Covid - >>> From Cold to Killer: How SARS-CoV-2 Evolved without Hemagglutinin Esterase to Agglutinate, Then Clot Blood Cells in Pulmonary and Systemic Microvasculature
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https://doi.org/10.2139/ssrn.3706347 PPR: PPR243457
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Abstract
The role of vascular occlusion in the morbidities, pulmonary and systemic, of COVID-19 has received increasing focus. Histological studies of lung tissue from COVID-19 patients have found extensively damaged endothelium of capillaries adjoining relatively intact alveoli, corresponding to hypoxemia accompanying normal breathing mechanics in such patients.
Essential to the study of vascular occlusion in COVID-19 are viral properties dating back to studies of Jonas Salk in the 1940s that have been positively established for SARS-CoV-2. First, SARS-CoV-2 binds to red blood cells (RBCs) in vitro and also clinically in COVID-19 patients. Second, although fusion and replication of SARS-CoV-2 occur via ACE2, such hemagglutinating viruses initially attach to infective targets and clump with blood cells via much more abundantly distributed glycoconjugate binding sites, notably those tipped with sialic acid (SA). SARS-CoV-2, in particular, attaches to these glycan binding sites. Third, certain enveloped viruses express an enzyme, hemagglutinin esterase (HE), that counteracts viral-RBC clumping. Notably, among betacoronaviruses, the common cold strains express HE while SARS-CoV-2, SARS-CoV-1 and MERS, the virulent strains, do not. The hemagglutinating properties of SARS-COV-2 establish a framework for “catch and clump” induction of microvascular occlusion proposed here. Ultramicroscopic studies of tissues from COVID-19 patients indicate a key role for hemagglutination early and mid-course in COVID-19, before such clumps develop into clots via the coagulation cascade.
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PPRID: PPR243457
EMSID: EMS106340
SSRN preprint, version 3, posted 2020 October 12
https://doi.org/10.2139/ssrn.3706347
Keywords: SARS-CoV-2, COVID-19, betacoronavirus, sialic acid, glycophorin A, CD147, hemagglutination, hemadsorption, hemagglutinin esterase (HE)
Background
Although COVID-19 typically gains an infectious foothold in the respiratory epithelium [1–3], circulatory system damage is frequently observed in lungs and other organ systems of COVID-19 patients, with such features as intravascular clotting, microvascular occlusion and peripheral ischemia [4–17]. In one study of 183 hospitalized COVID-19 patients, 71% of those who died had disseminated intravascular coagulation during their hospital stay [18]. One clinical reviewer characterized COVID-19 as “a systemic disease that primarily injures the vascular endothelium” [19].
Essential to the study of vascular occlusion in COVID-19 which, as will be considered, is in turn central to the morbidities of this disease, are viral properties dating back to classic experiments of the 1940s. Viruses fuse and replicate through host cell receptors specific to the viral strain, which for SARS-CoV-2 is ACE2 [20, 21]. But many enveloped viruses, including coronaviruses, also attach to host cell membranes via glycoprotein molecules, including those tipped with sialic acid (SA) [22–25], that term designating a class of monosaccharides [26–34], and via sulfated glycoproteins such as heparan sulfate proteoglycans (HSPG) [35–37]. In particular, SARS-CoV-2 binds to both SA [22, 23, 25, 38] as well as to HSPG and heparan sulfate [39, 40] surface molecules on host cells.
SA is densely distributed on red blood cells (RBCs) as terminal residues of both the CD147 receptor [31] and glycophorin A surface proteins [41–45]. Through viral bindings to SA surface molecules, SARS-CoV-2 agglutinates with red blood cells (RBCs), as established both in vitro [46] and clinically [47], to be considered below. Such hemagglutination occurs for other coronaviruses [46, 48–58] and several other viral strains [28, 48, 49, 54, 59] and is simulated in the classic viral hemagglutination assay. In this assay, developed in the 1940s [49, 60–62] and extended later in that decade by Jonas Salk [63–67], virus particles mix with RBCs to form a hemagglutinated sheet [68–71]. But for viruses that express SA cleaving enzymes, that sheet subsequently collapses as these enzymes cleave SA binding sites on the RBCs [49, 59, 69, 72].
Viral-SA binding, host decoy defense, and cleaving of SA binding via hemagglutinin esterase (HE)
For viruses that bind to SA, including SARS-CoV-2 as noted above, that binding plays a key role in viral infectivity, as SA typically serves as an initial point of attachment to host cells [27, 32, 33, 54, 58, 73–79]. The human host, reciprocally, defends against viral attachment to host cell infectious targets by presenting SA on an array of decoys, including red blood cells (RBCs), platelets and leukocytes [41, 80–82]. Expressed on the plasma membranes of all of these blood cells are SA binding sites [41–43, 83–89] and CD147 receptors [88, 90–96], the latter in turn expressing SA [31, 97]. SA is arrayed nanometers apart on glycophorin A molecules on the RBC membrane surface, with about one million GPA molecules per RBC [41, 43–45, 98–100]. SA is and also contained in CD147 [31], with 1,695 CD147 receptors per RBC [95].
Hemagglutinin esterase (HE) is an SA-cleaving enzyme expressed by certain viral strains, including some betacoronaviruses that release it from stubby projections half the length of their S-protein spikes [26, 29, 72, 73, 101, 102]. HE boosts the infectivity of these viral strains by facilitating release of replicated virions from SA binding sites on host cells [30, 32, 49, 59, 72, 74, 79, 101, 103–107] and by limiting viral snagging on SA binding sites of non-infectious targets such as mucins, plasma proteins and blood cells [26, 28, 29, 49, 72, 82, 103–105, 108]. Viral HE can both bond and cleave host cell SA binding sites [34, 109], but for betacoronaviruses, even those expressing HE, attachments to host cell SA binding sites are effected mainly through glycan binding sites on viral spike protein S1, in particular, those clustered on the N-terminal domain (NTD) [54, 101, 110–112]. In both wild and laboratory-transformed viral strains, a balance between the strength of viral-SA binding and the countering effect of HE-mediated release has been found important for infectious potency [32, 49, 72, 101, 103, 104, 106, 107].
SARS-CoV-2 adjusts for lack of HE through multivalent SA bonds
Genomic analysis of the five human betacoronaviruses reveals that the HE enzyme is expressed in those that cause the common cold, OC43 and HKU1, but not in SARS-CoV, SARS-CoV-2 and MERS, the three deadly strains [113–117]. To compensate for a missing release capability in those three betacoronavirus strains lacking HE, SA binding affinity is diminished, while multiplicity of such bonds allows for robust variation in attachment strength [26, 27, 38, 54, 58, 73]. These viral-SA bonds have weak individual affinity but greatly increased strength collectively [26, 27, 38, 54, 58, 75, 118]. The dissociation constant Kd for a multivalent viral-SA attachment increases exponentially in absolute value with the number of bonds [119, 120]. For example, the Kd of an individual bond of virus to a host SA binding site is in the low millimolar range [26, 27, 106, 121], but Kd would be in the nanomolar range for a triple bond [119]. A single SA bond for a virus particle first attaching to a cell would provide only a tentative foothold [27, 54, 90, 122–124], allowing motility to an ACE2 receptor for fusion and replication.
SARS-CoV-2 binds to SA
Because bindings from viral spike protein to SA are of limited strength when univalent, several coronaviruses strains, including SARS-CoV-2, require an experimental detection methodology that supports multivalent bindings to register viral attachment to hosts. Three studies tested chimerically constructed microarrays and nanoarrays of viral binding sites from human betacoronavirus strains OC43 [72], HKU1 [73] and MERS [58] for hemagglutination of RBCs. Each of these studies found that viral binding sites induced hemagglutination only when arrayed in nanoparticles capable of forming multivalent attachments to SA surface molecules on RBCs (with SA being arrayed nanometers apart in clusters on glycophorin A molecules on the RBC membrane surface, as noted above [41, 44, 45, 98–100]). Given indicated similarities between SARS-CoV-2 and other betacoronaviruses in binding to SA [22–25], it was not surprising that one study did not detect such binding by SARS-CoV-2 using conventional microarray procedures [125]. Using a gold nanoparticle array bearing SA derivatives, however, SARS-CoV-2 binding to SA was demonstrated [38]. The detection system used in this study is adaptable to mass COVID-19 screening, with viral spike protein detectable at a 5 nM concentration threshold.
SARS-CoV-2 binds to CD147
Another point of attachment for the SARS-CoV-2 virus on host cells is the CD147 receptor, which in turn contains SA at its terminal domains [31, 97]. CD147, also known as basigin, BSG, or EMMPRIN, appears throughout the human body [90], especially densely on blood and endothelial cells [11, 12, 88, 90–95, 126–130]. It is expressed in four different isoforms and with varying degrees of sialoglycosylation [31, 92, 93, 131, 132]. The possibility that the CD147 receptor might serve, like its component molecule SA, as a point of host cell attachment for SARS-CoV-2 was demonstrated in a multifaceted in vitro study by Wang et al. [133], supported by clinical findings [134]. Binding of SARS-CoV-2 spike protein to CD147 was shown by SPR, Co-IP and ELISA assays, and immuno-electron microscopy revealed colocalization of CD147 and viral spike protein on infected Vero E6 cells. Just as binding of SARS-CoV-2 [38, 125], OC43 [72], HKU1 [73] and MERS [58] to SA binding sites was detected using only nanoarray techniques, not microarrays techniques, as noted, so a study using microarray techniques failed to detect SARS-CoV-2 binding to CD147 [135].
Wang et al. further demonstrated binding of SARS-CoV-2 virus to CD147 receptors through significant in vitro inhibition of viral proliferation by meplazumab, a humanized anti-CD147 antibody [133]. Also, clinical benefits from masking of host cell CD147 receptors by meplazumab were indicated by markedly improved clinical outcomes in patients given this agent [134]. Seventeen hospitalized COVID-19 patients, six with severe disease and seven in critical status, had a median time to viral clearance of three days vs. 13 days for 11 controls. Similar statistically significant reductions in case severity, time to hospital discharge and chest CT abnormalities were achieved in the treatment group. Of note with respect to the clinical benefits obtained by masking of CD147 by meplazumab is the much greater density of SA-containing CD147 receptors, for example, than of ACE2 receptors found in lung and bronchial tissue [90].
SARS-CoV-2 attachments to RBCs, other blood cells and endothelial cells
Attachments of SARS-CoV-2 viral particles to blood and endothelial cells, mediated by binding to SA and SA-expressing CD147 receptors, will be a central focus of this continuing examination. As will be demonstrated through histological findings, these viral attachments, unconstrained by an HE release mechanism, may be key to the pathology of COVID-19, both in capillaries of smallest cross-sectional diameter which permeate the lung and in systemic microvasculature. SARS-CoV-2 binding to human RBCs is of particular interest, as demonstrated in vitro [46] and in COVID-19 patients [47]. Hemagglutination occurs more generally [28, 48, 49, 54, 59, 136] in eight families of viruses including SARS-CoV-2 and other coronaviruses [46, 48–58, 71, 137], and is inhibited by HE or the neuraminidase enzyme in those viral strains that express these [28, 51–56, 58, 136]. An electron micrograph of RBCs studded with virus particles that bridge RBCs is shown in Figure 1, and a schematic showing virus-mediated RBC clumping is shown in Figure 2.
Figure 1: Electron micrograph of chick erythrocytes hemagglutinated by influenza virus.
Reproduced with permission from Elsevier (Bossart et al., 1973 [138]).
Figure 2: Schematic of hemagglutination.
Reproduced with permission from Springer Nature (Killian, 2014 [68]). Hemagglutination is common for many strains of several families of viruses, including betacoronaviruses. It is the basis of a widely used viral assay technique developed by Hirst, McClelland and Hare and extended by Jonas Salk in the 1940s. For SARS-CoV-2, binding to RBCs is demonstrated using the related hemadsorption assay [46].
SA surface molecules that are densely distributed on RBCs (35 million per cell [43]), mainly as terminal residues of glycophorin A [41, 42], with glycophorin A serving no discernible physiological role other than as a decoy for pathogens [41, 81, 82]. RBCs indeed perform an active pathogen clearance role, described in 1953 as “immune adherence” [139, 140], attaching to microorganisms and then passing them off to leukocytes or delivering them to macrophages in the spleen or liver for phagocytosis [41, 80, 82, 141]. With bacteria, this clearance process requires both antibody and complement [139, 140]. For viruses, however, RBCs snag them directly [49, 59, 68–71, 139, 140], and antiviral antibodies inhibit viral-RBC binding [50, 59, 65–67, 139, 142]. Having no nucleus or other infrastructure to support viral replication and being relatively expendable, with trillions per human host [81], RBCs are well suited for this immune defense role, especially critical in the initial period of infection, prior to antibody formation [41, 81, 82].
Several other blood and vascular cells other than RBCs express surface SA glycoconjugates and can thus also attach to SARS-CoV-2 virus particles. SA and SA-rich CD147 are expressed on endothelial cells of blood vessel linings (luminal surface) [11, 12, 25, 85, 87, 88, 91–94, 127–130, 143, 144], platelets [85–89, 92–94], lymphocytes [90, 145, 146], macrophages [90, 147], and other types of white blood cells [85, 88, 90, 91, 93, 94]. Platelets, the second most copious blood component [148], serve a pathogen clearance role like that of RBCs, binding to viruses and assisting in their phagocytosis by leukocytes and sequestration in the liver and spleen [16, 149–152]. Platelets are often in the mix of clumps that develop in severe malaria between infected RBCs and other RBCs [153–156], as shown in Figure 3. These clumps, also called rosettes, along with cytoadhesion between infected RBCs and endothelial cells, cause vascular occlusion, which is the key morbidity of severe malaria [154, 157–160]. This clumping of RBCs and platelets and endothelial cytoadhesion by infected RBCs in severe malaria suggests parallels to COVID-19, the morbidities of which also often extend to the vasculature, as noted above.
Figure 3: Adhesion of erythrocytes infected with Plasmodium falciparum to human cells.
Reproduced (b-d) with permission from Cambridge University Press (Rowe et al., 2009 [153]). (b) Cytoadherence of infected erythrocytes to in-vitro-cultured brain endothelial cells, visualized by light microscopy after Giemsa staining. (c) Rosettes detected in in vitro P. falciparum cultures, observed after preparation of Giemsa-stained thin smears and light microscopy. (d) Platelet-mediated clumps of infected erythrocytes formed after in vitro co-incubation of parasite cultures with platelets, observed by Giemsa-stained thin smears and light microscopy.
“Catch and Clump” occlusion of the microvasculature
Just as the malaria parasite attaches to an RBC using the latter’s SA binding sites [82, 161, 162] and penetrates the RBC through the latter’s CD147 receptors [163, 164], so SARS-CoV-2 binds to both SA and CD147 on blood and endothelial cells, as noted above. These parallels between severe malaria and COVID-19 in both adhesive dynamics and morbidities lend support to a “catch and clump” scenario that has been proposed as a triggering or contributing factor to the vascular occlusion of COVID-19 [165]. The diameter of a SARS-CoV-2 virus particle is 0.10-0.12 µm [166], small with respect to either an RBC disk diameter of 8 µm [167, 168] or the 3-20 µm cross sectional diameter of a capillary [167, 169]. But viral attachments to an endothelial cell (a “catch”) or to RBCs could nevertheless cause Velcro-like snags on walls of alveolar capillaries, of average cross-sectional diameter 6 µm [170], against which RBCs flatten, with flow-induced shape changes [168, 171]. Also, virally mediated clumps of RBCs and other blood cells could create bottlenecks elsewhere in the vasculature as blood flows from arterioles into capillaries.
Such viral bonds would dynamically detach and reattach [27, 54, 90, 122] in turbulent blood flow yet would strengthen and become durable with increased multiplicity. Initially transient obstructions would reduce the speed of blood flow, in turn enabling formation of more catches and clumps in a cascade of microvascular occlusion. Highlighting the potential for such virally mediated hemagglutination is the aggregation of RBCs that can occur even without the presence of virus, joined by macromolecules in plasma, under conditions of low blood flow shear rates [172] such as in veins [173]. Such hemagglutination is reversible, the RBCs separating at higher shear rates [172].
Thrombi seen in serious cases of COVID-19 and postmortem in autopsies [5, 7, 9, 10, 174, 175] can provide only tenuous clues as to whether hemagglutination triggered by SARS-CoV-2 could have initially triggered coagulation, or whether these developed from an inflammatory cascade caused by viral infection [16, 174, 176–180]. Blurring the line between these two scenarios is the key role of CD147 in inflammatory processes involved in vascular occlusion. In response to immune stimuli, CD147 is upregulated in T cells [130, 181], platelets [88, 182] and endothelial cells [183], the latter upon exposure in vitro to the betacoronavirus MHV-4, either active or UV-deactivated. CD147 in turn has been observed to promote adhesion by RBCs [91, 96, 141, 184], leukocytes [88, 132, 185–187] and platelets [93, 186, 187] to other blood cells and endothelial cells. Cyclophilin A (CyPA) can activate platelets via its signaling partner CD147, resulting in adhesion and thrombus formation in vitro and in vivo, which was inhibited by anti-CD147 monoclonal antibody [188].
The role of CD147 as a key mediator of inflammatory response is implicated in the pathogenesis of a number of diseases, including lung inflammation, rheumatoid arthritis, atherosclerosis, heart failure, and ischemic myocardial injury and stroke [88, 91, 181, 187, 189–191]. Of particular interest are indicated pro-infectious roles of CD147 and its binding partner CyPA in SARS-CoV-2, SARS-CoV-1 and other viruses [94, 192, 193]. That role is highlighted by in vitro inhibition of SARS-CoV-2 by a CyPA antagonist [194] and statistically significant clinical benefits for COVID-19 patients treated with the anti-CD147 antibody Meplazumab [134].
In a mouse model of acute lung inflammation simulating acute lung injury in humans, anti-CD147 antibodies significantly reduced neutrophil infiltration of lung tissue and associated tissue pathology [91, 195]. Anti-CD147 antibody also inhibited the formation of leukocyte aggregates that caused liver injury in mice [132]. It substantially attenuated formation of platelet-monocyte aggregation and adhesion to vascular walls in vivo [186], and limited the adhesion of peripheral blood T lymphocytes to an extracellular matrix glycoprotein [185]. These reductions of blood cell clumping by anti-CD147 antibody, consistent with the pro-adhesive effects of CD147 on RBCs and other blood cells as noted above, indicate the potential for a CD147-neutralizing therapeutic to mitigate key morbidities of COVID-19.
The potential role of the cytoadhesive phenomena as proposed in microvascular occlusion associated with SARS-CoV-2 is illuminated by histological, immunochemical and other findings from COVID-19 patients. Notable is an immunofluorescence analysis of RBCs from the blood of nine hospitalized COVID-19 patients [47]. The mean percentage of RBCs having SARS-CoV-2 spike protein punctae was 41% at day 0 of hospital admission, with values ranging from 0% for one patient and 18% for two patients to 79% for another patient. C3 complement components on RBCs were also detected, with 41% of RBCs having these at day 0. (At day 7, these mean percentages were 44% for viral spike protein and 52% for C3 complement.)
This finding of viral traces on 41% of RBCs from COVID-19 patients is not surprising given the attachments of SARS-CoV-2 to RBCs seen in vitro in the hemadsorption assay [46] (similar to the hemagglutination assay [196, 197]) and given the hemagglutination observed for other coronavirus strains as noted above. While RBCs in free-flowing blood were marked by the noted viral traces, the disposition of other RBCs sequestered with endothelial and other blood cells in the vasculature is revealed in ultramicroscopic and immunochemical studies of lung, kidney, skin, and colon tissue from COVID-19 patients [198–204]. Microvascular occlusion was prominent in most tissue specimens from each study. Severe endothelial injury was seen in most of these studies, with viral spike protein detected along capillary walls and within endothelial cells in several tissue specimens [198, 199, 201–203]. Figure 4 shows localization of SARS-CoV-2 viral RNA in occluded capillaries along the alveolar septum from a patient who died of COVID-19 [199].
Figure 4: SARS CoV-2 viral RNA demonstrates striking capillary localization within the interalveolar septa of the lung of a patient who succumbed to COVID-19.
Licensed under creative commons 2.0 (Magro et al., 2020 [199]). SARS CoV-2 membrane protein demonstrated a similar pattern of microvascular localization.
Inconsistent results have been reported for viral load in blood of COVID-19 patients. Most of these were limited by detection using plasma or serum [205–215], subject to clearance of virus by agglutination with RBCs. But ultrasensitive detection technology in one study [205] and use of whole blood in another [206] yielded 74% and 72% positive detection rates for SARS-CoV-2 virus in COVID-19 patients, respectively. Ultrasensitive detection methods found the presence of viral spike S1 traces in plasma of 64% COVID-19 patients in another study [216]. Five studies found that viral blood load was strongly correlated with disease severity [205, 210–213, 216] (another study found no such correlation [209]). Circulatory system damage in multiple organs of COVID-19 patients, as cited above, indicates spread of disease by SARS-CoV-2 in blood, this viral load demonstrated directly by viral traces on RBCs and on vasculature within COVID-19 tissue specimens as noted.
Microvascular occlusion of alveolar septal capillaries and pulmonary morbidities of COVID-19
Septal capillaries in the lung are especially vulnerable to virally mediated occlusion of blood flow. Each RBC flows through these about once per minute for a duration of 700 milliseconds [171, 217], distorting its shape and flattening against capillary walls to fit the smallest cross-sectional diameter of those capillaries (6 µm) [168, 170, 171]. Some of the viral traces and damage seen in endothelial cells of alveolar septal capillaries from the cited COVID-19 histological studies [198, 201] would likely represent viral penetration through ACE2 receptors. But with 28,000 CD147 receptors vs. 175 ACE2 receptors per endothelial cell [127], most viral attachments to endothelium would likely engage CD147 and other binding sites containing terminal SA molecules that are abundantly distributed on endothelial cells [25, 87, 143]. As noted above, these bonds would be relatively weak, dynamically attaching and detaching but subject to considerable strengthening with multivalency. Under the constant flow of RBCs pressing against septal capillary walls, the cumulative effect of such viral snags and of virally mediated clumps of blood cells flowing into those capillaries could be occlusion consistent with the histological findings noted.
Such cytoadhesive attachments could obstruct a septal capillary but leave it intact, its walls lined with virus, as shown in Figure 4. Other septal capillaries, however, might be torn apart as surges of cardiac pressure pushed obstructed RBCs through their walls, a scenario that occurred for some tissues from two of the lung studies noted above [198, 201]. Figure 5 depicts these features: broken capillary walls, extravasated RBCs and interspersed fibrinous microthrombi. Red cell extravasation was also observed in seven biopsy specimens of “COVID toes” [198, 201, 202]. A third potential scenario for virally mediated cytoadhesion in septal capillaries is the disaggregation of snagged RBCs as viral attachments were broken by surges of cardiac pressure. This release would readily occur for viral bonds to blood or endothelial cells of low multiplicity and weak avidity. But for a stronger, multivalent such viral attachment to an RBC, the counterforce of a cardiac pressure surge might strip a viral spike off the viral envelope, like an arm of an octopus released in a predator, as may be reflected in the viral traces found on 41% of RBCs of COVID-19 patients. A closer examination of the potential for SARS-CoV-2 spike protein, either from its base virion or abortively released from a virally infected host cell during abortive viral replication [218, 219], to become attached to an RBC has been more closely explored [220].
Figure 5: Microthrombi in the Interalveolar Septa of a Lung from a Patient Who Died from Covid-19.
Reproduced with permission from the Massachusetts Medical Society (Ackermann et al., 2020 [201]). The interalveolar septum of this patient shows slightly expanded alveolar walls with multiple fibrinous microthrombi (arrowheads) in the alveolar capillaries. Extravasated erythrocytes and a loose network of fibrin can be seen in the intraalveolar space (hematoxylin– eosin staining; the scale bar corresponds to 50 µm).
A striking feature of several of these lung specimens was severe endothelial damage in septal capillaries with minimal damage to adjoining alveolar tissue [198, 200]. As was summarized for a series of five COVID-19 patients with respiratory failure in one of these studies, “no viral cytopathic changes were observed and the diffuse alveolar damage with hyaline membranes, inflammation, and type II pneumocyte hyperplasia, hallmarks of classic acute respiratory distress syndrome, were not prominent” [198]. This histological dichotomy of damaged capillaries adjoining relatively normal alveoli fits the anomalous features of ARDS often seen in COVID-19 patients, characterized by severe hypoxemia with poorly oxygenated blood from the lungs despite well-preserved lung mechanics and respiratory compliance [10, 16, 17, 19, 198, 221]. A similar pattern of microvascular damage was found in a high resolution MRI study of the brains of 13 patients with COVID-19 [222]. The lead investigator of that study summarized, “COVID-19 seems to have a propensity to damage small blood vessels in the brain, rather than the nerve cells themselves” [223].
A direct relationship between impaired breathing function and pulmonary microvascular occlusion in COVID-19 was demonstrated through high-resolution lung CT scans of 103 hospitalized patients and 107 controls [224, 225]. A partial redistribution in blood flow from smaller to larger diameter blood vessels in the lung (this analysis restricted to vessels with minimum diameter of 2.5 mm) was detected in the scans of the COVID-19 patients vs. controls. The investigators attributed this redistribution of blood flow to either dysregulated vasoconstriction or occlusion of the microvasculature, the latter explanation being in alignment with the histological findings noted above. For 51 of these COVID-19 patients for whom clinical data was accessible, the extent of redistribution of blood flow from smaller to larger pulmonary blood vessels was closely correlated (p=0.023) with the degree of oxygen supplementation they required.
Other cases from the histological studies of COVID-19 specimens cited provide further indications that initial triggers of microvasculature occlusion can be cytoadhesive rather than inflammatory. For six of 11 lung specimens exhibiting severe septal capillary congestion, immunochemical testing revealed no presence of fibrin [200]. In six cases of purpura skin lesions (“COVID toes”), microthrombi in capillaries were colocalized with SARS-CoV-2 spike and envelope protein and complement components, but interferon signaling and other inflammatory markers were essentially absent [199]. In kidney tissues from several COVID-19 patients, obstructive aggregates of RBCs were observed in capillaries without inflammation, fibrin or a significant platelet component [204].
The noted ultramicroscopic studies of tissues from COVID-19 patients are consistent with clinical manifestations of hemagglutination of SARS-CoV-2 as seen in vitro, with parallels to the cytoadhesive morbidities of severe malaria, in which CD147 and SA bindings are also a feature. Further linkage of these cytoadhesive phenomena with the morbidities of COVID-19 is indicated by three variations in infectious conditions that are correlated with reduced morbidities.
i). Much faster blood flow in younger subjects
Clinical studies of blood flow in different tissues have found much higher flow velocities in younger vs. older subjects, which would limit formation of cytoadhesive vascular occlusions and thus could account for much reduced COVID-19 morbidities in younger age groups [226, 227]. Rates of capillary flow under toe and finger nails for subjects of mean age 26 were almost double those for mean age 63[/b] [228]. In other studies of capillary flow in various tissues, older subjects had 23% [229] and 40% [230] diminished flow velocities vs. younger subjects and a 47% decrease in flux amplitude [230]. Differences in flow rates in arteries for older vs. younger subjects were significant but less pronounced: 26-27% lower [231–233]. A similar protective effect of greater blood flow turbulence is suggested by a 38% increased incidence of COVID-19 for systolic blood pressure < 90 mmHg [227] and decreased deaths for increased blood pressure with adjustments for other risk factors [226].
ii). Reduced COVID-19 incidence in subjects with less agglutination-prone blood type O
A genomic association study of 1,980 COVID-19 patients found that, with adjustments for age and sex, those with blood group O had a 35% lower risk of contracting COVID-19 than those of other blood groups [234]. Blood group A was associated with the highest risk of incidence, 45% higher than for other blood groups. Other studies reported similar reduced risks for blood group O [235, 236]. For malaria, in a matched case-control study of 567 patients, its results consistent with other studies [237, 238], blood group O was associated with a 66% reduction in the odds of developing the severe form of the disease [239]. This reduced incidence of severe malaria correlates with reduced rates of rosette formation from the blood of group O malaria patients [237, 239]. Both clinical [238] and in vitro [240, 241] studies also showed a lower reduction in risk of severe malaria for blood group A vs. B, again paralleling incidence rates for COVID-19 [234–236]. The group A and B risk factors for malarial rosette formation appear to be mediated by adhesive trisaccharides found on RBCs and white blood cells with these blood types [153, 158], making blood more prone to agglutination.
iii). The common cold betacoronaviruses but not the lethal strains express the HE enzyme
As noted above, for viruses that express the HE enzyme, the interlaced sheet that they form when intermixed with RBCs subsequently collapses as HE cleaves SA binding sites on the RBCs. Also as noted, the betacoronavirus strains that cause the common cold, OC43 and HKU1, express the HE enzyme while SARS-CoV, which first emerged in 2003, and the two other virulent strains MERS, and SARS-CoV-2, do not. With an OC43 or HKU1 infection, HE enzyme released by virus within a cytoadhesive clump or snag in formation would act to release viral bonds and clear the obstruction. Release by virus of significant quantities of HE over the course of minutes [51, 242] is consistent with an evolutionary advantage conferred for virus particles to migrate efficiently to penetration points of host cell targets rather than become snagged elsewhere for extended periods [32, 49, 72, 101, 103, 106, 107]. Thus, although OC43 and HKU1 follow the same respiratory entry route as SARS-CoV-2, HE-mediated release of viral bindings to SA surface molecules on host cells may protect infected subjects from the morbidities that are manifested in SARS, MERS and COVID-19.
It would be reasonable to extend this consideration of hemagglutination effects to the remaining two of seven human coronaviruses, NL63 and 229E, which cause the common cold. But these two viral strains are alphacoronaviruses that do not express HE [114] and also do not agglutinate erythrocytes [243–245] based upon different SA-binding properties [246–249]. Results are likewise indeterminate as to whether viral loads in blood for the four common cold coronavirus strains might be lower than for SARS-CoV, SARS-CoV-2 or MERS and thus contribute to their lesser virulence, even if expression of HE were a factor as well. Studies of viral load in blood for these four common cold coronaviruses are indeed sparse and inconclusive [250, 251] and difficult to compare with varying such results for SARS-CoV-2 as cited above.
Agents that competitively bind to SARS-CoV-2 spike protein
For hemagglutinating viruses, including SARS-CoV-2 and other coronaviruses, the antiviral activity of associated antibodies can be demonstrated and quantified in the hemagglutination inhibition assay. Whereas virions mixed with RBCs will clump, the addition of a sufficient concentration of antibodies against that virus will block hemagglutination by competitively binding to regions of the virus [65–67, 142]. This hemagglutination inhibition assay, as refined by Jonas Salk and used in his work on antiviral vaccines in the 1940s [63–67], is still used to determine viral antibody titers [67]. For coronaviruses and several other viral families, per the background above, SA glycoconjugate binding sites on viral spike protein are the attachment points of RBCs that are blocked by antibodies [34, 67, 74, 245, 252, 253]. SARS-CoV-2 vaccines and monoclonal antibodies have focused on deploying antibodies to epitopes on the receptor binding domain (RBD) [254–259] to inhibit viral replication via ACE2 fusion.
For coronaviruses and other viral strains, certain molecules such as gangliosides [51] and fetuin [34] can also block viral attachments to host cells. In the quest for therapeutics for SARS-CoV-2, investigators used molecular modeling to explore whether any existing drugs would likewise competitively bind to viral spike protein. Four such studies that collectively screening over 800 such molecules were conducted toward that goal [260–263]. The strongest or close to strongest binding affinity in each was obtained for ivermectin, a macrocyclic lactone that is a safe and commonly available drug, distributed in 3.7 billion doses worldwide since 1987 [264–266]. Additional molecular modeling studies of binding to SARS-CoV-2 spike protein sites that focused on IVM in particular, including Lehrer and Rheinstein (2020) [267], likewise found strong binding affinities for IVM [268–272].
Discussion and Conclusion
The biological mechanisms that activate the coagulation cascade in COVID-19 entail complex pathways that remain to be fully identified. Yet one viral property central to these vascular occlusive phenomena is that SARS-CoV-2 is a hemagglutinating virus, as established in vitro through the hemadsorption assay, in vivo from the presence of viral spike protein punctae on 41% of RBCs from COVID-19 patients, and biochemically from SARS-CoV-2 binding to SA glycoconjugate binding sites. As discussed above, the framework for these observations was established in the 1940s with the viral hemagglutination assay and its extension to the hemagglutination inhibition assay. The arrangement and composition of glycans on SARS-CoV-2 spike protein, arrayed at 22 N-glycosylation sites on each of its three monomers [38, 112, 273–275], have been more closely studied, and specific biological mechanisms by which competitive inhibition of those bindings could be achieved have been explored [220].
Acknowledgements
The author is grateful to David Hankins and Allen Hirsh for biochemical insights that helped shape this study and to Jennifer Hibberd and Jerome Dancis for help in editing and refining this manuscript.
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