key: cord-0698878-xq6flbbu authors: Althaus, Karina; Marini, Irene; Zlamal, Jan; Pelzl, Lisann; Singh, Anurag; Häberle, Helene; Mehrländer, Martin; Hammer, Stefanie; Schulze, Harald; Bitzer, Michael; Malek, Nisar; Rath, Dominik; Bösmüller, Hans; Nieswandt, Bernard; Gawaz, Meinrad; Bakchoul, Tamam; Rosenberger, Peter title: Antibody-induced procoagulant platelets in severe COVID-19 infection date: 2021-01-08 journal: Blood DOI: 10.1182/blood.2020008762 sha: 0e3fdc76886b8ee3eca6551501594bc5cfb27449 doc_id: 698878 cord_uid: xq6flbbu The pathophysiology of COVID-19 associated thrombosis seems to be multifactorial. We hypothesized that COVID-19 is accompanied by procoagulant platelets and platelet apoptosis with subsequent alteration of the coagulation system. We investigated depolarization of mitochondrial inner transmembrane potential (ΔΨm), cytosolic calcium (Ca2+) concentration, and phosphatidylserine (PS) externalization by flow cytometry. Platelets from intensive care unit (ICU) COVID-19 patients (n=21) showed higher ΔΨm depolarization, cytosolic Ca2+ concentration and PS externalization, compared to healthy controls (n=18) and COVID-19 non-ICU patients (n=4). Moreover significant higher cytosolic Ca2+ concentration and PS was observed compared to septic ICU control group (ICU control). In ICU control group (n=5; ICU non-COVID-19) cytosolic Ca2+ concentration and PS externalization was comparable to healthy control, with an increase in ΔΨm depolarization. Sera from ICU COVID-19 patients induced significant increase in apoptosis markers (ΔΨm depolarization, cytosolic Ca2+ concentration and PS externalization) compared to healthy volunteer and septic ICU control. Interestingly, immunoglobulin G (IgG) fractions from COVID-19 patients induced an Fc gamma receptor IIA dependent platelet apoptosis (ΔΨm depolarization, cytosolic Ca2+ concentration and PS externalization). Enhanced PS externalization in platelets from ICU COVID-19 patients was associated with increased sequential organ failure assessment (SOFA) score (r=0.5635) and D-Dimer (r=0.4473). Most importantly, patients with thrombosis had significantly higher PS externalization compared to those without. The strong correlations between procoagulant platelet and apoptosis markers and increased D-Dimer levels as well as the incidence of thrombosis may indicate that antibody-mediated platelet apoptosis potentially contributes to sustained increased thromboembolic risk in ICU COVID-19 patients. Accumulating evidence indicates an association between SARS-CoV-2 associated pneumonia and hypercoagulable state of patients with corona virus disease 2019 (COVID-19) who require intensive care (severe infection) 1 . It is becoming more and more clear that infections with SARS-CoV-2 do not meet the criteria of disseminated intravascular coagualation (DIC) according to the International Society of Thrombosis and Haemostasis (ISTH) and the patterns of coagulation seem to be different to severe infections 2 . Only in patients with severe aggravation of disease an overt DIC occurred 3 . At the same time, however, the incidence of acute pulmonary embolism (PE), deep-vein thrombosis, ischemic stroke, myocardial infarction and/or systemic arterial embolism in COVID-19 patients admitted to the intensive care unit (ICU) is as high as 49% 4 . The pathophysiology of COVID-19 associated-thromboembolic events seems to be complex and multifactorial, involving interplay between cellular and plasma elements of the hemostatic system and components of the innate immune response to the infecting pathogen. The phosphatidylserine (PS) externalization in apoptotic platelets, as a substrate, might be an initiation for multiple coagulation factors 5 . So a combination of several activation events initiated by exposure of the endothelium, platelets, and leukocytes to pathogen-and damage-associated molecular patterns might be responsible for the uncontrolled activation of the coagulation system in severely ill COVID-19 patients 6, 7 . To date most clinical reports on COVID-19-associated coagulopathy focused on an increased activation of the plasma coagulation system [1] [2] [3] 7 . Elevated D-Dimer levels were consistently reported, whereas their gradual increase during disease course is particularly associated with disease worsening 8 . Other coagulation abnormalities such as prothrombin time (PT), activated partial thromboplastin time (aPTT) prolongation, together with severe thrombocytopenia have been found to be associated with life-threatening DIC 9 . Platelets have been recently recognized as a mediator of inflammation and sensor of infectious agents through the interaction of surface receptors and pathogens or immune system derivatives 10 . Viral infections elicit the systemic inflammatory response that affects platelets, which become activated upon antigen specific recognition and interaction with white blood cells 11 . While platelet activation plays a critical role in the procoagulatory effect of viral infections 12, 13 , platelets derived from patients with HIV as well as secondary dengue virus infection have been shown to have increased upregulation of the intrinsic pathway of apoptosis [14] [15] [16] . In addition, it is well known that the survival and lifespan of platelets is finely regulated by the intrinsic or mitochondrial apoptosis pathway 17, 18 . We hypothesized that coagulation disturbances observed in ICU COVID-19 patients are accompanied by platelet apoptosis with subsequent alterations of the coagulation system. In platelets, apoptosis is mediated by the mitochondrial outer membrane permeabilization (MOMP) which is regulated by the members of the Bcl2 protein family that either promote (pro-apoptotic proteins: Bak, Bax and Bim) or inhibit (anti-apoptotic proteins: Bcl-xL and Bcl-w) the apoptosis 19 . The mitochondrial inner membrane potential (ΔΨm) collapse is followed by the efflux of cytochrome c into the cytoplasm which forms a multi-protein complex called apoptosome. The latter triggers the activation of the caspase 9 and the following downstream caspase cascade including caspase 3 and 7. Finally, the PS externalization on the extracellular membrane represents one of the late stage of the apoptosis pathway 20 . In this study, we found evidence that platelets from severe ICU COVID-19 patients have an upregulation of apoptotic markers. Most importantly, sera and IgGfractions isolated from COVID-19 patients were able to induce apoptosis in platelets from healthy donors. Furthermore, our data indicate that platelet apoptosis might be associated with thromboembolic complications and increased mortality in severe COVID-19. During the SARS-CoV-2 outbreak in Tuebingen (between March the1 st and April the 30 th 2020), 27 ICU COVID-19 and non-ICU patients were referred to our laboratory for extended investigations of the coagulations system. Blood samples from healthy donors (n=18) and septic ICU non-COVID-19 patients (n=5) were collected to serve as controls. Surgical patients who developed post-operative sepsis and needed passive ventilation due to respiratory failure were included in the ICU non-COVID-19 control group. Among them, two patients had pneumonia, two developed a systemic infection after trauma, and one patient had a septic reaction after perforation of the appendix. Electronic medical records were used to collect demographical data, clinical treatments and outcome. Diagnosis of thromboembolic complications was made when clinically or laboratory indicated based on computer tomography, ultrasound imaging or in case of death by postmortem pathology. To estimate the status of critical illness, the sequential organ failure assessment (SOFA) score system was employed as previously described 21 . Data were independently reviewed by two physicians (K.A. and S.H.). In case of disagreements, a third physician was consulted (M.M.). For more details see supplemental material. Platelets were isolated from citrated blood and tested within 3 hours (h*). In brief, after one centrifugation step (120g, 20 minutes [min*] at room temperature [RT], without brake), platelet rich plasma (PRP) was gently separated and used for further analysis. To detect changes in the ΔΨm, the tetramethylrhodamine ethyl ester (TMRE) assay kit (Abcam, Cambridge, United Kingdom) was used as previously described, with minor modification 22, 23 . In brief, PRP was stained with 10 µM TMRE (30 min* at RT) and directly measured by flow cytometry (FC) (Navios, Beckman-Coulter, USA). The complete depolarization of platelet mitochondrial potential was induced using the uncoupler of mitochondrial oxidative phosphorylation carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP) (10 µM, 1 h* at 37°C) 16 . Changes in the ΔΨm were determined as ratio of the mean of fluorescence intensity (MFI) signal TMRE from healthy donors' PRP compared to the MFI signal of platelets from COVID-19 and ICU non-COVID-19 patients. Cytosolic Ca 2+ concentration was determine using Fluo-3/AM (Sigma-Aldrich, St. Louis, USA) as previously described 24 , with minor modifications. In brief, PRP were incubated with Fluo3/AM (3 µM) in TRIS (trisaminomethane) buffer (10 mM TRIS, 0.9% NaCl, 1 mM CaCl 2 , pH 7.4) for 30 min* at 37°C. After washing once with TRIS buffer, cells were resuspended in TRIS containing 1 mM CaCl 2 . To exclude unspecific effects like the activation of platelets via complement or non-specific immune complexes, all sera were heat-inactivated (56°C for 30 min*), followed by a sharp centrifugation step at 5,000g. The supernatant was collected. All experiments involving patients' sera were performed after incubation of 5 µL serum with 25 µL washed platelets (7.5x10 6 , see supplemental material for more information on the preparation of Afterwards, samples were washed once (7 min*, 650g, RT, without brake) and gently resuspended in 75 µL of phosphate-buffered saline (PBS, Biochrom, Berlin, Germany). When indicated, immunoglobulin G (IgG) fractions were isolated from serum (for more details see supplemental material). Washed platelets were stained with 10 µM TMRE (30 min* at RT) and directly measured by FC. As positive control, cells were preincubated with FCCP (10 µM for 30 min* at 37°C). Serum-mediated apoptosis was quantified as ratio of ΔΨm depolarization comparing the MFI of washed platelets incubated with serum from healthy donors with sera from (non-ICU and ICU) COVID-19 or ICU non-COVID-19 patients. Test results were normalized to the mean MFI of patients' sera compared to sera from healthy donors tested in parallel. Upon incubation with patients' sera washed platelets were incubated with Fluo3/AM (3 µM) in TRIS buffer (30 min* at 37°C). After washing once with TRIS buffer, cells were resuspended in TRIS supplemented with 1 mM CaCl 2 . Subsequently, Ca 2+ -dependent fluorescence intensity of samples was measured by FC. Ionomycin (5 µM, 15 min* at RT) was used as positive control. Test results were normalized to the mean MFI of patients' sera compared to sera from healthy donors tested in parallel. Protein levels of cleaved-caspase 9 were determined by western blot. In brief, washed platelet were centrifuged (5 min*, 700g at 4°C) and the pellet was resuspended in RIPA lysis buffer (ThermoFisher Scientific, Paisley, UK). The proteins were separated by electrophoresis using 12% SDS-PAGE. See the supplemental material for further details. The study was conducted in accordance with the declaration of Helsinki. Written informed consent was obtained from all volunteers prior to any study-related procedure. All tests were performed with rest material from routine testing. The study protocol of patient material was approved by the Institutional Review Board of the University of Tuebingen. The statistical analysis was performed using GraphPad Prism, Version 7.0 (GraphPad, La Jolla, USA). Because potential daily variations in FC measurements might result in bias in data analysis, test results were normalized to two healthy donors tested in parallel at the same time point (raw data are available in the supplemental data). For further information see the supplemental material. Data may be requested for academic collaboration from the corresponding author. Blood samples were collected from 27 consecutive COVID-19 patients who were admitted to our hospital with severe acute respiratory distress symptoms (ARDS) requiring intensive care (ICU, n=23) and hospitalized patients (non-ICU n=4) without ARDS. Two blood samples were excluded due to insufficient material. In total, 21 ICU COVID-19 patients were enrolled in the study, of whom 18/21 (86%) patients were male, Table 1. The mean age was 60 years (range: 29-88 years, Table 1 ). 15/21 (71%) patients had known risk factors for severe COVID-19 infection as described previously 26 Table 1 ). On the ICU, all patients received controlled respiratory support and six of them had additionally venous-venous extracorporeal membrane oxygenation (vv-ECMO). Anticoagulation with heparin was administrated at prophylactic doses (400 IE/h) in 12/21 (57%) patients or at therapeutic doses (2 to 3 fold aPTT) in 9/21 (43%) patients, depending on patient's risk factors for thrombosis. Table 1 . During the 30-day follow-up, 7/21 (33%) deceased, 13/21 (62%) patients developed thrombocytopenia (range platelet count: 9-149x10 9 /L) and 12/21 (57%) patients had at least one thromboembolic complication including kidney, spleen and liver infarction (n=7), catheter associated thrombosis (n=2), clotting of extracorporeal circulation system (n=1), pulmonary embolism (n=1), cerebral infarction (n=1) and myocardial infarction (n=1), Supplemental Table 1 . To evaluate the mitochondrial function in platelets from COVID-19 patients, the mitochondrial ΔΨm, was assessed. As shown in Figure 1A To draw conclusion on potential clinical impact of our findings, we limited the analysis of the correlations apoptosis markers to platelet count, SOFA score, D-Dimer and COVID-19-antibody levels at the days of blood sampling. We found that platelet count is negatively correlated with PS externalization (r= -0.6177, p=0.0028, Figure 2A ) and with cytosolic Ca 2+ concentration (r= -0.5666; p=0.0299, Figure 2B ). Strong positive correlation was also observed for PS externalization with D-Dimer (r=0.4473; p=0.0420; Figure 2C ) as well as with the SOFA score (r=0.5635; p=0.0078, Figure 2D ) and with SARS-CoV2-antibodies (AB) (r=0.5745; p=0.0064, Supplemental Figure 3 ). Next, we analyzed the markers of apoptosis in relation to the incidence of thromboembolic complications and mortality during the 30-day follow-up. We found that ICU COVID-19 patients with thromboembolic complications had significantly higher levels of PS externalization compared to those without thrombosis (2.85 ±0.75 vs. 0.99 ±0.20, p=0.0340, Figure 2E ). In addition, cytosolic Ca 2+ concentration was significantly higher in non-survivors compared to survivors (4.07 ±0.39 vs. 2.05 ±0.30, p=0.0012, Figure 2F ). To explore the mechanism of platelet apoptosis in COVID-19, patients' sera were incubated with washed platelets from healthy donors. As shown in Figure 3A Figure 3C ; Supplemental Figure 2C ). In addition, significantly higher levels of cleaved-caspase 9 were found after incubation with sera from COVID-19 compared to healthy control (3.40 ±0.65 vs. 1.00 ±0.00, p=0.0030; Figure 3D -E). In the whole cohort, sera from 13/21 (62%) patients induced enhanced ΔΨm depolarization, 8/19 (42%) patients increased cytosolic Ca 2+ concentration and 13/21 (62%) patient ' s sera mediated increased PS externalization on platelets of healthy donors ( Figure 3F ). In order to further dissect the pathways of the antibody-mediated platelet apoptosis, IgGfractions were isolated from apoptosis-inducing sera tested in the absence or presence of a FcγRIIA-blocking mAb (IV.3). Interestingly, IgG fractions from COVID-19 sera induced significant upregulation in all three apoptosis markers. ΔΨm depolarization was found significantly increased in platelets isolated from ICU COVID-19 patients compared to healthy donors (1.54 ±0.14 vs. 1.00 ±0.00, p=0.0051, Figure 4A ) as well as to non-ICU COVID-19 patients (1.54 ±0.14 vs. 1.16 ±0.08, p=0.0249, Figure 4A ). Cytosolic Ca 2+ concentration was increased compared to healthy donors Figure 4C ) as well as to ICU non-COVID-19 patients (1.70 ±0.27 vs. 1.08 ±0.08, p=0.0547, Figure 4C ). In contrast, a significant reduction of platelet apoptosis was observed after blocking platelet Figure 4E ) and PS externalization on platelets (9.59 ±1.52 vs. 2.12 ±0.20, p=0.0371, Figure 4F ), respectively. Of note, protein staining of the isolated IgG showed no hint of IgG aggregates or immune complexes (data not shown). To elucidate whether apoptosis is driven by loss of mitochondrial integrity or not, we investigated the impact of CsA and the pan caspase-inhibitor Q-VD-OPh on IgG mediated platelet apoptosis. CsA efficiently blocked ΔΨm depolarization (2.55 ±0.27 vs. 1.16 ±0.15, p=0.0313, Figure 5A ) and PS externalization (5.91 ±0.84 v.s. 1.13 ±0.15 vs., p=0.0313, Figure 5B ) as compared to non-treated platelets. Q-VD-OPh showed no significant inhibition of ΔΨm depolarization (3.70 ±0.67 vs. 3.08 ±0.75, p=0.2188, Figure 5C ) but significantly reduced PS externalization (5.25 ±1.38 vs. 3.54 ±1.06, p=0.0313, Figure 5D ) compared to non-treated platelets. Taken together, with the observed cleavage of caspase-9, these data indicate that IgG fractions from severe COVID-19 patients induce procoagulant/apoptotic platelets leading to the expression of PS by different pathways. COVID-19-infected individuals have a heightened risk of developing thromboembolic complications and a link has been suggested between low platelet count and severity of disease and mortality [27] [28] [29] . Here, we show that severe COVID-19 is associated with antibody-mediated upregulation of platelet apoptosis. In addition, we found a correlation between platelet apoptosis markers and SOFA score, plasma levels of D-Dimer as well as the incidence of thromboembolic complications in severe COVID-19 patients. These data indicate that platelet apoptosis may contribute to sustained inflammation and increased thromboembolic risk in COVID-19 patients and could potentially present a potential therapeutic target. A subpopulation with severe COVID-19 in our study had an increase in platelet apoptosis markers. The exact mechanism of COVID-19 induced platelet apoptosis has not been studied so far. Our data show depolarization of mitochondrial inner transmembrane potential in a subgroup and enhanced cytosolic Ca 2+ concentration in most of the patients with severe (ICU) COVID-19. Together with the increased PS externalization and cleavage of caspase 9, these results suggest that platelet apoptosis in severe COVID-19 is activated via the intrinsic pathway. Apoptosis is also described for septic patients 30 . But apoptosis seem to be different from apoptosis in septic patients. Notably, we could find increased ΔΨm depolarization in platelets of septic patients, but we could not show any increase in cytosolic Ca 2+ concentration or in PS externalization patient platelets in the ICU non-COVID-19 control group. After incubation of serum from septic patients with platelets of healthy donors we observed no effect hinting towards an additional serum factor for apoptosis in COVID-19 patients. While, no enhanced apoptosis was observed in patients , platelets or in healthy platelets after incubation of sera isolated from ICU non-COVID-19 control patients suggesting that the activation of the apoptotic pathway may be a specific effect induced by the COVID-19 infection through a serum component. But it remain unclear whether apoptosis alone is sufficient to support thromboembolic status in these patients or if it is linked to platelet activation Of note, cytosolic Ca 2+ is a marker for platelet apoptosis as well as agonist-induced activation. Our data indicate therefore that IgGs from severe COVID-19 patients induce procoagulant platelets that might contribute to the increased risk for thromboembolic complications 31, 32 . In apoptotic platelets lifetime is shortened and platelet function is also reduced. In our cohort severe thrombocytopenia was not common among COVID-19 patients, which was also described for the SARS-CoV-1 in 2008 33 . In fact, SARS-CoV-1, which caused the epidemic outbreak in 2003, has also been previously shown to induce apoptosis in Vero cells in a virus replication-dependent manner 34 , despite missing thrombocytopenia. Downregulation of Bcl-2, the activation of effector caspase 3, as well as the upregulation of the pro-apoptotic protein Bax were detected, suggesting the involvement of the caspase family and the activation of the mitochondrial signaling pathway. In fact, our data showed also that platelets from severe COVID-19 patients have higher levels of cleaved-caspase 9. Prolonged exposure to higher concentrations of platelet agonists has been shown to induce the collapse of mitochondrial membrane potential and subsequent platelet apoptosis 35 . Enhanced platelet activation leading to platelet deposition in damaged pulmonary blood vessels has been demonstrated recently for COVID-19 36 . To exclude potential contribution of platelet hyperactive status in our cohort, we assessed the impact of patients' sera on platelet apoptosis. Recent data showed that autoantibodies from patients with autoimmune thrombocytopenia (ITP) are able to induce platelet apoptosis 37 . The incubation of sera as well as IgG fractions from severe COVID-19 patients with platelets from healthy donors induced significant changes in apoptosis markers including ΔΨm depolarization, increased cytosolic Ca 2+ concentration, caspase 9 cleavage, and finally PS externalization. More importantly, the antibody-mediated platelet apoptosis was inhibited by blocking the FcγRIIA receptors using a specifc mAb. These data indicate that IgG antibodies contribute to the increased PS expression on platelets of patients with severe COVID-19 infection. However, we cannot exclude other co-factor(s) that could also induce procoagulant platelets in vivo. Interestingly, we found an association between the IgG binding to the Spike-protein of SARS-CoV2 and the PS externalization after incubation with patients' sera.However, it still remains unclear, whether it is a direct IgG-virus complex interaction like previously described for the influenza virus H1N1 infection 38 and dengue fever 15 or an indirect effect after binding to platelet nonspecific targets. Nevertheless, our data indicate that severe COVID-19 is associated with antibody-mediated activation of the intrinsic pathway via crosslinking FcγRIIA receptors by IgG antibodies against tobe-identified target antigen(s). These findings might offer new therapeutic options like blockade of the FcγRIIA receptor sginaling by tyrosinkinase inhibitors, which have been suggested to have a potential use to prevent platelet activation in heparin-induced thrombocytopenia (HIT) 39 . The clinical relevance of platelet apoptosis is supported by the significant negative correlation between PS levels on platelet surface with platelet count, the positive correlation with D-Dimer plasma levels, and most importantly with the SOFA score. Meanwhile, it is well established that COVID-19 is associated with increased risk for thromboembolic complications. During the 30day follow-up, 57% of our patients had thromboembolic complications. Platelet from ICU COVID-19 patients with thromboembolic events showed significantly increased externalization of the apoptosis marker PS compared to those with no thrombosis. This finding suggests a link between thromboembolism and platelet apoptosis in COVID-19 as has been suggested for acute pulmonary embolism 40 . Our study is subjected to limitations. First, as an observational study, we cannot conclude that the reported associations between platelet markers and laboratory parameters as well as clinical outcomes are causal or specific for infection with SARS-CoV-2. Second, we cannot exclude the possibility of remaining residual confounding or unmeasured potential confounders. Third, the low number of patients does not enable a final and robust multivariate statistical analysis. In addition, we observed a small difference in age between ICU COVID-19 patients and controls. However, age of the patient is not classically associated, and little is known on the impact of age on platelet apoptosis. It should also be emphasized that it is not clear whether the platelet apoptosis described here are drivers of disease severity or a mere consequence of hypercoagulation in severe (ICU) COVID-19 patients. Indeed, the definitive pathophysiology of COVID-19 and answering questions of causality will likely await the development of model systems for the disease. Our data showed that severe COVID-19 infection induces procoagulant platelets (loss of the mitochondrial potential associated with phosphatidylserine exposure and caspase-stimulation). Despite the small number of PS+ platelets that was observed in our study, these cells have a remarkable potential to accelerate the thrombus formation as described in the literature 5 . In addition, in an ongoing studies IgG fractions from COVID-19 patients that indcued PS externalization, showed a marked ability to induce rheological changes in whole blood, leading to increased thrombus formation. We believe that our findings present another piece of the puzzle and will motivate further research into the role of platelet apoptosis in COVID-19 associated thromboembolic complications. Data presented in this study may build a basis for future studies to dissect platelet-mediated pathological mechanisms involved in the progression of COVID-19 and a larger and multi-center study is warranted to investigate the predictive power of platelet apoptosis markers in wellphenotyped longitudinal cohorts. To Summarize, sera from severe COIVD-19 patients increase phosphatidylserine exposure on platelets. This exposure is triggered by elevated cytosolic calcium, which induces procoagulant and apoptotic cells, and inhibited via caspase inhibition indicating a significant contribution of the apoptosis pathways 41, 42 . These data may indicate procoagulant platelet and platelet apoptosis as a potential target of COVID-19 treatment as has been suggested for ARDS 43 ΔΨm depolarization, cytosolic calcium concentration and PS externalization. In round brackets are reported the two sera tested only for: ΔΨm depolarization and PS externalization. Data are presented as mean ± standard error mean (SEM) of the measured fold increase compared to control, not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. The number of sera tested is reported in each graphic. Dot lines represent the cutoffs determined testing sera from healthy donors as mean of fold increase (FI) + 2x SEM. 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