Platelet and Endothelial Activation as Potential Mechanisms Behind the Thrombotic Complications of COVID-19 Patients

Abstract

The authors hypothesized that the cytokine storm described in COVID-19 patients may lead to consistent cell-based tissue factor (TF)-mediated activation of coagulation, procoagulant microvesicles (MVs) release, and massive platelet activation. COVID-19 patients have higher levels of TF⁺ platelets, TF⁺ granulocytes, and TF⁺ MVs than healthy subjects and coronary artery disease patients. Plasma MV-associated thrombin generation is present in prophylactic anticoagulated patients. A sustained platelet activation in terms of P-selectin expression and platelet-leukocyte aggregate formation, and altered nitric oxide/prostacyclin synthesis are also observed. COVID-19 plasma, added to the blood of healthy subjects, induces platelet activation similar to that observed in vivo. This effect was blunted by pre-incubation with tocilizumab, aspirin, or a P2Y₁₂ inhibitor.

Keywords: ADP, adenosine diphosphate; CAD, coronary artery disease; COVID-19; COVID-19, coronavirus disease-2019; CRP, C-reactive protein; GPA, granulocyte–platelet aggregates; HS, healthy subject; IL, interleukin; IL-6; IL-6R, interleukin-6 receptor; LMWH, low-molecular-weight heparin; MPA, monocyte–platelet aggregates; MV, microvesicle; NO, nitric oxide; NOS, nitric oxide synthase; PGI2, prostacyclin; PLA, platelet–leukocyte aggregates; PS, phosphatidylserine; SARS-CoV-2, severe acute respiratory syndrome-coronavirus-2; TF, tissue factor; antiplatelet drugs; circulating microvesicles; platelet activation; tissue factor.

Graphical abstract

Figure 1

Quantification of TF⁺-Circulating Cells Tissue factor (TF) expression in platelets (A and B), granulocytes (C), and monocytes (D) in coronavirus disease-2019 (COVID-19) patients and in healthy subjects (HS). (B) Percentage of TF⁺-platelets of COVID-19 patients grouped according to oxygen supplementation (COVID-19 group 1: oxygen treated; COVID-19 group 2: mechanical ventilation). The dotted line represents the median with 25th and 75th percentiles of TF⁺ platelets in HS.

Figure 2

Analysis of Platelet Activation Markers in COVID-19 Patients and HS Percentage of P-selectin⁺ platelets (A) and of the total and TF⁺-platelet–granulocyte (B and D) and TF⁺-monocyte (E and G) aggregates. Percentage of TF⁺-platelet–granulocyte (C) and TF⁺-monocyte (F) aggregates in group 1 and group 2 COVID-19 patients. aggr = aggregate; gran = granulocytes; mono = monocyte; PLT = platelet; Psel = P-selectin; other abbreviations as in Figure 1.

Figure 3

Evaluation of Endothelial Activation Markers in COVID-19 Patients and HS Plasma levels of l-arginine (Arg) (A), asymmetric dimethylarginine (ADMA) (B), global arginine bioavailability ratio (GABR) (C), L-ornithine (Orn) (D), L-citrulline (Cit) (F), and 6-keto-PGF_1α(H). L-ornithine (E), L-citrulline (G), and 6-keto-PGF_1α(I) in group 1 and group 2 COVID-19 patients. Abbreviations as in Figure 1.

Figure 4

Ex Vivo Effect of Plasma From COVID-19 Patients on HS Platelets Plasma-depleted blood from HS (n = 5) reconstituted with autologous plasma (white bars), a plasma pool from HS (grey bars), or with a plasma pool from COVID-19 patients (orange bars). Percentage of TF⁺ and P-selectin⁺ platelets (A and B), CD41⁺TF⁺ MVs (G), total and TF⁺ granulocyte–platelet aggregates (C and D) and monocyte–platelet aggregates (D and F). The effect of whole blood incubation with interleukin (IL)-6 (100 pg/ml), ADP (0.1 μmol/l), or ADP+IL-6 on the same parameters is reported (n = 5). ADP = adenosine diphosphate; other abbreviations as in Figures 1 and 2.

Figure 5

In Vitro Effect of Tocilizumab on Platelet Activation Induced by COVID-19 Plasma Plasma-depleted blood from HS (n = 5) has been reconstituted with COVID-19 plasma pool (orange bars) or COVID-19 plasma pool pre-incubated with tocilizumab (Toc; 100 or 300 μg/ml; blue and light blue bars, respectively) or with an irrelevant (Irrel) IgG (300 μg/ml; grey bars). Blood reconstituted with autologous plasma is reported for comparison (white bars). Percentage of TF⁺ and P-selectin⁺ platelets (A and D), CD41⁺TF⁺ MVs (I), total and TF⁺ granulocyte–platelet aggregates (E and F) and monocyte–platelet aggregates (G and H). (B) Time needed for platelet-associated thrombin generation (TG) (lag time). (C) Representative curves of TG were reported. Abbreviations as in Figures 1 and 2.

Figure 6

In Vitro Effect of Aspirin and AR-C69931MX on Platelet Activation Induced by COVID-19 Plasma Blood from HS (n = 5), pre-incubated with aspirin (100 μmol/l) or with AR-C69931MX (0.5 μmol/l) or both, was plasma-depleted and reconstituted with COVID-19 plasma pool. Percentage of TF⁺ and P-selectin⁺ platelets (A and B), CD41⁺TF⁺-MVs (G), total and TF⁺ granulocyte–platelet aggregates (C and D) and monocyte–platelet aggregates (E and F). Abbreviations as in Figures 1 and 2.