key: cord-1002709-5byvojjx authors: Zaid, Y.; Puhm, F.; Allaeys, I.; Naya, A.; Oudghiri, M.; Khalki, L.; Limami, Y.; Zaid, N.; Sadki, K.; Ben El Haj, R.; Maher, W.; Belayachi, L.; Belefquih, B.; Benouda, A.; Cheikh, A.; Cherrah, Y.; Flamand, L.; Guessous, F.; Boilard, E. title: Platelets can contain SARS-CoV-2 RNA and are hyperactivated in COVID-19 date: 2020-06-23 journal: nan DOI: 10.1101/2020.06.23.20137596 sha: 5a6fa9bef083c1e42144a732b828f33d3d5bd659 doc_id: 1002709 cord_uid: 5byvojjx ABSTRACT Rationale: In addition to the overwhelming lung inflammation that prevails in COVID-19, hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVID-19. Objective: To evaluate the contribution of platelets to inflammation and thrombosis in COVID-19 patients. Methods and Results: We document the presence of SARS-CoV-2 RNA in platelets of COVID-19 patients. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in COVID-19, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents and phosphatidylserine-exposing extracellular vesicles. Functionally, platelets were hyperactivated in COVID-19 subjects, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions. Conclusions: These data suggest that platelets could participate in the dissemination of SARS-CoV-2 and in the overwhelming thrombo-inflammation observed in COVID-19. Thus, blockade of platelet activation pathways may improve outcomes in this disease. Angiotensin a serious public health emergency of international concern and by June 2020, the 72 virus had infected over 7 million and killed more than 400 000 individuals those who died, relative to survivors. 17, 18, 20 While thrombosis and coagulation abnormalities predict worse outcomes in COVID- in platelets of all individuals tested (10 individuals/group) ( Table 2 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.23.20137596 doi: medRxiv preprint of 12 cytokines/chemokines that were unchanged (IL-1α, IL-4, IL-5, IL-8, IL-10, IL- Platelets produce inflammatory cytokines in COVID-19 228 We then determined the ability of COVID-19 patient platelets to produce 229 inflammatory mediators. Platelets derived from healthy individuals and from severely 230 and non-severely affected patients were stimulated with low doses of α-thrombin 231 (0.025 and 0.05 U/mL). Platelets were then lysed, and the amounts of IL-1β, IL-18, 232 sCD40L and the eicosanoid thromboxane B2 (TxB2) were determined. While IL-18 233 secretion and thromboxane production were unaffected in the disease, we found that 234 COVID-19 platelets were more potent at producing IL-1β and soluble CD40L upon 235 exposure to 0.05 U/mL of thrombin in comparison with healthy subjects (Figure 2A) . 236 Platelets from all subjects were similarly efficient at releasing the inflammatory 237 mediators when higher concentrations of thrombin were used, suggesting that 238 platelets are a source of inflammatory mediators and may be primed to release 239 certain inflammatory molecules during SARS-CoV-2 infection. Because they are anucleated, the platelet cytokine content can mirror that of 242 megakaryocytes, or may be regulated by translation upon platelet activation. 57 243 However, nothing is known regarding the platelet cytokine content in COVID-19. Thus, we monitored cytokines in lysates prepared using platelets from COVID-19 245 patients and included healthy subjects as controls. While a few cytokines were below All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.23.20137596 doi: medRxiv preprint and the coagulation process due to the exposure of phosphatidylserine 30,58,59 . 268 However, it is currently unknown whether EV are produced in COVID-19. EV smaller 269 than 1 µm and of platelet origin identified by the surface expression of CD41 were 270 detected by high sensitivity flow cytometry. To determine whether EV expressed 271 surface phosphatidylserine, we included annexin V conjugated with a fluorescent 272 probe in the analyses. EV were sensitive to detergent treatment, which confirmed 273 their membrane moiety. Moreover, no annexin V + EV were detected when Ca 2+ ions 274 were chelated by ethylenediaminetetraacetic acid (EDTA), which further ensured the 275 specificity of the detection approach (Online Figure VII) . We found a statistically 276 significant increase in platelet-derived EV and in phosphatidylserine-exposing 277 platelet EV in COVID-19 patients in comparison with healthy subjects (Figure 4) . 278 The levels of both types of EV were similar in non-severe and severe COVID-19 279 patients. The relationship between platelet activation and the severity of COVID-19 was then 297 investigated by functional assays. Platelets from severe and non-severe COVID-19 298 patients were collected and analyzed by optical aggregometry under arterial flow 299 conditions using various doses of α-thrombin. Platelets exposed to suboptimal 300 concentrations of α-thrombin (0.05 U/mL) were highly activated (≥2.5-fold increase) 301 in both severe and non-severe patients compared with controls ( Figure 6A and 6B) . 302 When platelets from all groups were stimulated with a higher concentration of α-303 thrombin (2 U/mL), no differences were observed, pointing to a lower platelet 304 stimulation threshold in COVID-19 patients (Figure 6A and 6B) . Moreover, the 305 formation of platelet aggregates was visualized by rhodamine-based 306 immunofluorescence on a collagen-coated surface under flow conditions, and the 307 ratio of adherent platelets to total platelets (activated vs non-activated platelets) was 308 calculated. The number of adherent platelets was significantly higher in severe 309 patients compared with non-severe patients and controls, implying the involvement 310 of the thrombotic events in the disease magnitude ( Figure 6C ). The results are visualized in a graphical summary in Figure 7 . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.23.20137596 doi: medRxiv preprint their potency at producing inflammatory cytokines, such as IL-1, when activated by 365 α-thrombin. From platelet degranulation and cytokine release analyses, we concluded that All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.23.20137596 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020 . . https://doi.org/10.1101 /2020 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020 . . https://doi.org/10.1101 /2020 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020 . . https://doi.org/10.1101 /2020 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. Wallis test *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. 681 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.23.20137596 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.23.20137596 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.23.20137596 doi: medRxiv preprint The data suggest that platelets could participate in the dissemination of SARS-CoV-720 2 and in the overwhelming thrombo-inflammation observed in COVID-19. Thus, 721 blockade of platelet activation pathways may improve outcomes in this disease. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10. 1101 /2020 SARS-CoV-2: a storm is raging Novel Coronavirus-Infected Pneumonia in Wuhan, China COVID-19: consider cytokine storm 465 syndromes and immunosuppression How does 467 coronavirus kill? Clinicians trace a ferocious rampage through the body, from brain to 468 toes Acute aorto-iliac and mesenteric arterial 470 thromboses as presenting features of COVID-19 High incidence of venous 474 thromboembolic events in anticoagulated severe COVID-19 patients Venous and arterial 477 thromboembolic complications in COVID-19 patients admitted to an academic 478 hospital in