Is Increased Platelet Aggregation Is a Risk Factor for Cardiovascular Disease in Women With Idiopathic Central Precocious Puberty

Background Early menarche in girls is associated with an increased risk of cardiovascular events later in life, but the role of platelets in this risk has not been investigated during puberty. Here, we evaluated the effects of idiopathic central precocious puberty (ICPP) on platelet aggregation in platelet-rich plasma samples from female patients. Methods The study included 40 girls diagnosed with ICPP between February 2012 and June 2016, and a control group consisting of 30 healthy females. Adenosine diphosphate (ADP) and collagen-induced platelet aggregation were studied with photometric aggregometry. There was no difference in the platelet count or volume between girls with ICPP and the control group. In addition, the ADP-induced maximum aggregation time, value, and slope did not signicantly differ between the study and control groups (p > 0.05). However, the collagen-induced maximum aggregation time, value, and slope were signicantly higher in the study group (p < 0.001).


Introduction
The whole blood count, ferritin level, prothrombin time (PTZ), activated partial thromboplastin time (aPTT), and brinogen and platelet aggregation were analyzed in both groups. Erythrocyte indices, and the platelet count and mean platelet volume (MPV), were obtained using an automatic device (Technicon H-1 System; Technicon Co, Tournai, Belgium) Blood samples taken from the antecubital vein were collected into plastic syringes containing 1/10 volume 3.8% trisodium citrate. Platelet-rich and platelet-poor plasma were prepared by centrifugation (21). Platelet aggregation was assessed by photometric aggregometry using a whole blood aggregometer (Model 560; Chrono-Log Corporation, Havertown, PA, USA).
Collagen (5 µg/mL, Chrono Par No: 385; Chrono-Log Corporation) and ADP (10 µmol, Chrono Par No: 384; Chrono-Log Corporation) were used as agonists. The maximum aggregation time (s), value (%) and slope (%/min) were determined from the aggregation curves. The effects of ADP and collagen on aggregation were evaluated in both the control and study groups considering the effect of iron de ciency on aggregation (22,23).

Statistical Analysis
Statistical analysis was performed with SPSS software (ver. 22; SPSS Inc., Chicago, IL, USA). Data are provided as mean, standard deviation, median, lowest, highest and percentage values. Normally distributed data were compared using the t-test for independent samples and Mann-Whitney U test in all other cases. Differences were considered statistically signi cant at p <0.05.

Results
Forty female patients diagnosed with ICPP were included in the study group (Group 1) and thirty healthy females comprised the control group (Group 2). The demographic characteristics of Groups 1 and 2 are given in Table 1. There were no signi cant differences between the groups in age, height, weight, or BMI (p > 0.05).
The whole blood parameters of Groups 1 and 2 are given in Table 2. There were no signi cant differences between the groups in terms of the white blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) level, hematocrit (HCT) level, mean erythrocyte volume (MCV), mean erythrocyte hemoglobin (MCH) level, mean erythrocyte hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet (PLT) count, or MPV (p > 0.05).
There was no signi cant difference between Groups 1 and 2 in the plasma ferritin level, PTZ, aPTT, or brinogen level (p > 0.05; Table 3).
The mean maximum aggregation time, value and slope induced by 10 µmol ADP and 5 µg/mL collagen in Groups 1 and 2 are shown in Table 4. In the study group, at 10 µmol ADP, the mean maximum aggregation time, value, and slope did not signi cantly differ from control group values (p > 0.05). However, in the study group, at a collagen concentration of 5 µg/mL, the mean maximum aggregation time, value, and slope were signi cantly higher than in the control group (p = 0.001, 0.002 and 0.04, respectively).

Discussion
The onset of puberty before the age of 8 years in girls and 9 years in boys is considered to be PP, and is more common in girls. The vast majority of cases are ICPP (2). The loss of effectiveness of central systems suppressing neurons that secrete gonadotropin-releasing hormone (GnRH), and activity of systems that stimulate the release of LH and FSH in response to pulsatile release of GnRH, results in ICPP (24). Stimulation of the gonads by LH and FSH increases circulating levels of sex steroid hormones (especially E2 in girls) (4). Although sexual maturity has been shown to affect platelet aggregation in pigs, no published study has shown the effect of puberty and/or ICPP on platelet aggregation in humans (25).
In this study, platelet aggregation stimulated by ADP was not different between the study and control groups, whose demographic characteristics, whole blood parameters, ferritin levels, PTZ, aPTT and brinogen levels were not signi cantly different. However, the maximum aggregation time, value, and slope for collagen-induced platelet aggregation were signi cantly higher in the study group. Collagen is a strong agonist, but ADP is only a weak one (26). In our study, ADP was used at a concentration of 10 µmol, which is su cient for platelet aggregation (26). We did not detect any change in aggregation at this level of ADP, but there was an increase in collagen, suggesting selectivity for collagen-induced platelet activation pathways. Similar to our ndings, Leng et al. (27) achieved an increase in collagen-induced, but not thrombin-induced, platelet aggregation by giving different estrogen derivatives to ovariectomized mice, which was done to examine the effects of estrogens on arterial thrombosis. The authors suggested that the in vivo effects of estrogen on platelet function may be agonist-speci c. In the same study, collagen induced glycoprotein-VI, a platelet surface glycoprotein, which initiated adhesion followed by aggregation. Collagen, unlike ADP, also stimulates the release of thromboxane A2, a strong aggregation agent (28). In our study, glycoprotein-VI and/or thromboxane A2 may explain why there was no change in platelet aggregation with ADP but an increase with collagen.
Early menarche has been associated with an increased risk of coronary heart disease in later life (6-9). Canoy et al. retrospectively examined over 1 million middle-aged women and compared girls with early menarche (aged ≤ 10 years) and late menarche (≥ 17 years) girls to girls with normal menarche (13 years) (10). They showed that early and late menarche increased the risk of coronary heart disease, whereas the risk of cerebrovascular disease, hypertension, and diabetes mellitus was signi cantly lower. In this study, impaired glucose homeostasis and hypertension may partly explain the associations among early menarche, coronary heart disease, and cerebrovascular disease. Although the increased risk of coronary heart disease in women with early menarche is partially explained by the excess adipose tissue present in these women in adulthood, Canoy et al. did not report an increased incidence of obesity in adult women with coronary heart disease after early puberty, unlike other studies (11,29,30). To explain this association, Hardy et al. investigated cardiovascular structure and function (carotid intima-media thickness, pulse wave velocity, and left ventricular structure and function) in older women (60-64 years) with early menarche, but did not show any relationship with early menarche (31). It is thought that a process beginning with childhood obesity may ultimately lead to cardiovascular diseases in later life, in association with early puberty and adult obesity (29,(32)(33)(34). However, no study has investigated the role of platelet aggregation in the increased risk of cardiovascular disease in later life, either in girls or older women with early menarche.
Estrogens, especially E2, exert effects on tissues through their receptors. Platelets and their bone marrow precursors, megakaryocytes, also express estrogen receptors (35)(36)(37). Although many factors affect platelet function, the effects of hormones on platelet activity are poorly de ned (35). Con icting results have been reported regarding the effects of estrogen administration on platelet activation in mice and women (38). There have been few studies on the role of E2 in megakaryopoiesis and platelet production. In a study by Bord et al. (39), a higher number of medullary megakaryocytes was reported, without any change in the total bone marrow cell count, after long-term oral or transdermal estrogen hormone. In another study of post-menopausal women (40), a slight increase in platelet number and volume was found with estrogen treatment. In the study by Valéra et al. (41), the platelet count in mice was not signi cantly affected by chronic subcutaneous administration of E2 compared to ovariectomized mice. In a study by Miller et al. (42), in postmenopausal women, the number of platelets did not signi cantly differ from baseline after 48 months of E2 treatment. Similarly, Kaplan et al. (43) reported no change in the platelet count of a small group of women after 3 months of E2 treatment. However, another study reported a signi cant decrease in platelet count after 3 months of transdermal treatment of E2 (44). In our study, we found no difference between the study and control groups in terms of platelet number and volume.
The risk of thromboembolic events increases with oral E2 treatments in women (45). In an animal study by Rosenblum et al. (46), E2-treated rats showed more rapid aggregation after mesenteric endothelial damage compared to placebo. The reason for this has not been fully explained, but there are some putative mechanisms. Miller et al. (42) reported that estrogen increased the level of hepatic-derived coagulation factors. Thijs et al. (47) showed that E2 hormone therapy in postmenopausal women increased the levels of P-selectin and glycoprotein 53, which are associated with increased platelet activation and platelet degradation. Rank et al. (48) reported higher levels of platelet-derived microparticles in postmenopausal women receiving hormone replacement therapy, indicating platelet activation rather than endothelium-derived microparticles. Garcia-Martinez et al. (49) reported that E2 may cause an increase in P-selectin or calcium in platelets, leading to platelet activation and, ultimately, thromboembolism. Another proposed mechanism is increased activated protein C resistance in women taking E2 (50, 51).
By contrast, there are studies suggesting that E2 decreases platelet aggregation. Valéra et al. (52) reported that washed platelets isolated from E2-treated mice showed reduced aggregation after stimulation by thrombin and collagen. In the same study, the expression of various platelet proteins, including β1-tubulin, which is the main component of microtubules that modulate platelet production and function, was reduced with E2 treatment. Geng et al. (53) found a decrease in collagen-induced platelet aggregation after E2 administration in ovariectomized mice, and attributed this to decreased platelet and megakaryocyte glycoprotein-VI expression after E2 administration. In platelet aggregation studies performed in both sexually matured and juvenile pigs, Jayachandran et al. (25) found that ADP and collagen-induced platelet aggregation decreased in mature female pigs and increased in male mature pigs, and that sexual maturity and platelet aggregation changed with maturity. In in vitro aggregation studies using different concentrations of E2 and thrombin and ADP agonists, Nakano et al. (54) found that these agonists reduced platelet aggregation, possibly due to the reduction of calcium associated with the increased production of cyclic guanosine monophosphate (which is dependent on NO). Similarly, Bar et al. (55) showed that, in postmenopausal women, a signi cant decrease in adrenaline-induced platelet aggregation and adenosine triphosphate release occurred due to E2 intake. These studies show that megakaryocytes and platelets are important targets of the prothrombotic or antithrombotic effects of estrogens.
Contrary to these studies, E2 was not found to have any effect on platelet aggregation in other studies performed using different agonists, in which various estrogen derivatives were given to postmenopausal women. (42,43,56,57).
It is clear that there are many con icting results regarding the relationship between E2 and platelet aggregation. In this study, we showed that collagen-induced platelet aggregation was increased in girls with ICPP, which may be due to increased E2 levels. As PP is associated with a higher risk of cardiovascular events later in life, early treatment of ICPP may be bene cial. However, our sample size was relatively small, and further studies with larger number of patients are needed. Con ict of interest: The authors declare that they have no con icts of interest.

Abbreviations
Informed consent: Informed consent was obtained from all individual participants included in the study.
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