Thrombosis Research 139 (2016) 50–55
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RhoA/ROCK signaling contributes to sex differences in the activation of human platelets Peter Schubert a,b,c, Danielle Coupland a,b, Marie Nombalais b, Geraldine M. Walsh a,b, Dana V. Devine a,b,c,⁎ a b c
Centre for Innovation, Canadian Blood Services, Vancouver, BC, Canada Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada
a r t i c l e
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Article history: Received 23 September 2015 Received in revised form 18 December 2015 Accepted 10 January 2016 Available online 12 January 2016 Keywords: Sex differences Blood Platelets Signal transduction GTPase
a b s t r a c t Studies of sex-dependent differences in platelet aggregation and glycoprotein (GP)IIb/IIIa activation have demonstrated that platelets from females are more sensitive to agonists than those from males. To date, there is little understanding of these differences at a molecular level. Here, sex differences in reactivity of platelets from 86 women and 86 men were investigated. Platelet degranulation (CD62P expression) and activation of GPIIb/IIIa (PAC-1 binding), with and without ADP, were assessed. Extent of shape change (ESC) in response to ADP was measured. Basal CD62P and PAC-1 expression did not differ between the sexes. In response to ADP activation, mean PAC-1 binding in platelets from female donors was 17.9 ± 3.5% vs. 14.0 ± 4.1% in platelets from male donors, and ESC was significantly greater in platelets from females (p b 0.05). Evaluation of basal expression of signaling molecules along the ADP receptor pathway leading to GPIIb/IIIa activation and subsequent RhoA/ROCK signaling via GPIIb/IIIa ‘outside-in’ signaling showed that platelets from females produce 3-fold greater levels of phosphorylated protein kinase C (PKC) substrates. There was a 2.5-fold greater level of activated RhoA, and platelet sub-fractionation analysis demonstrated 2.7-fold more RhoA in the membrane fraction of female vs. male platelets. Similarly, there was a 2.8-fold increase in levels of phosphorylated myosin light chain (MLC) in platelets from females vs. males. The increased signaling activity in platelets from females mirrors their greater sensitivity to agonists. These findings further our understanding of the molecular differences between platelets from males and females. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction A large body of epidemiologic data supports the presence of sex differences in morbidity and mortality associated with cardiovascular diseases [1–6]. Other studies reported sex differences in the regulation of body weight [7], insulin resistance, body composition and energy balance [8], in lipid metabolism and the effect of obesity [9] as well as in vascular function [10]. Furthermore, women generally have better cardiac function and survival than men do, suggesting a sex-based cardiac physiology [11]. Treatment of these diseases can be influenced or hampered by sex-specific differences in signal transduction as demonstrated by pharmacokinetic studies showing that females possess a higher risk of clinically relevant adverse drug reactions [12]. Although sex differences in the endocrine and immune systems probably contribute, studies suggest that sex-specific genetic architecture also influences human phenotypes, including physiological and disease traits [13]. ⁎ Corresponding author at: Canadian Blood Services, UBC Centre for Blood Research, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. E-mail address: [email protected] (D.V. Devine).
http://dx.doi.org/10.1016/j.thromres.2016.01.007 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
In 1975, Johnson et al. showed a sex difference in platelet reactivity of rats and guinea pigs with platelets from males being significantly more sensitive to aggregation stimuli than those from females [14]. In contrast to this initial report, however, the data of subsequent years have supported higher platelet reactivity in woman vs. men. Results from animal models and clinical studies suggest that female platelets in general are more prone to trigger and participate in the thrombotic process than male platelets. The observation that women have a higher platelet count compared to men [15], however, seems not to be responsible for higher platelet reactivity in women [16]. Furthermore, collagen- and adenosine diphosphate (ADP)-induced platelet aggregation is stronger in woman than in men [17], a difference that persists after administration of aspirin, and might in part explain the greater therapeutic benefit of aspirin for men. In an investigation of platelet function before and after administration of aspirin in humans, males had a higher collagen-induced release of thromboxane B2, equally persistent platelet reactivity and greater thrombotic tendency [18]. Aspirin pretreatment, however, resulted in a significantly higher inhibition of platelet aggregation in males than in females, a finding confirmed in other studies [19,20]. Aspirin inhibited collagen-induced platelet aggregation in both sexes, but spontaneous aggregation was
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only inhibited by aspirin in males. A male/female mouse litter-mates model showed that female platelets bound more fibrinogen in response to low concentrations of thrombin and collagen-related peptide. They also demonstrated higher levels of platelet aggregation in female than in male platelets. These differences were independent of platelet size and surface expression of glycoproteins GPIIb/IIIa and GPIb-IX-V and were not blocked by aspirin [21]. Subsequently, numerous investigations confirmed that females exhibit increased platelet reactivity resulting in higher platelet aggregation levels [22]. These earlier studies on sex-specific aspects of human blood platelets focused on differences in the response to agonists [23], and lacked investigations to unravel molecular mechanisms leading to sex-specific differences in platelet activity and activation. In this study, signaling mechanisms were investigated to explore one potential molecular mechanism contributing to the difference in basal activity between female and male human platelets.
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optical density reading from the platelet-poor plasma and the platelet sample with 10 μM ADP treatment with stirring. 2.5. RhoA and Rap1 activation assays The assays were carried out using kit systems according to the manufacturer's instructions (Thermo Scientific, Rockford, IL), Briefly, platelet lysates were incubated with a pull-down protein consisting of glutathione-S-transferase (GST) fused to the GTPase binding domain of the respective downstream effector protein, Rhotekin for RhoA and RalGDS for Rap1. Active, GTP-bound GTPase was purified via the specific interaction to the effector domain using glutathione resin. Unbound lysate proteins, including inactive or GDP-bound GTPase, were removed using the spin columns. The active GTPase population was recovered from the glutathione resin using SDS-PAGE loading buffer (10% v/v glycerol, 62.5 mM Tris HCl pH 6.8, 2% w/w SDS, 0.01 mg/mL bromophenol blue, 5% v/v β-mercaptoethanol) and analyzed by western blot.
2. Materials and methods 2.6. Membrane enrichment/platelet crude fractionation 2.1. Ethical approval and blood donation This study was approved by the research ethics board of Canadian Blood Services and informed consent was obtained from healthy volunteers prior to whole blood donation. Donors underwent standard blood donation questionnaire procedures and a brief health check which includes temperature and blood pressure measurement in order to exclude volunteers who had symptoms of illness, blood pressure issues, were on medication especially aspirin or were on a dietary regime. In this study 86 female and 86 male donors were included. All female donors participating in this study were not pregnant. Whole blood samples drawn into vacutainers supplemented with citrate as anticoagulant were taken from the pouch during the whole blood donation procedure which was conducted at the Canadian Blood Services netCAD facility (Vancouver, Canada). 2.2. Preparation of platelets from whole blood and platelet lysis Platelet-rich-plasma (PRP) was obtained by spinning the whole blood collected in citrate vacutainers at 160 ×g for 15 min and the top layer was carefully removed. For flow and extent of shape change (ESC) experiments, PRP was used immediately. For protein analyses, PRP from randomly picked donors was spun again at 200 × g for 10 min and washed with CGS buffer (10 mM sodium citrate, 30 mM glucose, 120 mM NaCl, pH 6.5) prior to lysis in PBS-buffered 1% Triton X-100 supplemented with protease and phosphatase inhibitors (Roche, Mississauga, ON, Canada). 2.3. Flow cytometry assay All flow experiments used single antibody staining as described before [24]. PRP samples were stimulated with 10 μM ADP (or PBS for the untreated control sample) for 15 min prior to staining. Conjugated antibodies CD62P-PE (Beckman-Coulter, Mississauga, ON, Canada) or PAC 1-FITC (BD Biosciences, San Jose, CA) were added and incubated for 15 min followed by fixation with formal saline and immediate sample analyses carried out on a FACSCanto II (BD Biosciences, San Jose, CA). Isotype controls were gated at 0.2% to calculate percent positive values. 2.4. Extent of shape change ESC assays were conducted in a SPA2000 aggregometer (Chronolog Corp., Havertown, PA) based on light scattering assessment. Briefly, platelet concentrations were adjusted to 300 × 109 platelets/L with platelet-poor plasma obtained by centrifugation of platelet concentrate at 2060 ×g for 15 min. The instrument calculated ESC by integrating the
Crude fractionation of platelet lysates was carried out by differential centrifugation at 4 °C as described before [25]. Briefly, the low-spin pellet (LSP) containing a fraction enriched in the actin cytoskeleton was obtained from a spin at 10,000 ×g for 10 min. The supernatant of this first spin is subjected to a 100,000 × g spin for 3 h resulting in the high-spin pellet (HSP) representing the fractions enriched in the membrane-skeleton and the high-spin supernatant (HSN) comprising most of the cytosolic proteins. 2.7. SDS-PAGE and western blot analysis/densitometry Platelet lysates or supernatant after pull-down experiments were separated on a SDS-PAGE gel and blotted onto nitrocellulose membranes (Bio-Rad, Mississauga, ON, Canada). The membrane was probed with primary antibodies against Rap1 and RhoA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MLC, phosphorylated MLC, cofilin, phosphorylated cofilin and phosphorylated PKC-substrates (Cell Signaling, Danvers, MA). β-Actin (Sigma-Aldrich, St. Louis, MO, USA), followed by their respective secondary antibodies (Licor, Lincoln, NE). Bands were analyzed by the LICOR system and protein band intensities were determined by densitometry using the LICOR software ODYSSEE. Protein band intensities were normalized to the respective band intensity of the actin loading controls. 2.8. Statistics Continuous variables were expressed as mean ± SD. All distributions were checked for normality. Differences in continuous variables were compared by independent Student's t-test (Excel, version 2013, Microsoft, Redmond, WA) or Mann–Whitney U test (Minitab, Inc., State College, PA), as appropriate. For the ADP titration, two-way ANOVA was used (Minitab, Inc., State College, PA). A p-value b 0.05 was considered significant. 3. Results 3.1. Assessment of degranulation, GPIIb/IIIa activation and extent of shape change of male and female platelets Platelets from female (n = 86) and male (n = 86) donors were analyzed for the level of platelet degranulation by CD62P binding to surface-exposed p-selectin (Fig. 1A, -ADP), GPIIb/IIIa activation via PAC-1 antibody binding (Fig. 1B, -ADP) and extent of shape change (ESC, Fig. 1C). Mean basal platelet activation (± standard deviation) by p-selectin expression was 10.6 ± 4.3% and 13.0 ± 5.9% for female and male platelets, respectively. Mean basal GPIIb/IIIa activation was
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Fig. 1. Sex-dependent (F: female; M: male) platelet activation in unstimulated platelets vs. platelets treated with the agonist ADP. (A) Degranulation monitored by CD62P expression in unstimulated female (♦) and male (+) platelets, and in response to ADP stimulation of female (▲) and male (×) samples is shown. (B) GPIIb/IIIa activation assessed by PAC-1 binding to unstimulated female (♦) and male (+) platelets, and in response to ADP stimulation of female (▲) and male (×) platelets. (C) Agonist-induced responsiveness as determined by extent of shape change (ESC) of female (♦) and male (+) platelets. n = 86 for each sex. *p b 0.05.
5.1 ± 2.5% and 4.8 ± 2.4% for female and male platelets, respectively, and was not significantly different between the sexes. ADP-induced platelet degranulation determined by CD62P expression was 58.1 ± 6.1% vs. 53.1 ± 6.5% on female vs. male platelets, respectively, exhibiting no difference between the sexes. However, ADP-induced PAC-1 expression was significantly higher (p b 0.05) in female platelets showing 17.9 ± 3.5% vs. 14.0 ± 4.1% for male platelets. Female platelets exhibited a significantly higher ability to change shape in response to 10 μM ADP (Fig. 1C) with 22.0 ± 3.5% vs. 17.9 ± 3.5% for male platelets. 3.2. Sex disparity in response to ADP is not age-dependent In many studies, sex disparity is explained by the age profile of participants. In order to assess the impact of the age on the parameters collected in this study, correlations were examined between donor age and both basal CD62P and PAC-1 expression and levels of these markers in response to ADP in both male and female platelets. For females, donor age ranged between 20 and 69 and for males, age ranged between 18 and 68. No correlation was found between donor age and either basal or activated levels of CD62P or PAC-1 binding (data not shown).
activated isoforms, Rap1 also showed no sex-specific difference; however, an almost 2.5-fold, significantly higher amount of RhoA seems to be activated in female compared to male platelets (Fig. 3). Activated RhoA has been shown to be localized mainly at the membrane. Crude fractionation analyses revealed that about 2.7-fold more RhoA was localized in the membrane (fraction, HSP) of female platelets which is significantly higher than the male platelets (Fig. 3). 3.5. RhoA/ROCK signaling analyses RhoA is activated in platelets via outside-in signaling of GPIIb/IIIa. Activated RhoA subsequently activates the kinase ROCK which phosphorylates a variety of substrate proteins including myosin and myosin light chain kinase (MLCK) or leads to cofilin phosphorylation. Myosin
3.3. Sex-specific changes in activation of PKC substrates PKC, which is downstream of the ADP family P2Y receptor, phosphorylates a variety of substrate proteins and some of them can be detected at the same time with a single antibody. The PKC substrate phosphorylation profile was analyzed in both female and male platelets and a representative western blot and relative intensity analysis are shown in Fig. 2. Female platelets exhibit a ~3-fold, significantly higher activation status of the selected PKC substrate proteins compared the male platelets. 3.4. GTPases exhibit sex-specific activation Both CD62P and GPIIb/IIIa activation assays revealed a different response to ADP between females and males. Therefore signaling pathways leading to degranulation and GPIIb/IIIa activation were investigated to determine if the differences seen between male and female platelets are reflected in these signaling pathways. GTPases are molecular switches in many pathways leading to platelet activation. RhoA plays a pivotal role in both degranulation and GPIIb/IIIa outside-in signaling and Rap1 is known to activate GPIIb/IIIa. The expression profile of these GTPases was analyzed revealing no significant differences between female and male platelets for both GTPases (Fig. 3). For the
Fig. 2. Representative western blot of sex-specific profiles of activated protein kinase C (PKC) substrates in resting platelet samples. Bar graph of relative intensity was calculated from n = 16 western blots for each sex. *p b 0.05.
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Fig. 3. Using the same donors, representative western blots from resting platelet samples of sex-specific profiles of Rap1 (A), activated Rap1 (B), RhoA (C), activated RhoA (D) in platelet lysates and RhoA in the membrane-cytoskeletal fraction (E, high speed pellet, HSP). Bar graphs of relative intensity were calculated from n = 16 western blots for each sex. *p b 0.05.
light chain (MLC) exhibited no significant difference in expression level between female and male platelets; however, female platelets showed a 2.8-fold, significantly higher phosphorylation level in female compared to male platelets (Fig. 4A, B). Analysis of cofilin expression and its phosphorylation status revealed no sex-specific difference (Fig. 4C, D). 3.6. Agonist sensitivity to GPIIb/IIIa activation The sensitivity of ADP to the activation status of GPIIb/IIIa complex was analyzed in a titration experiment for both female and male
platelets (Fig. 5). Female platelets showed a significantly higher sensitivity than male platelets. 4. Discussion Earlier studies have shown higher reactivity/responsiveness of female vs. male human platelets in aggregation and GPIIb/IIIa activation [23]. However, these studies lacked data without agonist stimulation in order to assess whether this difference arises from sex-specific differences in basal levels in these assays or are due to different sensitivities
Fig. 4. Using the same donors as in Fig. 3, representative western blots from resting platelet samples of sex-specific profiles of myosin light chain (A, MLC), phosphorylated MLC (B), cofilin (C) and phosphorylated cofilin (D). Bar graphs of relative intensity were calculated from n = 16 from each sex. *p b 0.05.
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Fig. 5. Titration of ADP and activation levels of GPIIb/IIIa by PAC-1 binding in female (straight line) and male (dotted line) platelets. SD represent n = 10 for each sex, *p b 0.05.
to agonists. In this study, platelet activation determined by the expression of P-selectin on the platelet surface and the activation status of the fibrinogen receptor with and without ADP agonist stimulation was determined. Additionally, the ability of the platelet to change shape in response to ADP was assessed. No differences in basal platelet activation and fibrinogen receptor activity could be detected. However, a significant difference in responsiveness in female vs. male platelets in GPIIb/IIIa activation in response to ADP was observed. Lastly, the extent of shape change assay also revealed a significantly stronger response by female platelets. Donors for this study were chosen based on stringent criteria in order not to jeopardize any conclusions. Exclusion criteria were any kind of medications known to inhibit platelets, disease or illness, dietary plans or pregnancy for female donors. From the pool of samples collected in this study, a subset was randomly chosen for further analyses. The preparation of the platelet-rich plasma was carried out by the same person in order to avoid handling variations. Analyses of underlying molecular mechanisms were focused on the sex-specific difference in basal levels of signaling molecules involved in GPIIb/IIIa activation in the response to ADP. This agonist binds and activates P2Y receptors on the surface of platelets and triggers signaling cascades via G-proteins leading to GPIIb/IIIa activation and granule secretion [26]. Granule secretion is regulated by PLC-dependent PKC activation [27] and this study demonstrated that female platelets seem to exhibit a significantly higher activation of PKC substrates which would explain the difference in sex-specific ADP-stimulated degranulation. On the other hand, the classical pathway of integrin activation is the activation of the GTPase Rap1 in a PI3K-dependent manner paralleled with talin recruitment to the membrane into the integrin complex [28]. Therefore, Rap1 and activated Rap1 levels were determined in a subset of these donors, but no significant difference between female and male platelets could be detected. The above mentioned mechanism leading to integrin activation is inside-out signaling [29]; subsequent binding of fibrinogen in the plasma to the activated integrin complex initiates the complementary outside-in signaling leading among others to the activation of the GTPase RhoA [30]. Thus, RhoA and activated RhoA levels were determined in a subset of the study participants. A significant difference between female and male platelets in activated RhoA levels could be detected which are not due to differences in RhoA expression. The latter observation is in agreement with a genomic analysis demonstrating similar levels of RhoA expression in human female and male platelets [31]. RhoA is known to play a key role in granule secretion as well as outside-in signaling and results here confirm that ADP stimulation led to increased degranulation, GPIIb/IIIa activation and shape change. The increased level of activated RhoA in resting female vs. male platelets which seems to be primarily associated with the platelet membrane most likely stems from an enhanced GPIIb/IIIa outside-in signaling leading to increased phosphorylation of ROCK substrates such as MLC affecting actin rearrangement and subsequent shape change. This sex disparity, however, could not be observed for
the ADP-stimulated degranulation, Studies have shown that RhoA is required for efficient secretion of alpha and dense granules downstream of G [13] and G(q) proteins [32]. Stimulation of the P2Y receptor by ADP like in this study, however, seems to signaling via G(i) proteins [33] and might explain our observation. Furthermore, previous studies have revealed that female and male platelets have a similar number of GPIIb/IIIa molecules on the platelet surface [23]. Additionally, it could be shown that plasma from human blood does not contain significantly different levels of fibrinogen [34]. This result excludes potentially increased outside-in signaling to enhance RhoA activation. RhoA regulates the activity of the kinase ROCK which phosphorylates its substrates such as cofilin via Lim domain kinase (LIMK) or MLC which regulates actin de-/polymerization or contraction of actin fibers, respectively [35]. Only phosphorylated MLC showed a significant difference between female and male donors while the basal expression of cofilin showed no sex-specific difference. From these investigations, it was concluded that gender-specific differences in ESC are most likely due to increased phosphorylated MLC levels. Since more integrin molecules are activated on the surface of female compared to male platelets, but the fibrinogen levels are fairly similar in female and male plasma, this might explain a higher rate of outside-in singling leading to RhoA/ ROCK activation. Lastly, other studies suggested different sensitivity of platelets to agonists [21]. A titration of ADP monitoring the activation status of GPIIb/IIIa revealed a significantly higher sensitivity of female vs. male platelets. Taken together, the findings reported here suggest a model in which female platelets exhibit a higher sensitivity to ADP or an increased RhoA/ROCK signaling responsible for enhanced degranulation and integrin activation. Further analyses are necessary to verify these results and confirm the proposed signaling model. Only ADP was used as an agonist in this study. Although sex differences in thrombin- or TRAP-stimulated platelet and GPIIbIIIa activation have been shown [14,21–23], it would be interesting to test the effects of these agonists and others such as collagen, arachidonic acid or thromboxane A2 in the context of the signaling model proposed here. We postulate that sex differences in response to other agonists would be seen based on the cross-talk among many of the platelet agonist signaling pathways. Furthermore, the activation status of GPIIb/IIIa, determined in this study by PAC-1 binding, could be complemented by an analysis of the receptor's ability to bind fibrinogen. Additionally, plasma components such as lipids or hormones may influence the activation status or responsiveness to agonists affecting the RhoA/ROCK activity. It is known that hormones can trigger post-translational modifications of proteins such as palmitoylation which can modulate the protein function affecting protein trafficking and protein-protein interactions [36]. Trafficking and subsequent involvement in cellular signaling is mediated by this moiety determined by its interaction with the lipid rafts in the membrane [37]. Among others, membrane proteins such as P2Y receptors and their associated G-proteins as well as GTPases such as Ras and Rho proteins are found to be palmitoylated [38]. Therefore, these kinds of protein modifications triggered by effectors such as hormones can also play a role in the sex-dependent platelet response to agonists such as ADP seen in this study. Therefore, similar analysis of washed platelet samples could complement and expand our findings. Lastly, the differences in the GPIIb/IIIa activation may contribute to the disparity in the observed gender-dependent platelet activation. Inhibitors such as eptifibatide could be used to in female samples to mimic the levels of male samples. These adjustments would further fine-tune the read-out and conclusion and may assist in interpretation of clinical findings when sex-based differences occur.
Funding This research was supported financially by Canadian Blood Services, funded by the federal (Health Canada), provincial and territorial
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ministries of health. The views expressed herein do not necessarily represent the view of the federal government. Author contributions PS, GMW and DVD designed the study; PS, DC, MN and GMW performed research; PS and DC analyzed and interpreted the data; and PS, GMW and DVD wrote the manuscript. Conflicts of interest None. Acknowledgements The authors like to thank the donors who participated in this study and the Canadian Blood Services Development Laboratory (netCAD) for provision of whole blood samples, respectively. References [1] P. Appelros, B. Stegmayr, A. Terent, Sex differences in stroke epidemiology: a systematic review, Stroke 40 (4) (2009) 1082–1090. [2] D.A. De Silva, M. Ebinger, S.M. Davis, Gender issues in acute stroke thrombolysis, J. Clin. Neurosci. 16 (4) (2009) 501–504. [3] P. Gopalakrishnan, M.M. Ragland, T. Tak, Gender differences in coronary artery disease: review of diagnostic challenges and current treatment, Postgrad. Med. 121 (2) (2009) 60–68. [4] M.J. Reeves, C.D. Bushnell, G. Howard, et al., Sex differences in stroke: epidemiology, clinical presentation, medical care, and outcomes, Lancet Neurol. 7 (10) (2008) 915–926. [5] Y.Y. Tan, G.C. Gast, Y.T. van der Schouw, Gender differences in risk factors for coronary heart disease, Maturitas 65 (2) (2010) 149–160. [6] C.L. Verheugt, C.S. Uiterwaal, E.T. van der Velde, et al., Gender and outcome in adult congenital heart disease, Circulation 118 (1) (2008) 26–32. [7] H. Shi, D.J. Clegg, Sex differences in the regulation of body weight, Physiol. Behav. 97 (2) (2009) 199–204. [8] E.B. Geer, W. Shen, Gender differences in insulin resistance, body composition, and energy balance, Gend. Med. 6 (Suppl. 1) (2009) 60–75. [9] F. Magkos, B. Mittendorfer, Gender differences in lipid metabolism and the effect of obesity, Obstet. Gynecol. Clin. N. Am. 36 (2) (2009) 245–265 vii. [10] I.C. Villar, A.J. Hobbs, A. Ahluwalia, Sex differences in vascular function: implication of endothelium-derived hyperpolarizing factor, J. Endocrinol. 197 (3) (2008) 447–462. [11] E.D. Luczak, L.A. Leinwand, Sex-based cardiac physiology, Annu. Rev. Physiol. 71 (2009) 1–18. [12] J.M. Nicolas, P. Espie, M. Molimard, Gender and interindividual variability in pharmacokinetics, Drug Metab. Rev. 41 (3) (2009) 408–421. [13] C. Ober, D.A. Loisel, Y. Gilad, Sex-specific genetic architecture of human disease, Nat. Rev. Genet. 9 (12) (2008) 911–922. [14] M. Johnson, E. Ramey, P.W. Ramwell, Sex and age differences in human platelet aggregation, Nature 253 (5490) (1975) 355–357.
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Full Length Article
RhoA/ROCK signaling contributes to sex differences in the activation of human platelets Peter Schubert a,b,c, Danielle Coupland a,b, Marie Nombalais b, Geraldine M. Walsh a,b, Dana V. Devine a,b,c,⁎ a b c
Centre for Innovation, Canadian Blood Services, Vancouver, BC, Canada Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada
a r t i c l e
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Article history: Received 23 September 2015 Received in revised form 18 December 2015 Accepted 10 January 2016 Available online 12 January 2016 Keywords: Sex differences Blood Platelets Signal transduction GTPase
a b s t r a c t Studies of sex-dependent differences in platelet aggregation and glycoprotein (GP)IIb/IIIa activation have demonstrated that platelets from females are more sensitive to agonists than those from males. To date, there is little understanding of these differences at a molecular level. Here, sex differences in reactivity of platelets from 86 women and 86 men were investigated. Platelet degranulation (CD62P expression) and activation of GPIIb/IIIa (PAC-1 binding), with and without ADP, were assessed. Extent of shape change (ESC) in response to ADP was measured. Basal CD62P and PAC-1 expression did not differ between the sexes. In response to ADP activation, mean PAC-1 binding in platelets from female donors was 17.9 ± 3.5% vs. 14.0 ± 4.1% in platelets from male donors, and ESC was significantly greater in platelets from females (p b 0.05). Evaluation of basal expression of signaling molecules along the ADP receptor pathway leading to GPIIb/IIIa activation and subsequent RhoA/ROCK signaling via GPIIb/IIIa ‘outside-in’ signaling showed that platelets from females produce 3-fold greater levels of phosphorylated protein kinase C (PKC) substrates. There was a 2.5-fold greater level of activated RhoA, and platelet sub-fractionation analysis demonstrated 2.7-fold more RhoA in the membrane fraction of female vs. male platelets. Similarly, there was a 2.8-fold increase in levels of phosphorylated myosin light chain (MLC) in platelets from females vs. males. The increased signaling activity in platelets from females mirrors their greater sensitivity to agonists. These findings further our understanding of the molecular differences between platelets from males and females. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction A large body of epidemiologic data supports the presence of sex differences in morbidity and mortality associated with cardiovascular diseases [1–6]. Other studies reported sex differences in the regulation of body weight [7], insulin resistance, body composition and energy balance [8], in lipid metabolism and the effect of obesity [9] as well as in vascular function [10]. Furthermore, women generally have better cardiac function and survival than men do, suggesting a sex-based cardiac physiology [11]. Treatment of these diseases can be influenced or hampered by sex-specific differences in signal transduction as demonstrated by pharmacokinetic studies showing that females possess a higher risk of clinically relevant adverse drug reactions [12]. Although sex differences in the endocrine and immune systems probably contribute, studies suggest that sex-specific genetic architecture also influences human phenotypes, including physiological and disease traits [13]. ⁎ Corresponding author at: Canadian Blood Services, UBC Centre for Blood Research, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. E-mail address: [email protected] (D.V. Devine).
http://dx.doi.org/10.1016/j.thromres.2016.01.007 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
In 1975, Johnson et al. showed a sex difference in platelet reactivity of rats and guinea pigs with platelets from males being significantly more sensitive to aggregation stimuli than those from females [14]. In contrast to this initial report, however, the data of subsequent years have supported higher platelet reactivity in woman vs. men. Results from animal models and clinical studies suggest that female platelets in general are more prone to trigger and participate in the thrombotic process than male platelets. The observation that women have a higher platelet count compared to men [15], however, seems not to be responsible for higher platelet reactivity in women [16]. Furthermore, collagen- and adenosine diphosphate (ADP)-induced platelet aggregation is stronger in woman than in men [17], a difference that persists after administration of aspirin, and might in part explain the greater therapeutic benefit of aspirin for men. In an investigation of platelet function before and after administration of aspirin in humans, males had a higher collagen-induced release of thromboxane B2, equally persistent platelet reactivity and greater thrombotic tendency [18]. Aspirin pretreatment, however, resulted in a significantly higher inhibition of platelet aggregation in males than in females, a finding confirmed in other studies [19,20]. Aspirin inhibited collagen-induced platelet aggregation in both sexes, but spontaneous aggregation was
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only inhibited by aspirin in males. A male/female mouse litter-mates model showed that female platelets bound more fibrinogen in response to low concentrations of thrombin and collagen-related peptide. They also demonstrated higher levels of platelet aggregation in female than in male platelets. These differences were independent of platelet size and surface expression of glycoproteins GPIIb/IIIa and GPIb-IX-V and were not blocked by aspirin [21]. Subsequently, numerous investigations confirmed that females exhibit increased platelet reactivity resulting in higher platelet aggregation levels [22]. These earlier studies on sex-specific aspects of human blood platelets focused on differences in the response to agonists [23], and lacked investigations to unravel molecular mechanisms leading to sex-specific differences in platelet activity and activation. In this study, signaling mechanisms were investigated to explore one potential molecular mechanism contributing to the difference in basal activity between female and male human platelets.
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optical density reading from the platelet-poor plasma and the platelet sample with 10 μM ADP treatment with stirring. 2.5. RhoA and Rap1 activation assays The assays were carried out using kit systems according to the manufacturer's instructions (Thermo Scientific, Rockford, IL), Briefly, platelet lysates were incubated with a pull-down protein consisting of glutathione-S-transferase (GST) fused to the GTPase binding domain of the respective downstream effector protein, Rhotekin for RhoA and RalGDS for Rap1. Active, GTP-bound GTPase was purified via the specific interaction to the effector domain using glutathione resin. Unbound lysate proteins, including inactive or GDP-bound GTPase, were removed using the spin columns. The active GTPase population was recovered from the glutathione resin using SDS-PAGE loading buffer (10% v/v glycerol, 62.5 mM Tris HCl pH 6.8, 2% w/w SDS, 0.01 mg/mL bromophenol blue, 5% v/v β-mercaptoethanol) and analyzed by western blot.
2. Materials and methods 2.6. Membrane enrichment/platelet crude fractionation 2.1. Ethical approval and blood donation This study was approved by the research ethics board of Canadian Blood Services and informed consent was obtained from healthy volunteers prior to whole blood donation. Donors underwent standard blood donation questionnaire procedures and a brief health check which includes temperature and blood pressure measurement in order to exclude volunteers who had symptoms of illness, blood pressure issues, were on medication especially aspirin or were on a dietary regime. In this study 86 female and 86 male donors were included. All female donors participating in this study were not pregnant. Whole blood samples drawn into vacutainers supplemented with citrate as anticoagulant were taken from the pouch during the whole blood donation procedure which was conducted at the Canadian Blood Services netCAD facility (Vancouver, Canada). 2.2. Preparation of platelets from whole blood and platelet lysis Platelet-rich-plasma (PRP) was obtained by spinning the whole blood collected in citrate vacutainers at 160 ×g for 15 min and the top layer was carefully removed. For flow and extent of shape change (ESC) experiments, PRP was used immediately. For protein analyses, PRP from randomly picked donors was spun again at 200 × g for 10 min and washed with CGS buffer (10 mM sodium citrate, 30 mM glucose, 120 mM NaCl, pH 6.5) prior to lysis in PBS-buffered 1% Triton X-100 supplemented with protease and phosphatase inhibitors (Roche, Mississauga, ON, Canada). 2.3. Flow cytometry assay All flow experiments used single antibody staining as described before [24]. PRP samples were stimulated with 10 μM ADP (or PBS for the untreated control sample) for 15 min prior to staining. Conjugated antibodies CD62P-PE (Beckman-Coulter, Mississauga, ON, Canada) or PAC 1-FITC (BD Biosciences, San Jose, CA) were added and incubated for 15 min followed by fixation with formal saline and immediate sample analyses carried out on a FACSCanto II (BD Biosciences, San Jose, CA). Isotype controls were gated at 0.2% to calculate percent positive values. 2.4. Extent of shape change ESC assays were conducted in a SPA2000 aggregometer (Chronolog Corp., Havertown, PA) based on light scattering assessment. Briefly, platelet concentrations were adjusted to 300 × 109 platelets/L with platelet-poor plasma obtained by centrifugation of platelet concentrate at 2060 ×g for 15 min. The instrument calculated ESC by integrating the
Crude fractionation of platelet lysates was carried out by differential centrifugation at 4 °C as described before [25]. Briefly, the low-spin pellet (LSP) containing a fraction enriched in the actin cytoskeleton was obtained from a spin at 10,000 ×g for 10 min. The supernatant of this first spin is subjected to a 100,000 × g spin for 3 h resulting in the high-spin pellet (HSP) representing the fractions enriched in the membrane-skeleton and the high-spin supernatant (HSN) comprising most of the cytosolic proteins. 2.7. SDS-PAGE and western blot analysis/densitometry Platelet lysates or supernatant after pull-down experiments were separated on a SDS-PAGE gel and blotted onto nitrocellulose membranes (Bio-Rad, Mississauga, ON, Canada). The membrane was probed with primary antibodies against Rap1 and RhoA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), MLC, phosphorylated MLC, cofilin, phosphorylated cofilin and phosphorylated PKC-substrates (Cell Signaling, Danvers, MA). β-Actin (Sigma-Aldrich, St. Louis, MO, USA), followed by their respective secondary antibodies (Licor, Lincoln, NE). Bands were analyzed by the LICOR system and protein band intensities were determined by densitometry using the LICOR software ODYSSEE. Protein band intensities were normalized to the respective band intensity of the actin loading controls. 2.8. Statistics Continuous variables were expressed as mean ± SD. All distributions were checked for normality. Differences in continuous variables were compared by independent Student's t-test (Excel, version 2013, Microsoft, Redmond, WA) or Mann–Whitney U test (Minitab, Inc., State College, PA), as appropriate. For the ADP titration, two-way ANOVA was used (Minitab, Inc., State College, PA). A p-value b 0.05 was considered significant. 3. Results 3.1. Assessment of degranulation, GPIIb/IIIa activation and extent of shape change of male and female platelets Platelets from female (n = 86) and male (n = 86) donors were analyzed for the level of platelet degranulation by CD62P binding to surface-exposed p-selectin (Fig. 1A, -ADP), GPIIb/IIIa activation via PAC-1 antibody binding (Fig. 1B, -ADP) and extent of shape change (ESC, Fig. 1C). Mean basal platelet activation (± standard deviation) by p-selectin expression was 10.6 ± 4.3% and 13.0 ± 5.9% for female and male platelets, respectively. Mean basal GPIIb/IIIa activation was
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Fig. 1. Sex-dependent (F: female; M: male) platelet activation in unstimulated platelets vs. platelets treated with the agonist ADP. (A) Degranulation monitored by CD62P expression in unstimulated female (♦) and male (+) platelets, and in response to ADP stimulation of female (▲) and male (×) samples is shown. (B) GPIIb/IIIa activation assessed by PAC-1 binding to unstimulated female (♦) and male (+) platelets, and in response to ADP stimulation of female (▲) and male (×) platelets. (C) Agonist-induced responsiveness as determined by extent of shape change (ESC) of female (♦) and male (+) platelets. n = 86 for each sex. *p b 0.05.
5.1 ± 2.5% and 4.8 ± 2.4% for female and male platelets, respectively, and was not significantly different between the sexes. ADP-induced platelet degranulation determined by CD62P expression was 58.1 ± 6.1% vs. 53.1 ± 6.5% on female vs. male platelets, respectively, exhibiting no difference between the sexes. However, ADP-induced PAC-1 expression was significantly higher (p b 0.05) in female platelets showing 17.9 ± 3.5% vs. 14.0 ± 4.1% for male platelets. Female platelets exhibited a significantly higher ability to change shape in response to 10 μM ADP (Fig. 1C) with 22.0 ± 3.5% vs. 17.9 ± 3.5% for male platelets. 3.2. Sex disparity in response to ADP is not age-dependent In many studies, sex disparity is explained by the age profile of participants. In order to assess the impact of the age on the parameters collected in this study, correlations were examined between donor age and both basal CD62P and PAC-1 expression and levels of these markers in response to ADP in both male and female platelets. For females, donor age ranged between 20 and 69 and for males, age ranged between 18 and 68. No correlation was found between donor age and either basal or activated levels of CD62P or PAC-1 binding (data not shown).
activated isoforms, Rap1 also showed no sex-specific difference; however, an almost 2.5-fold, significantly higher amount of RhoA seems to be activated in female compared to male platelets (Fig. 3). Activated RhoA has been shown to be localized mainly at the membrane. Crude fractionation analyses revealed that about 2.7-fold more RhoA was localized in the membrane (fraction, HSP) of female platelets which is significantly higher than the male platelets (Fig. 3). 3.5. RhoA/ROCK signaling analyses RhoA is activated in platelets via outside-in signaling of GPIIb/IIIa. Activated RhoA subsequently activates the kinase ROCK which phosphorylates a variety of substrate proteins including myosin and myosin light chain kinase (MLCK) or leads to cofilin phosphorylation. Myosin
3.3. Sex-specific changes in activation of PKC substrates PKC, which is downstream of the ADP family P2Y receptor, phosphorylates a variety of substrate proteins and some of them can be detected at the same time with a single antibody. The PKC substrate phosphorylation profile was analyzed in both female and male platelets and a representative western blot and relative intensity analysis are shown in Fig. 2. Female platelets exhibit a ~3-fold, significantly higher activation status of the selected PKC substrate proteins compared the male platelets. 3.4. GTPases exhibit sex-specific activation Both CD62P and GPIIb/IIIa activation assays revealed a different response to ADP between females and males. Therefore signaling pathways leading to degranulation and GPIIb/IIIa activation were investigated to determine if the differences seen between male and female platelets are reflected in these signaling pathways. GTPases are molecular switches in many pathways leading to platelet activation. RhoA plays a pivotal role in both degranulation and GPIIb/IIIa outside-in signaling and Rap1 is known to activate GPIIb/IIIa. The expression profile of these GTPases was analyzed revealing no significant differences between female and male platelets for both GTPases (Fig. 3). For the
Fig. 2. Representative western blot of sex-specific profiles of activated protein kinase C (PKC) substrates in resting platelet samples. Bar graph of relative intensity was calculated from n = 16 western blots for each sex. *p b 0.05.
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Fig. 3. Using the same donors, representative western blots from resting platelet samples of sex-specific profiles of Rap1 (A), activated Rap1 (B), RhoA (C), activated RhoA (D) in platelet lysates and RhoA in the membrane-cytoskeletal fraction (E, high speed pellet, HSP). Bar graphs of relative intensity were calculated from n = 16 western blots for each sex. *p b 0.05.
light chain (MLC) exhibited no significant difference in expression level between female and male platelets; however, female platelets showed a 2.8-fold, significantly higher phosphorylation level in female compared to male platelets (Fig. 4A, B). Analysis of cofilin expression and its phosphorylation status revealed no sex-specific difference (Fig. 4C, D). 3.6. Agonist sensitivity to GPIIb/IIIa activation The sensitivity of ADP to the activation status of GPIIb/IIIa complex was analyzed in a titration experiment for both female and male
platelets (Fig. 5). Female platelets showed a significantly higher sensitivity than male platelets. 4. Discussion Earlier studies have shown higher reactivity/responsiveness of female vs. male human platelets in aggregation and GPIIb/IIIa activation [23]. However, these studies lacked data without agonist stimulation in order to assess whether this difference arises from sex-specific differences in basal levels in these assays or are due to different sensitivities
Fig. 4. Using the same donors as in Fig. 3, representative western blots from resting platelet samples of sex-specific profiles of myosin light chain (A, MLC), phosphorylated MLC (B), cofilin (C) and phosphorylated cofilin (D). Bar graphs of relative intensity were calculated from n = 16 from each sex. *p b 0.05.
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Fig. 5. Titration of ADP and activation levels of GPIIb/IIIa by PAC-1 binding in female (straight line) and male (dotted line) platelets. SD represent n = 10 for each sex, *p b 0.05.
to agonists. In this study, platelet activation determined by the expression of P-selectin on the platelet surface and the activation status of the fibrinogen receptor with and without ADP agonist stimulation was determined. Additionally, the ability of the platelet to change shape in response to ADP was assessed. No differences in basal platelet activation and fibrinogen receptor activity could be detected. However, a significant difference in responsiveness in female vs. male platelets in GPIIb/IIIa activation in response to ADP was observed. Lastly, the extent of shape change assay also revealed a significantly stronger response by female platelets. Donors for this study were chosen based on stringent criteria in order not to jeopardize any conclusions. Exclusion criteria were any kind of medications known to inhibit platelets, disease or illness, dietary plans or pregnancy for female donors. From the pool of samples collected in this study, a subset was randomly chosen for further analyses. The preparation of the platelet-rich plasma was carried out by the same person in order to avoid handling variations. Analyses of underlying molecular mechanisms were focused on the sex-specific difference in basal levels of signaling molecules involved in GPIIb/IIIa activation in the response to ADP. This agonist binds and activates P2Y receptors on the surface of platelets and triggers signaling cascades via G-proteins leading to GPIIb/IIIa activation and granule secretion [26]. Granule secretion is regulated by PLC-dependent PKC activation [27] and this study demonstrated that female platelets seem to exhibit a significantly higher activation of PKC substrates which would explain the difference in sex-specific ADP-stimulated degranulation. On the other hand, the classical pathway of integrin activation is the activation of the GTPase Rap1 in a PI3K-dependent manner paralleled with talin recruitment to the membrane into the integrin complex [28]. Therefore, Rap1 and activated Rap1 levels were determined in a subset of these donors, but no significant difference between female and male platelets could be detected. The above mentioned mechanism leading to integrin activation is inside-out signaling [29]; subsequent binding of fibrinogen in the plasma to the activated integrin complex initiates the complementary outside-in signaling leading among others to the activation of the GTPase RhoA [30]. Thus, RhoA and activated RhoA levels were determined in a subset of the study participants. A significant difference between female and male platelets in activated RhoA levels could be detected which are not due to differences in RhoA expression. The latter observation is in agreement with a genomic analysis demonstrating similar levels of RhoA expression in human female and male platelets [31]. RhoA is known to play a key role in granule secretion as well as outside-in signaling and results here confirm that ADP stimulation led to increased degranulation, GPIIb/IIIa activation and shape change. The increased level of activated RhoA in resting female vs. male platelets which seems to be primarily associated with the platelet membrane most likely stems from an enhanced GPIIb/IIIa outside-in signaling leading to increased phosphorylation of ROCK substrates such as MLC affecting actin rearrangement and subsequent shape change. This sex disparity, however, could not be observed for
the ADP-stimulated degranulation, Studies have shown that RhoA is required for efficient secretion of alpha and dense granules downstream of G [13] and G(q) proteins [32]. Stimulation of the P2Y receptor by ADP like in this study, however, seems to signaling via G(i) proteins [33] and might explain our observation. Furthermore, previous studies have revealed that female and male platelets have a similar number of GPIIb/IIIa molecules on the platelet surface [23]. Additionally, it could be shown that plasma from human blood does not contain significantly different levels of fibrinogen [34]. This result excludes potentially increased outside-in signaling to enhance RhoA activation. RhoA regulates the activity of the kinase ROCK which phosphorylates its substrates such as cofilin via Lim domain kinase (LIMK) or MLC which regulates actin de-/polymerization or contraction of actin fibers, respectively [35]. Only phosphorylated MLC showed a significant difference between female and male donors while the basal expression of cofilin showed no sex-specific difference. From these investigations, it was concluded that gender-specific differences in ESC are most likely due to increased phosphorylated MLC levels. Since more integrin molecules are activated on the surface of female compared to male platelets, but the fibrinogen levels are fairly similar in female and male plasma, this might explain a higher rate of outside-in singling leading to RhoA/ ROCK activation. Lastly, other studies suggested different sensitivity of platelets to agonists [21]. A titration of ADP monitoring the activation status of GPIIb/IIIa revealed a significantly higher sensitivity of female vs. male platelets. Taken together, the findings reported here suggest a model in which female platelets exhibit a higher sensitivity to ADP or an increased RhoA/ROCK signaling responsible for enhanced degranulation and integrin activation. Further analyses are necessary to verify these results and confirm the proposed signaling model. Only ADP was used as an agonist in this study. Although sex differences in thrombin- or TRAP-stimulated platelet and GPIIbIIIa activation have been shown [14,21–23], it would be interesting to test the effects of these agonists and others such as collagen, arachidonic acid or thromboxane A2 in the context of the signaling model proposed here. We postulate that sex differences in response to other agonists would be seen based on the cross-talk among many of the platelet agonist signaling pathways. Furthermore, the activation status of GPIIb/IIIa, determined in this study by PAC-1 binding, could be complemented by an analysis of the receptor's ability to bind fibrinogen. Additionally, plasma components such as lipids or hormones may influence the activation status or responsiveness to agonists affecting the RhoA/ROCK activity. It is known that hormones can trigger post-translational modifications of proteins such as palmitoylation which can modulate the protein function affecting protein trafficking and protein-protein interactions [36]. Trafficking and subsequent involvement in cellular signaling is mediated by this moiety determined by its interaction with the lipid rafts in the membrane [37]. Among others, membrane proteins such as P2Y receptors and their associated G-proteins as well as GTPases such as Ras and Rho proteins are found to be palmitoylated [38]. Therefore, these kinds of protein modifications triggered by effectors such as hormones can also play a role in the sex-dependent platelet response to agonists such as ADP seen in this study. Therefore, similar analysis of washed platelet samples could complement and expand our findings. Lastly, the differences in the GPIIb/IIIa activation may contribute to the disparity in the observed gender-dependent platelet activation. Inhibitors such as eptifibatide could be used to in female samples to mimic the levels of male samples. These adjustments would further fine-tune the read-out and conclusion and may assist in interpretation of clinical findings when sex-based differences occur.
Funding This research was supported financially by Canadian Blood Services, funded by the federal (Health Canada), provincial and territorial
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ministries of health. The views expressed herein do not necessarily represent the view of the federal government. Author contributions PS, GMW and DVD designed the study; PS, DC, MN and GMW performed research; PS and DC analyzed and interpreted the data; and PS, GMW and DVD wrote the manuscript. Conflicts of interest None. Acknowledgements The authors like to thank the donors who participated in this study and the Canadian Blood Services Development Laboratory (netCAD) for provision of whole blood samples, respectively. References [1] P. Appelros, B. Stegmayr, A. Terent, Sex differences in stroke epidemiology: a systematic review, Stroke 40 (4) (2009) 1082–1090. [2] D.A. De Silva, M. Ebinger, S.M. Davis, Gender issues in acute stroke thrombolysis, J. Clin. Neurosci. 16 (4) (2009) 501–504. [3] P. Gopalakrishnan, M.M. Ragland, T. Tak, Gender differences in coronary artery disease: review of diagnostic challenges and current treatment, Postgrad. Med. 121 (2) (2009) 60–68. [4] M.J. Reeves, C.D. Bushnell, G. Howard, et al., Sex differences in stroke: epidemiology, clinical presentation, medical care, and outcomes, Lancet Neurol. 7 (10) (2008) 915–926. [5] Y.Y. Tan, G.C. Gast, Y.T. van der Schouw, Gender differences in risk factors for coronary heart disease, Maturitas 65 (2) (2010) 149–160. [6] C.L. Verheugt, C.S. Uiterwaal, E.T. van der Velde, et al., Gender and outcome in adult congenital heart disease, Circulation 118 (1) (2008) 26–32. [7] H. Shi, D.J. Clegg, Sex differences in the regulation of body weight, Physiol. Behav. 97 (2) (2009) 199–204. [8] E.B. Geer, W. Shen, Gender differences in insulin resistance, body composition, and energy balance, Gend. Med. 6 (Suppl. 1) (2009) 60–75. [9] F. Magkos, B. Mittendorfer, Gender differences in lipid metabolism and the effect of obesity, Obstet. Gynecol. Clin. N. Am. 36 (2) (2009) 245–265 vii. [10] I.C. Villar, A.J. Hobbs, A. Ahluwalia, Sex differences in vascular function: implication of endothelium-derived hyperpolarizing factor, J. Endocrinol. 197 (3) (2008) 447–462. [11] E.D. Luczak, L.A. Leinwand, Sex-based cardiac physiology, Annu. Rev. Physiol. 71 (2009) 1–18. [12] J.M. Nicolas, P. Espie, M. Molimard, Gender and interindividual variability in pharmacokinetics, Drug Metab. Rev. 41 (3) (2009) 408–421. [13] C. Ober, D.A. Loisel, Y. Gilad, Sex-specific genetic architecture of human disease, Nat. Rev. Genet. 9 (12) (2008) 911–922. [14] M. Johnson, E. Ramey, P.W. Ramwell, Sex and age differences in human platelet aggregation, Nature 253 (5490) (1975) 355–357.
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