Platelet activation by collagen provides sustained release of anabolic cytokines

Abstract

Background: Platelet-rich plasma (PRP) has been increasingly used in sports medicine applications. Platelets are thought to release growth factors important in wound healing, including transforming growth factor (TGF-β1), platelet-derived growth factor (PDGF-AB), and vascular endothelial growth factor (VEGF). However, little is known about the effect of platelet activator choice on growth factor release kinetics.

Hypothesis: The choice of platelet activator would affect the timing and level of growth factor release from PRP.

Study design: Controlled laboratory study.

Methods: Platelet-rich plasma aliquots were activated with either thrombin or collagen. A control group of whole blood aliquots was clotted with thrombin. Supernatant containing the released growth factors was collected daily for 1 week. Levels of TGF-β1, PDGF-AB, and VEGF were measured using enzyme-linked immunosorbent assay (ELISA).

Results: The use of thrombin as an activator resulted in immediate release of TGF-β1 and PDGF-AB, while the collagen-activated PRP clots released similar amounts each day for 5 days. The use of collagen as an activator resulted in an 80% greater cumulative release of TGF-β1 from the PRP aliquots over 7 days (P < .001). Concentrating platelets to 3 times the systemic blood level resulted in a 3-fold higher release of TGF-β1, 2.5-fold greater release of PDGF, and 5-fold greater release of VEGF (all P < .0001) when compared with whole blood control clots, but no significant differences in the timing of release were noted.

Conclusion: These experiments demonstrated that the choice of platelet activator can significantly influence the release kinetics of cytokines from PRP, with thrombin resulting in an immediate release and collagen having a more sustained release pattern.

Clinical relevance: The level and rate of growth factor release depends on the selected platelet activator, a factor that should be considered when selecting a PRP system for a given application.

Figures

Figure 1

Cumulative TGF-β1 release over time measured in the supernatant surrounding 4 types of blood clots. The naturally clotting whole blood (WB), thrombin-activated whole blood (WB + BT), and thrombin-activated platelet-rich plasma (PRP + BT) clots released TGF-β1 at the 2-hour time point and maintained the same level of cumulative TGF-β1 on the remaining 6 days. In contrast, the collagen-activated platelet-rich plasma (PRP + COL) clots released a relatively small amount of TGF-β1 at the 2-hour time point and showed an increase in the cumulative TGF-β1 levels in the supernatant between day 1 and 5, after which the concentration remained constant. This time-dependent release for PRP + COL was statistically significant by repeated-measures ANOVA (P < .0001) but not significant for WB (P = .999), WB + BT (P = .895), or PRP + BT (P = .532). The shapes at each time point represent average values (n = 9) of growth factor released, and error bars are standard errors of the mean (SEMs).

Figure 2

Cumulative PDGF-AB release over time in hours measured in the supernatant surrounding 4 types of blood clots. The PDGF-AB release trends were similar to those observed for TGF-β1 in Figure 1. The naturally clotted whole blood (WB) and thrombin-activated clots released a large amount of PDGF-AB at the 2-hour time point and maintained the same cumulative level of PDGF-AB in the supernatant on the remaining 6 days. In contrast, the collagen-activated platelet-rich plasma (PRP) released an increasing total amount of PDGF-AB in the supernatant between days 1 and 5. Repeated-measures ANOVA confirmed a statistically significant time-dependent PDGF-AB release for PRP + COL (P <.0001) but not for WB (P = .999), WB + BT (P = .864), or PRP + BT (P = .800). While the profile of release was different for the collagen and thrombin-activated release of PDGF-AB, there was no significant difference between the cumulative release of PDGF-AB by the fifth day after clot activation. The shapes at each time point represent average values (n = 9) of growth factor released, and error bars are standard errors of the mean (SEMs).

Figure 3

Cumulative VEGF release over time measured in the supernatant surrounding 4 types of blood clots (refer to Figure 1 for sample descriptions). The majority of the release of VEGF from the thrombin-activated whole blood clot was over the first 2 hours, while that from the collagen-activated platelet-rich plasma (PRP) clot occurred over a longer period of time. The release of VEGF from the naturally clotted whole blood (WB) and thrombin-activated PRP groups also occurred over 5 days. These trends were supported by repeated-measures ANOVA for PRP + COL (P < .0001) and PRP + BT (P < .0001) and WB (P <.0001) but not significant for WB + BT (P = .124). The shapes at each time point represent average values (n = 9) of growth factor released, and error bars are standard errors of the mean (SEMs).