In this study, we hypothesized that release kinetics of growth factors released from activated platelets is affected by both platelet activator and platelet concentration. Our results show that both factors have significant influence on growth factor release. We studied 3 different growth factors: TGF-β1, PDGF-AB, and VEGF. While there is a plethora of growth factors released from platelets, these 3 are best described for both their association with platelets and their effect in the wound healing cascade and will therefore serve as surrogates for growth factor release for the purpose of this study. TGF-β1 is released from platelets in a latent form and later becomes activated by any number of molecules involved in extracellular matrix perturbations.24
To enable measurement of TGF-β1 in vitro, an activation step is required during the ELISA procedure. As a result, the state of the TGF-β1 (active or latent) released into the supernatant was not specifically determined. However, this limitation is somewhat mitigated by the prior work reporting that in the in vivo environment where platelets are present, latent TGF-β1 released from the clot is activated by a furin-like enzyme, which is simultaneously released by the platelets.4
Thus, for evaluation of the performance of the 2 platelet activators, collagen and thrombin, the determination of latent versus activated TGF-β1 in vitro may have less of an effect on what occurs in vivo.
Our first hypothesis was that activator choice would significantly affect growth factor release. This hypothesis was found to be true when comparing thrombin and collagen activation of PRP. Thrombin activation resulted in an almost immediate release of PDGF-AB and TGF-β1 from the clots, whereas the collagen activator resulted in a gradual accumulation of the growth factors in the surrounding supernatant. For PDGF-AB, the activator changed only the release profile over time but not the final concentration. In contrast, the cumulative release of TGF-β1 from PRP was higher when collagen was used as the activator, suggesting a sustained release of growth factor. In this case, both release time and total amount were influenced by the activator choice.
Prior studies have reported almost complete release of growth factors from thrombin-activated PRP within the first few hours of activation.32,35
Our results in the thrombin group parallel this observation. However, the collagen group had a sustained release of cytokines over the first several days. This difference in timing of release may be due to the mechanisms by which thrombin and collagen activate platelets. For collagen to activate platelets, the platelets must first adhere to the collagen and then subsequently be activated by it through a second receptor.5
This may require a lengthier mechanism for platelet activation than the enzymatic cleavage process of thrombin-mediated platelet activation. In addition, in normal wound healing, collagen is often the initial activator of platelets, with a platelet monolayer forming over the exposed collagen. Once the initial platelet layer is formed, there is a secondary accumulation of additional platelets via the action of thrombin. Thus, collagen activation of platelets occurs earlier in wound healing, and thus, a delayed activation may be functionally helpful so that the release of growth factors does not occur prematurely before full formation of a provisional scaffold.
Furthermore, the cumulative release of growth factors was less from whole blood when thrombin was used as an activator than when the whole blood was allowed to clot without use of an activator. This effect is most evident in the release of VEGF but also present for PDGF-AB and TGF-β1. This is consistent with prior studies that have shown decreased levels of fibroblast growth factor activity due to cleavage by thrombin.36
Serine proteases, including thrombin, actively degrade growth factors in chronic wound fluid.18
As such, we hypothesize that the decreased levels of growth factors found in whole blood with thrombin compared with whole blood without thrombin are due to proteolytic cleavage, and further studies to validate this hypothesis are planned. In contrast to the influence on release time provided by collagen activation, thrombin activation of whole blood results in a reduction in total growth factor concentrations, suggesting an interaction between growth factor activator choice and platelet concentration.
An activated platelet delivery system that provides for the sustained release of growth factors from platelets could help stimulate a functional healing process similar to that seen in tissues that heal spontaneously.3,17
To date, bovine thrombin has been a commonly used activator of PRP and fibrin sealant.1,16
While bovine thrombin is a potent platelet activator,11
it also causes the development of antibodies against thrombin, prothrombin, factor V, and cardiolipin with resultant clinical problems that include severe postoperative bleeding to bypass graft thrombosis and an auto-immune syndrome similar to lupus in animal studies.19
The use of thrombin also results in impaired migration of anterior cruciate ligament (ACL) cells through collagen-PRP hydrogels as well as impaired strength of the hydrogels24
so alternatives to use of this material are desirable.
The release of VEGF did not appear to be affected significantly by activator choice and was incremental over the 7-day period for both activators. One possible reason for the delayed release of VEGF in all groups and the different response in the VEGF profile from the TGF-β1 and PDGF-AB profiles may be due to the other cell types present in the PRP. Not only were platelets concentrated in the PRP groups, but the number of white blood cells was doubled as well, due principally to a 5-fold increase in lymphocyte concentration (). Vascular endothelial growth factor has been shown to accumulate during blood storage in a time-dependent manner in nonleukodepleted whole blood.26,27
The accumulation was shown to occur for up to 35 days, including and extending beyond the 1-week time frame for this study. One suggested hypothesis for the accumulation is a gradual release of presynthesized VEGF from white blood cell granules.26
Synthesis of VEGF has been verified in both lymphocytes and neutrophils.12,14
Thus, if the overall VEGF release were affected by the concentration of white blood cells rather than only platelet activation, we might expect similar profiles in both the thrombin- and collagen-activated PRP groups as seen in this experiment. The gradual release of VEGF from the whole blood groups suggests a contribution from neutrophils as well because they accounted for the majority of white blood cells in whole blood, whereas lymphocytes were most populous in the PRP. It is unclear whether this white blood cell effect dominated the VEGF release profile or if it was simply an accessory to the release from platelets observed for the other 2 growth factors. Further studies evaluating the effect of lymphocyte concentration on cytokine release from PRP are planned.
We also hypothesized that concentrating platelets as PRP would result in increased cytokine release from a clot when compared with whole blood. This hypothesis was proven when comparing whole blood and PRP activated by the same activator, thrombin. We found increases in PDGF-AB and TGF-β1 that paralleled the increase in platelet count (approximately 3 times) and an increase in VEGF release that was 5-fold greater than the systemic blood clot. Our results are in line with previous findings that increasing platelet counts in blood may result in higher levels of growth factor release but not necessarily on through a linear correlation for every individual.8,23,38
The PRP used in this study contained 3 times more platelets than the baseline whole blood, which is similar to the increase in PDGF and TGF-β1. The higher release of VEGF may again be due to the increased concentration not only of platelets but of leukocytes as well within the PRP.12,26,27
There were several limitations of this study. First, we only measured the growth factor release in the supernatant. There may have been additional growth factors released from the platelets but bound to the collagen or other extracellular matrix proteins and thus not available in the supernatant. Further work examining the growth factor adhesion to the multiple extracellular matrix proteins would be required to define this. However, the majority of the global biological activity of a blood clot in the first week after injury is to emit cytokines and growth factors that stimulate cells within the surrounding tissues (macrophages, stem cells, and fibroblasts) rather than cells within the scaffold itself (red blood cells, small numbers of terminally differentiated white blood cells).7
Thus, the growth factors released into the surrounding milieu of the wound are also of some importance in understanding the activity of PRP placed in a wound site. Second, this is an in vitro study, and how the release kinetics would be different in vivo remains to be studied. A final limitation is that in this study, we used a PRP preparation method in which the PRP contains not only platelets but also red blood cells and white blood cells. The inclusion of these other cell types may have confounded results to some extent, even though the same numbers of all cell types were used in each PRP group. While the use of a platelet-only preparation would certainly have been of interest on a basic science level, it may be less useful for the clinician trying to understand how his or her PRP preparation method will perform in the operating room.
In summary, the use of thrombin to activate platelet clots results in an almost immediate release of all the platelet-associated anabolic growth factors studied here, while the use of collagen as an activator results in a more sustained release over a week of 2 of the 3 cytokines studied. In addition, using a higher concentration of platelets does result in a higher average growth factor release, but the increase in growth factor release may not correlate exactly with the increase in platelet count for every individual. The use of thrombin as an activator should be considered carefully, and the optimal concentration of platelets for any given clinical application also deserves further study. In applications where a more sustained growth factor release is desired, a collagen activator may be of some benefit.