Sialylated-Glycan Bindings between SARS-CoV-2 Spike Protein and Cells: Comparison
View Latest Version
Please note this is a comparison between Version 3 by Lindsay Dong and Version 2 by Lindsay Dong.

Consistent with well-established biochemical properties of coronaviruses, sialylated glycan attachments between SARS-CoV-2 spike protein (SP) and host cells are key to the virus’s pathology. SARS-CoV-2 SP attaches to and aggregates red blood cells (RBCs), as shown in many pre-clinical and clinical studies, causing pulmonary and extrapulmonary microthrombi and hypoxia in severe COVID-19 patients. SARS-CoV-2 SP attachments to the heavily sialylated surfaces of platelets (which, like RBCs, have no ACE2) and endothelial cells (having minimal ACE2) compound this vascular damage. Notably, experimentally induced RBC aggregation in vivo causes the same key morbidities as for severe COVID-19, including microvascular occlusion, blood clots, hypoxia and myocarditis.

  • SARS-CoV-2
  • spike protein
  • COVID-19
  • sialic acid
  • glycophorin A

1. Introduction

The Molecular Composition of Glycans on SARS-CoV-2 SP and the RBC

The arrangement and chemical composition of the SARS-CoV-2 SP glycans have been determined, with those at its 22 N-glycosylation sites having a total of nine sialic acid (SA) terminal residues [1][2][3][4][5][6][7][8][9][10][11] and its four O-glycans having a total of three SA terminal residues [11]. This provides a basis for exploring these viral SP attachments to host cells, notably red blood cells (RBCs), platelets, leukocytes and endothelial cells [1]. RBCs and platelets have densely distributed sialoglycoproteins but no ACE2 receptors on their surfaces [12][13]; the same holds for leukocytes and most other blood cells [14][15][16]. Endothelial cells likewise have a heavily sialylated surface coating (glycocalyx), with about 28,000 SA-tipped CD147 receptors but only about 175 ACE2 receptors per cell [17][18].
Of particular interest are attachments of SARS-CoV-2 SP to the RBC, the latter coated with one million SA-tipped glycophorin A (GPA) molecules and a total of 35 million SA monosaccharides per cell [19][20][21]. The heavily sialylated GPA strands are spaced about 14 nm apart on the RBC surface and extend out 5 nm [19]. Band 3 protein is another molecule on the RBC surface, with 1.2 million copies per RBC, which extends > 10 nm from the RBC surface [19][22] and is glycosylated by poly-N-acetyllactosamine, a sialylated branched-chain glycan [23][24][25][26]. GPA and poly-N-acetyllactosamine, the two most abundant glycans on the RBC membrane [25], have been found to mediate hemagglutination by various bacterial and viral pathogens [26][27][28][29].
Hemagglutination as caused by these pathogen–glycan attachments is of particular interest in view of a primal defense mounted by RBCs along with platelets against pathogens having SA terminal moieties by attaching to them and delivering them to leukocytes or conveying them to macrophages in the liver and spleen for phagocytosis [20][30][31][32][33][34][35][36]. 
A clear experimental demonstration of binding between SARS-CoV-2 SP and sialylated glycans on host cells was provided using NMR spectroscopy [37]. It was found, in particular, that a site on the SP N-terminal domain (NTD) binds to α2,3 and α2,6 sialyl N-acetyllactosamine, which are components or variants thereof of the sialylated poly-N-acetyllactosamine glycans of the band 3 strands extending from the RBC surface. Intriguingly, this SP-to-glycan binding was found to be much more pronounced for α2,3 than for α2,6 SA-linked N-acetyllactosamine [37], while α2,3 vs. α2,6-linked SA is likewise much more prevalent in sialylated poly-N-acetyllactosamine of adult (vs. fetal) RBCs [24].
Possibilities for binding are indicated as well between SARS-CoV-2 SP and/or glycans at its glycosylation sites and GPA on the RBC surface, with GPA, as noted, having no known physiological role other than this type of immune adherence. The positive electrostatic potential of SARS-CoV-2 SP [38] supports its binding to the negatively charged, densely distributed SA on the RBC surface, most on its million GPA strands [39][40]. SA in its predominant human form, Neu5Ac, is the most common terminal residue of GPA [19][22][41]. For the N- and O-glycans on SARS-CoV-2 SP, the most common terminal residues are galactose (Gal), with 27 total, and Neu5Ac (SA), with 12 total [9][10][11][42]. Through binding configurations proposed by Varki and Schnaar (2017) [43] and others [37][44][45][46], multivalent bonds can form via α2–3 and α2–6 linkages from Neu5Ac on GPA to Gal on glycans populating SARS-CoV-2 SP glycosylation sites.

2. In Vitro, In Vivo and Clinical Studies Demonstrate Induction of RBC Aggregation by SARS-CoV-2 SP

Many in vitro, in vivo and clinical studies demonstrate that SARS-CoV-2 SP attaches to RBCs and induces RBC aggregation. Boschi et al. (2022) found that SARS-CoV-2 SP from each of the Wuhan, Alpha, Delta and Omicron strains induced RBC clumping (hemagglutination) when mixed with human RBCs in phosphate-buffered saline (PBS) [38]. To explore whether bridging of adjacent RBCs by SARS-CoV-2 SP via glycan bonds might be the cause of this observed hemagglutination, an agent with indicated high-affinity binding to multiple SARS-CoV-2 SP glycan-binding sites [47], the macrocyclic lactone ivermectin (IVM), was added to the mix of SP and RBCs both before and after hemagglutination formed. IVM blocked the formation of hemagglutination when added to the initial mix and reversed hemagglutination over the course of 30 min when added after it formed [38]. The same SP-induced RBC clumping effect as noted above was demonstrated in zebrafish embryos, which have blood cell glycosylation patterns [48] and capillary diameters [49] similar to those of humans. When SARS-CoV-2 SP was microinjected into the common cardinal vein of a zebrafish embryo at a concentration similar to that obtained in critically ill COVID-19 patients, it caused the formation of small RBC clumps and an associated reduction in blood flow velocity within 3–5 min after injection, accompanied by thrombosis in capillaries, arteries and veins [50]. When SP was coinjected with a mixture of heparan sulfate and heparin (molecular mass of each ≤ 30 kDa), however, with both of these glycosaminoglycans having strong binding affinity to SARS-CoV-2 SP [50][51][52], the extent of thrombosis was markedly reduced [50]. SARS-CoV-2 SP attachments to RBCs were demonstrated directly by Lam et al. (2021) through immunofluorescence analysis of RBCs from the blood of nine hospitalized COVID-19 patients [53]. For these patients at hospital admission, the mean percentage of RBCs having SARS-CoV-2 SP traces was 41%. This finding suggests that concentrations of SARS-CoV-2 SP in blood as typically reported in other studies using plasma or serum may significantly understate actual values due to high-affinity binding to RBCs, which are removed from plasma and serum. SARS-CoV-2 SP and pseudovirus were each found to bind to nanoparticle arrays bearing SA derivatives [7] and to SA-tipped CD147 receptors, likewise detected using nanoarrays [54]. Nanoarray methods are required to detect SARS-CoV-2 SP glycan attachments, because these methods allow bindings to form multivalently, whereas univalent bindings are weak [1] and not detectable by microarray methods [55]. The presence of SARS-CoV-2 SP in the blood of COVID-19 patients and its induction of hemagglutination in vitro and in vivo would suggest that the same would occur clinically, which is indeed the case. In three publications that used scanning electron microscopy to examine blood from the cubital vein blood of patients with mild-to-moderate cases of COVID-19, all hospitalized but none requiring intensive care, a team of investigators observed blood cell clumping and other anomalies [56][57][58]. The first study found stacked RBC aggregates (rouleaux) ranging in size from 3–12 cells. Light microscopy examination of smears from the blood of 20 hospitalized COVID-19 patients with anemia detected large, stacked RBC clumps (rouleaux), in 85% of those patients [59]. Another study, which examined the sublingual microcirculation of 38 COVID-19 patients in intensive care using video microscopy, found that the mean number of RBC microaggregates detected in these patients was 15 times the mean number for 33 healthy volunteers [60]. These RBC microaggregates were found in two-thirds of the COVID-19 patients vs. two of the 33 healthy volunteers. Paralleling these studies that document RBC aggregation in severe COVID-19 are many that report microvascular occlusion. Postmortem examinations of hundreds of patients who died from COVID-19 in many studies consistently found microthrombi in the pulmonary microvasculature in most patients [61][62][63][64][65][66][67][68][69][70][71][72]. Microthrombi in alveolar capillaries were nine times as prevalent in postmortem COVID-19 patients compared to influenza patients [64]. RBC clumping and microthrombi in the lungs have been regarded as likely causes of hypoxemia in severe COVID-19 patients [56][58][73][74], which in turn is closely associated with mortal outcomes [75].

3. Glycan Bindings from SARS-CoV-2 SP to Platelets and Endothelial Cells Cause Endothelial Damage, Inflammation and Coagulation

Attachments of SARS-CoV-2 SP to the heavily sialylated [12][13][18] surfaces of platelets and endothelial cells cause endothelial damage, platelet activation and associated coagulation which, as with the attachments to RBCs, contribute to the severe morbidities of COVID-19. Platelets, having no ACE2 receptors, like RBCs [14][15], act with RBCs in a role that was termed “immune adherence” [33], attaching to and clearing pathogens [35][36], and are found enmeshed with RBCs in blood cell clumps in COVID-19 patients [57]. The degree of sialyation of the endothelial cell surface is exemplified by the 28,000 SA-tipped CD147 receptors vs. the 175 ACE2 receptors per endothelial cell [17]. For glomerular endothelial cells from a conditionally immortalized human cell line, the enzyme neuraminidase, which hydrolyzes SA, removed more than 50% of the cells’ surface coating (glycocalyx) [18]. The endothelial cell thus provides a prime target for the SARS-CoV-2 virus, and indeed, both whole virus and viral SP have been found on endothelial cells in clinical and in vivo COVID-19 infections [64][71][76][77][78][79][80][81]. These SARS-CoV-2 viral or SP attachments to the endothelium can be perilous to the human host, with trillions of RBCs each flowing once per minute through the lungs and then the extrapulmonary vasculature [82] and with the cross-sectional diameter of most capillaries so small that RBCs distort their shape to squeeze through [83]. Thus, SARS-CoV-2 virus particles or SP attached to endothelial cells or RBCs could create resistance to blood flow or even potentially rip off a piece of an endothelial cell or the entire cell [1]. Indeed, one study found that serum levels of circulating endothelial cells (CECs) in mild-to-moderate COVID-19 patients were up to 100 times the levels for matched controls. The study also found that each of these CECs from the COVID-19 patients typically had several holes in their membranes approximately the size of the SARS-CoV-2 viral capsid (the viral envelope) [56].

4. Experimentally Induced RBC Clumping In Vivo: Parallels to Severe COVID-19

Studies dating back to the 1940s in dogs, rabbits, mice, hamsters and other animals closely examined the effects of IV injection of high-molecular-weight dextran (HMWD), generally of molecular weight (MW, loosely equivalent to molecular mass) ≥ 100 kDa or other blood cell-agglutinating agents. In several studies, blood cell aggregation was induced within minutes to hours after IV injection of HMWD [84][85][86][87][88][89], with molecular bridging of RBCs by HMWD molecules being a hypothesized mechanism for this effect [90][91][92][93]. After HMWD injection in vivo, small clumps of RBCs formed and then enlarged into longer stacked clumps (rouleaux) and, in some cases, into vast trees with branches of hundreds of stacked RBCs [85][86][94]. In vitro, the addition of HMWD to blood likewise induced RBC aggregation [95][96] and did so as well when added to RBCs in PBS [97][98]. The same RBC disaggregating effect of LMWD was observed in vitro [99], possibly caused by competitive binding to RBCs that limited bridging between adjacent RBCs by larger molecules. Even in healthy humans or animals, RBC clumps can transiently form under conditions of slow blood flow, e.g., in deep veins of the lower limbs, but they typically disaggregate as they move into regions of faster blood flow [100][101][102][103][104][105][106][107][108][109][110] and are rarely problematical in healthy subjects [89][111][112]. Yet under pathological conditions in diseases such as diabetes, malignant hypertension and malaria [103][112][113][114][115], these RBC aggregates can persist and grow via a positive feedback loop whereby the clumps cause decreased blood flow velocity with a concomitant reduction in shear forces that in turn causes further aggregation [100][102][103][104][105][106][107][109][112]. The capability of LMWD to rapidly reverse RBC aggregation and associated microvascular occlusion caused by injection of HMWD, as noted above, distinguishes blood clumping, e.g., as induced by HMWD, from clotting, in which blood cell clumps harden into fibrin-enmeshed clots via the coagulation cascade. Indeed, several mammalian diseases are associated with increased levels of RBC aggregation and microvascular occlusion which do not typically cause blood clotting, although risks of this complication are increased [112][113]. Blood cell clumping and clotting are not completely unrelated phenomena, however, given the potential of RBC aggregation to trigger deep vein thrombosis [116][117] and the role of fibrinogen, an essential promoter of blood clotting, in blood cell clumping as well [100][118][119][120].

4.1. Induced RBC Aggregation Causes Microvascular Occlusion, Hypoxia, Blood Clots, and Redistribution of Blood Flow from Smaller to Larger Blood Vessels

When HMWD or other agglutinating agents were injected into animals at sufficient concentrations to overwhelm the host’s ability to safely sequester the RBC aggregates formed, these clumps caused microvascular occlusion as detected in a variety of host tissues [113], including the myocardium [94][121], muscle [122] and abdominal cavity [94] of rats; the conjunctival vessels of dogs, cats and rabbits [88][123]; the cheek pouch of hamsters [89][111]; and the kidney, liver, ear chamber, bone marrow and heart tissue of rabbits, including the myocardium and pericardium [85][86][87][124][125]. In the myocardium of rabbits and rats, the degree of myocardial tissue damage was correlated with the observed degree of intravascular aggregation of blood cells [85][87][94], with hypoxia resulting from vascular occlusion proposed to be the cause of tissue damage [85][87]. Associated with the microvascular occlusion that it triggered, experimentally induced RBC aggregation caused decreased velocity of blood flow [84][86][87][88][89][107][113][121], increased blood viscosity [84][113][123][126], increased incidence of blood clotting [85][103][113] and decreased oxygen tension in arteries, veins and tissues, with accompanying hypoxic damage to body organs [85][87][113][127][128]. Another effect caused by induced blood cell clumping as observed in the conjunctiva of cats, dogs and rabbits and bone marrow of rabbits was a reduction in blood flow in the capillaries and other small vessels having cross-sectional diameters of about 10 μm or smaller [88][124], indicative of a shift of blood flow into the larger vessels.

4.2. Corresponding Morbidities in Severe COVID-19

As considered above, SARS-CoV-2 SP, like HMWD dextran, induces RBC aggregation, and the same morbidities caused by experimentally induced RBC aggregation have been commonly observed for cases of severe COVID-19. These morbidities of severe COVID-19 include microvascular occlusion in the lungs and other organ systems [61][62][63][64][65][66][67][68][69][70][71][72][129][130], hypoxia [73][131], arterial and venous thromboembolisms [63][69][71][72][132][133][134][135], disseminated intravascular coagulation [69][132][133][134][135][136][137] and multiorgan damage associated with these vascular aberrations and hypoxia [61][137][138].

4.3. Redistribution of Blood Flow from Smaller to Larger Blood Microvessels in COVID-19 Patients

Another effect of experimentally induced RBC aggregation, the redistribution of blood flow from microvessels to blood vessels of larger cross-sectional diameter, as described above, is also paralleled in COVID-19 is. Osiaevi et al. (2023) compared videomicroscopic imaging of the sublingual microvasculature of 16 critically ill COVID-19 patients, 17 patients with long COVID and 15 healthy controls [139]. The density of functional capillaries (having flowing RBC content ≥ 50%) with cross-sectional diameter 4–10 μm was sharply reduced for active COVID-19 patients vs. controls, with values for long COVID patients roughly halfway between those for active COVID-19 patients and healthy controls. The study investigators concluded from these and other measures of microvascular health that the long COVID patients had significant microvasculature impairment, lasting even 18 months after infection for some [139]. Rovas et al. (2021) reported similar sharp reductions in densities of functional capillaries at the lower end of the 4–25 μm cross-sectional diameter range in the sublingual microvasculature of COVID-19 patients vs. healthy controls [138]. The extent of reduction in density of functional capillaries of diameter 4–6 μm in the COVID-19 patients correlated with their oxygenation index (PaO2/FiO2) and with an index of multiorgan failure and associated mortality risk. Rovas et al. concluded from these correlations that the observed reduction in sublingual small capillary density was another manifestation of the pathological clogging of capillaries as also observed in pulmonary microthrombi at autopsies of COVID-19 patients.

5. Major Risk Factors of Age, Diabetes and Obesity for COVID-19 Severity Correlate with Increased Propensity to RBC Aggregation

The most significant risk factor for severe COVID-19 is age, with several studies showing a multifold increased risk of fatal outcomes with older age [131][140][141]. One multivariate analysis of 17 million subjects in the UK reported a sixfold increased mortality for ages 70 through 79 vs. 50 through 59 years [142]. A meta-analysis of 612,000 subjects in several countries conducted in 2020 found a mortality rate of 22.8% for ages 70–79 years vs. 0.3% for ages ≤ 29 years [143]. This multifold increase in COVID-19 mortality with older age aligns with a much greater extent of microvascular occlusion in older vs. younger healthy subjects, linked to both a significantly greater propensity to RBC aggregation and slower blood flow with increased age. Microscopic examinations of the bulbar conjunctiva of healthy subjects found that 30% of those of ages 56–75 years had aggregation in the smaller venules and capillaries, as compared with a 3% rate of such aggregation of those of ages 16–35 years [144]. This tenfold increased rate of microvascular occlusion in the older subjects corresponds to much greater RBC aggregation and slower blood flow with increased age. One study that measured RBC aggregability by multiple detection methods found a statistically significant increase in this value in the blood of middle-aged versus young adults [145]. As noted, RBC aggregate formation in vivo depends not only on aggregability under static conditions but also on the degree of shear forces that promote disaggregation, as associated with velocity of blood flow [100][104][105][106]. It is, thus, noteworthy that blood flow is slower with increased age [146][147][148][149][150][151][152]. Mean velocity of capillary flow under fingernail and toenails for subjects of mean age 63 years was half of that for subjects of mean age 26 years [146]. Older subjects had 23% [147] and 40% [148] diminished flow velocities vs. younger subjects for capillary flow in other tissues. Arterial blood flow velocities were 26–27% lower for older vs. younger subjects in two studies [150][151]. 

6. SARS-CoV-2 SP Unattached to Whole Virus Induces Microvascular Occlusion In Vivo

6.1. Myocardial Damage as a Signal of Microvascular Occlusion

A clinical window into morbidities associated with RBC aggregation is provided by the myocardium—the heart muscle—which is among the tissues most susceptible to the damaging effects of experimentally induced RBC aggregation and ensuing microvascular occlusion. Several studies found that injection of HMWD (high-MW dextran) caused myocardial damage [85][87][113][153] and/or electrocardiogram (ECG) changes [94][113][126][153] characteristic of myocarditis. In one study, 40 min after HMWD injection, ECG abnormalities were apparent, and HMWD induced lasting myocardial damage [153]. Both the degree of myocardial damage [85][87] and of ECG abnormalities [94] correlated with the extent of microvascular occlusion. Clinically, for hospitalized patients with coronary heart disease, the number of microthrombi per field of observation in the bulbar conjunctival microcirculation was found to be correlated with both the extent of ECG and symptomatic abnormalities [94].

6.2. Myocardial Damage Experimentally Induced by SARS-CoV-2 SP in the Absence of Whole Virus

Induction of myocarditis by SARS-CoV-2 SP in the absence of whole virus was evidenced in two rodent studies by IV injection of BNT162b2, the Pfizer-BioNTech mRNA vaccine, an experimental system in which SP is generated by host cells, distinct from intramuscular (IM) injection used for clinically administered COVID-19 vaccinations. Clinical cases of SARS-CoV-2 SP found in endothelial cells after IV mRNA vaccination [154][155][156] support the possibility that SP could be generated by nucleated endothelial cells in blood vessels post-vaccination. In mice, after a second IV vaccine dose, 67% had grossly visible white patches over the visceral pericardium and all showed changes of myopericarditis, compared with only mild degenerative changes in the myocardium in the intramuscular (IM)-injection group [157].

6.3. Clinical Signs of Microvascular Occlusion and Myocarditis after Exposure to SARS-CoV-2 SP

Further insights into microvascular occlusion caused by SARS-CoV-2 SP in the absence of whole virus in a clinical setting were provided by optical coherence tomography angiography (OCT-A) imaging of the retinal microvasculature. Determinations of the vascular density (VD) of flowing blood vessels in various retinal layers of human subjects, an indicator of microvascular occlusion, found that the CoronaVac vaccine, made from inactivated whole virus, caused no changes after vaccination [158][159]. The Pfizer-BioNTech mRNA vaccine caused small but statistically significant reductions in VD vs. controls at three days [160] and at two and four weeks [159] after vaccination. Reductions in many of these VD values at two weeks after vaccination were statistically significant at p < 0.001; most of these resolved by four weeks after vaccination, but seven of these VD reductions persisted at statistically significant levels at that time [159]. The significance of these findings derives not from the occasional ocular adverse effects that have been reported after mRNA COVID-19 vaccinations [161][162] but rather from indications that ocular microvascular occlusion mirrors a pathology elsewhere in the body [112][163]. Myocardial injury is another indicator of microvascular occlusion, as noted above, which opens another diagnostic window, PET-CT scanning, since fluorodeoxyglucose F18 (FDG) uptake in myocardial tissue has been found to track myocardial injury [164][165]. 

7. Decreased Clinical Severity of COVID-19 by Agents That Inhibit RBC Aggregation

7.1. Fluvoxamine

Fluvoxamine (FLV), a selective serotonin reuptake inhibitor (SSRI), attracted interest from prominent medical researchers [166][167][168] after early clinical trials indicated promising results for COVID-19 treatment [169][170][171][172]. Although rapid recovery from severe illness was not generally observed, one study showed a significant reduction in residual symptoms of COVID-19 at 14 days after start of FLV treatment vs. untreated controls [169], and another showed significant reductions in emergency room visits or hospitalizations [171]. A plausible biochemical mechanism is the sharp reduction by FLV in serum levels of serotonin, which is a powerful inducer of RBC and platelet aggregation. In vitro, serotonin caused marked aggregation of RBCs, platelets and leukocytes [88]. In vivo, injection of serotonin resulted in blood cell aggregates being trapped in small venules and capillaries in the ocular conjunctival vasculature [88]. In dogs, a serotonin antagonist prevented an increase in pulmonary alveolar dead space, an indication of pulmonary vascular obstruction, after hemorrhagic shock [173].

7.2. Hydroxychloroquine (HCQ)

The application of HCQ, an aminoquinoline, for treatment of COVID-19, as developed by an infectious disease team at Aix-Marseilles University in France [174][175][176], has been the subject of significant controversy, a review of which is not attempted here. However, it is of note that HCQ has been found to have pronounced activity in reducing blood cell aggregation and associated microvascular occlusion. In 44 human subjects with vascular conditions including coronary artery and cerebrovascular disease, all having initial manifestations of microvascular occlusion, ocular conjunctival microvasculature was observed over a nine-month period following the start of HCQ treatment [177].

7.3. Ivermectin (IVM)

To identify potential therapeutics for COVID-19, four in silico studies collectively screened over 1000 molecules for binding to SARS-CoV-2 SP and other SARS-CoV-2 viral targets [52][178][179][180]. In each of these studies, the strongest or close-to-strongest binding affinity to SP was obtained for IVM, a macrocyclic lactone with multifaceted antiparasitic and antimicrobial activity, distributed in four billion doses for human diseases worldwide since 1987 [181][182][183]. Aminpour et al. (2022) found by molecular docking computations that IVM binds with high affinity (<−7.0 kcal/mol) to seven sialoside-binding sites or other glycan-binding sites on SARS-CoV-2 S1, six on the N-terminal domain (NTD) and one on the receptor-binding domain (RBD). These binding energy values of <−7.0 kcal/mol were obtained for the RBD in both the open (“up”) and closed (“down”) positions [47]. As a measure of significance of this binding energy value, binding energies of <−7.0 kcal/mol predicted efficacy for a large set of HIV inhibitors with 98% sensitivity and 95% specificity in another study [184]. Additional molecular modeling studies of IVM binding to SARS-CoV-2 SP [185][186][187][188][189], including one by Lehrer and Rheinstein (2020) [190], likewise found strong binding affinities for IVM. Competitive binding by IVM to SP glycan-binding sites is thus a likely biochemical mechanism for the in vitro inhibition and reversal by IVM of aggregation of human RBCs by SARS-CoV-2 SP as noted above [38]. In early 2020, two Florida physicians, Jean-Jacques and Juliana Rajter, were intrigued by an indication of a clinical parallel to this in vitro effect—observations that several COVID-19 patients with severe respiratory impairment and SpO2 deficits experienced normalized breathing function within 1–2 days after treatment with IVM [191][192]. Months later, Rajter et al. (2020) reported results of a propensity-matched case control study of COVID-19 patients treated with a low dose of IVM plus standard of care (SOC) at four Florida hospitals, which yielded a 40% reduction in mortality vs. controls given only SOC [193]. After that study was concluded, through mid-2021, more than 20 randomized controlled trials (RCTs) of IVM treatment of COVID-19 were conducted, with six of seven meta-analyses reporting notable reductions in deaths and with a mean 0.31 relative risk of mortality vs. controls (a 69% reduction) [194]. By 2022, however, several other RCTs reported that IVM yielded no statistically significant benefits for COVID-19 treatment, as summarized in an August 2022 editorial which declared that it was “time to stop using ineffective COVID-19 drugs” [195]. Curiously, however, the editorial prominently cited in support a June 2022 meta-analysis of ten RCTs for IVM treatment of COVID-19 encompassing 3472 patients [196] which actually reported as the first finding in its results section a twofold reduction in deaths in its pooled IVM treatment vs. placebo groups (pooled log odds ratio of −0.67, 95% CI = −1.20 to −0.13, with low heterogeneity). The mortality reduction was less (log OR = −0.12) for the RCTs rated as having a low risk of bias, but included in that group, weighted to account for 63% of that pooled log OR, was a study of dubious credibility. Coauthors of the aforementioned study, the TOGETHER trial [197], have repeatedly refused to disclose four of their key outcome numbers, namely per protocol deaths and hospitalizations, treatment vs. placebo [198], which are of key importance given critiques of the primary outcome used in all arms of the TOGETHER trial by the U.S. Food and Drug Administration [199] and National Institutes of Health [200]. Instead, a TOGETHER trial coauthor directed inquiring scientists to the ICODA data repository, listed as the data source in the study’s data sharing statement [297,300][198][201]. After two months of futile attempts by scientists to obtain the data from ICODA, however, on 7 June 2022, an ICODA manager reported that the TOGETHER trial data were never held by that data repository and that she had instructed its authors to stop citing it as their data source [297,301][198][202]. In another prominently cited RCT of IVM treatment of COVID-19 that reported negative conclusions [201203], IVM was substituted for placebo doses for 38 patients, a mistake caught a month later, and blinding was broken by use of sugar water as the placebo for one-third of the study’s patients (liquid IVM has a bitter taste). Adverse events that are distinctive to the high IVM dose used (transient and non-critical) occurred at almost identical rates in the placebo and IVM arms, while over-the-counter sales of IVM surged in the study region during the study period [202204]. The RCT evidence for IVM-based treatments of COVID-19 is thus mixed; however, in rare cases, efficacy of a drug has been conclusively established without RCT findings when it has achieved consistent major clinical benefits in the face of an established baseline of null effect. For example, the 96% cure rate for peptic ulcers by a triple therapy achieved in a 1990 clinical trial [203205] provided conclusive evidence of the therapy’s efficacy, given a baseline of palliative but rarely curative results for that chronic condition [204206][205207]. The associated discovery of H. pylori as the underlying pathogen for peptic ulcers was honored with the Nobel prize for medicine in 2005 [206208]. For penicillin, early in vitro and mouse studies provided convincing indications of marked antibacterial activity. Alexander Fleming found in 1929 that transparent regions formed around penicillin embedded in agar plates of several species of cultured bacteria, indicating inhibition of bacterial growth [207209]. In the absence of RCT evidence, penicillin production was then ramped up to industrial scale, saving the lives of thousands of soldiers during World War II [3[210]. For cases of moderate and severe COVID-19 in patients on room air, there is a consistent baseline of null effect in a 1–2 week timeframe: the magnitude of reductions in SpO2 levels correlate with the extent of pulmonary damage, and neither of these normalize in that timeframe [211][212][213][214][215][216][217]. With that backdrop of null effect, three studies of severe COVID-19 patients on room air treated with IVM-based regimens observed sharp increases in SpO2 after 1 day of treatment [218][219][220][221] while SpO2 decreased during the same 10]-day period in a fourth group of such patients under standard care.

For cases of moderate and severe COVID-19 in patients on room air, there is a consistent baseline of null effect in a 1–2 week timeframe: the magnitude of reductions in SpO2 levels correlate with the extent of pulmonary damage, and neither of these normalize in that timeframe [311–317]. With that backdrop of null effect, three studies of severe COVID-19 patients on room air treated with IVM-based regimens observed sharp increases in SpO2 after 1 day of treatment [318–321] while SpO2 decreased during the same 1-day period in a fourth group of such patients under standard care.

For one of these three studies, Stone et al. (2022) [318][218], taking into account some missing values for the 34 treated patients at <48 h post-treatment, paired t-test calculations were performed for post-treatment minus pre-treatment SpO2 values for the study patients at +12 h, +24 h and +48 h after the start of IVM administration. These paired t-test values were highly significant, with p < 10−6 in each case. One patient in the study had an increase in SpO2 from 79% recorded at the first IVM dose to 95% three hours later, and four other patients had increases of 12 or more in SpO2% within 12 h after the first IVM dose. These sharp, rapid improvements parallel the disaggregation of RBC clumps observed in vitro over the course of 30 min by Boschi et al. (2022) and can be explained by rapid clearance of RBC aggregates in the vasculature and corresponding increases in efficiency of oxygenation in pulmonary and extrapulmonary tissues. In 2020, Peru provided a unique setting to track clinical efficacy of IVM-based treatment for COVID-19 with close consideration of confounding factors, using excess deaths data from its national health system, which aligned with WHO monthly summary data [208222]. Treatment with IVM and adjunct agents was deployed at intensive, moderate or limited levels under semi-autonomous policies in its 25 states, enabling comparisons with reductions in excess deaths at 30 days after peak values, state by state. A Kendall tau calculation yields a two-tailed p-value of 0.002 for reductions in excess deaths correlated with level of IVM use in Peru’s 25 states. On a national scale, during four months of IVM use in 2020, before a new president of Peru elected on November 17 restricted its use, there was a 14-fold reduction in nationwide excess deaths, and then a 13-fold increase in the two months following the restriction of IVM use [208222]. This set of real-world national health data, accompanied by extensive additional data by which potential confounding influences can be tracked, provides another significant indication of efficacy of IVM treatment of COVID-19.

8. A Comparison of Degree of Clinical Susceptibility to COVID-19 and RBC Aggregability in Various Animal Species

Susceptibility to COVID-19 and severity of this disease have been tracked for dozens of mammalian species, as reported in a summary figure by Meekins et al. (2021) [209223]. RBC aggregability values and related values of blood viscosity at low shear velocity have been tracked for many mammalian species as well, as reported by Baskurt and Meiselman in 2013 [210224]. A correlation calculation between these two values, by species, provides a test of whether RBC aggregability is likely associated with COVID-19 morbidity. The COVID status of mammalian species was reported by Meekins et al. using designators for viral shedding, clinical signs, mortality and transmission. Scholars derived a composite COVID status index from the first three of these indicators (transmission was not used) with values of 0 for none of these three, 1 for viral shedding only, 2 for clinical signs and 3 for clinical signs and mortality. For RBC aggregability, an aggregation index shown in Baskurt and Meiselman for 22 mammalian species was used. They also reported values of blood viscosity under low-shear conditions for 27 mammalian species that were closely correlated with the corresponding RBC aggregation index for species having values shown in both figures. For a species tracked in Baskurt and Meiselman that reported blood viscosity but not RBC aggregability, the latter value was interpolated from the blood viscosity value. Correspondence between RBC aggregability and blood viscosity was established using the values of each for cattle and horses; these species had the minimum and maximum values of all species tracked by Baskurt and Meiselman, respectively, for both of these indices.

9. Conclusions

The central role of sialylated glycan attachments between SARS-CoV-2 SP and RBCs and other blood cells in the severe morbidities of COVID-19 is founded on well-established biochemistry of coronaviruses and RBCs and established here through multiple channels of substantiation. Many preclinical and clinical studies show that SARS-CoV-2 SP attaches to and aggregates RBCs. Experimentally induced RBC clumping in vivo causes the same morbidities and the same redistribution of blood flow from smaller to larger blood vessels as for severe COVID-19. The key risk factors of increased age, diabetes and obesity for COVID-19 morbidity are each associated with significantly increased RBC aggregation. SARS-CoV-2 SP in the absence of whole virus as generated experimentally by IV injection of mRNA COVID vaccines in vivo, which caused SP to be generated in the absence of whole virus, induced microvascular occlusion. Three generic agents which attracted prominent interest as COVID-19 therapeutics all yielded significant reductions in RBC aggregation. For mammalian species, the degree of clinical susceptibility to COVID-19 correlates with the aggregation propensity of RBCs with p = 0.033. These in vitro, in vivo and clinical findings, together, provide a convincing demonstration that RBC aggregation induced by SARS-CoV-2 SP through sialylated glycan attachments and resulting microvascular occlusion is key to the morbidities of severe COVID-19. These insights can support therapeutic and preventative strategies for evolving variants of this disease and for long COVID, while imaging of the retinal or sublingual microvasculature of active or long COVID patients can provide important support to these efforts.

References

  1. Scheim, D.E. A Deadly Embrace: Hemagglutination Mediated by SARS-CoV-2 Spike Protein at its 22 N-Glycosylation Sites, Red Blood Cell Surface Sialoglycoproteins, and Antibody. Int. J. Mol. Sci. 2022, 23, 2558.
  2. Koehler, M.; Delguste, M.; Sieben, C.; Gillet, L.; Alsteens, D. Initial Step of Virus Entry: Virion Binding to Cell-Surface Glycans. Annu. Rev. Virol. 2020, 7, 143–165.
  3. Ströh, L.J.; Stehle, T. Glycan Engagement by Viruses: Receptor Switches and Specificity. Annu. Rev. Virol. 2014, 1, 285–306.
  4. Shajahan, A.; Supekar, N.T.; Gleinich, A.S.; Azadi, P. Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2. Glycobiology 2020, 30, 981–988.
  5. Guo, W.; Lakshminarayanan, H.; Rodriguez-Palacios, A.; Salata, R.A.; Xu, K.; Draz, M.S. Glycan Nanostructures of Human Coronaviruses. Int. J. Nanomed. 2021, 16, 4813–4830.
  6. Choi, Y.K.; Cao, Y.; Frank, M.; Woo, H.; Park, S.-J.; Yeom, M.S.; Croll, T.I.; Seok, C.; Im, W. Structure, Dynamics, Receptor Binding, and Antibody Binding of the Fully Glycosylated Full-Length SARS-CoV-2 Spike Protein in a Viral Membrane. J. Chem. Theory Comput. 2021, 17, 2479–2487.
  7. Baker, A.N.; Richards, S.-J.; Guy, C.S.; Congdon, T.R.; Hasan, M.; Zwetsloot, A.J.; Gallo, A.; Lewandowski, J.R.; Stansfeld, P.J.; Straube, A.; et al. The SARS-CoV-2 Spike Protein Binds Sialic Acids and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device. ACS Cent. Sci. 2020, 6, 2046–2052.
  8. Lardone, R.D.; Garay, Y.C.; Parodi, P.; de la Fuente, S.; Angeloni, G.; Bravo, E.O.; Schmider, A.K.; Irazoqui, F.J. How glycobiology can help us treat and beat the COVID-19 pandemic. J. Biol. Chem. 2021, 296, 100375.
  9. Gao, C.; Zeng, J.; Jia, N.; Stavenhagen, K.; Matsumoto, Y.; Zhang, H.; Li, J.; Hume, A.J.; Mühlberger, E.; van Die, I.; et al. SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors. bioRxiv 2020.
  10. Chen, W.; Hui, Z.; Ren, X.; Luo, Y.; Shu, J.; Yu, H.; Li, Z. The N-glycosylation sites and Glycan-binding ability of S-protein in SARS-CoV-2 Coronavirus. bioRxiv 2020.
  11. Casalino, L.; Gaieb, Z.; Goldsmith, J.A.; Hjorth, C.K.; Dommer, A.C.; Harbison, A.M.; Fogarty, C.A.; Barros, E.P.; Taylor, B.C.; McLellan, J.S.; et al. Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein. ACS Cent. Sci. 2020, 6, 1722–1734.
  12. Hyvärinen, S.; Meri, S.; Jokiranta, T.S. Disturbed sialic acid recognition on endothelial cells and platelets in complement attack causes atypical hemolytic uremic syndrome. Blood 2016, 127, 2701–2710.
  13. Kasinrerk, W.; Tokrasinwit, N.; Phunpae, P. CD147 monoclonal antibodies induce homotypic cell aggregation of monocytic cell line U937 via LFA-1/ICAM-1 pathway. Immunology 1999, 96, 184–192.
  14. Campbell, R.A.; Boilard, E.; Rondina, M.T. Is there a role for the ACE2 receptor in SARS-CoV-2 interactions with platelets? J. Thromb. Haemost. 2021, 19, 46–50.
  15. Cosic, I.; Cosic, D.; Loncarevic, I. RRM Prediction of Erythrocyte Band3 Protein as Alternative Receptor for SARS-CoV-2 Virus. Appl. Sci. 2020, 10, 4053.
  16. Scheim, D.E.; From Cold to Killer: How SARS-CoV-2 Evolved without Hemagglutinin Esterase to Agglutinate, Then Clot Blood Cells in Pulmonary and Systemic Microvasculature. OSF Preprints. Available online: https://osf.io/sgdj2 (accessed on 30 October 2023).
  17. Ahmetaj-Shala, B.; Vaja, R.; Atanur, S.; George, P.; Kirkby, N.; Mitchell, J. Systemic analysis of putative SARS-CoV-2 entry and processing genes in cardiovascular tissues identifies a positive correlation of BSG with age in endothelial cells. bioRxiv 2020.
  18. Singh, A.; Satchell, S.C.; Neal, C.R.; McKenzie, E.A.; Tooke, J.E.; Mathieson, P.W. Glomerular endothelial glycocalyx constitutes a barrier to protein permeability. J. Am. Soc. Nephrol. 2007, 18, 2885–2893.
  19. Viitala, J.; Järnefelt, J. The red cell surface revisited. Trends Biochem. Sci. 1985, 10, 392–395.
  20. Baum, J.; Ward, R.H.; Conway, D.J. Natural selection on the erythrocyte surface. Mol. Biol. Evol. 2002, 19, 223–229.
  21. Levine, S.; Levine, M.; Sharp, K.A.; Brooks, D.E. Theory of the electrokinetic behavior of human erythrocytes. Biophys. J. 1983, 42, 127–135.
  22. Aoki, T. A Comprehensive Review of Our Current Understanding of Red Blood Cell (RBC) Glycoproteins. Membranes 2017, 7, 56.
  23. Zhou, D. Why are glycoproteins modified by poly-N-acetyllactosamine glyco-conjugates? Curr. Protein Pept. Sci. 2003, 4, 1–9.
  24. Fukuda, M.; Dell, A.; Oates, J.E.; Fukuda, M.N. Structure of branched lactosaminoglycan, the carbohydrate moiety of band 3 isolated from adult human erythrocytes. J. Biol. Chem. 1984, 259, 8260–8273.
  25. Bua, R.O.; Messina, A.; Sturiale, L.; Barone, R.; Garozzo, D.; Palmigiano, A. N-Glycomics of Human Erythrocytes. Int. J. Mol. Sci. 2021, 22, 8063.
  26. Liukkonen, J.; Haataja, S.; Tikkanen, K.; Kelm, S.; Finne, J. Identification of N-acetylneuraminyl alpha 2-->3 poly-N-acetyllactosamine glycans as the receptors of sialic acid-binding Streptococcus suis strains. J. Biol. Chem. 1992, 267, 21105–21111.
  27. Nycholat, C.M.; McBride, R.; Ekiert, D.C.; Xu, R.; Rangarajan, J.; Peng, W.; Razi, N.; Gilbert, M.; Wakarchuk, W.; Wilson, I.A.; et al. Recognition of Sialylated Poly-N-acetyllactosamine Chains on N- and O-Linked Glycans by Human and Avian Influenza|A Virus Hemagglutinins. Angew. Chem. Int. Ed. 2012, 51, 4860–4863.
  28. Paul, R.W.; Lee, P.W.K. Glycophorin is the reovirus receptor on human erythrocytes. Virology 1987, 159, 94–101.
  29. Korhonen, T.K.; Haahtela, K.; Pirkola, A.; Parkkinen, J. A N-acetyllactosamine-specific cell-binding activity in a plant pathogen, Erwinia rhapontici. FEBS Lett. 1988, 236, 163–166.
  30. Nelson, D.S. Immune Adherence. In Advances in Immunology; Dixon, F.J., Humphrey, J.H., Eds.; Academic Press: Cambridge, MA, USA, 1963; Volume 3, pp. 131–180.
  31. Anderson, H.L.; Brodsky, I.E.; Mangalmurti, N.S. The Evolving Erythrocyte: Red Blood Cells as Modulators of Innate Immunity. J. Immunol. 2018, 201, 1343–1351.
  32. Varki, A.; Gagneux, P. Multifarious roles of sialic acids in immunity. Ann. N. Y. Acad. Sci. 2012, 1253, 16–36.
  33. Nelson, R.A. The Immune-Adherence Phenomenon: An Immunologically Specific Reaction Between Microorganisms and Erythrocytes Leading to Enhanced Phagocytosis. Science 1953, 118, 733–737.
  34. De Back, D.Z.; Kostova, E.; Klei, T.; Beuger, B.; van Zwieten, R.; Kuijpers, T.; Juffermans, N.; van den Berg, T.; Korte, D.; van Kraaij, M.; et al. RBC Adhesive Capacity Is Essential for Efficient ‘Immune Adherence Clearance’ and Provide a Generic Target to Deplete Pathogens from Septic Patients. Blood 2016, 128, 1031.
  35. Siegel, I.; Lin Liu, T.; Gleicher, N. The Red-Cell Immune System. Lancet 1981, 318, 556–559.
  36. Stocker, T.J.; Ishikawa-Ankerhold, H.; Massberg, S.; Schulz, C. Small but mighty: Platelets as central effectors of host defense. Thromb. Haemost. 2017, 117, 651–661.
  37. Unione, L.; Moure, M.J.; Lenza, M.P.; Oyenarte, I.; Ereño-Orbea, J.; Ardá, A.; Jiménez-Barbero, J. The SARS-CoV-2 Spike Glycoprotein Directly Binds Exogeneous Sialic Acids: A NMR View. Angew. Chem. Int. Ed. 2022, 61, e202201432.
  38. Boschi, C.; Scheim, D.E.; Bancod, A.; Militello, M.; Bideau, M.L.; Colson, P.; Fantini, J.; Scola, B.L. SARS-CoV-2 Spike Protein Induces Hemagglutination: Implications for COVID-19 Morbidities and Therapeutics and for Vaccine Adverse Effects. Int. J. Mol. Sci. 2022, 23, 15480.
  39. Reid, M.E.; Mohandas, N. Red blood cell blood group antigens: Structure and function. Semin. Hematol. 2004, 41, 93–117.
  40. Pretini, V.; Koenen, M.H.; Kaestner, L.; Fens, M.H.A.M.; Schiffelers, R.M.; Bartels, M.; Van Wijk, R. Red Blood Cells: Chasing Interactions. Front. Physiol. 2019, 10, 945.
  41. Jaskiewicz, E.; Jodłowska, M.; Kaczmarek, R.; Zerka, A. Erythrocyte glycophorins as receptors for Plasmodium merozoites. Parasites Vectors 2019, 12, 317.
  42. Sikora, M.; von Bülow, S.; Blanc, F.E.C.; Gecht, M.; Covino, R.; Hummer, G. Computational epitope map of SARS-CoV-2 spike protein. PLoS Comput. Biol. 2021, 17, e1008790.
  43. Varki, A.; Schnaar, R.L.; Schauer, R. Chapter 15: Sialic Acids and Other Nonulosonic Acids. In Essentials of Glycobiology, 3rd ed.; Varki, A., Cummings, R., Esko, J., Eds.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 2017.
  44. Soares, C.O.; Grosso, A.S.; Ereño-Orbea, J.; Coelho, H.; Marcelo, F. Molecular Recognition Insights of Sialic Acid Glycans by Distinct Receptors Unveiled by NMR and Molecular Modeling. Front. Mol. Biosci. 2021, 8, 727847.
  45. Cohen, M.; Varki, A. Chapter Three—Modulation of Glycan Recognition by Clustered Saccharide Patches. In International Review of Cell and Molecular Biology; Jeon, K.W., Ed.; Academic Press: Cambridge, MA, USA, 2014; Volume 308, pp. 75–125.
  46. Cohen, M.; Varki, A. The sialome—Far more than the sum of its parts. Omics 2010, 14, 455–464.
  47. Aminpour, M.; Cannariato, M.; Safaeeardebili, M.E.; Preto, J.; Moracchiato, A.; Doria, D.; Donato, F.; Zizzi, E.A.; Deriu, M.A.; Scheim, D.E.; et al. In Silico Analysis of the Multi-Targeted Mode of Action of Ivermectin and Related Compounds. Computation 2022, 10, 51.
  48. Yamakawa, N.; Vanbeselaere, J.; Chang, L.-Y.; Yu, S.-Y.; Ducrocq, L.; Harduin-Lepers, A.; Kurata, J.; Aoki-Kinoshita, K.F.; Sato, C.; Khoo, K.-H.; et al. Systems glycomics of adult zebrafish identifies organ-specific sialylation and glycosylation patterns. Nat. Commun. 2018, 9, 4647.
  49. Au Sam, H.; Storey Brian, D.; Moore John, C.; Tang, Q.; Chen, Y.-L.; Javaid, S.; Sarioglu, A.F.; Sullivan, R.; Madden Marissa, W.; O’Keefe, R.; et al. Clusters of circulating tumor cells traverse capillary-sized vessels. Proc. Natl. Acad. Sci. USA 2016, 113, 4947–4952.
  50. Zheng, Y.; Zhao, J.; Li, J.; Guo, Z.; Sheng, J.; Ye, X.; Jin, G.; Wang, C.; Chai, W.; Yan, J.; et al. SARS-CoV-2 spike protein causes blood coagulation and thrombosis by competitive binding to heparan sulfate. Int. J. Biol. Macromol. 2021, 193, 1124–1129.
  51. Gupta, Y.; Maciorowski, D.; Zak, S.E.; Kulkarni, C.V.; Herbert, A.S.; Durvasula, R.; Fareed, J.; Dye, J.M.; Kempaiah, P. Heparin: A simplistic repurposing to prevent SARS-CoV-2 transmission in light of its in-vitro nanomolar efficacy. Int. J. Biol. Macromol. 2021, 183, 203–212.
  52. Dayer, M.R. Coronavirus (SARS-CoV-2) Deactivation via Spike Glycoprotein Shielding by Old Drugs: Molecular Docking Approach. J. Epigenet. 2021, 2, 31–38.
  53. Lam, L.K.M.; Reilly, J.P.; Rux, A.H.; Murphy, S.J.; Kuri-Cervantes, L.; Weisman, A.R.; Ittner, C.A.G.; Pampena, M.B.; Betts, M.R.; Wherry, E.J.; et al. Erythrocytes identify complement activation in patients with COVID-19. Am. J. Physiol. Lung Cell Mol. Physiol. 2021, 321, L485–L489.
  54. Wang, K.; Chen, W.; Zhang, Z.; Deng, Y.; Lian, J.-Q.; Du, P.; Wei, D.; Zhang, Y.; Sun, X.-X.; Gong, L.; et al. CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells. Signal Transduct. Target. Ther. 2020, 5, 283.
  55. Li, W.; Hulswit, R.J.G.; Widjaja, I.; Raj, V.S.; McBride, R.; Peng, W.; Widagdo, W.; Tortorici, M.A.; van Dieren, B.; Lang, Y.; et al. Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein. Proc. Natl. Acad. Sci. USA 2017, 114, E8508–E8517.
  56. Melkumyants, A.; Buryachkovskaya, L.; Lomakin, N.; Antonova, O.; Serebruany, V. Mild COVID-19 and Impaired Blood Cell–Endothelial Crosstalk: Considering Long-Term Use of Antithrombotics? Thromb. Haemost. 2022, 122, 123–130.
  57. Buryachkovskaya, L.; Lomakin, N.; Melkumyants, A.; Docenko, J.; Ermishkin, V.; Serebruany, V. Enoxaparin dose impacts blood cell phenotypes during mild SARS-CoV-2 infection: The observational single-center study. Rev. Cardiovasc. Med. 2021, 22, 1685–1691.
  58. Melkumyants, A.; Buryachkovskaya, L.; Lomakin, N.; Antonova, O.; Docenko, J.; Ermishkin, V.; Serebruany, V. Effect of Sulodexide on Circulating Blood Cells in Patients with Mild COVID-19. J. Clin. Med. 2022, 11, 1995.
  59. Berzuini, A.; Bianco, C.; Migliorini, A.C.; Maggioni, M.; Valenti, L.; Prati, D. Red blood cell morphology in patients with COVID-19-related anaemia. Blood Transfus. 2021, 19, 34–36.
  60. Favaron, E.; Ince, C.; Hilty, M.P.; Ergin, B.; van der Zee, P.; Uz, Z.; Wendel Garcia, P.D.; Hofmaenner, D.A.; Acevedo, C.T.; van Boven, W.J.; et al. Capillary Leukocytes, Microaggregates, and the Response to Hypoxemia in the Microcirculation of Coronavirus Disease 2019 Patients. Crit. Care Med. 2021, 49, 661–670.
  61. Li, H.; Deng, Y.; Li, Z.; Dorken Gallastegi, A.; Mantzoros, C.S.; Frydman, G.H.; Karniadakis, G.E. Multiphysics and multiscale modeling of microthrombosis in COVID-19. PLoS Comput. Biol. 2022, 18, e1009892.
  62. Rapkiewicz, A.V.; Mai, X.; Carsons, S.E.; Pittaluga, S.; Kleiner, D.E.; Berger, J.S.; Thomas, S.; Adler, N.M.; Charytan, D.M.; Gasmi, B.; et al. Megakaryocytes and platelet-fibrin thrombi characterize multi-organ thrombosis at autopsy in COVID-19: A case series. eClinicalMedicine 2020, 24, 100434.
  63. Wichmann, D.; Sperhake, J.-P.; Lütgehetmann, M.; Steurer, S.; Edler, C.; Heinemann, A.; Heinrich, F.; Mushumba, H.; Kniep, I.; Schröder, A.S.; et al. Autopsy Findings and Venous Thromboembolism in Patients With COVID-19. Ann. Intern. Med. 2020, 173, 268–277.
  64. Ackermann, M.; Verleden, S.E.; Kuehnel, M.; Haverich, A.; Welte, T.; Laenger, F.; Vanstapel, A.; Werlein, C.; Stark, H.; Tzankov, A.; et al. Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in COVID-19. N. Engl. J. Med. 2020, 383, 120–128.
  65. Fox, S.E.; Akmatbekov, A.; Harbert, J.L.; Li, G.; Quincy Brown, J.; Vander Heide, R.S. Pulmonary and cardiac pathology in African American patients with COVID-19: An autopsy series from New Orleans. Lancet Respir. Med. 2020, 8, 681–686.
  66. Hanff, T.C.; Mohareb, A.M.; Giri, J.; Cohen, J.B.; Chirinos, J.A. Thrombosis in COVID-19. Am. J. Hematol. 2020, 95, 1578–1589.
  67. Fahmy, O.H.; Daas, F.M.; Salunkhe, V.; Petrey, J.L.; Cosar, E.F.; Ramirez, J.; Akca, O. Is Microthrombosis the Main Pathology in Coronavirus Disease 2019 Severity?-A Systematic Review of the Postmortem Pathologic Findings. Crit. Care Explor. 2021, 3, e0427.
  68. Menter, T.; Haslbauer, J.D.; Nienhold, R.; Savic, S.; Hopfer, H.; Deigendesch, N.; Frank, S.; Turek, D.; Willi, N.; Pargger, H.; et al. Postmortem examination of COVID-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction. Histopathology 2020, 77, 198–209.
  69. McFadyen, J.D.; Stevens, H.; Peter, K. The Emerging Threat of (Micro)Thrombosis in COVID-19 and Its Therapeutic Implications. Circ. Res. 2020, 127, 571–587.
  70. Poh, K.C.; Jia Tay, V.Y.; Lin, S.H.; Chee, H.L.; Thangavelautham, S. A review of COVID-19-related thrombosis and anticoagulation strategies specific to the Asian population. Singap. Med. J. 2022, 63, 350–361.
  71. Bussani, R.; Schneider, E.; Zentilin, L.; Collesi, C.; Ali, H.; Braga, L.; Volpe, M.C.; Colliva, A.; Zanconati, F.; Berlot, G.; et al. Persistence of viral RNA, pneumocyte syncytia and thrombosis are hallmarks of advanced COVID-19 pathology. eBioMedicine 2020, 61, 103104.
  72. Overton, P.M.; Toshner, M.; Mulligan, C.; Vora, P.; Nikkho, S.; de Backer, J.; Lavon, B.R.; Klok, F.A.; the PVRI Innovative Drug Development Initiative. Pulmonary thromboembolic events in COVID-19—A systematic literature review. Pulm. Circ. 2022, 12, e12113.
  73. Gattinoni, L.; Gattarello, S.; Steinberg, I.; Busana, M.; Palermo, P.; Lazzari, S.; Romitti, S.; Quintel, M.; Meissner, K.; Marini, J.J.; et al. COVID-19 pneumonia: Pathophysiology and management. Eur. Respir. Rev. 2021, 30, 210138.
  74. Poor, H.D. Pulmonary Thrombosis and Thromboembolism in COVID-19. Chest 2021, 160, 1471–1480.
  75. Petrilli, C.M.; Jones, S.A.; Yang, J.; Rajagopalan, H.; O’Donnell, L.; Chernyak, Y.; Tobin, K.A.; Cerfolio, R.J.; Francois, F.; Horwitz, L.I. Factors associated with hospital admission and critical illness among 5279 people with coronavirus disease 2019 in New York City: Prospective cohort study. BMJ 2020, 369, m1966.
  76. Magro, C.; Mulvey, J.J.; Berlin, D.; Nuovo, G.; Salvatore, S.; Harp, J.; Baxter-Stoltzfus, A.; Laurence, J. Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases. Transl. Res. 2020, 220, 1–13.
  77. Nuovo, G.J.; Magro, C.; Shaffer, T.; Awad, H.; Suster, D.; Mikhail, S.; He, B.; Michaille, J.-J.; Liechty, B.; Tili, E. Endothelial cell damage is the central part of COVID-19 and a mouse model induced by injection of the S1 subunit of the spike protein. Ann. Diagn. Pathol. 2021, 51, 151682.
  78. Ko, C.J.; Harigopal, M.; Gehlhausen, J.R.; Bosenberg, M.; McNiff, J.M.; Damsky, W. Discordant anti-SARS-CoV-2 spike protein and RNA staining in cutaneous perniotic lesions suggests endothelial deposition of cleaved spike protein. J. Cutan. Pathol. 2021, 48, 47–52.
  79. Liu, F.; Han, K.; Blair, R.; Kenst, K.; Qin, Z.; Upcin, B.; Wörsdörfer, P.; Midkiff, C.C.; Mudd, J.; Belyaeva, E.; et al. SARS-CoV-2 Infects Endothelial Cells In Vivo and In Vitro. Front. Cell. Infect. Microbiol. 2021, 11, 701278.
  80. Magro, C.M.; Mulvey, J.J.; Laurence, J.; Seshan, S.; Crowson, A.N.; Dannenberg, A.J.; Salvatore, S.; Harp, J.; Nuovo, G.J. Docked severe acute respiratory syndrome coronavirus 2 proteins within the cutaneous and subcutaneous microvasculature and their role in the pathogenesis of severe coronavirus disease 2019. Hum. Pathol. 2020, 106, 106–116.
  81. Perico, L.; Benigni, A.; Remuzzi, G. SARS-CoV-2 and the spike protein in endotheliopathy. Trends Microbiol. 2023.
  82. Blom, J.A. Monitoring of Respiration and Circulation; CRC Press: Boca Raton, FL, USA, 2003; p. 27.
  83. Guest, M.M.; Bond, T.P.; Cooper, R.G.; Derrick, J.R. Red Blood Cells: Change in Shape in Capillaries. Science 1963, 142, 1319–1321.
  84. Shoemaker, W.C.; Brunius, U.; Gelin, L.-E. Hemodynamic and microcirculatory effects of high and low viscosity dextrans. Surgery 1965, 58, 518–523.
  85. Stalker, A.L. Histological changes produced by experimental erythrocyte aggregation. J. Pathol. Bacteriol. 1967, 93, 203–212.
  86. Stalker, A.L. The microcirculatory effects of dextran. J. Pathol. Bacteriol. 1967, 93, 191–201.
  87. Fajers, C.M.; Gelin, L.E. Kidney-, liver- and heart-damages from trauma and from induced intravascular aggregation of blood-cells: An experimental study. Acta Pathol. Microbiol. Scand. 1959, 46, 97–104.
  88. Swank, R.L.; Fellman, J.H.; Hissen, W.W. Aggregation of Blood Cells by 5-Hydroxytryptamine (Serotonin). Circ. Res. 1963, 13, 392–400.
  89. Cullen, C.F.; Swank, R.L. Intravascular Aggregation and Adhesiveness of the Blood Elements Associated with Alimentary Lipemia and Injections of Large Molecular Substances. Circulation 1954, 9, 335–346.
  90. Pribush, A.; Zilberman-Kravits, D.; Meyerstein, N. The mechanism of the dextran-induced red blood cell aggregation. Eur. Biophys. J. 2007, 36, 85–94.
  91. Zhu, R.; Avsievich, T.; Bykov, A.; Popov, A.; Meglinski, I. Influence of Pulsed He–Ne Laser Irradiation on the Red Blood Cell Interaction Studied by Optical Tweezers. Micromachines 2019, 10, 853.
  92. Lee, K.; Shirshin, E.; Rovnyagina, N.; Yaya, F.; Boujja, Z.; Priezzhev, A.; Wagner, C. Dextran adsorption onto red blood cells revisited: Single cell quantification by laser tweezers combined with microfluidics. Biomed. Opt. Express 2018, 9, 2755–2764.
  93. Chien, S.; Jan, K.-m. Ultrastructural basis of the mechanism of rouleaux formation. Microvasc. Res. 1973, 5, 155–166.
  94. Zhen, Z.-Y.; Guo, Y.-C.; Zhang, Z.-G.; Liang, Y.; Ge, P.-J.; Jin, H.-M. Experimental Study on Microthrombi and Myocardial Injuries. Microvasc. Res. 1996, 51, 99–107.
  95. Reinke, W.; Gaehtgens, P.; Johnson, P.C. Blood viscosity in small tubes: Effect of shear rate, aggregation, and sedimentation. Am. J. Physiol.-Heart Circ. Physiol. 1987, 253, H540–H547.
  96. Volger, E.; Schmid-Schönbein, H.; Gosen, J.v.; Klose, H.J.; Kline, K.A. Microrheology and light transmission of blood. Pflügers Arch. 1975, 354, 319–337.
  97. Bosek, M.; Ziomkowska, B.; Pyskir, J.; Wybranowski, T.; Pyskir, M.; Cyrankiewicz, M.; Napiórkowska, M.; Durmowicz, M.; Kruszewski, S. Relationship between red blood cell aggregation and dextran molecular mass. Sci. Rep. 2022, 12, 19751.
  98. Neu, B.; Wenby, R.; Meiselman, H.J. Effects of dextran molecular weight on red blood cell aggregation. Biophys. J. 2008, 95, 3059–3065.
  99. Engeset, J.; Stalker, A.L.; Matheson, N.A. Objective measurement of the dispersing effect of dextran 40 on red cells from man, dog, and rabbit. Cardiovasc. Res. 1967, 1, 385–388.
  100. Baskurt, O.K.; Meiselman, H.J. Erythrocyte aggregation: Basic aspects and clinical importance. Clin. Hemorheol. Microcirc. 2013, 53, 23–37.
  101. Barshtein, G.; Wajnblum, D.; Yedgar, S. Kinetics of linear rouleaux formation studied by visual monitoring of red cell dynamic organization. Biophys. J. 2000, 78, 2470–2474.
  102. Fung, Y.-C. Chapter 3: The flow properties of blood. In Biomechanics: Mechanical Properties of Living Tissues; Springer: New York, NY, USA, 1993; pp. 66–108.
  103. Bicher, H.I. Chapter I: Red cell aggregation in thrombotic disease, trauma and shock. In Blood Cell Aggregation in Thrombotic Processes; C. C. Thomas: Springfield, IL, USA, 1972; Volume I, pp. 5–18.
  104. Bicher, H.I. Chapter III: Mechanism of red cell aggregation. In Blood Cell Aggregation in Thrombotic Processes; C. C. Thomas: Springfield, IL, USA, 1972; Volume III, pp. 47–61.
  105. Brooks, D.E.; Evans, E.A. Rheology of blood cells. In Clinical Hemorheology: Applications in Cardiovascular and Hematological Disease, Diabetes, Surgery and Gynecology; Chien, S., Dormandy, J., Ernst, E., Matrai, A., Eds.; Springer: Dordrecht, The Netherlands, 1987; pp. 73–96.
  106. Lowe, G.D.O. Thrombosis and hemorheology. In Clinical Hemorheology: Applications in Cardiovascular and Hematological Disease, Diabetes, Surgery and Gynecology; Chien, S., Dormandy, J., Ernst, E., Matrai, A., Eds.; Springer: Dordrecht, The Netherlands, 1987; pp. 195–226.
  107. Bishop, J.J.; Nance, P.R.; Popel, A.S.; Intaglietta, M.; Johnson, P.C. Effect of erythrocyte aggregation on velocity profiles in venules. Am. J. Physiol.-Heart Circ. Physiol. 2001, 280, H222–H236.
  108. Chien, S.; Sung, L.A. Physicochemical basis and clinical implications of red cell aggregation. Clin. Hemorheol. Microcirc. 1987, 7, 71–91.
  109. Maeda, N.; Seike, M.; Kon, K.; Shiga, T. Erythrocyte Aggregation as a Determinant of Blood Flow: Effect of pH, Temperature and Osmotic Pressure. In Oxygen Transport to Tissue X; Mochizuki, M., Honig, C.R., Koyama, T., Goldstick, T.K., Bruley, D.F., Eds.; Springer US: New York, NY, USA, 1988; pp. 563–570.
  110. Sakariassen, K.S.; Orning, L.; Turitto, V.T. The impact of blood shear rate on arterial thrombus formation. Future Sci. OA 2015, 1, FSO30.
  111. Swank, R.L.; Cullen, C.F. Circulatory Changes in the Hamster’s Cheek Pouch Associated with Alimentary Lipemia. Proc. Soc. Exp. Biol. Med. 1953, 82, 381–384.
  112. Knisely, M.H.; Bloch, E.H.; Eliot, T.S.; Warner, L. Sludged Blood. Science 1947, 106, 431–440.
  113. Bicher, H.I. Chapter II: Pathological significance of intravascular red cell aggregation. In Blood Cell Aggregation in Thrombotic Processes; C. C. Thomas: Springfield, IL, USA, 1972; pp. 19–46.
  114. Barshtein, G.; Ben-Ami, R.; Yedgar, S. Role of red blood cell flow behavior in hemodynamics and hemostasis. Expert Rev. Cardiovasc. Ther. 2007, 5, 743–752.
  115. Ditzel, J.; Sagild, U. Morphologic and hemodynamic changes in the smaller blood vessels in diabetes mellitus. II. The degenerative and hemodynamic changes in the bulbar conjunctiva of normotensive diabetic patients. N. Engl. J. Med. 1954, 250, 587–594.
  116. Yu, F.T.; Armstrong, J.K.; Tripette, J.; Meiselman, H.J.; Cloutier, G. A local increase in red blood cell aggregation can trigger deep vein thrombosis: Evidence based on quantitative cellular ultrasound imaging. J. Thromb. Haemost. 2011, 9, 481–488.
  117. Byrnes, J.R.; Wolberg, A.S. Red blood cells in thrombosis. Blood 2017, 130, 1795–1799.
  118. Ami, R.B.; Barshtein, G.; Zeltser, D.; Goldberg, Y.; Shapira, I.; Roth, A.; Keren, G.; Miller, H.; Prochorov, V.; Eldor, A.; et al. Parameters of red blood cell aggregation as correlates of the inflammatory state. Am. J. Physiol.-Heart Circ. Physiol. 2001, 280, H1982–H1988.
  119. Wagner, C.; Steffen, P.; Svetina, S. Aggregation of red blood cells: From rouleaux to clot formation. C. R. Phys. 2013, 14, 459–469.
  120. Meiselman, H.J. Red blood cell aggregation: 45 years being curious. Biorheology 2009, 46, 1–19.
  121. Junxiu, Z.; Yu, F.; Shaodan, L.; Yi, L.; Yin, Z.; Yunxia, G.; Minghui, Y. Microvascular pathological features and changes in related injury factors in a rat acute blood stasis model. J. Tradit. Chin. Med. 2017, 37, 108–115.
  122. Kim, S.; Popel, A.S.; Intaglietta, M.; Johnson, P.C. Aggregate formation of erythrocytes in postcapillary venules. Am. J. Physiol.-Heart Circ. Physiol. 2005, 288, H584–H590.
  123. Swank, R.L. Suspension Stability of the Blood After Injections of Dextran. J. Appl. Physiol. 1958, 12, 125–128.
  124. Brånemark, P.-I. Experimental Investigation of Microcirculation in Bone Marrow. Angiology 1961, 12, 293–305.
  125. Engeset, J.; Stalker, A.L.; Matheson, N.A. Effects of Dextran 40 on Red Cell Aggregation in Rabbits. Cardiovasc. Res. 1967, 1, 379–384.
  126. Swank, R.L.; Escobar, A. Effects of Dextran Injections on Blood Viscosity in Dogs. J. Appl. Physiol. 1957, 10, 45–50.
  127. Bicher, H.I.; Bruley, D.; Knisely, M.H.; Reneau, D.D. Effect of microcirculation changes on brain tissue oxygenation. J. Physiol. 1971, 217, 689–707.
  128. Hysi, E.; Saha, R.K.; Kolios, M.C. Photoacoustic ultrasound spectroscopy for assessing red blood cell aggregation and oxygenation. J. Biomed. Opt. 2012, 17, 125006.
  129. Tang, N.; Li, D.; Wang, X.; Sun, Z. Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia. J. Thromb. Haemost. 2020, 18, 844–847.
  130. Pellegrini, D.; Kawakami, R.; Guagliumi, G.; Sakamoto, A.; Kawai, K.; Gianatti, A.; Nasr, A.; Kutys, R.; Guo, L.; Cornelissen, A.; et al. Microthrombi as a Major Cause of Cardiac Injury in COVID-19: A Pathologic Study. Circulation 2021, 143, 1031–1042.
  131. Mikami, T.; Miyashita, H.; Yamada, T.; Harrington, M.; Steinberg, D.; Dunn, A.; Siau, E. Risk Factors for Mortality in Patients with COVID-19 in New York City. J. Gen. Intern. Med. 2020, 36, 17–26.
  132. Lowenstein, C.J.; Solomon, S.D. Severe COVID-19 Is a Microvascular Disease. Circulation 2020, 142, 1609–1611.
  133. Sastry, S.; Cuomo, F.; Muthusamy, J. COVID-19 and thrombosis: The role of hemodynamics. Thromb. Res. 2022, 212, 51–57.
  134. Avila, J.; Long, B.; Holladay, D.; Gottlieb, M. Thrombotic complications of COVID-19. Am. J. Emerg. Med. 2021, 39, 213–218.
  135. Al-Samkari, H.; Karp Leaf, R.S.; Dzik, W.H.; Carlson, J.C.T.; Fogerty, A.E.; Waheed, A.; Goodarzi, K.; Bendapudi, P.K.; Bornikova, L.; Gupta, S.; et al. COVID-19 and coagulation: Bleeding and thrombotic manifestations of SARS-CoV-2 infection. Blood 2020, 136, 489–500.
  136. Dolhnikoff, M.; Duarte-Neto, A.N.; de Almeida Monteiro, R.A.; da Silva, L.F.F.; de Oliveira, E.P.; Saldiva, P.H.N.; Mauad, T.; Negri, E.M. Pathological evidence of pulmonary thrombotic phenomena in severe COVID-19. J. Thromb. Haemost. 2020, 18, 1517–1519.
  137. Maier, C.L.; Truong, A.D.; Auld, S.C.; Polly, D.M.; Tanksley, C.-L.; Duncan, A. COVID-19-associated hyperviscosity: A link between inflammation and thrombophilia? Lancet 2020, 395, 1758–1759.
  138. Rovas, A.; Osiaevi, I.; Buscher, K.; Sackarnd, J.; Tepasse, P.-R.; Fobker, M.; Kühn, J.; Braune, S.; Göbel, U.; Thölking, G.; et al. Microvascular dysfunction in COVID-19: The MYSTIC study. Angiogenesis 2021, 24, 145–157.
  139. Osiaevi, I.; Schulze, A.; Evers, G.; Harmening, K.; Vink, H.; Kümpers, P.; Mohr, M.; Rovas, A. Persistent capillary rarefication in long COVID syndrome. Angiogenesis 2023, 26, 53–61.
  140. Wu, R.; Ai, S.; Cai, J.; Zhang, S.; Qian, Z.M.; Zhang, Y.; Wu, Y.; Chen, L.; Tian, F.; Li, H.; et al. Predictive Model and Risk Factors for Case Fatality of COVID-19: A Cohort of 21,392 Cases in Hubei, China. Innovation 2020, 1, 100022.
  141. Zhou, F.; Yu, T.; Du, R.; Fan, G.; Liu, Y.; Liu, Z.; Xiang, J.; Wang, Y.; Song, B.; Gu, X.; et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: A retrospective cohort study. Lancet 2020, 395, 1054–1062.
  142. Williamson, E.J.; Walker, A.J.; Bhaskaran, K.; Bacon, S.; Bates, C.; Morton, C.E.; Curtis, H.J.; Mehrkar, A.; Evans, D.; Inglesby, P.; et al. OpenSAFELY: Factors associated with COVID-19 death in 17 million patients. Nature 2020, 584, 430–436.
  143. Bonanad, C.; García-Blas, S.; Tarazona-Santabalbina, F.; Sanchis, J.; Bertomeu-González, V.; Fácila, L.; Ariza, A.; Núñez, J.; Cordero, A. The Effect of Age on Mortality in Patients With COVID-19: A Meta-Analysis With 611,583 Subjects. J. Am. Med. Dir. Assoc. 2020, 21, 915–918.
  144. Ditzel, J. Angioscopic Changes in the Smaller Blood Vessels in Diabetes Mellitus and their Relationship to Aging. Circulation 1956, 14, 386–397.
  145. Manetta, J.; Aloulou, I.; Varlet-Marie, E.; Mercier, J.; Brun, J.F. Partially opposite hemorheological effects of aging and training at middle age. Clin. Hemorheol. Microcirc. 2006, 35, 239–244.
  146. Richardson, D.; Schwartz, R. Comparison of capillary blood flow in the nailfold circulations of young and elderly men. AGE 1985, 8, 70.
  147. Richardson, D.; Shepherd, S. The cutaneous microcirculation of the forearm in young and old subjects. Microvasc. Res. 1991, 41, 84–91.
  148. Tsuchida, Y. The effect of aging and arteriosclerosis on human skin blood flow. J. Dermatol. Sci. 1993, 5, 175–181.
  149. Ajmani, R.S.; Rifkind, J.M. Hemorheological changes during human aging. Gerontology 1998, 44, 111–120.
  150. Dinenno, F.A.; Jones, P.P.; Seals, D.R.; Tanaka, H. Limb Blood Flow and Vascular Conductance Are Reduced With Age in Healthy Humans. Circulation 1999, 100, 164–170.
  151. Krejza, J.; Mariak, Z.; Walecki, J.; Szydlik, P.; Lewko, J.; Ustymowicz, A. Transcranial color Doppler sonography of basal cerebral arteries in 182 healthy subjects: Age and sex variability and normal reference values for blood flow parameters. AJR Am. J. Roentgenol. 1999, 172, 213–218.
  152. Ackerstaff, R.G.A.; Keunen, R.W.M.; Pelt, W.v.; Swijndregt, A.D.M.v.; Stijnen, T. Influence of biological factors on changes in mean cerebral blood flow velocity in normal ageing: A transcranial Doppler study. Neurol. Res. 1990, 12, 187–191.
  153. Bicher, H.I.; Beemer, A.M. Induction of ischemic myocardial damage by red blood cell aggregation (sludge) in the rabbit. J. Atheroscler Res. 1967, 7, 409–414.
  154. Yamamoto, M.; Kase, M.; Sano, H.; Kamijima, R.; Sano, S. Persistent varicella zoster virus infection following mRNA COVID-19 vaccination was associated with the presence of encoded spike protein in the lesion. J. Cutan. Immunol. Allergy 2023, 6, 18–23.
  155. Sano, H.; Kase, M.; Aoyama, Y.; Sano, S. A case of persistent, confluent maculopapular erythema following a COVID-19 mRNA vaccination is possibly associated with the intralesional spike protein expressed by vascular endothelial cells and eccrine glands in the deep dermis. J. Dermatol. 2023, 50, 1208–1212.
  156. Morz, M. A Case Report: Multifocal Necrotizing Encephalitis and Myocarditis after BNT162b2 mRNA Vaccination against COVID-19. Vaccines 2022, 10, 1651.
  157. Li, C.; Chen, Y.; Zhao, Y.; Lung, D.C.; Ye, Z.; Song, W.; Liu, F.-F.; Cai, J.-P.; Wong, W.-M.; Yip, C.C.-Y.; et al. Intravenous Injection of Coronavirus Disease 2019 (COVID-19) mRNA Vaccine Can Induce Acute Myopericarditis in Mouse Model. Clin. Infect. Dis. 2022, 74, 1933–1950.
  158. Gedik, B.; Bozdogan, Y.C.; Yavuz, S.; Durmaz, D.; Erol, M.K. The assesment of retina and optic disc vascular structures in people who received CoronaVac vaccine. Photodiagn. Photodyn. Ther. 2022, 38, 102742.
  159. Saritas, O.; Yorgun, M.A.; Gokpinar, E. Effects of Sinovac-Coronavac and Pfizer-BioNTech mRNA vaccines on choroidal and retinal vascular system. Photodiagn. Photodyn. Ther. 2023, 43, 103702.
  160. Gedik, B.; Erol, M.K.; Suren, E.; Yavuz, S.; Kucuk, M.F.; Bozdogan, Y.C.; Ekinci, R.; Akidan, M. Evaluation of retinal and optic disc vascular structures in individuals before and after Pfizer-BioNTech vaccination. Microvasc. Res. 2023, 147, 104500.
  161. Da Silva, L.S.C.; Finamor, L.P.S.; Andrade, G.C.; Lima, L.H.; Zett, C.; Muccioli, C.; Sarraf, E.P. Vascular retinal findings after COVID-19 vaccination in 11 cases: A coincidence or consequence? Arq. Bras. Oftalmol. 2022, 85, 158–165.
  162. Haseeb, A.A.; Solyman, O.; Abushanab, M.M.; Abo Obaia, A.S.; Elhusseiny, A.M. Ocular Complications Following Vaccination for COVID-19: A One-Year Retrospective. Vaccines 2022, 10, 342.
  163. Bjoerk, V.O.; Intonti, F.; Nordlund, S. Correlation between sludge in the bulbar conjunctiva and the mesentery. Ann. Surg. 1964, 159, 428–431.
  164. Haider, A.; Bengs, S.; Schade, K.; Wijnen, W.J.; Portmann, A.; Etter, D.; Fröhlich, S.; Warnock, G.I.; Treyer, V.; Burger, I.A.; et al. Myocardial 18F-FDG Uptake Pattern for Cardiovascular Risk Stratification in Patients Undergoing Oncologic PET/CT. J. Clin. Med. 2020, 9, 2279.
  165. Yao, Y.; Li, Y.-M.; He, Z.-X.; Civelek, A.C.; Li, X.-F. Likely Common Role of Hypoxia in Driving 18F-FDG Uptake in Cancer, Myocardial Ischemia, Inflammation and Infection. Cancer Biother. Radiopharm. 2021, 36, 624–631.
  166. Prominent Researchers Look Deeper into Fluvoxamine and See Potential as COVID-19 Treatment. Trialsite News. 25 April 2021. Available online: https://trialsitenews.com/prominent-researchers-look-deeper-into-fluvoxamine-see-potential-as-covid-19-treatment/ (accessed on 30 October 2023).
  167. Johnson, C.K. Cheap Antidepressant Shows Promise Treating Early COVID-19. Yahoo News. 27 October 2021. Available online: News.yahoo.com/cheap-antidepressant-shows-promise-treating-223735055.html (accessed on 30 October 2023).
  168. Sukhatme, V.P.; Reiersen, A.M.; Vayttaden, S.J.; Sukhatme, V.V. Fluvoxamine: A Review of Its Mechanism of Action and Its Role in COVID-19. Front. Pharmacol. 2021, 12, 763.
  169. Seftel, D.; Boulware, D.R. Prospective Cohort of Fluvoxamine for Early Treatment of Coronavirus Disease 19. Open Forum Infect. Dis. 2021, 8, ofab050.
  170. Facente, S.N.; Reiersen, A.M.; Lenze, E.J.; Boulware, D.R.; Klausner, J.D. Fluvoxamine for the Early Treatment of SARS-CoV-2 Infection: A Review of Current Evidence. Drugs 2021, 81, 2081–2089.
  171. Reis, G.; Moreira Silva, E.A.; Medeiros Silva, D.C.; Thabane, L.; Milagres, A.C.; Ferreira, T.S.; dos Santos, C.V.Q.; de Souza Campos, V.H.; Nogueira, A.M.R.; de Almeida, A.P.F.G.; et al. Effect of early treatment with fluvoxamine on risk of emergency care and hospitalisation among patients with COVID-19: The TOGETHER randomised, platform clinical trial. Lancet Glob. Health 2022, 10, e42–e51.
  172. Lenze, E.J.; Mattar, C.; Zorumski, C.F.; Stevens, A.; Schweiger, J.; Nicol, G.E.; Miller, J.P.; Yang, L.; Yingling, M.; Avidan, M.S.; et al. Fluvoxamine vs Placebo and Clinical Deterioration in Outpatients With Symptomatic COVID-19: A Randomized Clinical Trial. JAMA 2020, 324, 2292–2300.
  173. Swank, R.L.; Edwards, M.J. Microvascular occlusion by platelet emboli after transfusion and shock. Microvasc. Res. 1968, 1, 15–22.
  174. Gautret, P.; Lagier, J.-C.; Parola, P.; Hoang, V.T.; Meddeb, L.; Mailhe, M.; Doudier, B.; Courjon, J.; Giordanengo, V.; Vieira, V.E.; et al. Hydroxychloroquine and azithromycin as a treatment of COVID-19: Results of an open-label non-randomized clinical trial. Int. J. Antimicrob. Agents 2020, 56, 105949.
  175. Gendrot, M.; Andreani, J.; Jardot, P.; Hutter, S.; Delandre, O.; Boxberger, M.; Mosnier, J.; Le Bideau, M.; Duflot, I.; Fonta, I.; et al. In Vitro Antiviral Activity of Doxycycline against SARS-CoV-2. Molecules 2020, 25, 5064.
  176. Million, M.; Cortaredona, S.; Delorme, L.; Colson, P.; Levasseur, A.; Hervé, T.-D.; Karim, B.; Salima, L.; Bernard La, S.; Laurence, C.-J.; et al. Early Treatment with Hydroxychloroquine and Azithromycin: A ‘Real-Life’ Monocentric Retrospective Cohort Study of 30,423 COVID-19 Patients. medRxiv 2023.
  177. Madow, B.P. Use of antimalarial drugs as “desludging” agents in vascular disease processes: Preliminary report. JAMA 1960, 172, 1630–1633.
  178. Nallusamy, S.; Mannu, J.; Ravikumar, C.; Angamuthu, K.; Nathan, B.; Nachimuthu, K.; Ramasamy, G.; Muthurajan, R.; Subbarayalu, M.; Neelakandan, K. Exploring Phytochemicals of Traditional Medicinal Plants Exhibiting Inhibitory Activity Against Main Protease, Spike Glycoprotein, RNA-dependent RNA Polymerase and Non-Structural Proteins of SARS-CoV-2 Through Virtual Screening. Front. Pharmacol. 2021, 12, 667704.
  179. Kalhor, H.; Sadeghi, S.; Abolhasani, H.; Kalhor, R.; Rahimi, H. Repurposing of the approved small molecule drugs in order to inhibit SARS-CoV-2 S protein and human ACE2 interaction through virtual screening approaches. J. Biomol. Struct. Dyn. 2020, 40, 1299–1315.
  180. Suravajhala, R.; Parashar, A.; Malik, B.; Nagaraj, V.A.; Padmanaban, G.; Kavi Kishor, P.B.; Polavarapu, R.; Suravajhala, P. Comparative Docking Studies on Curcumin with COVID-19 Proteins. Preprints.Org 2020.
  181. Yagisawa, M.; Foster, P.J.; Hanaki, H.; Omura, S. Global Trends in Clinical Studies of Ivermectin in COVID-19. Jpn. J. Antibiot. 2021, 74, 44–95.
  182. Juarez, M.; Schcolnik-Cabrera, A.; Dueñas-Gonzalez, A. The multitargeted drug ivermectin: From an antiparasitic agent to a repositioned cancer drug. Am. J. Cancer Res. 2018, 8, 317–331.
  183. Campbell, W.C. History of avermectin and ivermectin, with notes on the history of other macrocyclic lactone antiparasitic agents. Curr. Pharm. Biotechnol. 2012, 13, 853–865.
  184. Chang, M.W.; Lindstrom, W.; Olson, A.J.; Belew, R.K. Analysis of HIV Wild-Type and Mutant Structures via in Silico Docking against Diverse Ligand Libraries. J. Chem. Inf. Model. 2007, 47, 1258–1262.
  185. Dasgupta, J.; Sen, U.; Bakashi, A.; Dasgupta, A. Nsp7 and Spike Glycoprotein of SARS-CoV-2 Are Envisaged as Potential Targets of Vitamin D and Ivermectin. Preprints.Org 2020.
  186. Hussien, M.A.; Abdelaziz, A.E.M. Molecular docking suggests repurposing of brincidofovir as a potential drug targeting SARS-CoV-2 ACE2 receptor and main protease. Netw. Model. Anal. Health Inform. Bioinform. 2020, 9, 56.
  187. Kaur, H.; Shekhar, N.; Sharma, S.; Sarma, P.; Prakash, A.; Medhi, B. Ivermectin as a potential drug for treatment of COVID-19: An in-sync review with clinical and computational attributes. Pharmacol. Rep. 2021, 73, 736–749.
  188. Maurya, D. A Combination of Ivermectin and Doxycycline Possibly Blocks the Viral Entry and Modulate the Innate Immune Response in COVID-19 Patients. ChemRxiv 2020.
  189. Saha, J.K.; Raihan, J. The Binding mechanism of Ivermectin and levosalbutamol with spike protein of SARS-CoV-2. Struct. Chem. 2021, 32, 1985–1992.
  190. Lehrer, S.; Rheinstein, P.H. Ivermectin Docks to the SARS-CoV-2 Spike Receptor-binding Domain Attached to ACE2. In Vivo 2020, 34, 3023–3026.
  191. Rajter, J.J.; (Broward Health Medical Center, Fort Lauderdale, FL, USA). Personal communication, 28 May 2020.
  192. Local Doctor Tries New Coronavirus Drug Treatment. NBC Miami News. 14 April 2020. Available online: https://www.nbcmiami.com/news/local/local-doctor-tries-new-coronavirus-drug-treatment/2219465/ (accessed on 30 October 2023).
  193. Rajter, J.C.; Sherman, M.S.; Fatteh, N.; Vogel, F.; Sacks, J.; Rajter, J.-J. Use of Ivermectin is Associated with Lower Mortality in Hospitalized Patients with COVID-19 (ICON study). Chest 2020, 159, 85–92.
  194. Santin, A.D.; Scheim, D.E.; McCullough, P.A.; Yagisawa, M.; Borody, T.J. Ivermectin: A multifaceted drug of Nobel prize-honored distinction with indicated efficacy against a new global scourge, COVID-19. New Microbes New Infect. 2021, 43, 100924.
  195. Abdool Karim, S.S.; Devnarain, N. Time to Stop Using Ineffective COVID-19 Drugs. N. Engl. J. Med. 2022, 387, 654–655.
  196. Shafiee, A.; Teymouri Athar, M.M.; Kohandel Gargari, O.; Jafarabady, K.; Siahvoshi, S.; Mozhgani, S.-H. Ivermectin under scrutiny: A systematic review and meta-analysis of efficacy and possible sources of controversies in COVID-19 patients. Virol. J. 2022, 19, 102.
  197. Reis, G.; Moreira Silva, E.A.; Medeiros Silva, D.C.; Thabane, L.; Milagres, A.C.; Ferreira, T.S.; dos Santos, C.V.Q.; Campos, V.H.S.; Nogueira, A.M.R.; de Almeida, A.P.F.G.; et al. Effect of Early Treatment with Ivermectin among Patients with COVID-19. N. Engl. J. Med. 2022, 386, 1721–1731.
  198. Scheim, D.E.; Aldous, C.; Osimani, B.; Fordham, E.J.; Hoy, W.E. When Characteristics of Clinical Trials Require Per-Protocol as Well as Intention-to-Treat Outcomes to Draw Reliable Conclusions: Three Examples. J. Clin. Med. 2023, 12, 3625.
  199. U.S. Food & Drug Administration. Memorandum Explaining Basis for Declining Request for Emergency Use Authorization of Fluvoxamine Maleate. Available online: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2020/EUA%20110%20Fluvoxamine%20Decisional%20Memo_Redacted.pdf (accessed on 30 October 2023).
  200. NIH COVID-19 Treatment Guidelines. Fluvoxamine: Selected Clinical Data, Limitations and Interpretation. Table 4c. 16 December 2021. Available online: https://www.covid19treatmentguidelines.nih.gov/tables/fluvoxamine-data/ (accessed on 30 October 2023).
  201. López-Medina, E.; López, P.; Hurtado, I.C.; Dávalos, D.M.; Ramirez, O.; Martínez, E.; Díazgranados, J.A.; Oñate, J.M.; Chavarriaga, H.; Herrera, S.; et al. Effect of Ivermectin on Time to Resolution of Symptoms Among Adults With Mild COVID-19: A Randomized Clinical Trial. JAMA 2021, 325, 1426–1435. TOGETHER Trial DSS and Data Repository Screenshots. Date-Time Stamped Screenshots from Publications of the TOGETHER trial (NCT04727424). Available online: https://drive.google.com/file/d/1pBZ1GihxW_ROB3Aid6tFMplqAyMYOGDl/preview (accessed on 30 October 2023).
  202. Scheim, D.E.; Hibberd, J.A.; Chamie-Quintero, J.J. Protocol Violations in López-Medina et al.: 38 Switched Ivermectin (IVM) and Placebo Doses, Failure of Blinding, Ubiquitous IVM use OTC in Cali, and Nearly Identical AEs for the IVM and Control Groups. OSF Preprints. 2021. Email from Sarah Fullegar, Sent 7 June 2022 to Edmund Fordham, Screenshot, Email Addresses Redacted. Available online: https://drive.google.com/file/d/1lUsSRf1KX-pa9T5EX4HbegdK8mYNQ_Ty/preview (accessed on 30 October 2023).
  203. George, L.L.; Borody, T.J.; Andrews, P.; Devine, M.; Moore-Jones, D.; Walton, M.; Brandl, S. Cure of duodenal ulcer after eradication of Helicobacter pylori. Med. J. Aust. 1990, 153, 145–149. López-Medina, E.; López, P.; Hurtado, I.C.; Dávalos, D.M.; Ramirez, O.; Martínez, E.; Díazgranados, J.A.; Oñate, J.M.; Chavarriaga, H.; Herrera, S.; et al. Effect of Ivermectin on Time to Resolution of Symptoms Among Adults With Mild COVID-19: A Randomized Clinical Trial. JAMA 2021, 325, 1426–1435.
  204. Coghlan, J.G.; Gilligan, D.; Humphries, H.; McKenna, D.; Dooley, C.; Sweeney, E.; Keane, C.; O’Morain, C. Campylobacter pylori and recurrence of duodenal ulcers—A 12-month follow-up study. Lancet 1987, 2, 1109–1111. Scheim, D.E.; Hibberd, J.A.; Chamie-Quintero, J.J. Protocol Violations in López-Medina et al.: 38 Switched Ivermectin (IVM) and Placebo Doses, Failure of Blinding, Ubiquitous IVM use OTC in Cali, and Nearly Identical AEs for the IVM and Control Groups. OSF Preprints. 2021.
  205. Graham, D.Y.; Lew, G.M.; Klein, P.D.; Evans, D.G.; Evans, D.J., Jr.; Saeed, Z.A.; Malaty, H.M. Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. A randomized, controlled study. Ann. Intern. Med. 1992, 116, 705–708. George, L.L.; Borody, T.J.; Andrews, P.; Devine, M.; Moore-Jones, D.; Walton, M.; Brandl, S. Cure of duodenal ulcer after eradication of Helicobacter pylori. Med. J. Aust. 1990, 153, 145–149.
  206. Watts, G. Nobel prize is awarded to doctors who discovered H pylori. BMJ 2005, 331, 795. Coghlan, J.G.; Gilligan, D.; Humphries, H.; McKenna, D.; Dooley, C.; Sweeney, E.; Keane, C.; O’Morain, C. Campylobacter pylori and recurrence of duodenal ulcers—A 12-month follow-up study. Lancet 1987, 2, 1109–1111.
  207. Fleming, A. On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to their Use in the Isolation of B. influenzæ. Br. J. Exp. Pathol. 1929, 10, 226–236. Graham, D.Y.; Lew, G.M.; Klein, P.D.; Evans, D.G.; Evans, D.J., Jr.; Saeed, Z.A.; Malaty, H.M. Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. A randomized, controlled study. Ann. Intern. Med. 1992, 116, 705–708.
  208. Chamie, J.J.; Hibberd, J.A.; Scheim, D.E. COVID-19 Excess Deaths in Peru’s 25 States in 2020: Nationwide Trends, Confounding Factors, and Correlations With the Extent of Ivermectin Treatment by State. Cureus 2023, 15, e43168. Watts, G. Nobel prize is awarded to doctors who discovered H pylori. BMJ 2005, 331, 795.
  209. Meekins, D.A.; Gaudreault, N.N.; Richt, J.A. Natural and Experimental SARS-CoV-2 Infection in Domestic and Wild Animals. Viruses 2021, 13, 1993. Fleming, A. On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to their Use in the Isolation of B. influenzæ. Br. J. Exp. Pathol. 1929, 10, 226–236.
  210. Baskurt, O.K.; Meiselman, H.J. Comparative hemorheology. Clin. Hemorheol. Microcirc. 2013, 53, 61–70. Lobanovska, M.; Pilla, G. Penicillin’s Discovery and Antibiotic Resistance: Lessons for the Future? Yale J. Biol. Med. 2017, 90, 135–145.
  211. Annunziata, A.; Coppola, A.; Carannante, N.; Simioli, F.; Lanza, M.; Di Micco, P.; Fiorentino, G. Home Management of Patients with Moderate or Severe Respiratory Failure Secondary to COVID-19, Using Remote Monitoring and Oxygen with or without HFNC. Pathogens 2021, 10, 413.
  212. Aoki, R.; Iwasawa, T.; Hagiwara, E.; Komatsu, S.; Utsunomiya, D.; Ogura, T. Pulmonary vascular enlargement and lesion extent on computed tomography are correlated with COVID-19 disease severity. Jpn. J. Radiol. 2021, 39, 451–458.
  213. Ding, X.; Xu, J.; Zhou, J.; Long, Q. Chest CT findings of COVID-19 pneumonia by duration of symptoms. Eur. J. Radiol. 2020, 127, 109009.
  214. Metwally, M.I.; Basha, M.A.A.; Zaitoun, M.M.A.; Abdalla, H.M.; Nofal, H.A.E.; Hendawy, H.; Manajrah, E.; Hijazy, R.f.; Akbazli, L.; Negida, A.; et al. Clinical and radiological imaging as prognostic predictors in COVID-19 patients. Egypt. J. Radiol. Nucl. Med. 2021, 52, 100.
  215. Osman, A.M.; Farouk, S.; Osman, N.M.; Abdrabou, A.M. Longitudinal assessment of chest computerized tomography and oxygen saturation for patients with COVID-19. Egypt. J. Radiol. Nucl. Med. 2020, 51, 255.
  216. Quispe-Cholan, A.; Anticona-De-La-Cruz, Y.; Cornejo-Cruz, M.; Quispe-Chirinos, O.; Moreno-Lazaro, V.; Chavez-Cruzado, E. Tomographic findings in patients with COVID-19 according to evolution of the disease. Egypt. J. Radiol. Nucl. Med. 2020, 51, 215.
  217. Wang, Y.; Dong, C.; Hu, Y.; Li, C.; Ren, Q.; Zhang, X.; Shi, H.; Zhou, M. Temporal Changes of CT Findings in 90 Patients with COVID-19 Pneumonia: A Longitudinal Study. Radiology 2020, 296, e55–e64.
  218. Stone, J.C.; Ndarukwa, P.; Scheim, D.E.; Dancis, B.M.; Dancis, J.; Gill, M.G.; Aldous, C. Changes in SpO2 on Room Air for 34 Severe COVID-19 Patients after Ivermectin-Based Combination Treatment: 62% Normalization within 24 Hours. Biologics 2022, 2, 196–210.
  219. Hazan, S.; Dave, S.; Gunaratne, A.W.; Dolai, S.; Clancy, R.L.; McCullough, P.A.; Borody, T.J. Effectiveness of ivermectin-based multidrug therapy in severely hypoxic, ambulatory COVID-19 patients. Future Microbiol. 2022, 17, 339–350.
  220. Babalola, O.E.; Ndanusa, Y.; Adesuyi, A.; Ogedengbe, O.J.; Thairu, Y.; Ogu, O. A Randomized Controlled Trial of Ivermectin Monotherapy Versus HCQ, IVM, and AZ Combination Therapy in COVID-19 Patients in Nigeria. J. Infect. Dis. Epidemiol. 2021, 7, 233.
  221. Babalola, O.E.; (Bingham University, New Karu, Nigeria). Personal communication, 28 February 2022.
  222. Chamie, J.J.; Hibberd, J.A.; Scheim, D.E. COVID-19 Excess Deaths in Peru’s 25 States in 2020: Nationwide Trends, Confounding Factors, and Correlations With the Extent of Ivermectin Treatment by State. Cureus 2023, 15, e43168.
  223. Meekins, D.A.; Gaudreault, N.N.; Richt, J.A. Natural and Experimental SARS-CoV-2 Infection in Domestic and Wild Animals. Viruses 2021, 13, 1993.
  224. Baskurt, O.K.; Meiselman, H.J. Comparative hemorheology. Clin. Hemorheol. Microcirc. 2013, 53, 61–70.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)
Video Production Service
Feedback