Erythrocyte a Target for Covid-19 Infected Patients

A pandemic infection among humans appeared through contamination with the SARS-CoV2 Virus (Covid-19) around the world and the majority of dead`s occurs by acute respiratory syndrome followed by irreversible failure in all the organs [1]. Beyond the nose and mouth the eyes are targets for the Covid-19 entrance on the human been [2]. The variety of therapeutic drugs for ocular diseases treatment are used in eye drops formula. Besides its local application and its dimension those enter into the eyes microcirculation and rapidly appear in systemic blood circulation or macrocirculation [3]. The dimension of Covid-19 is lower than the eye drops formula composition known [4].

Recently erythrocyte membrane Band3 protein was identified as the entrance channel for SARS-CoV2 Virus (Covid-19) to inside the RBC [5]. Consequently, arise the question to know if the microscope observation of the human blood smears be the accurate, efficient, fast and cheaper test to identify with confidence the Covid-19 infected people and screening them from those asymptomatic but with the virus from those that have not ? What is the type of interactions occurring inside the RBC between hemoglobin (Hb) molecules or any other the internal biomolecules with each one components of the Corvid-19? These continue to be open questions yet without answer. However, they are also outside of the present opinion. The presence of the Covid-19 inside the erythrocytes increase its internal viscosity which decrease the RBC deformability allow vascular erythrocyte deposition, impair the efflux of oxygen to the endothelial cell wall originating tissue hypoxia installation and decrease of the blood perfusion compromising the blood flow in post capillaries venules [6].

The deoxygenated erythrocytes permanence in microvascular network will develop reactive oxygen and nitrogen species (ROS, NOS) formation with initiation of the acute phase of inflammatory response [7]. If there are a general inflammatory situation in all tissues and organs dead occurs [8]. We have verified in the last century that patients with arterial hypertension, well known as a typical inflammatory disease of blood macrocirculation, also showed microcirculation disturbances in the eyes [9]. The spread of the inflammation process in the systemic circulatory network gathers nowadays consent [10]. The first reported cases of dead with the pandemic disease mention that patients with SARS-CoV2 Virus present lungs destruction [11]. Otherwise in some symptomatic patients disseminated coagulation was evidenced while others were affected in kidney or in liver and others were infected in nervous system starting suffering with neurological diseases [12]. Those data need to be explained in due time, but all pathophysiological situations have in common the presence of the Covid-19 transported by blood flow inside RBC.

So, taking into account all described above and knowing that the eyes are able to receive the Covid-19 which pass to microcirculation where enter into erythrocyte through membrane band 3 protein comes up the question to know if the microscope observation of the human blood smears may be the accurate, efficient, fast and cheaper test to identify with confidence the presence of Covid-19 in infected people separating them from those asymptomatic where the virus is observed and certificate the absence in those not contaminated? The microscopic observation of blood smears must take into account the viral charge of the patients. So, some could show inside discocyte RBC clearly Covid-19 virus visualization. Otherwise different RBC shapes could be adopted like macrocytes, echinocytes, stomatocytes, spherocytes and acantocytes. Those abnormal RBC shapes have lower ability to reversible deform in order to travel in the capillaries. So, intravascular hemolysis if occurs exposed to the blood flow the RBC internal molecules influent to the disseminated coagulation mechanism observed in some Covid -19 patients [12]. Exovesiculation is a natural occurring process in RBC eryptosis but also can be induced [13,14]. It could be expected to observe in some blood smears RBC with decreased dimensions resulting from exovesicles liberation induced by the presence either of the virus or its own components meanwhile obtained by the action of internal degradative enzymes. Also, it can be observed the presence of reticulocytes as a result of a fast erythropoiesis to null the massive RBC destruction by the advance state of infection of patients without feeling symptoms [15]. If no change erythrocyte shape or neither Covid-19 virus molecules are observed inside RBC in blood smears, a fluorescence label for RNA must be add to the blood samples for fluorescence microscopic observation [16].

Mohandas [17] write that “erythrocyte is more red that dead. At lungs oxygen (O2) enters RBC concomitantly with nitric oxide (NO) and both are delivery from the capillaries at surrounding tissues with lower partial pressure of oxygen [18]. In inflammatory situation vascular endothelial cell are dysfunctional generating reactive oxygen species (ROS) because inducible NO synthase enzyme form substitutes the native constitutive and increasing the NO amount produced [19]. To avoid the oxidative environment, it is necessary to increase the NO scavenger ability of the RBC by reducing or maintaining normal the NO efflux from erythrocyte [20]. So is mandatory to quantify in the first blood samples taken from the Covid -19 patients the NO efflux from RBC using the amperometric method described for the first time, by us [21]. Making those measurements during the patients stay at intensive care units (ICU) help their prognostic evaluation, which could be reinforced by quantification of the blood hemodynamic parameters obtained with a sublingual microcirculatory device [22].

We have observed in patents with amyotrophic lateral sclerosis (ALS) an inverse association between lower levels of NO efflux from erythrocytes and the amelioration of their respiratory function [23]. High plasma fibrinogen (Fib) and high acetylcholine (ACh) concentrations are among other biomarkers of systemic acute phase of inflammation [24,25]. When in ex-vivo studies were performed ACh increase NO efflux from erythrocyte while higher [Fib] did not change it [26,27]. The values of NO efflux maintain in normal range when both Fib and ACh are present at high concentrations [28]. Others in ex-vivo studies showing that ditiotreitol (DTT) and velnacrine maleate both used as therapeutic drugs are able per se to decrease the NO liberation. efflux from RBC reinforcing its scavenger ability [26,29]. In case of high levels of NO efflux from RBC, DTT is better than velnacrine when Covid-19 patients are submitted to the ventilator’s machines at ICU. Velnacrine maleate is a strong acetylcholinesterase inhibitor which affect the human cognitive power [30].

Once inside the erythrocyte NO interacts with Hb and becomes able to regulate the uptake and delivery of O2 in function of the tissue oxygen partial pressure [31]. The studies done to modulate the RBC availability in NO, for example when the scavenger of NO is need in inflammatory situations, activators of adenylated cyclase such as forsfokolin (used in obesity and glaucoma treatment) and inhibitors of phosphodiesterase 3 (PDE3) such as phosphoinositol 3 kinase(PI3K) inhibitor wortmannin (WORT) are used [32,33].

NO enter and sort through RBC membrane band 3 protein known as anionic channel bicarbonate and chloride exchange in dependence for example of the of Heinz body formation which close it binding to the cytoplasmic domain of band 3 protein [34]. There are other lipophilic antioxidative drugs like malatonine and others to block the erythrocyte membrane band 3 protein contributing for new views for treatment of the patients infected with SARS-CoV2 Virus [35,36].

In this opinion erythrocyte is present as target for fast test the presence or absence of SARS-CoV2 Virus through observation of the blood smears. Also was suggest the follow-up of the infection progress at IUC looking to the microcirculation hemodynamic parameters using sublingual device in concomitance with the values of nitric oxide efflux from RBC. Some therapeutic drugs acting in the NO signal transduction mechanism erythrocytes by activation of the adenylyl cyclase or inhibition of the phosphodiesterase 3 were suggested. Also compounds that close the dimeric or tetrameric structural forms of membrane protein band 3 have also indicated. Some news scientific contributions are left to which want to forward.

1. Liu PP, Blet A, Smyth D, Li H (2020) The science underlying COVID-19. Implications for the cardiovascular system. Circulation 142(1): 68-78.
2. Varela-Fernández R, Díaz-ToméV, Luaces-Rodríguez A, Conde-Penedo A, García-Otero X, et al. (2020) Drug delivery to the posterior segment of the eye: Biopharmaceutical and pharmacokinetic considerations. Pharmaceutics 12(3): 269.
3. Vaajanen A, Vapaatalo H (2017) A Single drop in the eye: Effects on the whole body? The Open Ophthal J 11: 305-314.
4. Bar-on YN, Flamholz A, Phillipsp R, Milo R (2020) SARS-CoV-2 (COVID-19) by the numbers. Elife 9: e57309.
5. Cosic I, Cosic D, Loncarevic I (2020) RRM prediction of erythrocyte band3 protein as alternative receptor for SARS-CoV-2 virus. Appl Sci 10(11): 4053.
6. Lopes de Almeida, de Oliveira S, Saldanha C (2012) Erythrocyte as a biological sensor. Clin Hemorheol Microc 51(1): 1-20.
7. Silva-Herdade AS, Giulia A, Faggi C, Calado A, Saldanha C (2016) Erythrocyte deformability: A partner of the inflammatory response. Microvasc Res 107: 34-38.
8. Vincent JL, Nelson DR, Williams MD (2011) Is worsening multiple organ failure the cause of death in patients with severe sepsis? Critical Care Medicine 39(5): 1050-1055.
9. Nogueira JB, Freitas A, Moreira C, Saldanha C, J Martins Silva, et al. (1988) Hypertension in an accelerated phase: Cardiologic, ophthalmologic and hemorheologic study. Rev Port Cardiol 7(3): 305-318.
10. Boudier HAS, Cohuet GM, Baumann M, Safar ME (2003) The heart, macrocirculation and microcirculation in hypertension: A unifying hypothesis. J Hypertens 21(3): S19-S23.
11. Alqahtani JS, Oyelade T, Aldhahir AM, Alghamdi SM, Almehmadi M, et al. (2020) Prevalence, Severity and mortality associated with COPD and smoking in patients with COVID-19: A rapid systematic review and meta-analysis. PLoS ONE 15(5): e0233147.
12. Saldanha C, Messias A (2017) Sepsis needs follow-up studies in intensive care units: Another avenue for translational research. Nov Appro Drug Des Dev1(1): NAPDD.MS.ID. 555555.
13. Saldanha C, Santos NC, Martins-Silva J (2002) Fluorescent probes DPH, TMA-DPH, and C17HC induce erythrocyte exovesiculation. J Membrane Biol 190(1): 75-82.
14. Saldanha C, Santos NC, Martins-Silva J (2004) A colorimetric process to visualize erythrocyte exovesicles aggregates. Biochem and Molec Biol Educ 32: 250-253.
15. Paulson RF, Ruan B, Hao S, Chen Y (2020) Stress erythropoiesis is a key inflammatory response. Cells 9(3): 634.
16. Kocks C, Boltengagen A, Piwecka M, Rybak-Wolf A, Rajewsky N (2018) Single-molecule fluorescence in situ hybridization (FISH) of circular RNA CDR1as. Methods Mol Biol 1724: 77-96.
17. Mohandas N, An X (2012) Malaria and human red blood cells. Med Microbiol Immunol 201(4): 593-598.
18. Sonveaux P, Lobysheva II, Feron O, McMahon TJ (2007) Transport and peripheral bioactivities of nitrogen oxides carried by red blood cell hemoglobin: Role in oxygen delivery. Physiology 22: 97-112.
19. Mariarosaria Bucci, Fiorentina Roviezzo, Inmaculada Posadas, Jun Yu, Luca Parente, et al. (2005) Endothelial nitric oxide synthase activation is critical for vascular leakage during acute inflammation in vivo. Proc of the Nation Acad of Scienc 102(3): 904-908.
20. Huang K, Han T, Hyduke D, Vaughn M, Van Herle H, et al. (2001) Modulation of nitric oxide bioavailability by erythrocytes. Proc of the Nation Acad of Scienc 98(20): 11771-11776.
21. Carvalho FA, Martins-Silva J, Saldanha C (2004) Amperometric measurements of nitric oxide in erythrocytes. Biosens. Bioelectron 20(3): 505-508.
22. Boerma EC, Mathura KR, van der Voort PH, Spronk PE, Can Ince (2005) Quantifying bedside-derived imaging of microcirculatory abnormalities in septic patients: A prospective validation study. Crit Care 9(6): 601-606.
23. Duarte C, Pinto S, Napoleão P, Pronto-Laborinho AC, Barros MA, et al. (2016) Identification of erythrocyte biomarkers in amyotrophic lateral sclerosis. Clinical Hemorheol and Microc 63(4): 423-437.
24. Wyatt AR, Wilson MR (2013) Acute phase proteins are major clients for the chaperone action of α2-macroglobulin in human plasma. Cell Stress and Chaperones 18(2): 161-170.
25. Das U (2007) Acetylcholinesterase and butyrylcholinesterase as possible markers of low-grade systemic inflammation. MedSci Monit 13(12): RA214-RA221.
26. Carvalho FA, Almeida JP, Fernandes IO, Freitas-Santos T, Saldanha C (2008) Non-neuronal cholinergic system and signal transduction pathways mediated by band 3 in red blood cells. Clin Hemorheol Microcirc 40(3): 207-227.
27. Lopes de Almeida JP, Freitas-Santos T, Saldanha C (2009) Fibrinogen-dependent signaling in microvascular erytrocyte function: Implications on nitric oxide efflux. J Membr Biol 231(1):47-53.
28. Saldanha C, Freitas T, Almeida J (2012) Fibrinogen effects on erythrocyte nitric oxide mobilization in presence of acetylcholine. Life Sciences 91(21-22): 1017-1022.
29. Lopes de Almeida JP, Carvalho FA, Silva-Herdade AS, Santos-Freitas T, Saldanha C (2009) Redox thiol status plays a central role in the mobilization and metabolism of nitric oxide in human red blood cells. Cell Biol Intern 33(3): 268-275.
30. Anticolinergicos afecta capacidade de raciocinio.
31. Saldanha C (2013) Timolol modulates erythrocyte nitric oxide bioavailability. J Clin Exp Ophthalmol 4(3).
32. Saldanha C, Silva-Herdade AS (2017) Physiological properties of human erythrocytes in inflammation. J Cel Biotec 3(1) :15-20.
33. Silva-Herdade AS, Freitas T, Almeida JP, Saldanha C (2016) Fibrinogen signaling in erythrocyte nitric oxide mobilization in presence of PI3-K and adenylyl cyclase inhibitors. European J of Biomed and Pharmac Sci 3(4): 28-34.
34. Huang KT, Han TH, Hyduke DR, Vaughn MW, Herle HV, et al. (2001) Modulation of nitric oxide bioavailability by erythrocytes. Proc Natl Acad Sci USA 98(20): 11771-11776.
35. Morabito R, Remigante A, Marino A (2019) Melatonin protects band 3 protein in human erythrocytes against H₂O₂-induced oxidative stress. Molecules 24(15): 2741.
36. Mele L, Paino F, Papaccio F (2018) A new inhibitor of glucose-6-phosphate dehydrogenase blocks pentose phosphate pathway and suppresses malignant proliferation and metastasis in vivo. Cell Death Dis 9(5): 572.