The sex specific effects of extraneous testosterone on ADP induced platelet aggregation in platelet‐rich plasma from male and female subjects

Sir, Excessive platelet aggregation, particularly by ADP in humans [1], on the site of the atherosclerotic plaque rupture on the coronary artery wall of the heart has been reported to result in the development of coronary artery disease (CAD). Numerous investigators have reported that male gender and aging increase the rate of occurrence of CAD in middle-aged male [2]. The mechanism of action of the male sex hormone in the increased incidences of CAD in male remains obscure. The ethical clearance was obtained from the International Review Board from the institute for the collection of blood from both normal male and female volunteers (M = 20, F = 20, n = 40, age group: 30–45 years). The basal concentrations of testosterone in the blood collected from male and female in the morning measured by enzyme-linked immunosorbent assay (ELISA) were found to be 21.4 4.90 nM and 1.45 0.45 nM, respectively, which is found to be in between the physiological levels (9.36–37.1 nM in males and 0.21–2.98 nM in females). All participating volunteers were asked to stop to take any medication including acetyl salicylic acid (aspirin) for at least 4 weeks before donating blood. No female volunteers had ever used any contraceptive medications. The collected blood was anticoagulated by adding sodium citrate (final concentration of 13 mM). The platelet-rich plasma (PRP) was prepared as described before [3]. Testosterone (17bhydroxy-3-oxo-4-androstene, ≥98% pure) was obtained from Sigma Aldrich Co., and was dissolved in minimal quantity of absolute ethanol and appropriately diluted as required in 0.9%NaCl. Thromboxane A2 (TXA2) was determined in its thromboxane B2 (TXB2) form by ELISA using TXB2 antibody obtained from Sigma Aldrich Co. ADP and flutamide (2-methyl-N-(4-nitro-3-[trifluoromethyl] phenyl)propanamide), a testosterone receptor blocker [7], were obtained from Sigma Aldrich Co., and were dissolved in 0.9% NaCl. In some of the experiments, PRP was pre-incubated with 10 lM flutamide for 60 min at 37 °C followed by incubation with 40 nM testosterone for 40 min at 37 °C. Platelet aggregation was carried out in an aggregometer as described before [3]. Nitric oxide (NO) was determined by methemoglobin method as described before [4,8]. The significance of the results was determined by Student’s t-test. The P < 0.05 was considered to be significant. Correlation coefficient (r) was determined by Pearson’s test. When the PRP, either from male or from female volunteers, was treated with 2.0 lM ADP and the aggregation of platelets was studied, it was found that in both the cases, the aggregation of platelets followed typical and similar ADP-induced primary and secondary aggregation profiles (Figure 1A, Lines c, d). However, when the PRP preparations were pre-incubated with 40 nM testosterone and the aggregation of platelets was studied by using ADP, it was found that only in the case of male PRP, preincubation of the preparation with testosterone resulted in the significant stimulation of ADP-induced platelet aggregation (Figure 1A, Line a, P < 0.05, n = 20). In contrast, the PRP from female subjects pre-incubated with similar amount of the hormone and treated with ADP failed to show any enhancement of the platelet aggregation as shown in the case of the male PRP. Indeed, the aggregation profile of the female PRP in the presence of both testosterone and ADP was similar to the aggregation induced by ADP alone (Figure 1A, Line b). At a fixed time (i.e., at 5 min), extent of platelet aggregation in male PRP is shown in Figure 1B (●). Our results as presented in the text demonstrated a significant role of testosterone, a major male sex hormone, in the ADP-induced platelet aggregation. Although ADP-induced platelet aggregation is reported to be more important than that by other platelet-aggregating agents in the formation of thrombus [5, 6], no difference in ADP-induced platelet aggregation could be seen in male PRP or in the female PRP (Figure 1A, Lines c, d). Pre-incubation of the male PRP with 10 lM flutamide nullified the testosterone-induced amplification of the platelet aggregation induced by ADP (Figure 1A, Line e). Ethanol appropriately diluted with 0.9% NaCl (vehicle) similar to that in the testosterone