Fig. 5.

ACE2/MAPK mediates the 1 potentiating effects of SARS-CoV-2 on platelet activation. A SARS-CoV-2 virus and its Spike protein phosphorylate ACE2, Erk, p38, and JNK in human platelets. Platelets from healthy donors were pretreated with SARS-CoV-2 (1 × 10⁵ PFU) or its Spike protein (2 μg/mL) for various times, as 5 indicated. For ACE2 phosphorylation detection, cell lysates were prepared and subjected to immunoprecipitation (IP) with anti-phospho-Ser/Thr antibody, followed by immunoblotting analysis with anti-ACE2 antibody. Cell lysates without the process of immunoprecipitation (10% of input) were analyzed in parallel as loading controls. For p-Erk, p-p38, and p-JNK detection, cell lysates were prepared and directly subjected to immunoblotting. Representative results are presented from 4 experiments using platelets from different healthy donors and summary data is presented in the Additional file 1: Online Figure 7. B Enhanced platelet aggregation by SARS-CoV- 2 is abolished by MAPK inhibitors. The healthy human platelets were pretreated with 10 μM PD98059 (ERK1/2 inhibitor), 10 μM SB203580 (p38 inhibitor), or 10 μM SP600125 (JNK inhibitor) for 10 min, then treated with SARS-CoV-2 (1 × 10⁵ PFU, 30 min) before stimulation by 0.025 U/mL thrombin or 0.6 μg/mL collagen. Representative results are presented from 3 experiments using platelets from different donors. C Enhanced platelet aggregation by Spike protein is abolished by MAPK inhibitors. Human platelets were pretreated with 10 μM PD98059, 10 μM SB203580, or 10 μM SP600125 for 10 min, then treated with Spike protein (2 μg/mL, 5 min) or vehicle as control before stimulation by 0.025 U/mL thrombin or 0.6 μg/mL collagen. Representative results are presented from 3 experiments using platelets from different healthy donors. D Accelerated clot retraction by SARS-CoV-2 (D1) or its Spike protein (D2) is abolished by MAPK inhibitors. Platelets were pretreated with 10 μM PD98059, 10 μM SB203580, or 10 μM SP600125 for 10 min, and then incubated with SARS26 CoV-2 (1 × 10⁵ PFU, 30 min) or Spike protein (2 μg/mL, 5 min) as in B and C. Clot retraction was initiated as in Fig. 3d. Representative images and summary data of 3 experiments are presented using platelets from different healthy donors. Representative images and summary data are presented from 3 experiments using platelets from different donors. E Increased phosphorylation of Erk, p38, and JNK in platelets from COVID-19 patients, compared with healthy donors. Representative results are presented using platelets from 6 individuals from different COVID-19 patients (n = 3) and healthy donors (n = 3). Statistical analyses were performed using unpaired two34 tailed Student’s t test in (D1) and (D2). *P < 0.05; **P < 0.01. MAPK indicates mitogen activated protein kinase; PD, PD98059; SB, SB203580; SP, SP600125