key: cord-0910936-agh7qi4a
authors: Bye, Alexander P; Hoepel, Willianne; Mitchell, Joanne L; Jégouic, Sophie; Loureiro, Silvia; Sage, Tanya; Vidarsson, Gestur; Nouta, Jan; Wuhrer, Manfred; de Taeye, Steven; van Gils, Marit; Kriek, Neline; Cooper, Nichola; Jones, Ian; den Dunnen, Jeroen; Gibbins, Jonathan M
title: Aberrant glycosylation of anti-SARS-CoV-2 spike IgG is a pro-thrombotic stimulus for platelets
date: 2021-07-30
journal: Blood
DOI: 10.1182/blood.2021011871
sha: 648f82b4133cca957e61241cf49d37e9504aa251
doc_id: 910936
cord_uid: agh7qi4a

A subset of patients with COVID-19 become critically ill, suffering from severe respiratory problemsand also increased rates of thrombosis.The causes of thrombosis in severely ill COVID-19 patients are still emerging, but the coincidence of critical illness with the timing of the onset of adaptive immunity could implicate an excessive immune response. We hypothesised that platelets might be susceptible to activation by anti-SARS-CoV-2 antibodies and contribute to thrombosis. We found that immune complexes containing recombinant SARS-CoV-2 spike protein and anti-spike IgG enhanced platelet-mediated thrombosis on von Willebrand Factor in vitro, but only when the glycosylation state of the Fc domain was modified to correspond with the aberrant glycosylation previously identified in patients with severe COVID-19. Furthermore, we found that activation was dependent on FcγRIIA and we provide in vitro evidence that this pathogenic platelet activation can be counteracted by therapeutic small moleculesR406 (fostamatinib) and ibrutinib that inhibit tyrosine kinases Sykand Btkrespectively or by the P2Y12 antagonist cangrelor.

toward development of acute respiratory distress syndrome (ARDS). It is now believed that 51 multiple factors contribute to the thromboinflammatory state that results in high rates of 52 thrombotic complications. Evidence has indicated the presence of activated vascular 53 endothelial cells, macrophages, platelets, neutrophils and an activated coagulation system in 54 critically ill COVID-19 patients. The mechanistic trigger that causes the changes that 55 accompany an increase in severity in a subset of patients is still the subject of intense 56 research. However, the disparity between the time of peak viral load at 5-6 days after the 57 onset of symptoms and occurrence of ARDS after 8-9 days, implicate an excessive immune 58 response rather than direct actions of the virus itself. 5 59

Further evidence that the adaptive immune response is disturbed in severely ill COVID-19 60 patients has been provided by a study that found high levels of extrafollicular B cell 61 activation in critically ill patients which correlates with increased morbidity, antibody titres 62 and levels of inflammatory biomarkers. 6 Other studies have also noted the strong 63 association of high antibody titres with disease severity and survival. 7,8 However, antibodies 64 of severely ill COVID-19 patients have qualitative as well as quantitative differences 65 compared to those with mild illness. Anti-spike IgG in serum samples from severely ill 66 COVID-19 patients were found to have low levels of fucosylatation and elevated 67 galactosylation in the Fc domain. 9,10 68

Platelets express the antibody receptor FcγRIIA, but it is not known if immune complexes 69 containing afucosylated IgG might activate platelet FcγRIIA. Clustering of platelet FcγRIIA 70 induced by ligand binding triggers intracellular signalling via Syk and Btk activation and 71 promotes granule secretion and integrin α IIb β 3 activation. 11,12 Therefore, activation of FcγRIIA 72 by afucosylated anti-spike IgG might further exacerbate thromboinflammation in critically ill 73 In this study we investigate the effects of low fucosylation and high galactosylation of anti-75 spike IgG on platelet activation to find the significance of aberrant IgG glycosylation 76 identified in critically-ill COVID-19 patients on platelet-mediated thrombus formation. We 77 found that potent activation of platelets by immune complexes containing SARS-CoV-2 spike 78 and anti-spike IgG only occurs when the IgG expresses both low fucosylation and high 79 galactosylation in the Fc domain and an additional prothrombotic signal, we used vWF, is 80 also present. Enhanced platelet activation and thrombus formation, measured in vitro, was 81 sensitive to FcγRIIA inhibition and small molecules inhibitors of Syk, Btk and P2Y12, 82

suggesting that these therapeutic strategies might reduce platelet-mediated thrombosis in 83 critically-ill COVID-19 patients. 84

The sequence of SARS-CoV-2 S1 was obtained from the cloned full-length S sequence and 87 was cloned into the expression vector pTriEx1.1 (EMD Millipore, UK) and characterised as 88 described previously. 13 Sf9 cells were transfected with the baculovirus expression vector 89

FlashBAC Gold (Oxford Expression Technologies, UK) and with the SARS-CoV2-S1 transfer 90 vector to produce recombinant baculovirus. Large-scale protein expression was performed 91 by infecting 1L of T.nao38 cells with a high titre stock of the recombinant baculovirus and 92 incubated for 3-5 days at 27°C. After incubation the supernatant containing the secreted 93 protein was harvested, clarified by centrifugation at 4,300xg for 20min and filtered through a 94 0.45μm filter. The clear supernatant was supplemented with 0.5nM nickel sulphate before 95 being loaded onto the Bio-Scale Mini Profinity IMAC Cartridge (Bio-Rad, UK). The elution 96 was carried out at a flow rate of 2.5 ml/min with a gradient elution of 0.05-0.25M imidazole 97 over 60 min. Characterisation of the spike protein by western blot and ELISA showed that 98 the protein is not cleaved at the furin site (no S2 is detected), preventing the conformation 99 change required for the post-fusion form. This was confirmed by ELISA with human CV30 100 monoclonal anti-spike protein antibody and CR3022 anti-COVID-19 and SARS-CoV S 101 glycoprotein antibody (Absolute Antibody, UK) whose differential binding was consistent with 102 the prefusion trimer. 103

COVA1-18 IgG was produced in HEK 293F cells as described previously. 14 Antibodies with 106 modified glycosylation states were generated and validated as previously described 107

Validation of the modifications made to COVA1-18 glycosylation are included in table 1. 108

Blood samples were obtained from healthy donors that had given informed consent and 110 using procedures approved by the University of Reading Research Ethics Committee and 111 collected into vacutainers containing 3.8% (w/v) sodium citrate. Platelet-rich plasma (PRP) 112 was prepared by centrifuging whole blood at 100g for 20 minutes. Washed platelets were 113 prepared by adding acid citrate dextrose to PRP and centrifuging at 350g for 20 minutes and 114 resuspending the platelet pellet at 4 × 10 8 cells/mL in Tyrode's buffer (NaCl 134 mM, KCl 115 2.68 mM, CaCl 2 1.80 mM, MgCl 2 1.05 mM, NaH 2 PO 4 417 μM, NaHCO 3 11.9 mM, Glucose 116 5.56 mM, pH 7.4). 117

Glass 8 well microslides (Ibidi, Munich, Germany) were coated with 5μg/ml recombinant 119 SARS-COV-2 spike protein for 60 minutes at 37 o C, washed and then blocked with 10% fetal 120 calf serum (FCS) for 1h at 37 o C. The slides were then washed and treated with 10μg/ml 121 COVA1-18 antibodies (see above) for 1h at 37 o C. Washed platelets at 2 × 10 7 cells/mL 122 were incubated on the slides for 1h at 37 o C. Non-adherent platelets were washed off with 123

Tyrode's buffer and then slides were fixed with 10% formyl saline for 10 minutes. Wells were 124 then washed and the platelets were labelled with 2μM DiOC 6 . Fluorescence images of 125 adherent platelets were captured with the 20× objective lens of a confocal Ti2 microscope 126 (Nikon). 127

In vitro thrombus formation minutes at 37 o C, washed and then blocked with 10% fetal calf serum (FCS) for 1h at 37 o C. 131

The slides were then washed and treated with 10μg/ml COVA1-18 antibodies (see above) 132 for 1h at 37 o C and then 20μg/ml vWF (Abcam, UK) for 1h. Thrombus formation was 133 measured by perfusing citrated whole blood with 20μg/ml vWF through the flow chambers at 134 1000s -1 for 6 minutes before fixing with 10% formyl saline, staining with 2μM DiOC6 and 135 then imaged by acquiring z-stacks using the 20× objective lens of a confocal Ti2 136 fluorescence microscope (Nikon). 137

Aggregation was measured in washed platelets in half-area 96-well plates (Greiner) by 139

shaking at 1200 rpm for 5 minutes at 37°C using a plate shaker (Quantifoil Instruments) after 140 stimulating with collagen at a range of concentrations. Changes in light transmittance of 405 141 nm was measured using a FlexStation 3 plate reader (Molecular Devices). 142

Measurements of fibrinogen binding were performed using washed platelets pretreated with 144 immune complexes, created by incubating 5μg/ml recombinant SARS-COV-2 spike protein 145 with 10μg/ml COVA1-18 for 60 minutes at 37 o C, and then stimulated with 1 µg/mL CRP, 146

10μM ADP or 1μM TRAP-6 in the presence of fluorescein isothiocyanate-conjugated 147 polyclonal rabbit anti-fibrinogen antibody (Agilent Technologies LDA UK Limited, Cheadle, 148

United Kingdom), and then incubated for 20 minutes in the dark. Platelets were then fixed by 149 addition of filtered formyl saline (0.2% formaldehyde in 0.15M NaCl) and median 150 fluorescence intensities were measured for 5000 platelets per sample on an Accuri C6 Flow 151

Cytometer (BD Biosciences, Berkshire, United Kingdom). 152

Statistical testing as described in figure Severe COVID-19 is associated with increased levels of many prothrombotic plasma 177 proteins including von Willebrand Factor (vWF), which has been noted as up to five fold 178 higher (to approx. 5U/ml) in COVID-19 patients in intensive care. [16] [17] [18] We combined the immune complex coatings with vWF at 20μg/ml to simulate a modest increase in plasma 180 vWF levels of approximately 2U/ml. The combined immune complex and vWF coating 181 resulted in significantly more platelet adhesion to the Low Fuc High Gal IgG than the WT IgG 182 (Figure 1Bi-ii) . Adhesion to IgG with either Low Fuc or High Gal was not significantly different 183 to the WT IgG. This suggested that both hypofucosylation and hypergalactosylation are 184 IgG and vWF alone in stimulating thrombus formation under shear ( Figure 1D ). We found 204 that spike and IgG alone or combined to form immune complexes were insufficient to 205 stimulate thrombus formation in the absence of vWF. The vWF coating alone supported 206 formation of thrombi, but this was not further increased when combined with spike, IgG (WT 207 or low fuc, high gal) or immune complexes containing spike and WT IgG. However, a 208 significant increase in thrombus volume was found when the vWF coating was combined 209 with immune complexes containing spike and Low Fuc High Gal IgG This suggests that anti-210 spike IgG with aberrant glycosylation of the Fc domain synergises with vWF to enhance 211 thrombus formation. This replicates the synergy observed between platelet receptors that 212 predominantly mediate adhesion, such as GPIb and integrin α2β1, with receptors that 213 strongly activate platelet signalling such as GPVI and CLEC-2. 19 214

FcγRIIA is the only Fc receptor expressed in platelets and activates intracellular signalling 215 via Syk activation. 20 We hypothesised FcγRIIA may play the role of a signalling receptor to 216 synergise with the vWF adhesion receptor GPIb to enhance thrombus formation. To test this, 217 whole blood was preincubated with FcγRIIA blocking antibody IV.3 before perfusion through 218 vWF-coated flow chambers with either WT or Low Fuc High Gal immune complexes ( Figure  219 2Ai-ii). Blockade of FcγRIIA resulted in a significant reduction in the volume of thrombi 220 formed on the Low Fuc High Gal IgG-containing immune complexes. A modest, non-221 significant increase in thrombus volume observed with WT IgG, was also reversed by IV.3. 222

To understand if potentiation of thrombus formation on vWF also occurred at lower shear 223 more representative of those found in small veins and venules, we measured thrombus 224 formation at 200s -1 ( Figure 2B ). We found that thrombi formed on vWF at a shear rate of 225 200s -1 were smaller than those formed at 1000s-1, but Low Fuc High Gal IgG-containing 226 immune complexes again caused significant potentiation of thrombus volume relative to WT 227

To understand the signalling processes underpinning the enhancement of thrombus 232 formation on vWF and Low Fuc High Gal IgG and identify potential treatment strategies to 233 counteract pathogenic platelet activation we studied the effects of small molecule inhibitors 234 ( Figure 3 ). FcγRIIA signals through the tyrosine kinase Syk, so we treated whole blood with 235 the Syk inhibitor R406 which is the active metabolite of the FDA-approved drug fostamatinib. 236

Treatment with R406 significantly reduced the volume of thrombi formed on Low Fuc High 237

Gal IgG (373k ± 42k μm 3 ) relative to vehicle (820k ± 172k μm 3 ), indicating that activation of 238

Syk is important to the prothrombotic effects of aberrantly glycosylated anti-spike IgG and 239 that treatment with fostamatinib might be beneficial for patients with severe COVID19 240 through suppression of IgG-driven platelet activation. The FcγRIIA signalling pathway is also 241 dependent on Btk 12 and we therefore treated platelets with the Btk inhibitor ibrutinib, which is 242 an FDA and EMA approved drug for treatment of B cell cancers. We found that ibrutinib 243 treatment reduced the volume of thrombi formed on the Low Fuc High Gal IgG (348k ± 68k 244 μm 3 ) to levels similar to the WT IgG (478k ± 76k μm 3 ).Platelet activation stimulated by 245

FcγRIIA triggers secretion of ADP which activates the P2Y12 receptor and provides positive 246 feedback signalling required for integrin α IIb β 3 activation and aggregation. 21 We hypothesised 247 that inhibition of P2Y12 using an antagonist might also help reduce thrombotic tendency in 248 severely ill COVID-19 patients and we treated platelets with the P2Y12 antagonist cangrelor, 249 an active drug molecule that does not require metabolism, to this in vitro.was We found that 250 cangrelor reduced the volume of the thrombi formed on WT IgG, although this reduction was 251 non-significant. Cangrelor treatment significantly reduced thrombi formed on Low Fuc High 252

Gal IgG (389k ± 40k μm 3 ) to comparable levels to those observed with WT IgG. 253

Platelet aggregability is enhanced in patients with severe COVID-19 22 and we hypothesised 255 that this might be due to the presence of immune complexes containing anti-spike IgG with 256 aberrant glycosylation of the IgG Fc domain. To test this hypothesis we preincubated 257 recombinant SARS-COV-2 spike protein with the same four COVA1-18 IgG variants used in 258 previous experiments to enable formation of immune complexes in suspension. We then 259 treated washed human platelets with the immune complexes and stimulated with a range of 260 collagen (type I) concentrations to induce aggregation ( Figure 4A ). We found that none of 261 the immune complexes enhanced the potency (EC 50 ) of collagen-evoked aggregation 262 ( Figure 4B ) or caused aggregation on their own. We also assessed the ability of the immune 263 complexes to potentiate activation of integrin α IIb β 3 by measuring fibrinogen binding 264 stimulated with ADP, TRAP-6 or CRP-XL by flow cytometry (Figure 4Ci-iii) . We observed no 265 significant difference between integrin activation stimulated by agonists in the presence of 266 spike protein alone or immune complexes containing both spike and IgG. These data 267

suggest that the manner of presentation of immune complexes may be an important part of 268 the mechanism by which they activate platelets. Clustering of FcγRIIA induces intracellular 269 signalling in platelets 20 and it is possible that immune complexes presented in suspension, 270 rather than immobilised on a surface, are below the concentration threshold required to 271 cause clustering of the receptor within our experimental system. Plasma samples from a 272 subset of COVID-19 patients positive for anti-spike IgG trigger platelet activation in 273 suspension, 23 and it is therefore possible that immune complexes of sufficient size and 274 concentration to activate platelets may occur. 275

There is a growing body of evidence that multiple complications arise in severely ill 19 patients that increase rates of thrombosis. These include damage to vascular endothelial 278 cells following direct infection with SARS-CoV-2, resulting in disruption of barrier function, 279 exposure of subendothelial collagen as well as release of prothrombotic plasma proteins 280 including vWF from activated endothelial cells. 17 The prothrombotic environment is 281 exacerbated by a cytokine storm that may be driven by activation of macrophages by 282 immune complexes containing afucosylated anti-spike IgG. 9 Hypofucosylated, 283 hypergalactosylated IgG has been identified in the plasma of severely ill COVID-19 patients 284 relative to patients with mild COVID-19 infection and correlated with disease severity. 9,15 In the present study we showed that immobilised immune complexes containing recombinant 286 anti-spike IgG with low fucosylation and high galactosylation activate platelets to enhance 287 thrombus formation on vWF, which is also elevated in severely ill COVID-19 patients. 16-18 288 Vascular endothelial cells can be infected by SARS-CoV-2 24 and we hypothesise that 289 subsequent expression of spike protein and formation of large immune complexes on the 290 cell surface might combine with secreted vWF to form a highly pro-thrombotic surface 291 ( Figure 5) . A similar mechanism for thrombosis induced by viral infection in the pulmonary 292 circulation has been identified in severe H1N1 infection, whereby immune complexes 293 present in the lungs activate platelets via FcγRIIA. 25 The role of aberrant IgG glycosylation in 294 stimulating this response was not investigated, but it has been suggested that afucosylated 295

IgG may be common to immune responses against all enveloped viruses. 9 Another study 296 that investigated platelet activation mediated by plasma samples from severely ill COVID-19 297 patients also reported that platelet activation was dependent on FcγRIIA. 23 The plasma 298 samples were positive for anti-spike antibodies, but the glycosylation status was not 299 measured. Another report identified a link between afucosylated IgG and FcγR-dependent 300 activation of macrophages in severe COVID-19 illness, in which high antibody titres 301 combined with altered glycosylation resulted in excessive secretion of cytokines. 15 Direct 302 binding of spike protein to platelet ACE2 has been reported as a potential mechanism for 303 platelet hyper reactivity in severe COVID-19 infection, 26 however, expression of ACE2 in 304 platelets is controversial 27 and we did not find evidence for direct platelet activation by spike 305

The role of platelets in COVID-19 is still emerging but platelet rich thrombi have been 307 identified in both large arteries and microthrombi. 4 The platelets of severely ill COVID-19 308 patients express markers of activation 28 and exposure of platelets from healthy donors to 309 plasma from these patients evokes activation. 29 Platelets contain many inflammatory 310 mediators within granules that might contribute toward the flood of cytokines present in 311 critically ill COVID-19 patients. Large numbers of platelet-monocyte and platelet-granulocyte aggregates have been identified in the blood of COVID-19 patients as well as development 313 of a pro-inflammatory phenotype in which expression of cytokines is increased. 30 There is 314 still scant information regarding the efficacy of antiplatelet drugs in COVID-19 patients but 315 one study has suggested that patients receiving antiplatelet therapy with aspirin prior to 316 hospital admission for COVID-19 appear to be partially protected and have better outcomes, 317 while a separate study found that in-hospital treatment of patients with aspirin reduced 318 mortality. 31,32 Other non-antiplatelet drugs in trials for COVID-19 therapy target proteins also 319 expressed in platelets and could therefore inhibit the contribution of platelets to 320 thromboinflammation. The Bruton's tyrosine kinase (Btk) inhibitor acalabrutinib has been 321 evaluated in clinical trials on the basis of its potential to block macrophage activation, 33 322 however, the potential of Btk inhibitors to reduce the contribution of platelets to thrombosis in 323 COVID-19 infection 34 and more generally in thomboinflammation 35 have also been noted. 324

We found that the Btk inhibitor ibrutinib reversed the enhancement of thrombus formation on 325 vWF caused by IgG with low fucosylation and high galactosylation, supporting the 326 hypothesis that this strategy might have dual benefits on macrophage and platelet activation. 327

The Syk inhibitor fostamatinib was identified as a potential COVID-19 therapeutic in a high 328 content screen of drugs that might protect against acute lung injury. 36 The active metabolite 329 of fostamatinib, R406 inhibits release of neutrophil extracellular NETS 37 and macrophage 330 activation induced by plasma from COVID-19 patients. 15 R406 is also known to have 331 inhibitory effects on signalling downstream of platelet GPVI and CLEC-2 receptors, 38 332 although maximal collagen-evoked aggregation is unaffected by oral administration of 333 R406, 39 and is currently in clinical trials for COVID-19 therapy in the US (NCT04579393) 334 and UK (NCT04581954). We found that R406 reversed the potentiation of thrombus 335 formation on vWF. This suggests that potential COVID-19 therapies such as fostamatinib or 336 acalabrutinib, targeting Syk or Btk respectively, may be effective not only in limiting the 337 inflammatory response, but also in reducing platelet-mediated thrombosis. 338 

Incidence of thrombotic complications in critically 355 ill ICU patients with COVID-19

Abnormal coagulation parameters are associated with poor 357 prognosis in patients with novel coronavirus pneumonia

Incidence of venous thromboembolism in 359 hospitalized patients with COVID-19

Megakaryocytes and platelet-fibrin thrombi 361 characterize multi-organ thrombosis at autopsy in COVID-19: A case series

The trinity of COVID-19: immunity, 364 inflammation and intervention

Extrafollicular B cell responses correlate with 366 neutralizing antibodies and morbidity in COVID-19

COVID-19-neutralizing antibodies predict 370 disease severity and survival

Afucosylated IgG characterizes enveloped viral 372 responses and correlates with COVID-19 severity

Proinflammatory IgG Fc structures in patients 374 with severe COVID-19

Human platelet IgG Fc receptor FcgammaRIIA in immunity and 376 thrombosis

Oral Bruton tyrosine kinase inhibitors block activation of 378 the platelet Fc receptor CD32a (FcgammaRIIA): a new option in HIT?

Recombinant SARS-CoV-2 spike 381 proteins for sero-surveillance and epitope mapping

Potent neutralizing antibodies from 383 COVID-19 patients define multiple targets of vulnerability

High titers and low fucosylation of early human anti-385 SARS-CoV-2 IgG promote inflammation by alveolar macrophages

Hypercoagulability of COVID-19 patients in 387 intensive care unit: A report of thromboelastography findings and other parameters of hemostasis

Severe COVID-19 infection associated with endothelial 393 activation

Identification of platelet function defects by multi-395 parameter assessment of thrombus formation

Clustering 397 of the platelet Fc gamma receptor induces noncovalent association with the tyrosine kinase p72syk. 398

CalDAG-GEFI deficiency protects mice in a novel model 400 of Fcgamma RIIA-mediated thrombosis and thrombocytopenia

Platelet gene expression and function in patients 402 with COVID-19

Platelet-activating immune complexes identified in 404 critically ill COVID-19 patients suspected of heparin-induced thrombocytopenia

Endothelial cell infection and endotheliitis in COVID-19

Influenza virus H1N1 activates platelets through 409 FcgammaRIIA signaling and thrombin generation

SARS-CoV-2 binds platelet ACE2 to enhance thrombosis in 411 COVID-19

Is there a role for the ACE2 receptor in SARS-CoV-2 413 interactions with platelets?

SARS-CoV-2 infection is associated with a pro-415 thrombotic platelet phenotype

Platelet activation and platelet-417 monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19

Mechanical Ventilation, ICU Admission, and In-Hospital Mortality in Hospitalized Patients with 423 COVID-19

Intermediate-dose anticoagulation, aspirin, and in-425 hospital mortality in COVID-19: a propensity score-matched analysis

Inhibition of Bruton tyrosine kinase in 427 patients with severe COVID-19

A rationale for 429 blocking thromboinflammation in COVID-19 with Btk inhibitors

Selective inhibition of thromboinflammation in 431 COVID-19 by Btk inhibitors

A High-Content Screen for Mucin-1-Reducing 433

Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury

Extracellular Traps Induced by COVID-19 Patient Plasma: A Potential Therapeutic

The novel Syk inhibitor R406 438 reveals mechanistic differences in the initiation of GPVI and CLEC-2 signaling in platelets

R406, an orally available spleen tyrosine kinase 441 inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation

This work was supported by Imperial College NIHR bioresources and grants from the British 351Heart Foundation (RG/15/2/31224 and RG/20/7/34866), ZonMw (10430 01 201 0008) and 352an Amsterdam Infection & Immunity COVID-19 grant (24184). 353