A strong correlation exists between platelet consumption and platelet hyperactivation in COVID-19 patients. Pilot study of the patient cohort from CCH RAS Hospital (Troitsk).

https://doi.org/10.1016/S0140-6736(20)30183-5

]

. In patients with the severe form of the disease, there is an association with interstitial pneumonia, thrombosis, and subsequent pulmonary failure

[

2,

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

C. Huang, Y. Wang, X. Li, L. Ren, J. Zhao, Y. Hu, L. Zhang, G. Fan, J. Xu, X. Gu, Z. Cheng, T. Yu, J. Xia, Y. Wei, W. Wu, X. Xie, W. Yin, H. Li, M. Liu, Y. Xiao, H. Gao, L. Guo, J. Xie, G. Wang, R. Jiang, Z. Gao, Q. Jin, J. Wang, B. Cao

The Lancet. 2020, 395, 497-506

3

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

https://doi.org/10.1111/jth.14848

]

. Thrombocytopenia was also shown to be common in COVID-19 patients, and lower platelet count correlated with disease severity and higher mortality rates

[

4,

Thrombocytopenia and its association with mortality in patients with COVID‐19

X. Yang, Q. Yang, Y. Wang, Y. Wu, J. Xu, Y. Yu, Y. Shang

Journal of Thrombosis and Haemostasis. 2020, 18, 1469-1472

https://doi.org/10.1016/j.cca.2020.03.022

5,

Thrombocytopenia is associated with severe coronavirus disease 2019 (COVID-19) infections: A meta-analysis

G. Lippi, M. Plebani, B. Henry

Clinica Chimica Acta. 2020, 506, 145-148

https://doi.org/10.24287/1726-1708-2021-20-1-184-191

6

Platelets in COVID-19: “innocent by-standers” or active participants?

O. An, A. Martyanov, M. Stepanyan, A. Boldova, S. Rumyantsev, M. Panteleev, F. Ataullakhanov, A. Rumyantsev, A. Sveshnikova

Pediatric Hematology/Oncology and Immunopathology. None, 20, 184-191

https://doi.org/10.1016/j.jinf.2020.03.037

]

. COVID-19 is known to cause a strong inflammatory response, the cytokine storm characterized by increased concentrations of interleukins 1β, 6, and 8, among others

[

7

The pathogenesis and treatment of the `Cytokine Storm' in COVID-19

Q. Ye, B. Wang, J. Mao

Journal of Infection. 2020, 80, 607-613

https://doi.org/10.1002/ajh.25829

]

. This inflammatory state can be one of the causes of serious hemostatic disorders observed in patients with severe forms of the disease

[

8

Hematological findings and complications of COVID ‐19

E. Terpos, I. Ntanasis‐Stathopoulos, I. Elalamy, E. Kastritis, T. Sergentanis, M. Politou, T. Psaltopoulou, G. Gerotziafas, M. Dimopoulos

American Journal of Hematology. 2020, 95, 834-847

https://doi.org/10.1111/jth.14850

]

. Pulmonary embolism and deep vein thrombosis of the lower extremities are among the leading causes of death in COVID-19

[

9

Hypercoagulability of COVID‐19 patients in intensive care unit: A report of thromboelastography findings and other parameters of hemostasis

M. Panigada, N. Bottino, P. Tagliabue, G. Grasselli, C. Novembrino, V. Chantarangkul, A. Pesenti, F. Peyvandi, A. Tripodi

Journal of Thrombosis and Haemostasis. 2020, 18, 1738-1742

https://doi.org/10.1111/jth.14817

]

. In less severe cases, microthrombi were still present in the lungs of patients

[

10,

Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy

N. Tang, H. Bai, X. Chen, J. Gong, D. Li, Z. Sun

Journal of Thrombosis and Haemostasis. 2020, 18, 1094-1099

https://doi.org/10.1016/j.thromres.2020.04.013

11

Incidence of thrombotic complications in critically ill ICU patients with COVID-19

F. Klok, M. Kruip, N. van der Meer, M. Arbous, D. Gommers, K. Kant, F. Kaptein, J. van Paassen, M. Stals, M. Huisman, H. Endeman

Thrombosis Research. 2020, 191, 145-147

]

.

The mechanism of thrombosis' initiation in COVID-19 is often proposed to be based on macrophages, the cells central to the inflammatory response in lungs

[

12,

The trinity of COVID-19: immunity, inflammation and intervention

M. Tay, C. Poh, L. Rénia, P. MacAry, L. Ng

Nature Reviews Immunology. 2020, 20, 363-374

https://doi.org/10.1038/s41577-020-00470-2

13

Leukocyte trafficking to the lungs and beyond: lessons from influenza for COVID-19

R. Alon, M. Sportiello, S. Kozlovski, A. Kumar, E. Reilly, A. Zarbock, N. Garbi, D. Topham

Nature Reviews Immunology. 2021, 21, 49-64

https://doi.org/10.1038/s41577-020-00470-2

]

. Macrophages, as well as lung epithelial cells, are infected by the SARS-CoV-2 virions, get activated, and thus induce the inflammatory response. Consequently, the blood vessel endothelial cells also get activated

[

13,

Leukocyte trafficking to the lungs and beyond: lessons from influenza for COVID-19

R. Alon, M. Sportiello, S. Kozlovski, A. Kumar, E. Reilly, A. Zarbock, N. Garbi, D. Topham

Nature Reviews Immunology. 2021, 21, 49-64

https://doi.org/10.1038/s41577-021-00536-9

14

Endothelial dysfunction and immunothrombosis as key pathogenic mechanisms in COVID-19

A. Bonaventura, A. Vecchié, L. Dagna, K. Martinod, D. Dixon, B. Van Tassell, F. Dentali, F. Montecucco, S. Massberg, M. Levi, A. Abbate

Nature Reviews Immunology. 2021, 21, 319-329

https://doi.org/10.1016/j.tcm.2015.12.001

]

. Activated endothelial cells express on their surface a potent activator of the plasma coagulation cascade, tissue factor (TF)

[

15

Tissue factor as a link between inflammation and coagulation

M. Witkowski, U. Landmesser, U. Rauch

Trends in Cardiovascular Medicine. 2016, 26, 297-303

https://doi.org/10.3389/fimmu.2019.02884

]

, and secrete von Willebrand Factor (vWF), which adheres platelets to the inflamed endothelium

[

16

von Willebrand Factor and Platelet Glycoprotein Ib: A Thromboinflammatory Axis in Stroke

F. Denorme, K. Vanhoorelbeke, S. De Meyer

Frontiers in Immunology. None, 10, 2884

]

. Thus, COVID-19 induced endothelial cell activation results in activation of both platelet and plasma coagulation, leading to pathologic thrombosis

[

12

The trinity of COVID-19: immunity, inflammation and intervention

M. Tay, C. Poh, L. Rénia, P. MacAry, L. Ng

Nature Reviews Immunology. 2020, 20, 363-374

https://doi.org/10.1378/chest.119.1_suppl.64S

]

. Additional evidence for the involvement of blood coagulation in the development of COVID-19 comes from the effectiveness of treatment with heparin and its derivatives

[

17

Heparin and Low-Molecular-Weight Heparin Mechanisms of Action, Pharmacokinetics, Dosing, Monitoring, Efficacy, and Safety

J. Hirsh, T. Warkentin, S. Shaughnessy, S. Anand, J. Halperin, R. Raschke, C. Granger, E. Ohman, J. Dalen

Chest. 2001, 119, 64S-94S

https://doi.org/10.1002/ajh.25829

]

. Heparins are also known for their anti-inflammatory effect

[

8,

Hematological findings and complications of COVID ‐19

E. Terpos, I. Ntanasis‐Stathopoulos, I. Elalamy, E. Kastritis, T. Sergentanis, M. Politou, T. Psaltopoulou, G. Gerotziafas, M. Dimopoulos

American Journal of Hematology. 2020, 95, 834-847

https://doi.org/10.1182/blood.2020006000

18

COVID-19 and its implications for thrombosis and anticoagulation

J. Connors, J. Levy

Blood. 2020, 135, 2033-2040

https://doi.org/10.1136/jim-2020-001628

]

. Although heparin effectiveness in COVID-19 treatment has been demonstrated several times

[

19,

Higher heparin dosages reduce thromboembolic complications in patients with COVID-19 pneumonia

C. Carallo, F. Pugliese, E. Vettorato, C. Tripolino, L. Delle Donne, G. Guarrera, W. Spagnolli, S. Cozzio

Journal of Investigative Medicine. 2021, 69, 884-887

https://doi.org/10.3389/fmed.2021.647917

20,

Risk of Clinically Relevant Venous Thromboembolism in Critically Ill Patients With COVID-19: A Systematic Review and Meta-Analysis

J. Gratz, M. Wiegele, M. Maleczek, H. Herkner, H. Schöchl, E. Chwala, P. Knöbl, E. Schaden

Frontiers in Medicine. 2021, 8, None

https://doi.org/10.1111/jth.13442

]

.

The key driving enzyme of blood plasma coagulation, thrombin, could induce platelet hyperactivation and procoagulant response

[

22

Systems biology insights into the meaning of the platelet's dual-receptor thrombin signaling

A. Sveshnikova, A. Balatskiy, A. Demianova, T. Shepelyuk, S. Shakhidzhanov, M. Balatskaya, A. Pichugin, F. Ataullakhanov, M. Panteleev

Journal of Thrombosis and Haemostasis. 2016, 14, 2045-2057

https://doi.org/10.1111/jth.13442

]

. Procoagulant platelets are characterized by the presence of phosphatidylserine (PS) on their surface, thus enhancing plasma coagulation

[

22,

Systems biology insights into the meaning of the platelet's dual-receptor thrombin signaling

A. Sveshnikova, A. Balatskiy, A. Demianova, T. Shepelyuk, S. Shakhidzhanov, M. Balatskaya, A. Pichugin, F. Ataullakhanov, M. Panteleev

Journal of Thrombosis and Haemostasis. 2016, 14, 2045-2057

https://doi.org/10.1182/blood-2016-02-696898

23

Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting

N. Podoplelova, A. Sveshnikova, Y. Kotova, A. Eckly, N. Receveur, D. Nechipurenko, S. Obydennyi, I. Kireev, C. Gachet, F. Ataullakhanov, P. Mangin, M. Panteleev

Blood. 2016, 128, 1745-1755

https://doi.org/10.1080/09537104.2018.1513473

]

. PS-positive platelets were proposed to be one of the key markers of the enhanced platelet activation in the circulation

[

24,

Flow cytometry for pediatric platelets

A. Ignatova, E. Ponomarenko, D. Polokhov, E. Suntsova, P. Zharkov, D. Fedorova, E. Balashova, A. Rudneva, V. Ptushkin, E. Nikitin, A. Shcherbina, A. Maschan, G. Novichkova, M. Panteleev

Platelets. 2019, 30, 428-437

https://doi.org/10.1152/physrev.00016.2011

25,

New Fundamentals in Hemostasis

H. Versteeg, J. Heemskerk, M. Levi, P. Reitsma

Physiological Reviews. 2013, 93, 327-358

https://doi.org/10.1161/ATVBAHA.118.311390

26

Clot Contraction Drives the Translocation of Procoagulant Platelets to Thrombus Surface

D. Nechipurenko, N. Receveur, A. Yakimenko, T. Shepelyuk, A. Yakusheva, R. Kerimov, S. Obydennyy, A. Eckly, C. Léon, C. Gachet, E. Grishchuk, F. Ataullakhanov, P. Mangin, M. Panteleev

Arteriosclerosis, Thrombosis, and Vascular Biology. 2019, 39, 37-47

https://doi.org/10.1093/infdis/jis180

]

. It should be noted that PS+ platelets are rapidly removed from circulation by macrophages

[

27

Platelet Apoptosis and Apoptotic Platelet Clearance by Macrophages in Secondary Dengue Virus Infections

M. Alonzo, T. Lacuesta, E. Dimaano, T. Kurosu, L. Suarez, C. Mapua, Y. Akeda, R. Matias, D. Kuter, S. Nagata, F. Natividad, K. Oishi

The Journal of Infectious Diseases. 2012, 205, 1321-1329

https://doi.org/10.1080/09537104.2018.1513473

]

. Therefore, mean platelet age can drop in case of platelet activation in circulation. It is noteworthy that younger platelets, recently produced by the megakaryocytes in the bone marrow, are larger in size

[

24

Flow cytometry for pediatric platelets

A. Ignatova, E. Ponomarenko, D. Polokhov, E. Suntsova, P. Zharkov, D. Fedorova, E. Balashova, A. Rudneva, V. Ptushkin, E. Nikitin, A. Shcherbina, A. Maschan, G. Novichkova, M. Panteleev

Platelets. 2019, 30, 428-437

]

and more prone to activation than older platelets

[

28

The Impact of COVID-19 Disease on Platelets and Coagulation

G. Wool, J. Miller

Pathobiology. 2021, 88, 15-27

https://doi.org/10.1152/physrev.00016.2011

]

, those are sequestered in the spleen or liver after 7-10 days of circulation

[

25

New Fundamentals in Hemostasis

H. Versteeg, J. Heemskerk, M. Levi, P. Reitsma

Physiological Reviews. 2013, 93, 327-358

]

. Thus, mean platelet size, as well as the percentage of PS+ platelets, could be indicators of platelet activation in circulation.

The mechanisms of SARS-CoV-2 influence on platelet functioning and the role of the platelet hemostasis in the pathology of COVID-19, in general, are currently actively studied. Younes et al. have shown that viral RNA was present in patients' platelets in 22% of cases, both severe and non-severe

[

29

Platelets Can Associate With SARS-CoV-2 RNA and Are Hyperactivated in COVID-19

Y. Zaid, F. Puhm, I. Allaeys, A. Naya, M. Oudghiri, L. Khalki, Y. Limami, N. Zaid, K. Sadki, R. Ben El Haj, W. Mahir, L. Belayachi, B. Belefquih, A. Benouda, A. Cheikh, M. Langlois, Y. Cherrah, L. Flamand, F. Guessous, E. Boilard

Circulation Research. 2020, 127, 1404-1418

]

. Conversely, Manne et al. have shown that no viral RNA was found in COVID-19 patients (3 from ICU and one non-ICU) using transmission electron microscopy, while mRNA expression of the SARS-CoV-2 N1 gene was present in 2 out of 25 patients. Both of them were in ICU

[

3

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

https://doi.org/10.1002/ajh.25829

]

. The angiotensin-converting enzyme 2 (ACE-2) receptor

[

8

Hematological findings and complications of COVID ‐19

E. Terpos, I. Ntanasis‐Stathopoulos, I. Elalamy, E. Kastritis, T. Sergentanis, M. Politou, T. Psaltopoulou, G. Gerotziafas, M. Dimopoulos

American Journal of Hematology. 2020, 95, 834-847

]

is the key route for SARS-CoV-2 entrance into human cells. However, it is not clear if this receptor is present on platelets

[

3,

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

https://doi.org/10.24287/1726-1708-2021-20-1-184-191

6,

Platelets in COVID-19: “innocent by-standers” or active participants?

O. An, A. Martyanov, M. Stepanyan, A. Boldova, S. Rumyantsev, M. Panteleev, F. Ataullakhanov, A. Rumyantsev, A. Sveshnikova

Pediatric Hematology/Oncology and Immunopathology. None, 20, 184-191

29,

Platelets Can Associate With SARS-CoV-2 RNA and Are Hyperactivated in COVID-19

Y. Zaid, F. Puhm, I. Allaeys, A. Naya, M. Oudghiri, L. Khalki, Y. Limami, N. Zaid, K. Sadki, R. Ben El Haj, W. Mahir, L. Belayachi, B. Belefquih, A. Benouda, A. Cheikh, M. Langlois, Y. Cherrah, L. Flamand, F. Guessous, E. Boilard

Circulation Research. 2020, 127, 1404-1418

https://doi.org/10.1182/blood-2012-04-416594

30

The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways

J. Burkhart, M. Vaudel, S. Gambaryan, S. Radau, U. Walter, L. Martens, J. Geiger, A. Sickmann, R. Zahedi

Blood. 2012, 120, e73-e82

]

. However, ACE2 presence on platelets is controversial, as there are both works confirming

[

29,

Platelets Can Associate With SARS-CoV-2 RNA and Are Hyperactivated in COVID-19

Y. Zaid, F. Puhm, I. Allaeys, A. Naya, M. Oudghiri, L. Khalki, Y. Limami, N. Zaid, K. Sadki, R. Ben El Haj, W. Mahir, L. Belayachi, B. Belefquih, A. Benouda, A. Cheikh, M. Langlois, Y. Cherrah, L. Flamand, F. Guessous, E. Boilard

Circulation Research. 2020, 127, 1404-1418

https://doi.org/10.1186/s13045-020-00954-7

31

SARS-CoV-2 binds platelet ACE2 to enhance thrombosis in COVID-19

S. Zhang, Y. Liu, X. Wang, L. Yang, H. Li, Y. Wang, M. Liu, X. Zhao, Y. Xie, Y. Yang, S. Zhang, Z. Fan, J. Dong, Z. Yuan, Z. Ding, Y. Zhang, L. Hu

Journal of Hematology & Oncology. 2020, 13, None

]

and denying

[

3

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

]

it. Finally, resting platelets of COVID-19 patients were increased in size

[

28

The Impact of COVID-19 Disease on Platelets and Coagulation

G. Wool, J. Miller

Pathobiology. 2021, 88, 15-27

]

and had increased expression of of P-selectin

[

3,

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

32

Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia

F. Taus, G. Salvagno, S. Canè, C. Fava, F. Mazzaferri, E. Carrara, V. Petrova, R. Barouni, F. Dima, A. Dalbeni, S. Romano, G. Poli, M. Benati, S. De Nitto, G. Mansueto, M. Iezzi, E. Tacconelli, G. Lippi, V. Bronte, P. Minuz

Arteriosclerosis, Thrombosis, and Vascular Biology. 2020, 40, 2975-2989

]

. However, it is noteworthy that an increase in thrombopoietin – a major inducer of platelet thrombopoiesis – has been reported in COVID-19 patients

[

32

Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia

F. Taus, G. Salvagno, S. Canè, C. Fava, F. Mazzaferri, E. Carrara, V. Petrova, R. Barouni, F. Dima, A. Dalbeni, S. Romano, G. Poli, M. Benati, S. De Nitto, G. Mansueto, M. Iezzi, E. Tacconelli, G. Lippi, V. Bronte, P. Minuz

Arteriosclerosis, Thrombosis, and Vascular Biology. 2020, 40, 2975-2989

]

. Therefore, the observed increase in platelets’ size could be due to increased thrombopoientin-dependent production of the new (younger) cells

[

29

Platelets Can Associate With SARS-CoV-2 RNA and Are Hyperactivated in COVID-19

Y. Zaid, F. Puhm, I. Allaeys, A. Naya, M. Oudghiri, L. Khalki, Y. Limami, N. Zaid, K. Sadki, R. Ben El Haj, W. Mahir, L. Belayachi, B. Belefquih, A. Benouda, A. Cheikh, M. Langlois, Y. Cherrah, L. Flamand, F. Guessous, E. Boilard

Circulation Research. 2020, 127, 1404-1418

https://doi.org/10.1080/09537104.2018.1513473

]

.

To summarize the above platelets of the COVID-19 patients are defective due to reasons that are poorly understood. Here, based on the experimental observations of platelet necrosis and size, as well as computational modeling, we suggest that the observed changes in platelets may be explained by their increased activation in circulation in COVID-19 patients.

Materials and Methods

Patients

32 patients, diagnosed with coronavirus infection, who were treated at the Federal State Budgetary Healthcare Institution Hospital of the Russian Academy of Sciences (Troitsk), as well as five healthy donors, aged from 21 to 45 years, who have not been ill and have not taken any medications within the last month, were enrolled in the study. All patients' condition was described as "mild severity" by physicians and did not require mechanical ventillation. All procedures comply with the ethical standards of the National Research Ethics Committee and the 1964 Declaration of Helsinki and its subsequent amendments or comparable ethical standards. Informed voluntary consent was obtained from each of the participants included in the study. All patients were subsequently discharged from the hospital within 2 weeks due to improvement in their condition. Blood samples of five patients were analyzed twice on different days. The study was approved by the decision of the Independent Ethics Committee of the Dmitry Rogachev National Research Center No. 3/2020 dated May 19, 2020.

Materials

Annexin-Alexa647, lactadherin-FITC (Sony Biotechnology, San-Jose, USA), HEPES, bovine serum albumin (BSA), D(+)glucose (Sigma, USA); NaCl; Na₂HPO₄; KCl; NaHCO₃; MgCl₂; CaCl₂ (Agat-Med, Moscow, Russia).

Flow Cytometry

Blood was collected in 3 mL tubes containing sodium citrate (3,8%) and was kept at room temperature for 30 minutes. During that time, red blood cells sedimented into the lower layers of the blood sample. Blood was then collected from the top 10 percent of the tube’s volume, was diluted in Tyrode’s buffer (134 мМ NaCl; 0.34 мМ Na₂HPO₄; 2.9 мМ KCl; 12 мМ NaHCO₃; 20 мМ HEPES; 5 мМ glucose; 1 мМ MgCl₂; 2 мМ CaCl₂; BSA 2% by weight; pH 7.3) to platelet concentration of 1×10³ per 1 mL. After that, AnnexinV-Alexa647 (2% v/v) and Lactadherin-FITC (2% v/v) were added to each sample, and cells were incubated for 10 minutes, as previously described

[

24

Flow cytometry for pediatric platelets

A. Ignatova, E. Ponomarenko, D. Polokhov, E. Suntsova, P. Zharkov, D. Fedorova, E. Balashova, A. Rudneva, V. Ptushkin, E. Nikitin, A. Shcherbina, A. Maschan, G. Novichkova, M. Panteleev

Platelets. 2019, 30, 428-437

https://doi.org/10.1038/311419a0

]

. Samples were analyzed using Beckman Coulter Navios flow cytometer. The platelet and vesicle populations were identified by the forward and side light scattering (Fig. 1A). Annexin-V, Lactadherin, and double-positive events were identified as shown in Fig. 1B. An event was considered PS-positive (PS+) if it was detected in lact+, anV+ or lact+anV+ gates (see Fig. 1B).

Computational model

The computational model was constructed on the principles of cellular automata models

[

33

Cellular automata as models of complexity

S. Wolfram

Nature. 1984, 311, 419-424

https://doi.org/10.1172/JCI26891

]

when each cell is considered as an object in the program. In the model, two types of cells were present: platelets and megakaryocytes. Each platelet has two features: age and size. Each megakaryocyte has only one feature: platelet production = 3500 (platelets per megakaryocyte,

[

34

The biogenesis of platelets from megakaryocyte proplatelets

S. Patel

Journal of Clinical Investigation. 2005, 115, 3348-3354

https://doi.org/10.1002/ajh.23562

]

). The parameter age is the number of days (model time step) for which the object exists. At each time step, M new megakaryocytes are produced in the bone marrow. \( M \) is a random number from \( N(\mu=10 ; \sigma=1) \), where N(_; _) is the normal distribution. A megakaryocyte could produce platelets only once. The model describes events related to 1 µL of blood. The platelet production is also influenced by the concentration of thrombopoetin in the following manner: \begin{equation} P=(\text { platelet production })^{*} n^{*} T P O \text { , }\tag{1}\end{equation}

where P is the number of platelets produced by the megakaryocyte, n is the random value from \( N(\mu=1 ; \sigma=0.1) \), TPO is the parameter reflecting thrombopoietin concentration according to equation (2): \begin{equation} TPO=1-\frac{1}{1+\left(\frac{N_{T P O}}{P l t}\right)^{h}}\tag{2}\end{equation}

where \( N_{TPO} \) is the approximate number of platelets below which TPO production in the liver is initiated, and Plt is the number of platelets at the given time point, the parameters \( N_{TPO}=120000 \) and h = 6 were adjusted to describe experimental data on the relationship between platelet counts and [TPO] concentration in blood from Makar et al.

[

35

Thrombopoietin levels in patients with disorders of platelet production: Diagnostic potential and utility in predicting response to TPO Receptor agonists

R. Makar, O. Zhukov, M. Sahud, D. Kuter

American Journal of Hematology. 2013, 88, 1041-1044

https://doi.org/10.1016/S0049-3848(16)30370-X

]

. In human organism platelets can be cleared from the circulation in the liver and spleen, and the probability of platelet clearance increases with platelet ageing

[

36

Platelet clearance by the hepatic Ashwell-Morrell receptor: mechanisms and biological significance

K. Hoffmeister, H. Falet

Thrombosis Research. 2016, 141, S68-S72

https://doi.org/10.1080/09537104.2021.1922659

]

. On the model on each time step, each platelet has a removal probability: \begin{equation} p_{\text {clear }}=n_{\text {nat }}+n_{\text {thr }}\tag{3}\end{equation}

where n_nat reflects the natural platelet removal and is a random value from N(0.625‧age; 0.0125‧age), and n_thr reflects the platelets removal due to their participation in thrombus formation and is a random value from N(0.625‧K; 0.0125‧K), where K is the consumption index, which can be varied to simulate severity of thrombosis. Platelet size is known to be related to the platelet's age and RNA content

[

37,

Platelet reticulated forms, size indexes, and functional activity. Interactions in healthy volunteers

V. V. Bodrova, O. N. Shustova, S. G. Khaspekova, and A. V. Mazurov

Platelets. 2021, None, 1-6

https://doi.org/10.1111/jth.14895

38,

Ultrastructural, transcriptional, and functional differences between human reticulated and non‐reticulated platelets

L. Hille, M. Lenz, A. Vlachos, B. Grüning, L. Hein, F. Neumann, T. Nührenberg, D. Trenk

Journal of Thrombosis and Haemostasis. 2020, 18, 2034-2046

https://doi.org/10.1016/j.eclinm.2020.100624

39

Investigation of the efficacy and safety of eltrombopag to correct thrombocytopenia in moderate to severe dengue patients - a phase II randomized controlled clinical trial

S. Chakraborty, S. Alam, M. Sayem, M. Sanyal, T. Das, P. Saha, M. Sayem, B. Byapari, C. Tabassum, A. Kabir, M. Amin, A. Nabi

EClinicalMedicine. 2020, 29-30, 100624

https://doi.org/10.3389/fmed.2017.00146

]

. To simulate the distribution of platelet sizes, we have introduced equation (3) into the model, where the size parameter is recalculated from the age parameter: \begin{equation} \text { size }=50\left(1-\frac{\left(1-\frac{M i n}{M a x}\right)}{1+\left(\frac{M e a n}{a g e}\right)^{h}}\right)\tag{4}\end{equation}

where s₀ reflects the initial platelet size and is a value from N(12; 1) in [fL], parameters Max = 11 [fL] and Min = 6 [fL] reflect the platelet size distribution found in healthy donors

[

40

Mean Platelet Volume and Immature Platelet Fraction in Autoimmune Disorders

D. Schmoeller, M. Picarelli, T. Paz Munhoz, C. Poli de Figueiredo, H. Staub

Frontiers in Medicine. 2017, 4, None

https://doi.org/10.1016/j.eclinm.2020.100624

]

, Mean = 3 [days] reflects median platelet age

[

39

Investigation of the efficacy and safety of eltrombopag to correct thrombocytopenia in moderate to severe dengue patients - a phase II randomized controlled clinical trial

S. Chakraborty, S. Alam, M. Sayem, M. Sanyal, T. Das, P. Saha, M. Sayem, B. Byapari, C. Tabassum, A. Kabir, M. Amin, A. Nabi

EClinicalMedicine. 2020, 29-30, 100624

https://doi.org/10.1080/09537104.2021.1922659

]

. The formula (3) and coefficient h = 3 were adjusted to describe data on the relationship between the immature platelet fraction, platelet size and platelet counts

[

37,

Platelet reticulated forms, size indexes, and functional activity. Interactions in healthy volunteers

V. V. Bodrova, O. N. Shustova, S. G. Khaspekova, and A. V. Mazurov

Platelets. 2021, None, 1-6

https://doi.org/10.1111/jth.14895

38,

Ultrastructural, transcriptional, and functional differences between human reticulated and non‐reticulated platelets

L. Hille, M. Lenz, A. Vlachos, B. Grüning, L. Hein, F. Neumann, T. Nührenberg, D. Trenk

Journal of Thrombosis and Haemostasis. 2020, 18, 2034-2046

https://doi.org/10.1016/j.eclinm.2020.100624

39

Investigation of the efficacy and safety of eltrombopag to correct thrombocytopenia in moderate to severe dengue patients - a phase II randomized controlled clinical trial

S. Chakraborty, S. Alam, M. Sayem, M. Sanyal, T. Das, P. Saha, M. Sayem, B. Byapari, C. Tabassum, A. Kabir, M. Amin, A. Nabi

EClinicalMedicine. 2020, 29-30, 100624

]

. The model was constructed in Python 3.7.

Clinical data

The results of complete blood count, biochemical analysis, blood clotting test, CT scans, data of daily objective examinations, age, and diagnoses of patients were kindly provided by the hospital with the patients' consent.

Data processing

Raw flow cytometry data were processed using FlowJo^TM Software. Statistical analysis was performed utilizing GraphPad Prizm.

Results and Discussion

Platelets size and phosphatidylserine exposure are increased in patients with COVID-19

The patients with mild Covid-19 disease examined in this study did not suffer from thrombocytopenia, as the platelets numbers in all patients were between 139x10³ and 519x10³ platelets per μl. To examine the size and activation status of the platelets we performed flow cytometry analyisis of cells in patients’ blood stained with lactadherin and Annexin V (Fig. 1). Analysis of the platelet’s forward scattering (FS-A) revealed a significant increase in patients FS-A compared to healthy donors (mean value ± SD: 213±25‧10³ a.u. for patients, 185±20‧10³ a.u. for healthy donors, p=0.027, Fig. 1C). These findings are in line with previously published data

[

3,

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

32

Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia

F. Taus, G. Salvagno, S. Canè, C. Fava, F. Mazzaferri, E. Carrara, V. Petrova, R. Barouni, F. Dima, A. Dalbeni, S. Romano, G. Poli, M. Benati, S. De Nitto, G. Mansueto, M. Iezzi, E. Tacconelli, G. Lippi, V. Bronte, P. Minuz

Arteriosclerosis, Thrombosis, and Vascular Biology. 2020, 40, 2975-2989

https://doi.org/10.1080/09537104.2017.1414175

]

. Interestingly, Kovacz et al. have reported that increased platelet size is characteristic for patients suffering from venous thromboembolism

[

41

Characteristics of platelet count and size and diagnostic accuracy of mean platelet volume in patients with venous thromboembolism. A systematic review and meta-analysis

S. Kovács, Z. Csiki, K. Zsóri, Z. Bereczky, A. Shemirani

Platelets. 2019, 30, 139-147

https://doi.org/10.1186/s12575-021-00142-y

]

, which allows us to suspect that some thrombotic events may occur in our cohort of COVID-19 patients

[

42

Anatomical and Pathological Observation and Analysis of SARS and COVID-19: Microthrombosis Is the Main Cause of Death

W. Chen, J. Pan

Biological Procedures Online. 2021, 23, None

https://doi.org/10.1007/s11239-020-02105-8

]

. The observed increase in platelet’s size may not be exclusive for patients with SARS-COV-2 virus. A couple of recent studies have demonstrated that platelet count is significantly reduced in influenza-induced pneumonia, and the mean platelet volume is significantly increased

[

43,

Difference of coagulation features between severe pneumonia induced by SARS-CoV2 and non-SARS-CoV2

S. Yin, M. Huang, D. Li, N. Tang

Journal of Thrombosis and Thrombolysis. 2021, 51, 1107-1110

https://doi.org/10.1002/jmv.26645

44

The value of the platelet count and platelet indices in differentiation of COVID‐19 and influenza pneumonia

N. Ozcelik, S. Ozyurt, B. Yilmaz Kara, A. Gumus, U. Sahin

Journal of Medical Virology. 2021, 93, 2221-2226

https://doi.org/10.1007/BF03040424

]

. Severe thrombocytopenia is extremely rare for the Epstein-Barr virus, but this infection often presents a mild reduction in platelet counts as well as moderate increase in platelet size for uncomplicated cases

[

45

Severe thrombocytopenia as a complication of acute Epstein-Barr virus infection

R. Likic, D. Kuzmanic

Wiener Klinische Wochenschrift. 2004, 116, 47-50

https://doi.org/10.1111/j.1537-2995.2008.01933.x

]

.

The percentage of PS+ platelets in patients was more than two times higher than that in healthy donors (mean value ± SD: 0.74±0.37% for patients, 0.29±0.07% for healthy donors; p<0.0001, Fig. 1D-F). The average percentage of PS+ events was higher for lactadherin than for annexin V (p=0.0372, Fig. 1E, F), which is consistent with the literature data on the higher effectivity of lactadherin for PS+ cells detection

[

46

Measurement of phosphatidylserine exposure during storage of platelet concentrates using the novel probe lactadherin: a comparison study with annexin V

A. Albanyan, M. Murphy, J. Rasmussen, C. Heegaard, P. Harrison

Transfusion. 2009, 49, 99-107

]

. The values obtained for both PS+ platelet markers correlated well with each other (r=0.64, p<0.0001), and the total percentage of PS+ events (r=0.74, p<0.0001 for annexin V, r=0.96, p<0.0001 for lactadherin, see Table 1). Therefore, we observe platelet hyperactivation in COVID-19, which is in line with previously published data

[

32

Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia

F. Taus, G. Salvagno, S. Canè, C. Fava, F. Mazzaferri, E. Carrara, V. Petrova, R. Barouni, F. Dima, A. Dalbeni, S. Romano, G. Poli, M. Benati, S. De Nitto, G. Mansueto, M. Iezzi, E. Tacconelli, G. Lippi, V. Bronte, P. Minuz

Arteriosclerosis, Thrombosis, and Vascular Biology. 2020, 40, 2975-2989

https://doi.org/10.1055/a-1401-2706

]

.

The PS+ platelets are known to form in response to strong stimulation, for example, dual collagen and thrombin

[

47,

Procoagulant Platelets: Mechanisms of Generation and Action

N. Podoplelova, D. Nechipurenko, A. Ignatova, A. Sveshnikova, M. Panteleev

Hämostaseologie. 2021, 41, 146-153

https://doi.org/10.1182/blood.V90.7.2615

48

Collagen But Not Fibrinogen Surfaces Induce Bleb Formation, Exposure of Phosphatidylserine, and Procoagulant Activity of Adherent Platelets: Evidence for Regulation by Protein Tyrosine Kinase-Dependent Ca2+ Responses

J. Heemskerk, W. Vuist, M. Feijge, C. Reutelingsperger, T. Lindhout

Blood. 1997, 90, 2615-2625

https://doi.org/10.1161/STROKEAHA.114.006492

]

. The observed increase in percentage of PS+ platelets (0.5-1.5%) in Covid-19 patients could reflect an ongoing platelet activation in circulation

[

49

Coated-Platelets Improve Prediction of Stroke and Transient Ischemic Attack in Asymptomatic Internal Carotid Artery Stenosis

A. Kirkpatrick, A. Tafur, A. Vincent, G. Dale, C. Prodan

Stroke. 2014, 45, 2995-3001

]

. An inflammatory process in the COVID-19 patient's body is inextricably linked with the activation of cells in adjacent tissues

[

12

The trinity of COVID-19: immunity, inflammation and intervention

M. Tay, C. Poh, L. Rénia, P. MacAry, L. Ng

Nature Reviews Immunology. 2020, 20, 363-374

]

and may lead to platelets’ activation due to their contact with the products of thrombus-forming processes (such as the contents of platelet granules, thrombin, ADP, etc.)

[

3

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

https://doi.org/10.1016/j.jinf.2020.03.037

]

. Thus, in case of moderate ongoing lung microthrombosis, weak increase of PS+ platelet number could be observed. This is in line with the clinical picture of the studied patients. None of them suffered from severe thrombotic complications during the course of the study.

Comparison of the percentage of procoagulant platelets and their size in patients with Covid-19 and healthy donors. A – Gating of the area for forward (FS-A; ) and sight (SS-A) light scattering: events in the red oval correspond to platelets, events in the red square correspond to platelets and vesicles. B – Estimation of the percentage of events positive for annexin (anV +), lactadherin (lact +), and phosphatidylserine-positive events (falling into both or one of the areas). C – Comparison of the obtained average platelet sizes from healthy donors (green) and patients with Covid-19 (red); p = 0.0269. D – Comparison of the obtained values of the percentage of phosphatidylserine-positive events in healthy donors (green) and patients with Covid-19 (red), p = 7.523x10-5 - platelets and p = 0.0003 - platelets and vesicles. E – Comparison of the obtained values of the percentage of annexin-positive events in healthy donors (green) and patients with Covid-19 (red), p = 6.466x10-5 - platelets and p = 0.0001 - platelets and vesicles. F – Comparison of the obtained values of the percentage of lactadherin-positive events in healthy donors (green) and patients with Covid-19 (red); p = 4.467x10-5 - platelets, p = 0.0001 - platelets and vesicles. G – General information about the Covid-19 patients included in the study. The Mann-Whitney test was used for significance; three asterisks denote p <0.001, four - p <0.0001.

Figure 1. Comparison of the percentage of procoagulant platelets and their size in patients with Covid-19 and healthy donors. A – Gating of the area for forward (FS-A; ) and sight (SS-A) light scattering: events in the red oval correspond to platelets, events in the red square correspond to platelets and vesicles. B – Estimation of the percentage of events positive for annexin (anV +), lactadherin (lact +), and phosphatidylserine-positive events (falling into both or one of the areas). C – Comparison of the obtained average platelet sizes from healthy donors (green) and patients with Covid-19 (red); p = 0.0269. D – Comparison of the obtained values of the percentage of phosphatidylserine-positive events in healthy donors (green) and patients with Covid-19 (red), p = 7.523x10-5 - platelets and p = 0.0003 - platelets and vesicles. E – Comparison of the obtained values of the percentage of annexin-positive events in healthy donors (green) and patients with Covid-19 (red), p = 6.466x10-5 - platelets and p = 0.0001 - platelets and vesicles. F – Comparison of the obtained values of the percentage of lactadherin-positive events in healthy donors (green) and patients with Covid-19 (red); p = 4.467x10-5 - platelets, p = 0.0001 - platelets and vesicles. G – General information about the Covid-19 patients included in the study. The Mann-Whitney test was used for significance; three asterisks denote p <0.001, four - p <0.0001.

Correlation of platelet parameters with clinical data

Platelets are not only the critical players in coagulation but are also an essential source of inflammatory mediators. In addition to anticoagulant therapy, applied to COVID-19 patients, anti-inflammatory therapy is also carried out using various approaches: these include immunosuppressants (for example, actemra), antimalarial drugs (hydroxychloroquine) and corticosteroids, IL-6 antagonists (tocilizumab)

[

7

The pathogenesis and treatment of the `Cytokine Storm' in COVID-19

Q. Ye, B. Wang, J. Mao

Journal of Infection. 2020, 80, 607-613

https://doi.org/10.1189/jlb.0607373

]

. Therefore we analyzed the correlation between platelet parameters, the applied therapy, and the concurrent patients' diseases.

The proportion of PS+ platelets correlated (r=0.69, p<0.01) with the lung damage (according to CT scan performed no later than 2 days from the date of platelet examination, Table 1). These results are in good agreement with the hypothesis about the influence of the inflammatory process occurring in the lungs on the coagulation system since an increase in the area of affected alveoli leads to activation of adjacent endothelial tissues

[

50,

Inflammation, endothelium, and coagulation in sepsis

M. Schouten, W. Wiersinga, M. Levi, T. van der Poll

Journal of Leukocyte Biology. 2008, 83, 536-545

https://doi.org/10.1016/j.antiviral.2013.05.003

51

The lung microvascular endothelium as a therapeutic target in severe influenza

S. Armstrong, S. Mubareka, W. Lee

Antiviral Research. 2013, 99, 113-118

]

. In addition, there is a negative correlation (r=-0.49, p<0.05) between the total number of platelets and the number of procoagulant ones. We presume that high consumption of platelets can cause these changes (Table 1). The percentage of PS+ platelets and platelet count correlated with blood fibrinogen levels (p<0.05 and p<0.001 respectively, Table 1). This result may be explained by thrombus formation leading to increased consumption of clotting factors and platelets. Owing to this, the liver compensates for protein intake

[

28,

The Impact of COVID-19 Disease on Platelets and Coagulation

G. Wool, J. Miller

Pathobiology. 2021, 88, 15-27

https://doi.org/10.1111/jth.14848

52

Thrombocytopenia and its association with mortality in patients with COVID‐19

X. Yang, Q. Yang, Y. Wang, Y. Wu, J. Xu, Y. Yu, Y. Shang

Journal of Thrombosis and Haemostasis. 2020, 18, 1469-1472

https://doi.org/10.1111/jth.14810

]

. No correlation was found between procoagulant platelets and C-reactive protein or D-dimer (see Table 1).

Correlation of platelet parameters with the applied therapy

In the hospital, all patients with suspected COVID-19 were prescribed thromboprophylaxis

[

53

ISTH interim guidance on recognition and management of coagulopathy in COVID‐19

J. Thachil, N. Tang, S. Gando, A. Falanga, M. Cattaneo, M. Levi, C. Clark, T. Iba

Journal of Thrombosis and Haemostasis. 2020, 18, 1023-1026

https://doi.org/10.1056/NEJM200104263441704

]

. The use of heparins can lead to transient heparin-induced thrombocytopenia (HIT)

[

54,

Temporal Aspects of Heparin-Induced Thrombocytopenia

T. Warkentin, J. Kelton

New England Journal of Medicine. 2001, 344, 1286-1292

https://doi.org/10.1056/NEJM199505183322003

55

Heparin-Induced Thrombocytopenia in Patients Treated with Low-Molecular-Weight Heparin or Unfractionated Heparin

T. Warkentin, M. Levine, J. Hirsh, P. Horsewood, R. Roberts, M. Gent, J. Kelton

New England Journal of Medicine. 1995, 332, 1330-1336

https://doi.org/10.1007/s11239-021-02436-0

]

. However, it was not observed in our study. The amount of PS+ platelets for patients receiving heparins also was not altered compared to other COVID-19 patients (Fig. 2A). Sometimes, antiplatelet drugs are prescribed for COVID-19 patients

[

56

Use of antiplatelet drugs and the risk of mortality in patients with COVID-19: a meta‐analysis

C. S. Kow and S. S. Hasan

Journal of Thrombosis and Thrombolysis. 2021, None, None

https://doi.org/10.1093/infdis/jiv392

]

. For two out of three patients receiving antiplatelet drugs in our study because of previous prescriptions, the amount of PS+ platelets was significantly lower than for other COVID-19 patients (Fig. 2A).

According to previously published recommendations, other therapeutic agents are prescribed for the patients in the hospital, including the antiretroviral drug lopinavir

[

57,

Treatment With Lopinavir/Ritonavir or Interferon-β1b Improves Outcome of MERS-CoV Infection in a Nonhuman Primate Model of Common Marmoset

J. Chan, Y. Yao, M. Yeung, W. Deng, L. Bao, L. Jia, F. Li, C. Xiao, H. Gao, P. Yu, J. Cai, H. Chu, J. Zhou, H. Chen, C. Qin, K. Yuen

Journal of Infectious Diseases. 2015, 212, 1904-1913

https://doi.org/10.1016/S0140-6736(20)31042-4

58

Triple combination of interferon beta-1b, lopinavir–ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial

I. Hung, K. Lung, E. Tso, R. Liu, T. Chung, M. Chu, Y. Ng, J. Lo, J. Chan, A. Tam, H. Shum, V. Chan, A. Wu, K. Sin, W. Leung, W. Law, D. Lung, S. Sin, P. Yeung, C. Yip, R. Zhang, A. Fung, E. Yan, K. Leung, J. Ip, A. Chu, W. Chan, A. Ng, R. Lee, K. Fung, A. Yeung, T. Wu, J. Chan, W. Yan, W. Chan, J. Chan, A. Lie, O. Tsang, V. Cheng, T. Que, C. Lau, K. Chan, K. To, K. Yuen

The Lancet. 2020, 395, 1695-1704

https://doi.org/10.1016/j.thromres.2020.06.033

]

. There were no differences in the number of procoagulant platelets between the groups of patients who took and did not take the lopinavir-ritonavir combination (Fig. 2A).

When comparing groups of patients for the presence of chronic diseases, statistically significant differences (p=0.027) were found only for the group of patients with chronic lung diseases. The percentage of PS+ events in the presence of the disease was lower than in the absence (mean value ± SD: 0.81±0.37% for absence, 0.41±0.17% for presence, Fig. 2B), which may be due to the presence of a compensatory mechanism in these patients' coagulation system. The patient cohort included in this study is highly heterogenic in terms of age and health status. However, the lack of significant differences between different patients' groups (Fig. 2B, Fig. S1) and the absence of the observed altered platelet phenotype in chronic diseases

[

59,

Small procoagulant platelets in diabetes type 2

M. Edvardsson, M. Oweling, P. Järemo

Thrombosis Research. 2020, 195, 1-7

https://doi.org/10.1097/MOG.0000000000000635

60,

Coagulation testing and management in liver disease patients

M. Stotts, J. Davis, N. Shah

Current Opinion in Gastroenterology. 2020, 36, 169-176

https://doi.org/10.1080/09537104.2018.1499890

61,

Platelet and coagulation disorders in newly diagnosed patients with pulmonary arterial hypertension

E. Vrigkou, I. Tsangaris, S. Bonovas, P. Kopterides, E. Kyriakou, D. Konstantonis, A. Pappas, A. Anthi, A. Gialeraki, S. Orfanos, A. Armaganidis, A. Tsantes

Platelets. 2019, 30, 646-651

https://doi.org/10.3402/jev.v3.25625

62

Levels of procoagulant microvesicles are elevated after traumatic injury and platelet microvesicles are negatively correlated with mortality

N. Curry, A. Raja, J. Beavis, S. Stanworth, P. Harrison

Journal of Extracellular Vesicles. 2014, 3, 25625

]

allows us to claim that this phenotype is the result of COVID-19.

Association of phosphatidylserine-positive events with clinical parameters of patients, the presence of chronic diseases, and ongoing therapy. A – Comparison of the results of patients on the therapy they take (3 different groups by the amount of low molecular weight heparins, a group was taking anticoagulants and antiplatelet agents, groups taking anti-HIV drugs and not taking them). When using the Mann-Whitney criterion statistics no significant differences were found between the groups. B – Comparison of the percentage of phosphatidylserine-positive events for groups of patients suffering from various chronic diseases (type II diabetes mellitus (Diabetes), diseases of the gastrointestinal tract (Digestive), lungs (Lungs; p = 0.0274), cardiovascular (Cardiovascular), chronic arterial hypertension (CAH)). The green rectangle shows the normal range obtained for healthy donors (mean ± 2 standard deviations). The Mann-Whitney test was used for significance; one asterisk indicates p <0.05.

Figure 2. Association of phosphatidylserine-positive events with clinical parameters of patients, the presence of chronic diseases, and ongoing therapy. A – Comparison of the results of patients on the therapy they take (3 different groups by the amount of low molecular weight heparins, a group was taking anticoagulants and antiplatelet agents, groups taking anti-HIV drugs and not taking them). When using the Mann-Whitney criterion statistics no significant differences were found between the groups. B – Comparison of the percentage of phosphatidylserine-positive events for groups of patients suffering from various chronic diseases (type II diabetes mellitus (Diabetes), diseases of the gastrointestinal tract (Digestive), lungs (Lungs; p = 0.0274), cardiovascular (Cardiovascular), chronic arterial hypertension (CAH)). The green rectangle shows the normal range obtained for healthy donors (mean ± 2 standard deviations). The Mann-Whitney test was used for significance; one asterisk indicates p <0.05.

The computational model predicts that the enhanced platelet consumption underlies thrombocytopathy in COVID-19 patients

Platelet phenotype of COVID-19 patients appeared to be distinctive: while no significant thrombocytopenia was detected in our study as well as in the literature

[

3,

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

28,

The Impact of COVID-19 Disease on Platelets and Coagulation

G. Wool, J. Miller

Pathobiology. 2021, 88, 15-27

29,

Platelets Can Associate With SARS-CoV-2 RNA and Are Hyperactivated in COVID-19

Y. Zaid, F. Puhm, I. Allaeys, A. Naya, M. Oudghiri, L. Khalki, Y. Limami, N. Zaid, K. Sadki, R. Ben El Haj, W. Mahir, L. Belayachi, B. Belefquih, A. Benouda, A. Cheikh, M. Langlois, Y. Cherrah, L. Flamand, F. Guessous, E. Boilard

Circulation Research. 2020, 127, 1404-1418

32

Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia

F. Taus, G. Salvagno, S. Canè, C. Fava, F. Mazzaferri, E. Carrara, V. Petrova, R. Barouni, F. Dima, A. Dalbeni, S. Romano, G. Poli, M. Benati, S. De Nitto, G. Mansueto, M. Iezzi, E. Tacconelli, G. Lippi, V. Bronte, P. Minuz

Arteriosclerosis, Thrombosis, and Vascular Biology. 2020, 40, 2975-2989

]

, increased platelet size and increased PS+ platelet subpopulation were also noted. In order to understand, whether such phenotype could be caused by the consumption of platelet due to thrombosis, we have constructed the model of the platelet production, ageing, and clearance (Fig. 3A).

In the model, in the absence of additional platelet consumption, the average platelet counts are between 155x10³ platelets/μl and 177x10³ platelets/μl (Fig. 3B). The number of platelets and platelet size were stochastically fluctuating (Fig. S2 A and B correspondingly). Introduction of additional consumption, which did not significantly alter platelet counts (∆Plt < 25 000/μl), results in a significant increase in the platelet size (Fig. 3B,C; S2A,B). Increased consumption also resulted in the platelets becoming younger (Fig. S2C,D). Enhancement of the platelet consumption above 2 resulted in mild thrombocytopenia and platelets becoming younger (Fig. 3B). Severe thrombocytopenia appeared at platelet consumption above K = 5, which corresponded to more than half of the produced platelets being consumed (Fig. 3B, S2). Additional sensitivity analysis revealed that model outcome was most sensitive to the TPO synthesis, platelet consumption, and platelet clearance parameters. These reactions are highlighted in red (Fig. 3A).

It is noteworthy that in patients the PS+ platelet fraction weakly correlated with the mean FS-A values (Table 1). This finding supports the hypothesis that in COVID-19, platelets are consumed due to thrombosis which leads to the increase in PS+ platelet fraction, younger overall age of platelets and therefore larger size. The weakness of the correlation could be caused by the enhanced PS+ platelet consumption by the liver and spleen [36]. However, it should also be kept in mind that the cells were resting during the experiment, and no comparison of the degree of activation for patients and healthy donors was carried out.

Computational model of platelet production in the presence of COVID-19 induced thrombosis. A – Detailed scheme of the model (most sensitive reactions are highlighted in red). B – Dependence of the average platelet count (green curve and dots) and platelet size (red curve and dots) from the platelet consumption index in the model. Platelet number and size in the absence of consumption lie in the areas, highlighted by green and red rectangles correspondingly. C – Platelet size distribution in the absence (green bars) and the presence (red bars) of consumption (with consumption index set to 2). Whiskers on all plots represent SD.

Figure 3. Computational model of platelet production in the presence of COVID-19 induced thrombosis. A – Detailed scheme of the model (most sensitive reactions are highlighted in red). B – Dependence of the average platelet count (green curve and dots) and platelet size (red curve and dots) from the platelet consumption index in the model. Platelet number and size in the absence of consumption lie in the areas, highlighted by green and red rectangles correspondingly. C – Platelet size distribution in the absence (green bars) and the presence (red bars) of consumption (with consumption index set to 2). Whiskers on all plots represent SD.

Conclusions

Here we observed a significant increase in the fraction of PS+ platelets in COVID-19 patients (Fig. 1). This phenomenon does not correlate either with therapeutic interventions carried out in the hospital or with chronic diseases in the patients (Fig. 2). Therefore, we assume that the observed increase in platelet size and phosphatidylserine exposure is mainly caused by COVID-19 and associated pneumonia. Previously it has been reported that such changes could be caused by active thrombosis [26], [41]. The proposed computational model demonstrates that moderate consumption, which does not result in pronounced thrombocytopenia, could result in a 1.3-fold increase in mean platelet volume observed for the COVID-19 patients (Fig. 1).

Based on our experimental results and theoretical findings, we propose the following scheme of the COVID-19 impact on human platelets:

· SARS-CoV-2 induces lung damage. This results in the blood vascular endothelium activation and tissue factor (TF) exposure to the blood flow

[

12,

The trinity of COVID-19: immunity, inflammation and intervention

M. Tay, C. Poh, L. Rénia, P. MacAry, L. Ng

Nature Reviews Immunology. 2020, 20, 363-374

https://doi.org/10.1038/s41577-020-00470-2

13

Leukocyte trafficking to the lungs and beyond: lessons from influenza for COVID-19

R. Alon, M. Sportiello, S. Kozlovski, A. Kumar, E. Reilly, A. Zarbock, N. Garbi, D. Topham

Nature Reviews Immunology. 2021, 21, 49-64

]

;

· TF induces thrombus formation and platelet consumption;

· Fraction of PS+ platelets is increased (Fig. 1) as a result of strong platelet activation, and platelets production is enhanced by the megakaryocytes (Fig. 3)

[

3,

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

29,

Platelets Can Associate With SARS-CoV-2 RNA and Are Hyperactivated in COVID-19

Y. Zaid, F. Puhm, I. Allaeys, A. Naya, M. Oudghiri, L. Khalki, Y. Limami, N. Zaid, K. Sadki, R. Ben El Haj, W. Mahir, L. Belayachi, B. Belefquih, A. Benouda, A. Cheikh, M. Langlois, Y. Cherrah, L. Flamand, F. Guessous, E. Boilard

Circulation Research. 2020, 127, 1404-1418

32

Platelets Promote Thromboinflammation in SARS-CoV-2 Pneumonia

F. Taus, G. Salvagno, S. Canè, C. Fava, F. Mazzaferri, E. Carrara, V. Petrova, R. Barouni, F. Dima, A. Dalbeni, S. Romano, G. Poli, M. Benati, S. De Nitto, G. Mansueto, M. Iezzi, E. Tacconelli, G. Lippi, V. Bronte, P. Minuz

Arteriosclerosis, Thrombosis, and Vascular Biology. 2020, 40, 2975-2989

]

due to the increased TPO synthesis by the liver

[

3

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

]

;

· A mild reduction of the platelet count occurs

[

3,

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

https://doi.org/10.1111/jth.14848

52

Thrombocytopenia and its association with mortality in patients with COVID‐19

X. Yang, Q. Yang, Y. Wang, Y. Wu, J. Xu, Y. Yu, Y. Shang

Journal of Thrombosis and Haemostasis. 2020, 18, 1469-1472

]

, while platelet size increases significantly

[

3,

Platelet gene expression and function in patients with COVID-19

B. Manne, F. Denorme, E. Middleton, I. Portier, J. Rowley, C. Stubben, A. Petrey, N. Tolley, L. Guo, M. Cody, A. Weyrich, C. Yost, M. Rondina, R. Campbell

Blood. 2020, 136, 1317-1329

28

The Impact of COVID-19 Disease on Platelets and Coagulation

G. Wool, J. Miller

Pathobiology. 2021, 88, 15-27