key: cord-0713662-7z2r2dkp
authors: Zlamal, J.; Althaus, K.; Jaffal, H.; Pelzl, L.; Singh, A.; Witzemann, A.; Haeberle, H.; Mirakaj, V.; Rosenberger, P.; Bakchoul, T.
title: cAMP prevents antibody-mediated thrombus formation in COVID-19
date: 2020-12-17
journal: nan
DOI: 10.1101/2020.12.15.20247775
sha: b36cd4f2ffc8e539d34cbe61074f7307c4123c6d
doc_id: 713662
cord_uid: 7z2r2dkp

Thromboembolic events are frequently reported in patients infected with the SARS-CoV-2 virus. However, the exact mechanisms of thromboembolic events remain elusive. In this work, we show that immunoglobulin G (IgG) subclass in patients with COVID-19 trigger the formation of procoagulant PLTs in a Fc-gamma-RIIA dependent pathway leading to increased thrombus formation in vitro. Most importantly, these events were significantly inhibited via Fc-gamma-RIIA blockade as well as by the elevation of PLTs intracellular cyclic-adenosine-monophosphate (cAMP) levels by the clinical used agent Iloprost. The novel findings of Fc-gamma-RIIA mediated prothrombotic conditions in terms of procoagulant PLTs leading to higher thrombus formation as well as the successful inhibition of these events via Iloprost could be promising for the future treatment of the complex coagulopathy observed in COVID-19 disease.

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Protein levels of cleaved-caspase 3 were determined by western blot. After 173 serum/IgG incubation, cells were washed with PBS for 7 min, 700xg at 4°C. 174

Subsequently, the pellet was resuspended in 100 μL of ice-cold RIPA lysis buffer 175 containing HALT TM protease and phosphatase inhibitor-cocktail (both ThermoFisher 176

Scientific, Paisley, UK). Protein concentrations were determined using the NanoDrop 177

One spectrophotometer (VWR, Bruchsal, Germany). 100 µg of protein was 178 solubilized in sample buffer (Invitrogen TM , Carlsbad, USA) at 95ºC for 10 min. overnight. After washing with TBS-T buffer, the membranes were incubated with the 187 appropriate secondary anti-rabbit or anti-mouse antibody conjugated with IRDye ® 680 188 / IRDye ® 800 (1:3000, LI-COR ® , Lincoln, USA) for 1 h at RT. Protein bands were 189 detected after additional washes (TBS-T) with Odyssey infrared imaging system COR ® , Lincoln, USA). Western blots were analyzed by ImageJ software (NIH, 191 Bethesda, USA). The results are shown as the ratio of total cleaved-caspase 3 to 192 procaspase 3 (full fragment) and normalized to wPLTs that were treated with healthy 193 control serum/IgG. 194

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To assess the impact of ICU COVID-19 IgG-induced effects on clot formation, an ex 213 vivo model for thrombus formation was established. A microfluidic system (BioFlux, 214

Fluxion Biosciences, Alameda, USA) was used at a shear rate of 1500 -1 (60 dyne) 215

according to the recommendations of the ISTH standardization committee for 216 biorheology (14). Briefly, microfluidic channels were coated with collagen (100 217 µg/mL, Collagen Horm, Takeda, Linz, Austria) overnight at 4°C and blocked with 218 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Finally, reconstituted whole blood samples were run at a shear rate of 1500s -1 234 (60 dyne) for a maximum of 5 min. Immunofluorescence and bright field images were 235 taken from 3-5 randomly chosen microscopic fields (x20, Olympus IX73, Olympus 236

GmbH, Hamburg, Germany). Clot formation was assessed by measuring the % of 237 surface area coated by thrombus (SAC) of 3-5 images via ImageJ (NIH, Bethesda, 238 USA) and normalized to the whole area. 239

Statistical analyses were performed using GraphPad Prism 7 (La Jolla, USA). T-test 241 was used to analyze normally distributed results. Non-parametric test (Mann-Whitney 242 test) was used when data failed to follow a normal distribution as assessed by 243 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in who were first admitted to normal ward and later to the ICU for mechanical 262 ventilation. As ICU control group, 5 patients who were admitted to ICU due to non-263 COVID-19 related causes were included in this study. 264

To investigate whether sera of ICU COVID-19 patients have the potential to induce 267 an increased Δψ depolarization as well as PS externalization on the PLT surface, 268 wPLTs from healthy individuals were incubated with sera from 26 ICU COVID-19 269 patients with a severe course of disease as well as 5 ICU non-COVID-19 patients. 270

Based on the calculated cuttoffs (mean+2xSD of healthy controls), 19/26 (73%) sera 271 from patients with severe COVID-19 disease induced significantly higher Δψ 272 depolarization in PLTs from healthy donors compared to ICU controls (FI in % Δψ 273 depolarization±SEM: 6.10±1.12 vs. 0.67±0.10, p value <0.0001, Fig. 1 A) . In addition, 274 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in Fig. 1 B) . Fig. 1 E+F) . Notably, the rise of PLT markers was associated with 290 increasing levels of detected IgGs against the spike S protein of SARS-CoV-2 in the 291 corresponding ICU COVID-19 patients' follow up sera but not in the total IgG 292 contents of isolated IgG fractions (Suppl. Fig. 2 A and B , respectively). Moreover, 293 declining PLT-counts were observed as Δψ depolarization as well as PS 294 externalization increased, vice versa (Suppl. Fig. 3 A and B , respectively). 295

To further verify the impact of sera from severe COVID-19 patients on PLTs, the 298 expression of the alpha granule release and PLT activation marker CD62p was 299 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.15.20247775 doi: medRxiv preprint

To further determine the underlying mechanistic pathways leading to ICU COVID-19 327

IgG induced formation of procoagulant PLTs, we next considered a potential ligation FcγRIIA. 346

procoagulant PLTs 348 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

PLTs 371

The interplay between the signalling pathways of the intracellular second 372 messengers, cAMP and calcium, has been shown to have an important role in 373 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. The alterations in PLTs that were observed in our study after incubation with 431 sera from patients with severe COVID-19 infection, such as Δψ disruption, caspase 3 432 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.15.20247775 doi: medRxiv preprint cleavage and PS externalization, could be found in apoptotic as well as procoagulant 433

PLTs. The involvement of PLT apoptosis to promote prothrombotic conditions has 434 been controversially discussed. In fact, recent data suggests that apoptotic PLTs are 435 unable to promote prothrombotic conditions (10). However, a clear dissection of the 436 molecular events leading to prothrombotic PLTs is challenging since activation of the 437 apoptosis caspase pathway has been described in the late phase of agonist-induced 438 While this findings are novel for COVID-19-associated coagulopathy, the correlation 458 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.15.20247775 doi: medRxiv preprint between FcγRIIA ligation and increased risk for thromboembolic events is well 459 established for heparin-induced thrombocytopenia (HIT) (24). Interestingly, the IgG 460 antibody formation peaks in HIT within 5-10 day after exposure to heparin and is 461 associated with PLT consumption and increased risk for thrombosis (25). Similarly, in 462 our study the ability of ICU COVID-19 sera to induce procoagulant PLTs was most 463 pronounced between day 3 and 7. Of note, antibody-induced alterations in PLT in COVID-19 antibody-mediated coagulopathy. Another minor finding from our 521 microfluidic system was that Iloprost, although significantly inhibited antibody-522 mediated thrombus formation, did not affect the CD62p-single positive population. 523

Since Iloprost prevented clot formation, this finding might indicate that PS rather than 524

CD62p exposure on the PLT surface is pivotal to trigger the onset of thromboembolic 525

Our study is subjected to some limitations. First, as an observational, 527 monocentric study, we cannot conclude that the reported associations between IgG 528 antibodies and changes in activation/apoptosis markers in COVID-19 are causal for 529 the thrombosis or specific for the disease. Second, we cannot exclude the possibility 530 of remaining residual confounding or unmeasured potential confounders in our 531 mechanistic studies. Third, the low number of patients does not enable a final and 532 robust statistical analysis to assess clinical outcomes in patients with increased 533 procoagulant PLTs. Nevertheless, data presented in this study may provide a 534 background for future studies to dissect mechanisms related to PLT activation that 535 are involved in the progression of COVID-19. 536 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.15.20247775 doi: medRxiv preprint

Taken together, our study shows that IgG antibodies from patients with severe 537 COVID-19 are able to stimulate FcγRIIA leading to the induction of procoagulant 538

PLTs with an increased ability of clot formation. These processes are dependent on 539 calcium and can be efficiently inhibited by cAMP inducers suggesting that ADC might 540 represent a potentially promising target to prevent thromboembolic complications in 541 COVID-19 disease. 542 543 544 545 546 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.15.20247775 doi: medRxiv preprint absence of moAb IV.3 or isotype control (moAb). ns, not significant; *p<0.05, 643 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.15.20247775 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.15.20247775 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

levels in wPLTs that were pretreated with vehicle or Iloprost (20 nM) prior to ICU

COVID-19 IgG incubation. α-Tubulin served as loading control. (C) Densitometric 719 analysis of cleaved caspase 3/procaspase 3 ratios from the WB data

SSC properties after ICU COVID-19 IgG incubation in vehicle or Iloprost

B+D) (E+G) Gate distribution of CD42a positive vehicle 723 or Iloprost (20 nM) pretreated wPLTs that were incubated with ICU COVID-19 IgG

Clinical course and risk factors for mortality of 747 adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Thrombosis in COVID-19

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Platelet gene 760 expression and function in patients with COVID-19

Insights into platelet-based control of 762 coagulation

Integrating platelet and 764 coagulation activation in fibrin clot formation

Procoagulant platelets: generation, function, and therapeutic targeting 766 in thrombosis

Procoagulant Phosphatidylserine-Exposing Platelets in vitro and in vivo

Procoagulant platelets and the pathways leading to cell death

A rapid and sensitive test for diagnosing 772 heparin-associated thrombocytopenia

In vitro flow based 776 systems to study platelet function and thrombus formation: Recommendations for standardization: 777 Communication from the SSC on Biorheology of the ISTH

Clinical features of patients infected with 779 2019 novel coronavirus in Wuhan

Cyclic nucleotide-dependent inhibitory signaling interweaves with 783 activating pathways to determine platelet responses

Protein kinase A determines platelet life span 785 and survival by regulating apoptosis

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the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted

Abnormal coagulation parameters are associated with poor 789 prognosis in patients with novel coronavirus pneumonia

Role of caspase in a subset of human platelet activation 794 responses

Increased platelet phosphatidylserine 796 exposure and caspase activation in chronic uremia

Heparin-induced thrombocytopenia: 798 mechanism of interaction of the heparin-dependent antibody with platelets

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Functional significance of the platelet immune receptors 804 GPVI and CLEC-2

Regulation of phosphatidylserine 809 transbilayer redistribution by store-operated Ca2+ entry: role of actin cytoskeleton

R93W mutation in Orai1 812 causes impaired calcium influx in platelets

Collagen but not 814 fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity 815 of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses

Autoantigenic epitopes on platelet glycoproteins

Two distinct 820 pathways regulate platelet phosphatidylserine exposure and procoagulant function

Dual 823 mechanism of integrin alphaIIbbeta3 closure in procoagulant platelets

Platelet factor XIII and calpain negatively regulate integrin 826 alphaIIbbeta3 adhesive function and thrombus growth

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.15.20247775 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.15.20247775 doi: medRxiv preprint