Anticoagulants Interfere With the Angiogenic and Regenerative Responses Mediated by Platelets

Abstract

Background and aims: Platelet rich plasma (PRP) obtained from blood anticoagulated with acid-citrate-dextrose (ACD) or sodium-citrate (SC) is used for regenerative medicine as source of platelet-derived growth factors. Allergic reactions against citrate were reported in patients after local injection of PRP allowing us to hypothesize that anticoagulants exert a harmful and local effect that interferes with the regenerative proprieties of platelets. Herein we test this hypothesis by analyzing the effect of ACD and SC on angiogenic and regenerative responses mediated by platelets. Methods: PRP was obtained from SC-or ACD-anticoagulated blood; platelets were lysed to release growth factors; and PRP releasates (PRPr) were used to induce in vitro endothelial proliferation and 2D-migration, and regeneration of mouse skin wounds. Results: We first compared proliferation and migration of endothelial cells mediated by anticoagulated-PRPr supplemented or not with CaCl 2. Alteration of endothelial adhesion and impediment of proliferation and migration was observed without CaCl 2. Although endothelial morphology was normalized in SC-and ACD-PRPr after calcium restitution, angiogenic responses were only markedly induced by SC-PRPr. In vivo studies revealed a delay in mouse skin regeneration after treatment with anticoagulated-PRPr without CaCl 2. Healing was only induced after calcium restitution in SC-but ACD-PRPr. Moreover, the development of inflammatory intradermal papules was evidenced after injection of ACD-PRPr. Supplementation of SC-PRPr with the equivalent concentration of dextrose (D-Glucose, 18 mM) present in ACD-PRPr resulted in reduction of endothelial proliferation and migration, delay of mouse skin regeneration and development of intradermal papules. Finally, collecting blood with half amount of SC significantly improved all the angiogenic and regenerative responses mediated by PRPr. In contrast, the delay of skin regeneration and the development of inflammatory papules remained stable after dilution of ACD. Conclusion: Our findings indicate that (1) calcium restitution is required to impair the cellular and tissue alterations induced by citrated-anticoagulants contained in PRP; (2) ACD-derived dextrose confers anti-angiogenic, anti-regenerative and pro-inflammatory proprieties to PRP; and (3) half concentration of SC improves the angiogenesis and regeneration mediated by PRP.

Use of platelet-rich fibrin for the treatment of periodontal intrabony defects: a systematic review and meta-analysis

“…One strategy that was proposed several years ago for the regeneration of intrabony defects was the use of platelet concentrates [10]. While platelet-rich plasma (PRP) was proposed as a first-generation platelet concentrate, the use of anticoagulants has since been shown to interfere with the angiogenic and regenerative responses mediated by platelets [11]. For these reasons, a second-generation platelet concentrate, termed platelet-rich fibrin (PRF), has more been introduced in regenerative medicine and dentistry [10,[12][13][14][15].…”

Isolation of Platelet-Derived Exosomes from Human Platelet-Rich Plasma: Biochemical and Morphological Characterization

“…With that purpose, we isolated exosomes exclusively released by PRP platelets, which were free of contaminants from other components present in PRP, allowing us to analyze pure PLT-Exos and to evaluate the influence of platelet activation by CaCl 2 on their molecular composition. It must be taken into consideration that the activation method chosen may modify the molecular release [ 30 ]. The activation method employed here based on CaCl 2 addition, is one of the most commonly used procedures to induce the release of molecular mediators from PRP platelets and avoids the use of exogenous biomolecules such as thrombin.…”

“…At the same time, CaCl 2 -activation prevents local hypocalcemia caused by calcium chelating anticoagulants required for blood collection in the PRP preparation. A recent study showed that calcium restitution is necessary to prevent cellular and tissue alterations due to the anticoagulants used in some PRP preparations [ 30 ].…”

Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential

“…In addition, growth factors can induce both anabolic and catabolic effects on cells [43] thus potentially affecting the cellular response. Another important aspect to take into account is the contribution of citrate deriving from anticoagulant ACD-A presents in the PRP units, primarily for its role as calcium chelator, which has been demonstrated to interfere with the angiogenic and regenerative proprieties of platelets [44]. On the other hand, some authors studying the specific effect of different concentrations of citrate supplementation in promoting bone regeneration, observed that exogenous supplementation of citrate is dose-dependent and that 200 µM is the optimal concentration able to induce the elevation of osteogenic markers [45].…”