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

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 Fc{gamma}RIIa-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. Cover illustration O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY One-sentence summaryThe Fc{gamma}RIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19

Determinants of PF4/heparin immunogenicity.

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder that occurs with variable frequency in patients exposed to heparin. HIT antibodies preferentially recognize large macromolecular complexes formed between PF4 and heparin over a narrow range of molar ratios, but the biophysical properties of complexes that initiate antibody production are unknown. To identify structural determinants underlying PF4/heparin immunogenicity, we characterized the in vitro interactions of murine PF4 (mPF4) and heparin with respect to light absorption, size, and surface charge (zeta potential). We show that PF4/heparin macromolecular assembly occurs through colloidal interactions, wherein heparin facilitates the growth of complexes through charge neutralization. The size of PF4/heparin macromolecules is governed by the molar ratios of the reactants. Maximal complex size occurs at molar ratios of PF4/heparin at which surface charge is neutral. When mice are immunized with complexes that differ in size and/or zeta potential, antibody formation varies inversely with heparin concentration and is most robust in animals immunized with complexes displaying a net positive zeta-potential. These studies suggest that the clinical heterogeneity in the HIT immune response may be due in part to requirements for specific biophysical parameters of the PF4/heparin complexes that occur in settings of intense platelet activation and PF4 release.