Thrombin

uPA and L-Arg were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Cy5-NHS (N-hydroxysuccinimide), DAF-FM diacetate (4-amino-5-methylamino-2,7-difluorofluorescein diacetate), FDA (fluorescein diacetate), DiD (1,1ʹ-WGA-Alexa 488ctadecyl-3,3,3ʹ,3ʹ-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt dye), WGA-Alexa 488 (3,3ʹ-WGA-Alexa 488ctadecyloxacarbocyanine perchlorate), thrombin, ADP (adenosine diphosphate), fibrinogen, rhodamine 6G, ferric chloride (FeCl₃) and phosphate-buffered saline (PBS) were purchased from Meilun Biology Technology Co., Ltd. (Dalian, China). NHS-PEG₃₄₀₀-DSPE was purchased from Shanghai ToYongBio Tech. Inc. (Shanghai, China). Bovine serum albumin (BSA), glucose, dimethyl sulfoxide (DMSO), dichloromethane (DCM) was from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Other chemicals used in this project are analytical reagents. ICR mice (male, 20–25 g body weight) were purchased from the Sippr-BK Laboratory Animal Co., Ltd. (Shanghai, China) and kept under SPF conditions. All animal experiments were conducted in accordance with guidelines evaluated and approved by the Ethics Committee of Fudan University.

A previously established fibrin gel bead sprouting assay was optimized to study angiogenesis (Nakatsu et al., 2007 ; Nakatsu and Hughes, 2008 (link)). A total of 2500 Cytodex microcarrier beads (Sigma-Aldrich, C3275) were coated with 1 × 10⁶ EA.hy926 in medium at 37°C and 5% CO₂. The tube was shaken gently every 20 min for 4 h. The coated beads were then transferred to a dish containing 5 ml EGM2 medium and left overnight. The next day, the EA.hy926-coated beads were resuspended at a concentration of 500 beads/ml in 2 mg/ml fibrinogen (Sigma-Aldrich, F3879) solution containing 0.15 U/ml aprotinin (Meilunbio, MB3095). Then, 0.625 U/ml thrombin (Meilunbio, MB1368) was added to a 24-well plate, followed by the addition of the fibrinogen/bead solution. The plate was left at room temperature for 5 min and then placed in an incubator at 37°C and 5% CO₂ for 15 min to generate a clot. Finally, fibroblast NIH-3T3 cells were seeded on top of the fibrin gel at a concentration of 2 × 10⁴ cells/well in 1 ml EGM2 medium. The medium was changed every other day. Sprouting images were captured after 2–4 days.