Influence of hydrocortisone on platelet receptor expression and aggregation in vitro

Introduction

Hydrocortisone differentially modulates inflammatory mediators and expression of adhesion molecules. Previously, a randomised controlled trial showed that hydrocortisone attenuates cell interactions in septic shock [1]. However, the effect of 'low'-dose hydrocortisone treatment on platelet activation has not yet been evaluated systematically.

Objective

To evaluate the influence of hydrocortisone levels on platelet activation in vitro.

Methods

Citrated blood samples were drawn from healthy blood donors (n = 25, 36% male, age: 40 ± 10 [mean ± standard deviation]). Exclusion criteria were smoking, diabetes mellitus, diseases and drugs interfering with cortisone levels or platelets. After measuring the morning cortisol level, blood samples were adjusted with hydrocortisone (Pharmacia, Erlangen, Germany) to final concentrations of 4.5 μg/ml (group 1), 9 μg/ml (group 2, low dose) and 90 μg/ml (group 3), respectively. The control group received no additional hydrocortisone. Samples were incubated for 10 min at 37°C with fluorescence-labeled monoclonal antibodies against CD62P, CD41, CD45, CD42b (all: Beckman-Coulter, Krefeld, Germany) or PAC-1 (Becton Dickinson, San Jose, CA, USA). To evaluate platelet reactivity 5 μM thrombin-receptor-agonist-peptide-6 (TRAP-6; Bachem, Germany) or 2.5 μM adenosine-di-phosphate (ADP; Sigma, Germany) were added. Analyses were performed in a flow cytometer. The mean fluorescence intensity (MFI) and percentage of CD45/41⁺ complexes was calculated. Determination of platelet aggregation was performed by turbidimetric procedure (BCT; Dade Behring, Marburg, Germany). Aggregation was induced with ADP (200 μM/l; Dade Behring), collagen (2 mg/l; Dade Behring) and epinephrine (100 μM/l; Dade Behring). Intergroup differences were compared by one-way analysis of variance.

Results

The initial cortisol level was 11.6 ± 3.5 μg/dl. Hydrocortisone administration had no significant influence on expression of PAC-1, CD62P and CD45/41⁺ complexes, with and without stimulation. In contrast, we observed a significant lower expression of CD42b in group 3 compared with the control group only without activation (P = 0.047). A similar observation was made for CD41 expression (group 3 vs control: unstimulated: P = 0.035). Differences between the control and groups 1 and 2 were not significant with either activator. Aggregometry showed significant later onset of maximum aggregation after activation with ADP and collagen (group 3 vs control: collagen: P = 0.001; ADP: P = 0.048).

Conclusion

This in vitro study demonstrates that administration of low-dose hydrocortisone neither reduces expression of investigated platelet receptors nor attenuates begin of maximum aggregation in doses recommended in septic shock. We therefore conclude that 'low'-dose hydrocortisone might not result in impaired platelet function.