Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia

Abstract

Objective: Pulmonary thrombosis is observed in severe acute respiratory syndrome coronavirus 2 pneumonia. Aim was to investigate whether subpopulations of platelets were programmed to procoagulant and inflammatory activities in coronavirus disease 2019 (COVID-19) patients with pneumonia, without comorbidities predisposing to thromboembolism. Approach and Results: Overall, 37 patients and 28 healthy subjects were studied. Platelet-leukocyte aggregates, platelet-derived microvesicles, the expression of P-selectin, and active fibrinogen receptor on platelets were quantified by flow cytometry. The profile of 45 cytokines, chemokines, and growth factors released by platelets was defined by immunoassay. The contribution of platelets to coagulation factor activity was selectively measured. Numerous platelet-monocyte (mean±SE, 67.9±4.9%, n=17 versus 19.4±3.0%, n=22; P<0.0001) and platelet-granulocyte conjugates (34.2±4.04% versus 8.6±0.7%; P<0.0001) were detected in patients. Resting patient platelets had similar levels of P-selectin (10.9±2.6%, n=12) to collagen-activated control platelets (8.7±1.5%), which was not further increased by collagen activation on patient platelets (12.4±2.5%, P=nonsignificant). The agonist-stimulated expression of the active fibrinogen receptor was reduced by 60% in patients (P<0.0001 versus controls). Cytokines (IL [interleukin]-1α, IL-1β, IL-1RA, IL-4, IL-10, IL-13, IL, 17, IL-27, IFN [interferon]-α, and IFN-γ), chemokines (MCP-1/CCL2 [monocyte chemoattractant protein 1]), and growth factors (VEGF [vascular endothelial growth factor]-A/D) were released in significantly larger amounts upon stimulation of COVID-19 platelets. Platelets contributed to increased fibrinogen, VWF (von Willebrand factor), and factor XII in COVID-19 patients. Patients (28.5±0.7 s, n=32), unlike controls (31.6±0.5 s, n=28; P<0.001), showed accelerated factor XII-dependent coagulation.

Conclusions: Platelets in COVID-19 pneumonia are primed to spread proinflammatory and procoagulant activities in systemic circulation.

Keywords: blood platelets; inflammation; interferons; monocytes; thrombosis.

Figure 1.

Computed tomography perfusion angiography (CTPA) scans. Axial CTPA images with mediastinal (A) and lung (B) window showing filling defects involving the proximal tract of left pulmonary artery (arrow). Diffuse ground glass opacifications together with diffuse thickening of interlobular septa are visible (B). MIP (maximum intensity projection) reformatted images on coronal plane (C) show filling defects both in some segmental and subsegmental branches of left pulmonary artery (small arrow) and in corresponding next venous branches (large arrow).

Figure 2.

Representative blood smear from patients with severe acute respiratory syndrome coronavirus 2 pneumonia showing platelet anisopoichilocytosis (A–D), platelet satellitism (E–H), and platelet engulfment by lymphocytes (F).

Figure 3.

Platelet phenotype. Whole-blood analysis of monocytes and neutrophil-platelet aggregates shows higher percentage of plateletmonocyte aggregates (A) and platelet-neutrophil aggregates (B) in citrated whole blood from coronavirus disease 2019 (COVID-19) patients (n=17) than healthy controls (n=22). The percentage of resting platelets expressing P-selectin in COVID-19 patients (n=12) is similar to that observed in platelets from healthy controls (n=22) stimulated with collagen (C). P-selectin expression does not further increase when platelets are stimulated with collagen (C). The expression of the active form of fibrinogen receptor α_IIbβ₃, as detected by the monoclonal antibody PAC-1, is similar under resting conditions in patients and healthy controls and lower in patients (n=16) in platelets stimulated with collagen (D). The number of platelet-derived microvesicles (PMV) is slightly higher in patients (n=15) than in controls (n=22; E) and correlates with the surface expression of P-selectin in COVID-19 patients (F). CD62P (P-selectin) indicates cluster of differentiation 62P; and PTL, platelets.

Figure 4.

Coagulation and coagulation factors assays. Activated partial thromboplastin time (APTT) was tested using plasma and platelet-rich plasma (PRP) from coronavirus disease 2019 (COVID-19) patients (n=32) and healthy controls (n=28; A). The activity of the coagulation factor VIII is similarly higher in plasma and PRP in COVID-19 patients, correlates with APTT (B and C), and is not stored in platelets, as demonstrated by the effects of platelets from patients added to control plasma (B). Factor XII activity does not differ in patients (n=20) and controls (n=20; D) but correlates with APTT only in patients (F) and increases when platelets from patients were suspended in control plasma (n=12; D and E). Plasma VWF (von Willebrand factor) antigen (Ag), collagen binding (CB), and ristocetin cofactor (RCo) is increased in COVID-19 patients (n=9) compared with controls (n=20; G). Fibrinogen activity is higher in plasma and PRP from patients (n=20) than controls (n=20; H). PTL indicates platelets.