Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC108

Poster Communications

Immunophilin proteins participate in platelet aggregation by regulating granule secretion and calcium homeostasis.

A. Berna-Erro1, E. Lopez1, J. M. Hernández-Cruz2, N. Bermejo3, J. G. Casado4, R. Tarrazona4, G. M. Salido1, J. A. Rosado1, P. C. Redondo1

1. Physiology (PHYCELL group), University of Extremadura, C

The immunophilin family has been targeted by using macrolide lactone derivates, such as cyclosporin A (CsA), tacrolimus (FK506) and everolimus (Rapamycin), in order to prevent organ rejection in transplanted patients (1,2). FK506 and CsA have been presented as efficient immunosupressor by altering lymphocyte T function, since both immunophilin blockers prevent calcineurin (CNa) activation (3). Here we analyze in in vitro assays whether inhibition of immunophilins would deregulate cytosolic free calcium concentration ([Ca2+]c) homeostasis, granules secretion and, subsequently, impair human platelets aggregability. Human platelets were isolated from healthy donors following the guidelines of Helsinki’s Declaration, as previously described elsewhere (4) and then were incubated with 2 μM of fura-2 AM for 45 min at 370C or left untreated. Changes in [Ca2+]c were monitored using a spectrofluorophotometer exciting the cells alternatively at 340/380 nm and emission was detected at 510 nm. Alpha- and dense-granules secretion was determined by flow cytometry (FACScan cytometer); meanwhile platelet aggregation experiments were performed in a Chrono-log aggregometer. Human platelets were incubated for 5 min with either the vehicle or FK506 (50 μM). FK506 increased Ca2+ release evoked by thrombin (Thr, 0.1 U/ml) by a 60.0 ± 22.4% (P<0.001 Student’s t-test, n=4-6) compared to control FK506-untreated platelets. However, immunohilin antagonists reduced Thr-evoked Ca2+ entry by 67.8 ± 11.5% (P<0.001, n=6). Stimulation of human platelets with different Thr concentrations (0.001-0.5 U/ml) reported a different degranulation pattern of alpha- and dense-granules, which were monitored by flow cytometry using PE-labeled anti-P-selectin antibody (anti-CD62) and mepacrine, respectively. Additionally, incubation of platelets with 50 μM of FK506 for 5 min significantly reduced Thr (0.1 U/ml) evoked both alpha- and dense-granule secretion. Finally, we have found a reduction in Thr-evoked aggregation in the presence of FK506, compared with control untreated cells but stimulated with Thr (0.01 U/ml), perhaps as consequence of the previous inhibitory effects of FK506 on granule secretion. In summary, we provide evidences for an important role of immunophilins in platelet secretion and aggregation.

Where applicable, experiments conform with Society ethical requirements