Additional file 1 of SARS-CoV-2 binds platelet ACE2 to enhance thrombosis in COVID-19
Creators: name: Zhang, Si
name: Liu, Yangyang
name: Wang, Xiaofang
name: Yang, Li
name: Li, Haishan
name: Wang, Yuyan
name: Liu, Mengduan
name: Zhao, Xiaoyan
name: Xie, Youhua
name: Yang, Yan
name: Zhang, Shenghui
name: Fan, Zhichao
name: Dong, Jianzeng
name: Yuan, Zhenghong
name: Ding, Zhongren
name: Zhang, Yi
name: Hu, Liang
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Mental Health
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 1: Expanded materials and methods.. Online Table 1. Antibodies, staining chemicals and dilutions used in this study. Online Table 2. The primers used for RT-PCR. Online Table 3. Characteristics of Healthy, Non-COVID-19 patients, Mild and moderate type COVID-19 patients and Severe and critically severe type COVID-19 patients groups before propensity score matching. Online Table 4. Patient Characteristics after propensity score matching Sever and critically severe type COVID-19 patients group to healthy group (1:2), Non-COVID-19 patients group (1:1) or Mild and moderate type COVID-19 patients group (1:2). Online Table 5. Patient Characteristics after propensity score matching healthy group to Mild and moderate type COVID-19 group (1:1) or Non-COVID-19 patients group to Mild and moderate type COVID-19 group (1:2). Online Figure 1. The flowchart showing the strategy of groups enrollment. Online Figure 2. Analysis of platelet activation in COVID-19 patients after propensity score matching. A and B, Dot plot showing increased platelet t acti activation (A) and CD62P expression (B) in severe and critically severe COVID-19 patients compared with healthy donors (1:2 matching), non-COVID-19 patients (1:1 matching) or mild and moderate COVID-19 patients (1:2 matching). C and D, Dot plot showing increased PAC-1 binding (C) and CD62P expression (D) in mild and moderate COVID-19 patients compared with healthy donors (1:1 matching), non-COVID-19 patients (2:1 matching). **P &lt; 0.01 vs control. Statistical analyses were performed using Mann-Whitney U test. *P &lt; 0.05; **P &lt; 0.01. Online Figure 3. SARS-CoV-2 RNA positive platelet-poor plasma enhanced platelet aggregation compared with SARS-CoV-2 RNA negative platelet-poor plasma and healthy platelet-poor plasma. Healthy platelets were incubated with healthy platelet-poor plasma (PPP), SARS-COV-2 RNA positive PPP and SARS-COV-2 RNA negative PPP from severe and critically severe COVID-19 patients at the concentration of 2oor plasma (PPP), SARS-COV-2 RNA positive PPP and SARS-COV-2 RNA ne experiment. Statistical analyses were performed using one-way ANOVA followed by Tukeyiment. Statistical analP &lt; 0.05, n = 4. Online Figure 4. SARS-CoV-2 and its Spike protein did not induce platelet aggregation in the absence of agonist. Platelets were treated with SARS-CoV-2 ((1telets were treated with SARS-CoV-2 not induce plateletway ANOVA followed bygat indicated time in aggregometry. Online Figure 5. Platelet ACE2 is reduced upon SARS-CoV-2 treatment. A, Platelets from healthy donors were pretreated with SARS-CoV-2 (1×105 PFU) for various times, as indicated. The ACE2 protein level was detected by Western blot. Representative images and summary data of 3 experiments are presented using platelets from different healthy donors. Statistical analyses were performed using two-way ANOVA and Tukey's post hoc test. **P &lt; 0.01 vs control. B, Decreased ACE2 expression in platelets from COVID-19 patients. Representative images of 12 different individuals from healthy group (n = 4), mild and moderate COVID-19 group (n = 4) and severe and critically severe COVID-19 group (n = 4) are presented. Online Figure 6. SARS-CoV-2 Spike protein dose-dependently enhance platelet activation. Washed platelets from healthy donors were incubated with Spike protein in the indicated concentration for 5 min, then stimulated with collagen (0.6 n = 4)e thrombin (0.025 U/mL), or ADP (5 min, then stimulated with collagen (0.6 n = 4)ere were assessed under stirring at 1200 rpm. Representative results and summary data of 4 experiments are presented. Statistical analyses were performed using One-way ANOVA, followed by Tukeyted. Statistical analyses were performed using One-wayata o 0.01. Online Figure 7. SARS-CoV-2 and its Spike protein phosphorylates ACE2, Erk, p38, and JNK in human platelets. A, Platelets were challenged with SARS-CoV-2 (1 x 105 PFU) or Spike protein (2 h SARS-CoV-2 (1tes ACE2, Erk,rmed using One-wayata ACE2 normalized to input ACE2, corresponding to Figure 5a, are provided from 4 experiments using platelets from different donors. B, Platelets were challenged with SARS-CoV-2 (1 x 105 PFU) or Spike protein (2 nors. CE2, Erk,rmed using One-wayata p-Erk normalized to Erk, p-p38 normalized to p38, and p-JNK normalized to JNK, corresponding to Figure 5a, are provided from 4 experiments using platelets from different donors. Statistical analyses were performed using One-way ANOVA, followed by Tukeyt donors. Statistical anaA) and (B). NS, no significance; *P &lt; 0.05; **P &lt; 0.01. Online Figure 8. Spike protein enhances in vitro platelet activation and in vivo thrombosis potential in hACE2 transgenic mice. A, Quantitative analysis of platelets aggregation and ATP release corresponding to Figure 6C. Data are provided from 4 experiments using platelets from different mice. B, After intravenous injection 200 ter intravenous injectionelets from different mice. ing One-way ANOVA, f formation was initiated, and the thrombus area was recorded. Representative image of thrombus formation and the relative fluorescence at different time points are shown. Statistically analysis of FeCl3-induced thrombosis by assessing thrombus area at 8 min (n = 5). Statistical analyses were performed using unpaired two-tailed Studentbus test. NS, no significance; **P &lt; 0.01. WT indicates wild-type; hACE2 indicates hACE2 transgenic. Online Figure 9. SARS-CoV-2 directly stimulates platelets for coagulation factor release. A and B, SARS-CoV-2 induced release of Factor V (A) and Factor XIII (B) from platelets. Washed platelets from healthy donors were incubated with or without SARS-CoV-2 (1×105 PFU) for 1 h at room temperature, and subject to ELISA assays. Thrombin at 0.025 U/mL served as the positive control. Summary data of 4 experiments using platelets from different healthy donors are presented. C, SARS-CoV-2 had no effect on platelet phosphatidylserine (PS) exposure. Washed platelets from healthy donors were incubated with or without SARS-CoV-2 (1×105 PFU) for 1 h at room temperature, and then stained with Annexin V-FITC for 30 min. PS exposure was analyzed using flow cytometry. Thrombin at 0.05 U/mL served as the positive control. Representative images and summary data are presented from 4 experiments using platelets from different healthy donors. D and E, Spike protein (2 erent healthy donor release of Factor V (D) and XIII (E) from platelets. Factor V and XIII in supernatant were assessed using commercial ELISA kits. Thrombin at 0.025 U/mL served as the positive control. Summary data of 4 experiments using platelets from different healthy donors are presented. F, Spike protein (2 d. ary data of 4 experiments using platelets after 1 h incubation. Thrombin at 0.05 U/mL served as the positive control. Representative images and summary data are presented from 4 experiments using platelets from different healthy donors. Statistical analyses were performed using One-way ANOVA, followed by Tukeylthy donors. Statistical A), (B), (C), (D), (E) and (F). NS, no significance; **P &lt; 0.01. Online Figure 10. SARS-CoV-2 directly potentiates PF4 and inflammatory cytokines secretion from platelets. A, SARS-CoV-2 and its Spike protein stimulated PF4 release from platelets. Summary data of at 3-4 experiments using platelets from different healthy donors are presented. B, C and D, SARS-CoV-2 and its Spike protein stimulated TNF-α, IL-8 and IL-1d its Spike proteinnted. of at 3-4 experim experiments using platelets from different healthy donors are presented. Washed platelets were separated from whole blood of healthy donors and adjusted to 3edfromO in platelet-poor plasma. Platelets were then challenged with SARS-CoV-2 (1×105 PFU), Spike protein (2 . Platelets were then challenged withh. E, Increased expression of PF4 in plasma from severe and critically severe COVID-19 patients, compared with healthy donors or mild and moderated COVID-19 patients. Concentrations of PF4 were measured in plasma from healthy donors, mild and moderated COVID-19 patients and severe and critically severe COVID-19 patients (n = 5). PF4 and TNF-α, IL-8 and IL-1-19 patients and severe and critically sever commercial ELISA kits. Statistical analyses were performed using One-way ANOVA, followed by Tukeykits. Sthoc analysis in (A), (B), (C), (D) and (E). *P &lt; 0.05; **P &lt; 0.01. Online Figure 11. SARS-CoV-2 directly stimulates leukocyte-platelet aggregates (LPAs) formation. A and B, SARS-CoV-2 (A) and Spike protein (B) increased leukocyte-platelet aggregates (LPAs, CD45+CD41+), monocyte-platelet aggregates (MPAs, CD14+CD41+) and neutrophil-platelet aggregates (NPAs, CD65+CD41+). After stimulation with SARS-CoV-2 (1hil-platelet aggregates (NPAs, CD65+CD41+). After(no with different antibodies for about 30 min, red blood cells were removed and the blood samples were then subjected to flow cytometry. Summary data of 5 experiments using blood samples from different healthy donors are presented. Statistical analyses were performed using unpaired two-tailed Student are pres in (A) and (B). **P &lt; 0.01.