Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent pathogen responsible for the coronavirus disease 2019 (COVID-19). Since its emergence, the novel coronavirus has rapidly achieved pandemic proportions causing remarkably increased morbidity and mortality around the world. A hypercoagulability state has been reported as a major pathologic event in COVID-19, and thromboembolic complications listed among life-threatening complications of the disease. Platelets are chief effector cells of hemostasis and pathological thrombosis. However, the participation of platelets in the pathogenesis of COVID-19 remains elusive. This report demonstrates that increased platelet activation and platelet-monocyte aggregate formation are observed in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome. In addition, exposure to plasma from severe COVID-19 patients increased the activation of control platelets ex vivo. In our cohort of COVID-19 patients admitted to the intensive care unit, platelet-monocyte interaction was strongly associated with tissue factor (TF) expression by the monocytes. Platelet activation and monocyte TF expression were associated with markers of coagulation exacerbation as fibrinogen and D-dimers, and were increased in patients requiring invasive mechanical ventilation or patients who evolved with in-hospital mortality. Finally, platelets from severe COVID-19 patients were able to induce TF expression ex vivo in monocytes from healthy volunteers, a phenomenon that was inhibited by platelet P-selectin neutralization or integrin αIIb/β3 blocking with the aggregation inhibitor abciximab. Altogether, these data shed light on new pathological mechanisms involving platelet activation and platelet-dependent monocyte TF expression, which were associated with COVID-19 severity and mortality.

Graphical abstract

Figure 1.

Increased platelet activation in critically ill COVID-19 patients. (A-B) The percentage of P-selectin (CD62P) (A) and CD63 (B) surface expression on platelets from SARS-CoV-2⁻ control participants, SARS-CoV-2⁺ asymptomatic subjects, or symptomatic patients presenting mild to severe COVID-19 syndrome. (C) The concentration of TXB₂ in plasma from control subjects or patients with COVID-19 presenting mild to severe syndrome. (D) Quantification of TXB₂, PF4, and PDGF in tracheal aspirates from severe COVID-19 patients under mechanical ventilation (n = 19). The horizontal lines in the box plots represent the median, the box edges represent the interquartile ranges, and the whiskers indicate the minimal and maximal value in each group. *P < .05 between selected groups. ns, nonsignificant.

Figure 2.

Inflammatory mediators generated in severe COVID-19 contribute to platelet activation. (A-B) The percentage of P-selectin (CD62P) (A) and CD63 (B) expression on platelets was plotted against the concentration of fibrinogen in plasma. Linear regression and Spearman correlation were calculated according to the distribution of the dots. (C-D) Platelets from healthy volunteers were incubated with plasma from severe COVID-19 patients (severe, n = 16) or from SARS-CoV2⁻ subjects (control, n = 11) for 2 hours. The percentages of P-selectin (C) and CD63 (D) are shown. All experiments were repeated with platelets from 3 independent healthy volunteers with similar results, and representative data from 1 of the platelet donors are shown. The horizontal lines in the box plots represent the median, the box edges represent the interquartile ranges, and the whiskers indicate the minimal and maximal value in each group. *P < .05 between selected groups.

Figure 3.

Increased platelet-monocyte aggregates in severe COVID-19 patients. (A) Gating strategy for analysis of platelet-monocyte aggregates in patients with COVID-19 or control subjects. (B) Percentage of platelet-monocyte complexes (CD14⁺CD41⁺) among monocytes from SARS-CoV-2⁻ control volunteers, SARS-CoV-2⁺ asymptomatic subjects, or symptomatic patients presenting mild to severe COVID-19 syndrome. The horizontal lines in the box plots represent the median, the box edges represent the interquartile ranges, and the whiskers indicate the minimal and maximal value in each group. *P < .05 between selected groups. (C) The percentage of platelet P-selectin (CD62P) surface expression was plotted against the percentage of platelet-monocyte complexes in the same infected subjects. Linear regression and Pearson correlation were calculated according to the distribution of the dots. (D) Phalloidin-labeled cytospin blood samples from control subjects or COVID-19 patients. Platelets and leukocytes obtained from whole blood after lysis of erythrocytes were labeled with Alexa 488–phalloidin (F actin, green) and DAPI (nuclei, blue). Scale bar, 10 μm; inset, 5 μm. Representative images of platelet-monocyte aggregates are shown. The fourth panel shows orthogonal views from different planes of the confocal image. The green line generated a 2-dimensional (2D) image that is shown in the top green rectangle whereas the red line–generated 2D image is shown in the red rectangle on the right. SSC-H, side scatter height.

Figure 4.

Platelet-monocyte complexes express TF in severe COVID-19 patients. (A-B) The percentage of TF surface expression on monocytes (A) or platelets (B) from SARS-CoV2⁻ control volunteers, SARS-Cov2⁺ asymptomatic subjects, or symptomatic patients presenting mild to severe COVID-19 syndrome. (C) The percentage of TF surface expression on monocytes that were complexed with platelets (CD14⁺CD41⁺) or circulating freely (CD14⁺CD41⁻) in severe COVID-19 patients. The horizontal lines in the box plots represent the median, the box edges represent the interquartile ranges, and the whiskers indicate the minimal and maximal value in each group. *P < .05 between selected groups.

Figure 5.

Increased platelet activation and TF expression by monocytes associate with severity and mortality in COVID-19. (A-E) Severe COVID-19 patients admitted to the ICU were stratified between those requiring invasive mechanical ventilation or noninvasive O₂ supplementation. The percentages of P-selectin (CD62P) (A) and CD63 (B) expression on platelets, the concentration of TXB₂ in plasma (C), the percentage of monocytes forming aggregates with platelets (D), and TF expression on monocytes (E) are shown. (F-K) Severe COVID-19 patients were stratified according to the 28-day mortality outcome as survivors or nonsurvivors. The percentages of P-selectin (CD62P) (F) and CD63 (G) expression on platelets, the concentration of TXB₂ in plasma (H), the quantification of PDGF-BB in tracheal aspirates (I), and the percentage of monocytes forming aggregates with platelets (J) and expressing TF (K) are shown. The horizontal lines in the box plots represent the median, the box edges represent the interquartile ranges, and the whiskers indicate the minimal and maximal value in each group. The gray box indicates the interquartile ranges and the dotted line represents the median of the SARS-CoV2⁻ control group. *P < .05 between selected groups.

Figure 6.

Platelets from COVID-19 patients induce TF expression in monocytes through mechanisms depending on P-selectin and integrin α_IIb/β₃. (A) Monocytes from healthy volunteers were incubated with platelets from severe COVID-19 patients (COVID-19 platelets) or from heterologous healthy volunteers (control platelets) for the indicated time points. The percentage of TF-expressing monocytes is shown. (B-D) Control monocytes were exposed to platelets from severe COVID-19 patients for 2 hours in the presence of anti–P-selectin (anti-CD62P) neutralizing antibody, the anti-α_IIb/β₃ antibody abciximab, or isotype-matched IgG. The percentage of TF-expressing monocytes (B), platelet-monocyte complexes (C), and the percentage and the mean fluorescence intensity (MFI) of P-selectin expression on CD14⁺CD41⁺ monocytes (D) are shown in each condition. (E-F) Control monocytes were exposed to platelets from COVID-19 patients for 2 hours in the presence of aspirin (100 µM) or clopidogrel (300 µM). Bars represent mean plus or minus standard error of the mean of monocytes exposed to platelets from 6 independent COVID-19 patients. All experiments were repeated with monocytes from 2 (B-D), 3 (E-F), or 4 (A) independent healthy volunteers with similar results, and representative data from 1 of the donors are shown. *P < .05 between selected groups.