Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19

Abstract

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection.

One-sentence summary: The FcγRIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19.

Figure 1.. Platelets from hospitalized COVID-19 patients exhibit increased activation at baseline and diminished functional reserve.

A–B) Representative histograms (A) and cumulative data (B) for CD62P surface expression of ex vivo isolated platelets assayed with or without TRAP stimulation for 20mins. The CD62P gMFI ratio of basal/TRAP-treated is also shown. The three patient cohorts shown are COVID-19 inpatient, COVID-19 convalescent and healthy donors. Kruskal-Wallis non-parametric testing was used and the p-values are depicted. C) Cumulative data for CD62P surface expression of ex vivo isolated platelets at baseline (basal), after TRAP activation (TRAP) and their ratio for hospitalized COVID-19 patients on full-dose or prophylactic/intermediate dose of anticoagulation. Mann-Whitney non-parametric testing was used and the p-values are depicted. D) Cumulative data for CD62P surface expression of ex vivo isolated platelets at baseline for hospitalized COVID-19 patients with or without cardiovascular disease risk factors. Mann-Whitney non-parametric testing was used, and the p-value is depicted. FMO control: Fluorescence minus one control, gMFI: geometric mean fluorescence intensity, TRAP: Thrombin Receptor Activation Peptide.

Figure 2.. The ability of COVID19 plasma to activate platelets is increased for patients with high circulating levels of ferritin and at timepoints preceding a thrombotic event.

A) Plasma from COVID-19 inpatient, COVID-19 convalescent and healthy donors was incubated with platelets isolated from healthy volunteers. The gMFI levels of CD63, CD62P, CD32 and C3aR are shown in these three groups. Kruskal-Wallis non-parametric testing was used to compare all three groups and pair-wise comparisons were also performed; p-values are depicted. B) Spearman correlation of the gMFI surface levels of the markers used in (A) and selected clinical parameters of the COVID-19 inpatient group. r correlation coefficient shown on the key (right). Asterisks *, ** and *** denote p values less than 0.05, 0.01 and 0.001 respectively. Highlighted squares denote FDR values less than 0.05. C) Representative scatter plots for ferritin with CD63, CD32 and C3aR. D) CD63 gMFI surface levels of control platelets induced by plasma derived from COVID-19 patients with thrombosis drawn at different timepoints relative to the thrombotic event. E) CD63 gMFI surface levels of control platelets induced by plasma derived from COVID-19 patients prior to thrombosis and after thrombosis, COVID-19 patients without thrombosis and COVID-19 patients in convalescence.

Figure 3.. COVID-19 plasma induced platelet activation is associated with markers of inflammation and cardiovascular disease.

A) Volcano plot with the x-axis representing the correlation coefficient of the different analytes with CD63 gMFI induction on platelets by COVID-19 plasma and the y-axis depicting the −log10 transformation of the corresponding p-value. B) Representative scatter plots for CD63 with CCL15, VEGFA, ADA and PCSK9.

Figure 4.. FcγRIIa activation and complement anaphylatoxins mediate platelet activation in COVID-19.

A) Heatmap of gMFI expression of CD62P, CD63, CD32 and C3aR on the surface of control platelets incubated with COVID-19 plasma (n=10 patients) in the presence or absence of neutralizing antibodies to FcγRIIa, IL6, C3a and C5a, as indicated. B) Boxplots of gMFI expression of CD63 on the surface of control platelets incubated with COVID-19 plasma (n=5 patients, depicted with distinct colors) with different combinations of neutralizing antibodies to FcγRIIa, IL6, C3a and C5a, as indicated. C) Boxplots of gMFI expression of CD63 on the surface of control platelets incubated with COVID-19 plasma (n=9 patients, depicted with distinct colors) with different combinations of IgG depletion and neutralizing antibodies to C3a and C5a.

Figure 5.. Fostamatinib ameliorates the heightened activation of platelets induced by COVID-19 plasma.

A) Phospho-Syk and total Syk gMFI were measured with flow cytometry of control platelets incubated with COVID-19 plasma (n=3 patients) for 1 and 5 minutes. Incubation with healthy control plasma for 5 mins was used to normalize the data. B) Boxplots of gMFI expression of CD62P on the surface of control platelets incubated with COVID-19 plasma (n=4 patients) in the absence or presence of fostamatinib or fostamatinib and neutralizing antibodies to C3a and C5a. Kruskal-Wallis non-parametric testing was used to compare the groups and the p-value is depicted. C) Representative studies of a hematoporphyrin-induced photochemical injury model in an endothelial-lined microfluidic channel. Images show platelet adhesion (in white) immediately after infusion of isolated washed healthy donor platelets, and 5 min, 10 min and 15 min after infusion. Shown is a representative study where plasma from a severe COVID-19 patient was added to the platelets. In the samples indicated, the Syk inhibitor R406, an anti-FcγRIIa antibody, or an anti-C5a antibody were added prior to infusion. Direction of blood flow is indicated by arrows. Size bar indicates 100μ. D) Overall data analysis from studies with plasma derived from 7 patients with severe COVID-19. The Y-axis shows accumulated platelets in each microfluidic lane done immediately after the isolated platelet suspension was added and at immediate, 5, 10 and 15 minutes (log10 scale, fold change over healthy control plasma flown in uninjured channels). E) All 7 sets of experiments from D are shown at the 10-minute timepoint.