title: In vitro study of the role of thrombin in platelet rich plasma (PRP) preparation: utility for gel formation and impact in growth factors release.
abstract: BACKGROUND
  The use of PRP has been studied for different fields, with promising results in regenerative medicine. Until now, there is no study in the literature evaluating thrombin levels in serum, used as autologous thrombin preparation. Therefore, in the present study we evaluated the role played by different thrombin concentrations in PRP and the impact in the release of growth factors. Also, different activators for PRP gel formation were evaluated.
  
  
  METHODS
  Thrombin levels were measured in different autologous preparations: serum, L-PRP (PRP rich in leukocytes) and T-PRP (thrombin produced through PRP added calcium gluconate). L-PRP was prepared according to the literature, with platelets and leukocytes being quantified. The effect of autologous thrombin associated or not with calcium in PRP gel was determined by measuring the time of gel formation. The relationship between thrombin concentration and release of growth factors was determined by growth factors (PDGF-AA, VEGF and EGF) multiplex analysis.
  
  
  RESULTS
  A similar concentration of thrombin was observed in serum, L-PRP and T-PRP (8.13 nM, 8.63 nM and 7.56 nM, respectively) with a high variation between individuals (CV%: 35.07, 43 and 58.42, respectively). T-PRP and serum with calcium chloride showed similar results in time to promote gel formation. The increase of thrombin concentrations (2.66, 8 and 24 nM) did not promote an increase in growth factor release.
  
  
  CONCLUSIONS
  The technique of using serum as a thrombin source proved to be the most efficient and reproducible for promoting PRP gel formation, with some advantages when compared to other activation methods, as this technique is easier and quicker with no need of consuming part of PRP. Noteworthy, PRP activation using different thrombin concentrations did not promote a higher release of growth factors, appearing not to be necessary when PRP is used as a suspension.
authors.affiliation: Hemocentro, Haemostasis Laboratory, State University of Campinas UNICAMP, Brazil.
authors.forename: Stephany Cares
authors.initials: SC
authors.lastname: Huber
authors.affiliation: Hemocentro, Haemostasis Laboratory, State University of Campinas UNICAMP, Brazil.
authors.forename: José Luiz Rosenberis
authors.initials: JL
authors.lastname: Cunha Júnior
authors.affiliation: Hemocentro, Haemostasis Laboratory, State University of Campinas UNICAMP, Brazil.
authors.forename: Silmara
authors.initials: S
authors.lastname: Montalvão
authors.affiliation: Hemocentro, Haemostasis Laboratory, State University of Campinas UNICAMP, Brazil.
authors.forename: Letícia Queiroz
authors.initials: LQ
authors.lastname: da Silva
authors.affiliation: Hemocentro, Haemostasis Laboratory, State University of Campinas UNICAMP, Brazil.
authors.forename: Aline Urban
authors.initials: AU
authors.lastname: Paffaro
authors.affiliation: Hemocentro, Haemostasis Laboratory, State University of Campinas UNICAMP, Brazil.
authors.forename: Francesca Aparecida Ramos
authors.initials: FA
authors.lastname: da Silva
authors.affiliation: Hemocentro, Haemostasis Laboratory, State University of Campinas UNICAMP, Brazil.
authors.forename: Bruno Lima
authors.initials: BL
authors.lastname: Rodrigues
authors.affiliation: Hemocentro, Haemostasis Laboratory, State University of Campinas UNICAMP, Brazil.
authors.forename: José Fabio Santos Duarte
authors.initials: JF
authors.lastname: Lana
authors.affiliation: Hemocentro, Haemostasis Laboratory, State University of Campinas UNICAMP, Brazil.
authors.forename: Joyce Maria
authors.initials: JM
authors.lastname: Annichino-Bizzacchi
keywords: Growth factor; Leukocytes; Platelet Rich Plasma; Thrombin; gel