The Activation Step holds the final, fundamental part of the platelet preparation: it is where we set the platelets and get them ready to work.
As previously mentioned in the Blood Extraction Step, the sodium citrate anticoagulant is the most commonly used, and it prevents clotting by forming chelates from calcium ions.
The activation consists, generally speaking, in the reversion of this process by returning calcium to the platelets, since it intervenes in the transformation of prothrombin into thrombin, as well as in the platelet aggregation and adhesion, through the membrane receptors of the platelets.
There are two different types of platelet activation
- Endogenous. This activation occurs naturally. It is also known as “physiological activation”, and it happens as the platelets react to the collagen fibers inside the body. Some authors recommend applying the platelets directly and letting them act on their own.
- Exogenous. This type of activation is pharmacologically induced before applying the PRP onto the patient. The most commonly used products for veterinary use are Calcium Gluconate at 10% (CG), Calcium Chloride at 10% (CaCl2) and Bovine Thrombin (BT).
How to choose the appropriate activation method?
Different research studies have determined that CaCl2 10% and CG 10% are the easier ones to obtain, regarding availability and cost.
When using CaCl2 10%, the dilution should be 0.1 cc of CaCl2 for every 0.9 cc of PRP, so that neutralization of the anticoagulant effect of the citrate can be achieved. In the case of CG 10%, the dilution should be 0.3 cc of CG for every 0.7 cc of PRP. Taking these numbers into account, we could say that CaCl2 10% is more efficient—dosage-wise— than CG 10%, but, in some countries it is easier and a lot cheaper to find CG, so availability is a very important element to observe when choosing between these two activators.
Various articles report that Bovine Thrombin dilution should be 5U/cc, but there are still no defined protocols for its use for PRP activation in veterinary medicine. It is important to highlight that BT is a little bit more expensive than CaCl2.
Other products are available, such as thromboxane and by-products, but there are much more expensive and have still not been tested appropriately in animals to offer a fair dosage protocol or opinion.
Now, speaking outside of dilutions, availability and costs, here goes one of the most important questions: which one works the best? Unfortunately, we still don’t have a full positive answer, since there are so many different animal species with different platelet functions and morphology, so there is a really wide research field waiting to be looked into to establish protocols for each species. However, the few studies that do offer close answers can be useful for the veterinary clinician.
There is an interesting variation in opinions regarding the best platelet activator for veterinary used. Some veterinarians prefer an endogenous activation, that is, not using pharmacological activation at all, and just applying the PRP in its natural form. Other experienced physicians believe endogenous activation is not the most effective way to really exploit every single one of the concentrated platelets. The most popular opinion is that, if one has already gone through “all the trouble” of the preparation steps, one might as well benefit from it fully, maximizing the platelet functions with the artificial available methods.
Research Study in Brazil
A research study made in Brazil in 2012 by Zandim et al., evaluated the effect of BT, CaCl2, and physiological activation in horse platelets, by analyzing the platelet morphology and activation state after the activation process.
In this study, BT showed not suitable for equine platelet activation, since the platelets where seen as irregularly shaped, as well ruptured granules both inside and outside the remaining cytoplasm. CaCl2 showed a full platelet activation of 24% of the platelets in the samples, 52% of activation state uncertain, 2% of irreversibly damaged platelets and 20% of resting platelets (the resting platelets CAN become active physiologically after application to the patient, through contact with the injured tissue); the platelets that were not treated with any activator were 49% uncertain, 41% resting, 9% fully activated and 1% irreversibly damaged.
The conclusions in this investigation recommend physiological activation when PRP is administered immediately after preparation and in its liquid form, since the resting percentage of resting platelets are very significant; but also recognizes that CaCl2 is a very efficient activator that offers a 24% certainty of activation, and should be considered if the PRP will not be administered immediately, or if will be processed to a gel form, rather than a liquid form.
Other Studies Done
Another study made in feline platelets by Silva et al. was published in BMC Veterinary Research also in 2012, and compared BT against CG for platelet activation. The results measured platelet efficiency, and showed 61.36% efficiency for CG activated PRP, and 65.64% for BT activation. The author’s conclusion was that both activators provide a very useful platelet concentration and efficiency. It is interesting how the results regarding BT are so different from each other, being the species the main variant.
So, even if we can still not answer the question with a 100% certainty for each species, at least we have a very useful insight regarding what works better in some species, we are aware that the variations are many, and we have made a significant approach to the action mechanism of the whole “PRPology”, in a way that our own criteria as veterinary clinicians can guide us to make the best decision for our patients. Let’s not forget that the commercial PRP Kits are extremely practical and can also be a wise option. Now all we can do is START using PRP and open the door for more research and clinical studies!