key: cord-0983865-bjoojunn
authors: Shen, Shu; Zhang, Jingyuan; Fang, Yaohui; Lu, Sihong; Wu, Jun; Zheng, Xin; Deng, Fei
title: SARS-CoV-2 interacts with platelets and megakaryocytes via ACE2-independent mechanism
date: 2021-04-29
journal: J Hematol Oncol
DOI: 10.1186/s13045-021-01082-6
sha: 633a57894304100539ce2dd79c8764cde26d88b2
doc_id: 983865
cord_uid: bjoojunn

Evidence suggests that platelets may directly interact with SARS-CoV-2, raising the concern whether ACE2 receptor plays a role in this interaction. The current study showed that SARS-CoV-2 interacts with both platelets and megakaryocytes despite the limited efficiency. Abundance of the conventional receptor ACE2 and alternative receptors or co-factors for SARS-CoV-2 entry was characterized in platelets from COVID-19 patients and healthy persons as well as human megakaryocytes based on laboratory tests or previously reported RNA-seq data. The results suggest that SARS-CoV-2 interacts with platelets and megakaryocytes via ACE2-independent mechanism and may regulate alternative receptor expression associated with COVID-19 coagulation dysfunction. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13045-021-01082-6.

Associated with coagulative disorders, COVID-19 patients have increased platelet activation and aggregation, and platelet-monocyte aggregation [1] [2] [3] , which highlights the critical role of platelets in SARS-CoV-2 infection and immunopathology [4] . Consistent with previous reports [1] [2] [3] , our retrospective survey of plasma samples from a cohort of 62 cases (severe or fatal and moderate COVID-19 patients, Additional file 1: Table S1 ) showed that COVID-19 was associated with mild thrombocytopenia (platelet count < 150 × 10 9 /L) and increased thrombosis (elevated D-dimer levels), and patients had increased platelet activation (elevated sP-selectin and sGPVI levels) and cytokine (PF4 and RANTES) release upon platelet activation (Fig. 1a) . Direct interaction of SARS-CoV-2 with human platelets was suggested based on increased P-selectin translocation on platelet surface (Fig. 1b) , and elevated levels of GPVI, PF4, and RANTES in platelet culture supernatants (Fig. 1c) . However, the characteristics and mechanisms of the direct interaction between SARS-CoV-2 and platelets are not well elucidated, and the role of platelet receptors in the interaction remains to be clarified [4, 5] .

SARS-CoV-2 infection in human platelets and its progenitor megakaryocyte cell line MEG-01 in vitro was subsequently characterized. SARS-CoV-2 N expression was observed in some platelets and MEG-01 cells (Fig. 2a) . SARS-CoV-2 RNA was detected in both culture supernatant and MEG-01 cells after SARS-CoV-2 incubation and could be maintained with a slight increase until 48 h p.i. (Fig. 2b) . This suggests that SARS-CoV-2 may infect and replicate in megakaryocytes despite insufficient efficiency. However, we failed to observe any viral particles in and culture supernatant (10 3 -10 4 copies/mL), which diminished after 12 h (data not shown). Therefore, we speculate that platelets may not support SARS-CoV-2 replication. This echoes recent studies which have shown that SARS-CoV-2 entry in platelets may not be common in COVID-19 patients: SARS-CoV-2 RNA was detected in platelets from a few severe (2/25, 8% [2] ; 2/11, 18.2% [6] ) and non-severe (9/38, 23.7% [6] ) patients and was not detected in platelets from patients (0/24 [7] ). The evidence of direct interaction between SARS-CoV-2 and platelets or megakaryocytes raised the concern whether ACE2 plays a role in the process. The IFA and western blot assays showed a lack of ACE2 expression in both human platelets and megakaryocytes (Fig. 2c, d) . The RNA abundance of 14 receptors or co-factors including ACE2 in human platelets and megakaryocytes was subsequently inspected based on RNA-seq data reported in previous studies [2, 8] (Additional file 1: Table S2 and S3). As summarized in Fig. 2e , the abundance order in platelets was: CD147 > GRP78 > KREMEN1 > ADAM17 > cathepsin L > NRP1 > ASGR1 > CD209L/L-SIGN > CD301 > CD26 > CD206, but CD209/ DC-SIGN, ACE2, and TMPRSS2 were not identified.

Human megakaryocytes had similar receptor profiles, coupled with the detection of CD209/DC-SIGN. We also verified receptor abundance in MEG-01 and human platelets using qRT-PCR. In MEG-01 cells, CD147, GRP78, KREMEN1, cathepsin L, NRP1, and ASGR1 were detected, while in platelets, CD147, GRP78, KREMEN1, and ASGR1 were detected. ACE2 was not detected in MEG-01 cells or platelets (Fig. 2f ) . These results indicate that SARS-CoV-2 may use receptors other than ACE2 to interact with platelets or megakaryocytes.

Further analysis using the RNA-seq data showed unchanged GRP78, ADAM1, cathepsin L, GRP1, and ASGR1 abundance in platelets between ICU and non-ICU COVID-19 patients and healthy persons and revealed elevated CD147 and KREMEN1 levels and reduced NRP1 levels in patients (Fig. 2g) . This was also observed in MEG-01 cells with increased CD147 and KREMEN1 levels and slightly reduced NRP1 levels after SARS-CoV-2 incubation (Fig. 2h) . These data suggest that SARS-CoV-2 infection may alter gene transcription in platelets and megakaryocytes, which is similar to DENV infection that markedly changes the platelet and megakaryocyte transcriptome [8] .

Owing to their roles in binding to spike protein and facilitating virus entry [9] [10] [11] , CD147, KREMEN1, and NRP1 triggering of SARS-CoV-2 entry in human platelets and megakaryocytes requires in-depth investigation. Moreover, based on the original functions of CD147 in signaling pathways via cell-cell interactions (See figure on previous page.) Fig. 1 Increased platelet activation in COVID-19 patients and that stimulated by SARS-CoV-2. a Platelet counts and D-dimer levels of patients with severe/fatal and moderate COVID-19 are shown as the medians and interquartile ranges. The normal range of platelet count (150-400 × 10 9 /L) is shaded, and the upper limit value of D-dimer (0.5 mg/L) is indicated with dotted line. Soluble P-selectin levels (sP-selectin), soluble GPVI levels (sGPVI), PF4, and RANTES in plasma of patients with severe/fatal or moderate COVID-19, and healthy controls were measured through ELISA. b Platelet activation was investigated using platelets from healthy donors incubated with SARS-CoV-2, thrombin, or virus culture medium (Mock) for 3 h at 37 °C. P selectin surface translocation was measured using flow cytometry and results using platelets from two healthy donors are shown. c Levels of GPVI, PF4, and RANTES in the incubation supernatants were determined through ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 

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 [9] and of NRP1 in cardiovascular, neuronal, and immune systems [10] , SARS-CoV-2 interaction with platelets is suspected to regulate platelet-mediated immune response [12] and promote coagulation dysfunction in COVID-19 [10] . 

The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13045-021-01082-6.

Authors' contributions SS and DF designed and conceived the project. SS, ZJY, and FYH performed the research, analyzed the data, and wrote the manuscript. LSH, WJ, and ZX collected and analyzed the clinical data. All authors have contributed to and approved the manuscript. 

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

The study protocol was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (number: 2020/0042-02-02).

Not applicable.

The authors declare that they have no competing interests.