Fluorescence intensity (FI) of Ca2+: 10 mL of human blood was collected in an anticoagulation tube (Sodium citrate). The blood was centrifuged at 200 r min−1 for 15 min to obtain platelet-rich plasma (PRP). 500 μL of PRP was added to centrifuge tube (1.5 mL), centrifuged at 1500 r min−1 for 15 min, and the supernatant was removed. Then, 10 μL of Fluo-4 AM was mixed with the platelets and incubated for 15 min at 37 °C in dark conditions. Subsequently, sample (10 mg) was added to the tube and incubated at 37 °C for 15 min, and then 200 μL of 1% Triton-100 was added. The mixtures were centrifuged at 12,000 rpm, and 100 μL of supernatant in each tube was transferred to one of the holes in a 96-well plate. FI was measured using a Spectral Scanning Multimode Reader (Thermo Scientific Varioskan Flash, USA) at an excitation of 494 nm and emission of 516 nm. The effect of Ca2+ in the material had been taken into account when calculating the FI and the percentage of intracellular Ca2+. The control group (PRP only) was tested using the same method as above. Each group was measured in triplicates.

The sample was first immersed in the PRP and incubated at 37 °C for 30 min. Then, the sample was rinsed with PBS solution for three times and incubated in PBS with 1% Triton X-100 at 37 °C for 1 h. Subsequently, the sample was incubated in 2.5% glutaraldehyde, dehydrated using a gradient ethanol solution (30, 50, 70, 90 and 100%) and went through freeze-drying. The morphology of platelets on the sample after incubation were observed through FE-SEM (FV1000, Olympus, Japan). The number of platelets on the samples was also quantified using a Lactate dehydrogenase (LDH) assay kit (Sigma, Aldrich) by measuring the LDH released from the lysed platelets [35].

Concentration of CD61 and CD62P (P-selection): The whole blood was centrifuged at 200 r min−1 for 15 min, and 100 μL of the upper PRP was taken in each EP tube. Then, sample (10 mg) was added and incubated at 37 °C for 30 min. Next, 1 mL PBS was added to each tube, and the sample was rinsed and taken out from the tube. The remaining mixture was centrifuged at 1500 r min−1 for 15 min, and the supernatant was removed. 50 μL of FITC (Shanghai Macklin Biochemical Co., Ltd) labeled anti-CD61 and anti-CD62p (Sino Biological, China) were added after dilution, and the mixture was kept in the dark at 4 °C for 20 min. The concentration CD61 and CD62p was detected using BD Accuri C6 flow cytometer.

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