PB0587 - Platelet reactivity to SARS-CoV-2 Spike protein and domains.

Background: COVID-19 has affected millions of people worldwide over the past two years. SARS-CoV- 2 uses the Spike (S) protein to infect target cells. An active immunothrombotic state has been described in severe stages of infection. Platelets are cells implicated in the pathophysiology of CoVID-19, presumably by contributing to the release of inflammatory cytokines and exhibiting a procoagulant phenotype, although the platelet response in the disease has been studied, information on the cellular response to S protein domains is scarce.

Aims: To study platelet reactivity response to SARS-CoV-2 virus Spike protein and RBD domain.

Methods: Blood samples were obtained from healthy volunteers by venipuncture after signing an informed consent form, using 3.2% sodium citrate as anticoagulant. Platelet-rich plasma (PRP) was obtained by slow centrifugation (100 g x 10 minutes). PRP was separated and resuspended in Tyrodes buffer. Platelet stimulation kinetics (1X107 cells/ml) was performed with S full protein and protein S receptor binding domain (RBD) [2 ug/ml], 37°C. PRP was also incubated with plasma from COVID-19 patients [20 ul] for different times. Platelet activity was assessed by flow cytometry: CD41-PECy7, CD62-PE and PAC1-FITC. We used ADP 20µM, collagen 0.19mg/mL and epinephrine 100µM as platelet activation controls.

Results: We observed platelet reactivity after stimulation with protein S, highest activation was observed at 90 min with full protein and at 120 min with RBD domain when compared to the basal expression of selected markers and is similar to the observed with the positive control agonists. Stimulation of PRP with plasma from COVID-19 patients show the presence of activation markers at 60 min. However, activation is lower than that observed with known activation agonists.

Conclusion(s): There is platelet reactivity to Spike protein, the RBD domain and with plasma from COVID-19 affected subjects.