Prednisolone exerts exquisite inhibitory properties on platelet functions

Abstract

We have previously reported presence of the glucocorticoid (GC) receptor (GR) alpha on blood platelets, and its ability to modulate platelet aggregation when activated by the synthetic GC prednisolone (Pred). In the present study we investigated the effects of Pred on broader aspects of platelet functions to unveil novel non-genomic actions on this cell type. Using whole blood assay we demonstrated that Pred was the only GC able to inhibit platelet aggregation and platelet-monocyte interactions. This latter effect was due to regulation of platelets, not monocytes. We next examined the effects of Pred on physiological actions of platelets, observing inhibition of platelet adhesion and spreading on collagen under static conditions. Moreover Pred inhibited thrombus formation under flow, suggesting potential important effects in haemostasis and thrombosis. Pred was unable to regulate platelet reactivity under conditions where the effects of platelet-derived ADP and TxA₂ were blocked, suggesting that the GC targeted the activation-dependent component of the adhesion and aggregation response. The effects of Pred were not mediated through cyclic nucleotide signaling, but rather seemed to evolve around selective regulation of P2Y₁₂ ADP receptor signaling, intimating a novel mode of action. This study details the actions of Pred on platelets unveiling novel properties which could be relevant for this GC in controlling unwanted vascular and thrombotic diseases.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Graphical abstract

Fig. 1

Prednisolone inhibits ADP-induced platelet aggregation in whole blood. (A) Representative platelet aggregation curves as determined with the whole blood aggregometer, allowing quantification of the area under the curve (AUC). Curves are from ADP (3.25 μM) with or without Pred (10 μM), showing duplicate readings. (B) Concentration–response curve for Pred against ADP (3.25 μM) as stimulus; n = 5, *P < 0.05 compared with untreated samples. (C) Lack of tachyfilaxis to Pred effect. The GC was incubated with the blood for 3 min, 60 min or 3 + 60 min (added twice) prior to ADP-induced aggregation. Data, mean ± SEM of 3 experiments, are reported as % of inhibition as compared to appropriate control samples, were vehicle was added; *P < 0.05 compared to control. (D) The reported GC were tested in the whole blood assay, using a 3 min pre-incubation period and ADP (3.25 μM) as stimulus. Prednisolone (Pred, 10 μM), dexamethasone (Dex, 10 μM), Nitro-dexamethasone (Nitro-Dex, 10 μM), triamcinolone (Triam, 10 μM), fludrocortisol (Fludro, 10 μM) and a combination of Fludro and Triam (5 μM each); data are mean ± SEM of 5 distinct experiments; *P < 0.05 compared to vehicle. (E) As in (D) except experiments were conducted with platelet rich plasma (PRP). Data are mean ± SEM of 4 distinct experiments; *P < 0.05 compared to vehicle.

Fig. 2

Prednisolone preferentially targets the platelet to inhibit platelet/monocyte aggregate formation. Formation of platelet/monocyte aggregates was investigated using flow cytometry. (A) Top let panel: scatter plot indicating the monocyte population (gate). Top right panel, plot for CD42b and CD14 in unstimulated identifies the quadrant for double positive events. Bottom right, blot after ADP (3.25 μM) addition. Bottom left, Pred (5 μM) was incubated for 5 min with the blood prior to ADP addition: note the lower extent of double positive events. (B) Concentration response for Pred inhibition of aggregates formation upon stimulation with 1.56 (closed squares) or 3.25 (closed triangles) μM ADP. Data are mean ± SEM of 5 experiments performed in triplicate; *P < 0.05 compared to vehicle. (C) Identification of the platelet as the main target for Pred. Monocytes and platelets were exposed, contemporarily or separately, to 500 nM Pred for 3 min prior to reconstitution and stimulation with 1.56 μM ADP. Data are mean ± SEM of 5 experiments performed in triplicate; *P < 0.05 compared to vehicle.

Fig. 3

Prednisolone potently inhibits platelet adhesion and spreading on collagen. Glass slides were coated overnight at 4 °C with collagen (100 μg/ml), or heat-denatured humans serum. Uncoated surfaces were blocked by overlay with heat inactivated human serum albumin (HSA, 5%) for 30 min at 20 °C. Washed platelets (5 × 107 platelets/ml) were pre-incubated with Pred (5 min) (0.1, 1 and 10 μM) in the presence and absence of RU486 and adhered for 30 min, stained with TRITC-phalloidin and viewed using the 60× magnification. (A) Representative images. Bar, 20 μm. (B) Platelets from 8 random visual fields with a total area of 0.1 mm2 were counted and expressed as number of adherent platelets/0.1 mm2. (C) These images were used to determine platelet surface area, presented the mean surface area (μm2) ± SEM. All data shown are mean ± SEM, n = 5 with separate blood donors. *P < 0.05 compared to collagen alone.

Fig. 4

Prednisolone effect on platelet adhesion under conditions that abrogate ADP and TxA2 activity. Glass slides were coated overnight at 4 °C with collagen (100 μg/ml), or heat-denatured humans serum. Uncoated surfaces were blocked by overlay with heat inactivated human serum albumin (HSA, 5%) for 30 min at 20 °C. Washed platelets (5 × 107 platelets/ml) were pre-incubated with Pred (5 min; 10 μM) in the presence and absence of apyrase (Ap; 2 U/ml) and indomethacin (Indo; 10 μM), and then adhered for 30 min, stained with TRITC-phalloidin and viewed using the 60× magnification. (A) Representative images. Bar, 20 μm. (B) Platelets from 8 random visual fields with a total area of 0.1 mm2 were counted and expressed as number of adherent platelets/0.1 mm2. All data shown are mean ± SEM, n = 4 with separate blood donors. *P < 0.05 compared to collagen alone. (C) Washed platelets (2.5 × 108 platelets/ml) pre-incubated with Pred (1 μM) and then stimulated with ADP (5 μM) alone or in the presence of A3P5P (300 μM) or MRS2395 (MRS; 10 μM), and the aggregation response was monitored for 4 min under constant stirring. All data shown are mean ± SEM, n = 4 with separate blood donors. *P < 0.05 compared to ADP in the presence individual inhibitors, §P < 0.05 compared aggregation with either Pred or MRS2395 alone.

Fig. 5

Role of Pred on the cGMP-signaling pathway. Glass slides were coated overnight at 4 °C with collagen (100 μg/ml), or heat-denatured humans serum. Uncoated surfaces were blocked by overlay with heat inactivated human serum albumin (HSA, 5%) for 30 min at 20 °C. Washed platelets (5 × 107 platelets/ml) were pre-incubated with Pred (5 min; 10 μM) in the presence and absence of either ODQ (20 μM) or myrPKI (500 nM), and then adhered for 30 min, stained with TRITC-phalloidin and viewed using the 60× magnification. Platelets from 8 random visual fields with a total area of 0.1 mm2 were counted and expressed as number of adherent platelets/0.1 mm2 (A) and platelet surface area (μm2) (B), all data shown are mean ± SEM, n = 4 with separate blood donors. *P < 0.05 compared to collagen alone. (C) Washed platelets were pre-treated with either ODQ (20 mM) or myrPKI (500 nM) for 10 min and then incubated with either the nitric oxide donor, S-nitrosoglutathione (GSNO; 10 mM) or prostaglandin E1 (1 mM) for 2 min. Platelets were lysed and immunoblotted with either anti-VASP-phospho239 or anti-VASP-phospho157 antibodies.

Fig. 6

Prednisolone inhibits thrombus formation on collagen in vitro. Washed platelets were stained with DiOC6 (1 μM), incubated with Pred (10 μM) or vehicle for 2 min and reconstituted blood was flowed over immobilized collagen (100 μg/ml) for 4 min at a indicated shear rate of 1000/s−1. Platelet deposition viewed by fluorescence microscopy . Images from 8 random fields of view were captured under fluorescence using cell^P imaging software (Olympus, Japan). Magnification, 60×. (A) Representative images. Bar, 20 μm. (B) Data are shown as percentage area coverage and are mean ± SD of n = 5 with separate blood donors. *P < 0.01 compared to collagen alone.