Hormones, age, and sex affect platelet responsiveness in vitro

Abstract

Background: Female sex confers a survival advantage following severe injury in the setting of trauma-induced coagulopathy, with female platelets having heightened responsiveness likely due to estrogen. The effects of testosterone on platelet biology are unknown, and platelets express both estradiol and androgen receptors on the plasma membrane. We hypothesize testosterone decreases platelet responses in vitro, and there are baseline differences in platelet function and metabolism stratified by sex/age.

Study design and methods: Apheresis platelets were collected from: older males (OM) ≥45 years, younger males (YM) <45 years, older females (OF) ≥54 years, and younger females (YF) <54 years, and testosterone and estradiol were measured. Platelets were incubated with testosterone (5.31 ng/ml), estradiol (105 pg/ml) or vehicle and stimulated with buffer, adenosine diphosphate (20 μM), platelet activating factor (2 μM), or thrombin (0.3 U/ml). Aggregation, CD62P surface expression, fibrinogen receptor surface expression, and platelet mitochondrial metabolism were measured.

Results: Testosterone significantly inhibited aggregation in OF and OM (p < .05), inhibited CD41a expression in YF, YM, and OM (p < .05), and affected a few of the baseline amounts of CD62P surface expression but not platelet activation to platelet-activating factor and adenosine diphosphate, and variably changed platelet metabolism.

Discussion: Platelets have sex- and age-specific aggregation, receptor expression, and metabolism. Testosterone decreases platelet function dependent on the stimulus, age, and sex. Similarly, platelet metabolism has varying responses to sex hormones with baseline metabolic differences dependent upon sex and age.

Keywords: estradiol; mitochondria; platelets; sex dimorphisms; testosterone.

Figure 1.. The effects of testosterone- and estradiol- pre-treatments on the aggregation profile of apheresis platelets vs. naïve (0.0001% DMSO/no sex hormone) NS-treated platelets, compared within and between sex and age groups.

Platelet aggregations were measured in response to ADP, PAF, or thrombin and only the significant values are mentioned: Panel A) ADP: naïve OF, OM vs. testosterone-treated platelet aggregation, naïve YM vs. OF, older males, Panel B) PAF: testosterone-pretreated platelets vs naïve in all groups, Panel C) Thrombin: no significance, Panel D) ADP: naïve OM vs. estradiol-treated, OM estradiol-pretreated platelets vs. estradiol-treated platelets from YF, Panel E) PAF: No significance, Panel F) Thrombin: testosterone-treated OM from YF and YM; naïve OM vs. naïve YF and OF, estradiol-treated OM vs. all estradiol groups. Designation of symbols: * versus naïve NS-treated, † versus YF, ‡ versus OM, § versus OF, p<0.05 (n=10 in all groups)

Figure 2.. The effects of estradiol and testosterone pretreatment on the activation (CD62P expression) and surface expression of CD41a.

The % CD62P expression from CD41a+ platelets were measured by flow cytometry in apheresis platelets pre-treated with buffer (naïve), testosterone, or estradiol and the stimulated with NS (control), ADP, or PAF. A. The % CD62P was significantly increased in all stimulated (ADP and PAF) platelets. The control (unstimulated) platelets from YF, both naïve and testosterone-treated had significantly lower % CD62P expression than YM and OF (testosterone only). B. CD41a surface expression (MFI) was measured and ADP and PAF caused significant increases from control in naïve, testosterone, and estradiol pretreated groups. In YM and OM testosterone pre-treatment increased CD41a surface expression vs. the naïve. Estradiol pre-treatment increased the CD41a MFI in OF and OM naïve and ADP-stimulated platelets. Designation of symbols: * versus control; # versus Naïve, p<0.05 (n=10–11)

Figure 3.. A compilation of mitochondrial respiration in platelets from donors stratified by sex and age.

The mitochondrial respiration in platelets from YF, OF, YM, and OM are illustrated in Seahorse tracings from naïve (media-treated) platelets (Panel A), testosterone-treated platelets (Panel B), and estradiol-treated platelets (Panel C). Oxygen consumption rate (OCR, mean±SEM) of platelets from different ages/sexes is shown as a function of different sequential measurements of mitochondrial stress treatment: baseline and hormone/vehicle additions (basal respiration), oligomycin (ATP-linked respiration), FCCP (maximum respiration) and Rot/AA (spare respiratory capacity); n=10 for each group.

Figure 4.. Platelet respiration stratified by donor sex and age.

Panel A The basal respiration of platelets from YF was significantly less than the basal respiration of platelets from both OF and YM (p<0.05) (). Panel B. Estradiol pretreatment significantly decreased the basal respiration of platelets from OM (p<0.05). Testosterone pretreatment increased the basal respiration in OF and YM vs OM and estradiol pre-treatment increased the basal respiration of platelets in both OF and YM compared to younger females and older males (p<0.05). Panel C. ATP-linked production was significantly higher in older females in naïve, testosterone- and estradiol-treated platelets vs YF but only estradiol was significant from OMs. YM naïve platelets were significant from both YF and OM. Panel D. Hormone treatment caused a significant decrease amongst all groups for the maximum respiration. The maximum respiration of naïve platelets from OF and YM was significantly higher than YF, but only YM were statistically different from OM. Panel E. The spare respiratory capacity was only affected by estradiol treatment in YF and OF from naïve. Panel F. The non-mitochondrial respiration is significantly lower in YM versus the platelets from OM with hormone treatment, both testosterone and estradiol. Testosterone also caused a significant increase from naïve in the platelets from OM. Designation of symbols: * versus naïve, † versus YF, and ‡ versus OM, p<0.05 (n=10 in all groups).

Figure 5.. The platelet extracellular acidification rate (ECAR).

Pre-incubation with testosterone or estradiol did not affect the ECAR within the age/sex groups (Panel A). The naïve, testosterone- and estradiol-treated platelets from OF had significantly increased ECARs from YF and OM, respectively (p<0.05) (Panel A). Oligomycin-sensitive ECAR in platelets from OF was significantly increased from the platelet from both YF and OM in naïve, testosterone- and estradiol-treated platelets, and testosterone treatment also had a significant increase from naïve platelets (Panel B). The naïve platelets from YM were also significantly higher than the platelets from YF and OM (Panel B). Designation of symbols: * versus naïve, † versus YF, ‡ versus OM, p<0.05 (n=10 in all groups).