Role of microvesicles and netosis in coagulopathy in patients with SARS-CoV-2 infection: a randomized clinical trial

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Abstract

BACKGROUND: The high incidence of thrombotic complications in patients with SARS-CoV-2 infection significantly worsens the prognosis of the disease. The primary issue is limited comprehension on the mechanisms by which systemic inflammation, neutrophil activation, extracellular neutrophil traps, and microvesicle-mediated hemostasis disorders are interconnected.

AIM: This study aimed to investigate the link between microvesicles and extracellular neutrophil traps and the occurrence of coagulopathies in patients with SARS-CoV-2 infection. The quantitative and qualitative characteristics of these phenomena were assessed based on the severity of the patients’ condition.

METHODS: The study included 213 patients with SARS-CoV-2 infection (138 patients with moderate disease and 75 with severe disease) and 20 healthy donors. The patients underwent blood chemistry, coagulation, and hematology tests. A quantitative analysis of microvesicles was performed using flow cytometry with monoclonal antibodies specific for surface markers (CD45, CD3, CD14, CD15, and CD61). The interaction of microvesicles with extracellular neutrophil traps was observed by confocal microscopy (Leica TCS SP5) using fluorescent labels (DAPI, FITC, APC, and PE) and subsequently analyzed for colocalization using a LAS AF package. Statistical analysis was conducted using the Student’s t-test, Spearman’s correlation coefficient, and linear regression methods.

RESULTS: The patients with moderate SARS-CoV-2 infection had hypercoagulation (fibrinogen, 4.8 [4.00; 5.60] g/L; D-dimer, 0.78 [0.30; 1.28] mg/L), with increased levels of neutrophil-derived (CD15⁺, 53.34% ± 6.92%) and platelet-derived (CD61⁺, 59.74% ± 11.22%) microvesicles. Tissue factor-enriched filamentous extracellular neutrophil traps with microvesicles were identified. The severe cases were associated with decreased levels of neutrophil-derived (CD15⁺, 10.32% ± 4.29%) and platelet-derived (CD61⁺, 20.9% ± 6.01%) microvesicles, thrombocytopenia (139.5 [104.25; 177.75] × 10⁹/L), hypocoagulation (international normalized ratio: 2.60 [2.32; 3.58]), and CD62⁺-positive aggregates.

CONCLUSION: Microvesicles and extracellular neutrophil traps play a key role in dysregulated hemostasis in patients with SARS-CoV-2 infection. Early hypercoagulation is mediated by their procoagulant activity, and severe cases are associated with decreased levels of peripheral microvesicles, which is indicative of consumptive coagulopathy.

About the authors

Elena S. Gracheva

Kazan State Medical University

Author for correspondence.
Email: Gracheva020688@mail.ru
ORCID iD: 0000-0002-8543-6529
SPIN-code: 3290-5459

Postgrad. Student, Assistant, Depart. of Biochemistry and Clinical Laboratory Diagnostics

Russian Federation, Kazan

Il’shat G. Mustafin

Kazan State Medical University

Email: ilshat.mustafin@kazangmu.ru
ORCID iD: 0000-0001-9683-3012
SPIN-code: 1588-6988

MD, Dr. Sci. (Medicine), Professor, Head, Depart. of Biochemistry and Clinical Laboratory Diagnostics

Russian Federation, Kazan

Dmitry V. Samigullin

Kazan Institute of Biochemistry and Biophysics — Kazan Scientific Center of Russian Academy of Sciences; Kazan National Research Technical University named after A.N. Tupolev-KAI

Email: samid75@mail.ru
ORCID iD: 0000-0001-6019-5514
SPIN-code: 4751-7443

Cand. Sci. (biology), Head, Lab. of Biophysics and Synaptic Processes

Russian Federation, Kazan; Kazan

Diana I. Abdulganieva

Kazan State Medical University

Email: diana-s@mail.ru
ORCID iD: 0000-0001-7069-2725
SPIN-code: 6676-4270

MD, Dr. Sci. (Medicine), Professor, Head, Hospital therapy depart.

Russian Federation, Kazan

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Supplementary files

Supplementary Files
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2. Fig. 1. Scatter plots representative of each study group: a, moderate SARS-CoV-2 infection; b, severe SARS-CoV-2 infection; and c, controls.

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3. Fig. 2. Scatter plots (a, e) and cytofluorograms of microvesicles expressing CD15 (b), CD45 (c), CD61 (d), CD14 (f), CD4 (g), and CD3 (h) attributed to patients with moderate SARS-CoV-2 infection. The analysis was performed within the gate characteristic of microvesicles in SARS-CoV-2 infection.

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4. Fig. 3. Scatter plots (a, e) and cytofluorograms of microvesicles expressing CD15 (b), CD45 (c), CD61 (d), CD14 (f), CD4 (g), and CD3 (h) attributed to patients with severe SARS-CoV-2 infection. The analysis was performed within the gate characteristic of microvesicles in SARS-CoV-2 infection.

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5. Fig. 4. Fluorescence of DNA fragments and microvesicles in platelet-free blood plasma of patients with moderate SARS-CoV-2 infection: a, DAPI-stained DNA fragments; b, FITC-labeled anti-CD15 monoclonal antibodies; c, PE-labeled CD13; d, APC-labeled CD62-positive microparticle aggregates; and e, overlapping of four dye channels.

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6. Fig. 5. Fluorescence of DNA fragments and microvesicle aggregates in platelet-free blood plasma of patients with moderate SARS-CoV-2 infection: a, DAPI-stained DNA fragments; b FITC-labeled anti-CD15 monoclonal antibodies; c, PE-labeled CD13; d, APC-labeled CD62-positive microparticle aggregates; and e, overlapping of four dye channels.

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7. Fig. 6. Fluorescence of DNA fragments and microvesicles in platelet-free blood plasma of patients with moderate SARS-CoV-2 infection: a, DAPI-stained DNA fragments; b, FITC-labeled CD61-positive microvesicles; c, PE-labeled TFs; d, APC-labeled CD15-positive microparticle aggregates; and e, overlapping of four dye channels.

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8. Fig. 7. Fluorescence of DNA fragments and microvesicles in platelet-free blood plasma of patients with severe SARS-CoV-2 infection: a, DAPI-stained DNA fragments; b, FITC-labeled anti-CD15 monoclonal antibodies; c, PE-labeled CD13; d, APC-labeled CD62-positive microparticle aggregates; and e, overlapping of four dye channels.

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