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Recombinant human erythropoietin (rhEPO) is included in the World Antidoping Agency list of prohibited substances 1. Its misuse to improve athletic performance can result in serious health consequences. It can elevate blood pressure, increase blood viscosity and, of interest here, it can bolster platelet reactivity, which is usually assessed by evaluating the expression of P‐selectin at the platelet surface 2. Hence, on this basis, the present work has been performed to study the ex vivo effects of rhEPO in a small cohort of young and healthy individuals (15 males and 14 females). Platelet reactivity and aggregation patterns which, in turn, may cause thrombotic disorders 3, have been evaluated.
Treatments with rhEPO 0.2IU and 0.4IU were performed. Platelet aggregation induced by adenosine diphosphate (ADP) or collagen, was evaluated as stated in the literature (see Supporting Information) 4. ADP is an important physiological platelet agonist released from erythrocytes and platelets that, under physiological conditions, triggers platelet clotting via the P2Y12 receptor, i.e.via purinergic signalling 5. Collagen is another natural platelet agonist that, under physiological conditions, triggers platelet aggregation via the glycoprotein (GP) VI transmembrane receptor. Furthermore, to investigate whether rhEPO was able to induce homotypic (platelet–platelet) or heterotypic (platelet–leucocytes) aggregation, experiments were also conducted on platelet rich plasma (see Supporting Information). Homotypic aggregation is confined to the vascular wall and sustains local thrombotic events. It requires activation of membrane glycoprotein complex α2bβ3, an integrin adhesion protein expressed on the surface of platelets, which forms molecular bridges between aggregating platelets via its binding with fibrinogen or von Willebrand factor. Heterotypic aggregation has been observed in the peripheral circulation and it is associated with high blood thrombogenicity. This has been demonstrated to take place via P‐selectin expression at the platelet surface, seemingly due to platelet reactivity 6. P‐selectin is an adhesion molecule which, in unstimulated platelets, can constitutively be detected within the α‐granules, i.e.in granules containing several growth factors and clotting proteins (such as thrombospondin, fibronectin, factor V and von Willebrand factor). At variance, upon platelet stimulation, P‐selectin is phosphorylated and translocates to the plasma membrane via a secretory pathway. In this work, we evaluated the percentage of platelets with activated α2bβ3 and the percentage of platelets showing P‐selectin at their surface. To perform these studies, flow cytometry analyses were carried (see Supporting Information).
In whole blood we found that, in both male and female donors, rhEPO increased platelet aggregation induced by ADP (Figure (Figure1A).1A). Interestingly, in males the most significant (P<0.05) effect was detectable at the lower dose (0.2IU), whereas in females platelet aggregation increased in a dose dependent manner and the most significant effect (P<0.05) was detectable at the dose of 0.4IU. Concerning the aggregation induced by collagen (Figure (Figure1B),1B), a gender difference in response to rhEPO was found. An increased aggregation was detected in platelets from males at the low dose (0.2IU), whereas, in females, a reduced aggregation was found at both doses.
Ex vivo studies carried out with platelet‐rich plasma showed that rhEPO increased the number of platelets with activated α2bβ3 in both males and females. The most significant effect (P<0.05) was detectable at the dose of 0.2IU (Figure (Figure1C).1C). These data suggested that rhEPO was able to induce homotypic aggregation in both males and females. However, surprisingly, as depicted in Figure Figure1D,1D, both rhEPO doses were able to induce a significant (P<0.05) increase of P‐selectin expressing platelets in females only.
This study ‘mimics’ ex vivo what could occur in the peripheral blood after intake of rhEPO. Bearingin mind that i) platelet collagen‐interaction is confined to the vascular wall and sustains the formation of platelet‐rich thrombi, ii) α2bβ3 activation plays a central role in thrombus stabilization and iii) P‐selectin, interconnecting platelets and leucocytes, were associated with high blood thrombogenicity, we can hypothesize that rhEPO intake could expose the athletes to a prothrombotic risk, which differs in males and females. In males, the effects of rhEPO could be confined to the vascular wall and contribute to the formation and stabilization of the thrombi. In females, its effects could be observed in the peripheral circulation and could be associated with high blood thrombogenicity. These data support the hypothesis that the consequences of rhEPO in athletes could be detrimental depending on the gender of the consumer and should merit more careful attention from the medical community.
All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare no support from any organization for the submitted work, no financial relationships with any organizations that might have an interest in the submitted work in the previous 3years and no other relationships or activities that could appear to have influenced the submitted work.
Supporting info item
Gambardella L., Vona R., Pichini S., Pacifici R., Malorni W., and Straface E. (2016) Gender difference in platelet aggregation and reactivity induced by recombinant human erythropoietin. Br J Clin Pharmacol, 81: 789–791. doi: 10.1111/bcp.12835.