This study aimed to optimize the preparation of platelet-rich plasma (PRP) for regenerative medicine by comparing different anticoagulants (sodium citrate [SC] and ethylenediaminetetraacetic acid [EDTA]) and resuspension media (plasma versus sodium chloride [NaCl]). The researchers found that EDTA resulted in higher platelet recovery rates (51.04% vs. 29.85%, p = 0.005) and preserved platelet functionality, with higher expression of platelet surface markers CD62p and PAC-1. In contrast, SC led to higher VEGF concentrations (628.73 pg/mL vs. 265.44 pg/mL, p = 0.013). Additionally, PRP resuspended in plasma showed higher platelet recovery rates (61.60% vs. 48.61%, p = 0.011) and VEGF levels (363.32 pg/mL vs. 159.83 pg/mL, p = 0.005) compared to NaCl. The study concludes that EDTA is superior as an anticoagulant and plasma is preferred for resuspension, enhancing platelet recovery and functionality. Further research is needed to evaluate the impact of these factors on the release and concentration of other growth factors and their therapeutic effects.This study aimed to optimize the preparation of platelet-rich plasma (PRP) for regenerative medicine by comparing different anticoagulants (sodium citrate [SC] and ethylenediaminetetraacetic acid [EDTA]) and resuspension media (plasma versus sodium chloride [NaCl]). The researchers found that EDTA resulted in higher platelet recovery rates (51.04% vs. 29.85%, p = 0.005) and preserved platelet functionality, with higher expression of platelet surface markers CD62p and PAC-1. In contrast, SC led to higher VEGF concentrations (628.73 pg/mL vs. 265.44 pg/mL, p = 0.013). Additionally, PRP resuspended in plasma showed higher platelet recovery rates (61.60% vs. 48.61%, p = 0.011) and VEGF levels (363.32 pg/mL vs. 159.83 pg/mL, p = 0.005) compared to NaCl. The study concludes that EDTA is superior as an anticoagulant and plasma is preferred for resuspension, enhancing platelet recovery and functionality. Further research is needed to evaluate the impact of these factors on the release and concentration of other growth factors and their therapeutic effects.