma-dependent platelet activation mediated by SARS-CoV-2. This was achieved by incubating SARS-CoV-2-contained plasma in the presence or absence of a thrombin inhibitor called D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK).     
   
The research findings showed that SARS-CoV-2 induced a fast and substantial rise in the proportion of CD62P platelets and increased the intensity of αIIbβ3* expression.
 
Significantly, after 60 minutes of SARS-CoV-2 exposure, most of the platelets expressed CD62P, while the levels of EVs improved significantly. However, SARS-CoV-2 did not activate the platelets in the presence of C.Med and the absence of plasma. In contrast, remarkable changes were observed in the thrombin levels, irrespective of the presence of plasma.
 
The detailed visualization of platelets in a fibrinogen-coated microfluidics chamber showed that SARS-CoV-2 induced the formation of large clots in the presence of plasma, while these clots were absent in the presence of C.Med.
 
Furthermore, the study team observed robust platelet activation in the presence of SARS-CoV-2 at concentrations of 1:12.5 to 1:50 v/v. Moreover, the EVs did not significantly increase when the concentration of SARS-CoV-2 was at 1:200 v/v dilution; however, this concentration level sufficiently induced the surface-level expression of αIIbβ3* and CD62P platelets. In contrast, platelet activation was not observed in the presence of C. Med.   
 
Importantly, SARS-CoV-2 did not induce platelet activation in the absence of FII, FX, and FVII. Moreover, these activation levels were comparable to those observed in the negative control samples.
 
Hence, this indicated that tissue factor (TF)-mediated coagulation was involved in SARS-CoV-2 induction of platelet activation.
 
The study team also observed that platelet activation was entirely blocked due to thrombin inhibition.
 
Interestingly, SARS-CoV-2-mediated activation of platelets in naive wild-type mice needed the presence of plasma as well as thrombin activation. Moreover, since the SARS-CoV-2 S protein does not detect murine ACE2 and there is no FcyRIIA expression by murine platelets, the team concluded that platelet activation was dependent on neither the ACE2- nor the Fc-receptor.
 
The blockage of activation due to a thrombin inhibitor also showed that the coagulation initiation, as well as thrombin production, was not a result of viral fixation.
 
The study team also found that high concentrations of untreated C.Med or beta-propiolactone-treated C.Med did not activate platelets even after incubating for 60 minutes. Overall, this showed that platelet activation induced by infectious and fixed SARS-CoV-2 was dependent on the levels of both plasma and thrombin.
 
In addition, significant levels of TF activity were observed on SARS-CoV-2 preparations, while TF activity was not found in C.Med. This suggested that SARS-CoV-2-infected cells generated the production of active TF and the presence of active TF was associated with SARS-CoV-2 infection. Notably, TF activity was higher in patients with severe and non-severe COVID-19 as compared to healthy participants. Among these two patient groups, TF activity was significantly higher in severe patients. This indicated SARS-CoV-2-induced TF expression, which further led to platelet activation. 
   
Importantly, the study findings showed the importance of differentiating viral components in understanding the viral mechanism involved in infections. The study team believed that this study could serve as a foundation for developing new therapies against COVID-19.
 
Thailand Medical News would further like to add that a number of phytochemicals such as Curcumin, Berberine and Quercetin are able to inhibit platelet activation in SARS-CoV-2 infections. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107428/
 
For more on Platelet activation and SARS-CoV-2, keep on logging to Thailand Medical News.