Yatim et al. Ann. Intensive Care (2021) 11:113 https://doi.org/10.1186/s13613-021-00899-1 RESEARCH Open Access Platelet activation in critically ill COVID-19 patients Nader Yatim1,2† , Jeremy Boussier1† , Richard Chocron12,14, Jérôme Hadjadj2,3, Aurélien Philippe4,5,6, Nicolas Gendron4,5,6, Laura Barnabei3, Bruno Charbit7, Tali‑Anne Szwebel2, Nicolas Carlier8, Frédéric Pène9,10, Célia Azoulay2, Lina Khider6,11, Tristan Mirault11,12, Jean‑Luc Diehl6,8,13, Coralie L. Guerin4, Frédéric Rieux‑Laucat3, Darragh Dufy1,7, Solen Kernéis15,16,17, David M. Smadja4,5,6† and Benjamin Terrier2,12,18*† Abstract Background: Microvascular, arterial and venous thrombotic events have been largely described during severe coro‑ navirus disease 19 (COVID‑19). However, mechanisms underlying hemostasis dysregulation remain unclear. Methods: We explored two independent cross‑sectional cohorts to identify soluble markers and gene‑expression signatures that discriminated COVID‑19 severity and outcomes. Results: We found that elevated soluble (s)P‑selectin at admission was associated with disease severity. Elevated sP‑ selectin was predictive of intubation and death (ROC AUC 0.67, p 0.028 and AUC 0.74, p 0.0047, respectively). An optimal cutof value was predictive of intubation with 66%= negative= predictive value= (NPV)= and 61% positive pre‑ dictive value (PPV), and of death with 90% NPV and 55% PPV. An unbiased gene set enrichment analysis revealed that critically ill patients had increased expression of genes related to platelet activation. Hierarchical clustering identifed ITG2AB, GP1BB, PPBP and SELPLG to be upregulated in a grade‑dependent manner. ROC curve analysis for the predic‑ tion of intubation was signifcant for SELPLG and PPBP (AUC 0.8, p 0.046 for both). An optimal cutof value for PBPP was predictive of intubation with 100% NPV and 45% PPV, and= for SELPLG= with 100% NPV and 50% PPV. Conclusion: We provide evidence that platelets contribute to COVID‑19 severity. Plasma sP‑selectin level was associ‑ ated with severity and in‑hospital mortality. Transcriptional analysis identifed PPBP/CXCL7 and SELPLG as biomarkers for intubation. These fndings provide additional evidence for platelet activation in driving critical COVID‑19. Specifc studies evaluating the performance of these biomarkers are required. Keywords: COVID‑19, Primary hemostasis, Platelets, Thrombo‑infammation Background (ARDS), respiratory failure and death in about 1% of Severe acute respiratory syndrome coronavirus 2 (SARS- cases [2, 3]. Most important risk factors for severe dis- CoV-2) is the causative agent of the coronavirus disease ease include age, overweight, diabetes, hypertension and 2019 (COVID-19) pandemic [1]. Severe disease is char- history of cardiovascular disease [4, 5]. Severe and criti- acterized by an acute respiratory distress syndrome cal patients were shown to develop arterial and venous thrombotic complications, such as pulmonary embo- lism, stroke and myocardial infarction [6, 7]. Markers of *Correspondence: [email protected] †Nader Yatim and Jeremy Boussier have contributed equally to this work coagulation activation, in particular increased D-dimer, †David M. Smadja and Benjamin Terrier are co‑senior authors fbrinogen and von Willebrand factor (vWF) levels were 2 Department of Internal Medicine, National Reference Center for Rare found to be associated with critical illness, whereas Systemic Autoimmune Diseases, AP‑HP, APHP.CUP, Hôpital Cochin, 75014 Paris, France only minor changes were noted in prothrombin time Full list of author information is available at the end of the article and platelet counts [8–11]. In addition, autopsy series © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Yatim et al. Ann. Intensive Care (2021) 11:113 Page 2 of 12 described multiple thrombosis in deceased COVID-19 were retrieved from the medical records using a stand- patients [12, 13]. Tese fndings suggest vascular micro- ardized data collection. As a control non-COVID-19 thrombotic disease as a primary factor for mortality in septic ICU cohort, we included plasma samples from a critically ill COVID-19 [6, 14]. Terefore, some authors previously described pre-pandemic cohort [25, 26]. supported the systematic use of curative anticoagula- Cohort 2 [27] was conducted between March 19, 2020 tion upon admission to intensive care unit (ICU) [15], a and April 3, 2020 in Cochin Hospital (Paris, France), in strategy that has been reported to decrease mortality in the setting of the local RADIPEM biological samples col- severe COVID-19 [8, 16–18]. lection derived from samples collected in routine care. Mechanisms underlying increased thrombotic events Inclusion criteria for COVID-19 inpatients were: age are still unclear but accumulating evidence point to a key between 18 and 80 years, diagnosis of COVID-19 accord- role for endothelial and platelet activation [19, 20]. As ing to WHO interim guidance and positive SARS-CoV-2 viral inclusions were described in endothelial cells, it has RT-PCR testing on a respiratory sample (nasopharyngeal been hypothesized that endothelial cell injury and activa- swab or invasive respiratory sample). Detailed clinical tion could drive platelet activation and subsequent coag- and immunological characterization of the cohort was ulopathy [13, 21]. Terefore, dissecting the contribution previously described in [27]. Epidemiological, demo- of platelets to COVID-19 critical illness is key to under- graphic, clinical, laboratory, treatment, and outcome data stand SARS-CoV-2 infection pathogenesis and identify were extracted from electronic medical records using a novel therapeutic strategies. standardized data collection form. Platelet P-selectin is a key thromboinfammatory mol- Te severity of COVID-19 was classifed at the time of ecule involved in platelet activation and function. It has inclusion based on the adaptation of the Sixth Revised been demonstrated to play a crucial role in primary Trial Version of the Novel Coronavirus Pneumonia Diag- hemostasis by regulating platelet–leukocyte interactions, nosis and Treatment Guidance and described in [27]. fbrin and tissue factor recruitment into platelet aggre- gates and thrombus formation [22]. Its soluble form, sP- Soluble P‑selectin measurement selectin, is released upon platelet and/or endothelial cell sP-selectin quantifcation was performed on Cohort activation and measurement of sP-selectin has been pro- 1. Plasmas were collected on 0.129 M trisodium citrate posed as a reliable marker of in vivo platelet activation tubes (9NC BD Vacutainer, Plymouth, UK). Plasma poor [23]. Moreover, sP-selectin levels have been shown to platelet was obtained after centrifugation twice at 2500 g correlate with acute lung injury severity score and related for 15 min. PPP was frozen after a second centrifugation death [24]. at 2500 g for 15 min and stored at − 80 °C until analysis Te aim of our study was to assess, in hospitalized of vascular markers. Soluble P-selectin were quantifed COVID-19 patients, the ability of sP-selectin to predict in PPP with a Human Magnetic Luminex Assay from requirement for mechanical ventilation and in-hospital R&D systems (Lille, France). Data were assessed with the mortality. Next, using whole-blood transcriptional data, Bio-Plex 200 using the Bio-Plex Manager 5.0 software we uncovered a platelet activation transcriptional signa- (Bio-Rad, Marnes-la-Coquette, France). Normalized ture associated with critical illness. concentration (NC) used to calculate receiver operating characteristic (ROC) area under curve (AUC) p values Methods represents sP-selectin concentration in pg/mL normal- Cohorts ized to platelet numbers (106/mL). Two independent cohorts were analyzed for this study. Data used for the analysis of soluble P-selectin’s ability Gene expression analysis to predict admission to ICU were extracted from a non- Detailed methods was previously reported in Hadjadj interventional study that was conducted at European et al. [27]. Briefy, we analyzed 100 ng (5 μL) of total RNA Georges Pompidou Hospital (Paris, France) and partially from each sample using the Nanostring Human Immu- described in [25] (Cohort 1). Briefy, Cohort 1 included nology kit v2 according to manufacturer’s instructions. consecutive patients with SARS-CoV-2 infection. Inclu- Raw RNA counts were adjusted using fve housekeeping sion criteria were patients over 18 years of age, with a genes selected from the 15 candidate control genes pro- proven SARS-CoV-2 infection, which presented to the vided by Nanostring, following the geNorm method. For emergency department with hospitalization criteria. gene set enrichment analysis (GSEA), genes were ordered Patients were then hospitalized into conventional