Sex differences in antiplatelet therapy: state-of-the art

Abstract Antiplatelet therapy is a cornerstone of secondary prevention of cardiovascular diseases (CVDs). However, current guidelines are based on data derived primarily from men, as women are generally underrepresented in trials. Consequently, there are insufficient and inconsistent data on the effect of antiplatelet drugs in women. Sex differences were reported in platelet reactivity, patient management, and clinical outcomes after treatment with aspirin, P2Y12 inhibitor, or dual antiplatelet therapy. To evaluate whether sex-specific antiplatelet therapy is needed, in this review we discuss (i) how sex affects platelet biology and response to antiplatelet agents, (ii) how sex and gender differences translate into clinical challenges and (iii) how the cardiological care in women might be improved. Finally, we highlight the challenges faced in clinical practice regarding the different needs and characteristics of female and male patients with CVD and address issues requiring further investigation. Plain Language Summary Antiplatelet therapy is a crucial part of cardiovascular disease prevention. Guidelines are based on data from men, as too few women participate in trials. We reviewed differences between women and men in antiplatelet therapy. Sex differences occur in platelet reactivity and in the response to the therapy with aspirin and/or P2Y12 inhibitors. In primary prevention with aspirin, sex should be considered. In secondary prevention, aspirin, clopidogrel, ticagrelor, and prasugrel should be used in women as in men, according to individual patient needs. Female and male cardiac patients might present with different symptoms and risk factors. Healthcare professionals often treat women differently than men and do not satisfy women’s needs. Education of researchers and healthcare professionals is required to provide highest standard of cardiological care to women. This review summarizes the evidence on sex differences in antiplatelet therapy – from diagnosis, through patient management, to treatment outcomes. It describes how molecular aspects translate into clinical practice and how to provide effective antiplatelet therapy to female patients. Abbreviations: ACS: acute coronary syndrome; ADP: adenosine 5’-diphosphate; BARC: Bleeding Academic Research Consortium; CAD: coronary artery disease; cAMP: cyclic adenosine 5’-monophosphate; COX-1: cyclooxygenase-1; CYP: cytochrome P; CVD(s): cardiovascular disease(s); DAPT: dual antiplatelet therapy; DES: drug-eluting stent; DM: Diabetes mellitus; GP: glycoprotein; MI: myocardial infarction; PCI: percutaneous coronary intervention; PGH2: prostaglandin H2; PKA: protein kinase A; TR: thromboxane receptor; TXA2: thromboxane A2; VASP: vasodilator-stimulated phosphoprotein; VWF: von Willebrand factor


Introduction
Remarkable advances in the prevention and treatment of cardiovascular diseases (CVDs) by means of coronary interventions led to the establishment of an essential role of antiplatelet agents in clinical practice.European Society of Cardiology advocates monotherapy with aspirin or alternatively clopidogrel as a secondary prevention measure in patients with coronary artery disease (CAD).Additionally, dual antiplatelet therapy (DAPT), consisting of aspirin and an inhibitor of the platelet P2Y 12 receptor, is the cornerstone of treatment in patients with chronic or acute coronary syndromes (ACS) who undergo percutaneous coronary intervention (PCI).Furthermore, antiplatelet therapy is recommended in patients with non-cardioembolic ischemic stroke, transient ischemic attack or symptomatic lower extremity artery disease.Inhibition of platelet reactivity reduces the rate of ischemic events, thus contributing to reduced overall CVD risk and better prognosis [1,2].
Despite the improvements in prevention strategies, CVDs remain the leading cause of death among women and men worldwide.Current guidelines are based on data derived primarily from men, as women are generally underrepresented in trials [3].Consequently, there are insufficient and inconsistent data on the effect of antiplatelet drugs in women.Although modern therapies are designed to improve treatment of both, women and men, numerous studies have revealed differences in platelet reactivity and clinical outcomes, suggesting that antiplatelet therapy in women may not be as beneficial as in men [4].Disparities may arise due to sex, defined as biological and physiological characteristics.Moreover, gender -social construct reflecting cultural norms, cannot be neglected, as lifestyle, socio-economic class, and several other factors have crucial impact on CVD and its pathophysiology [5,6].To evaluate whether sex-specific antiplatelet therapy is needed, in this review we discuss (i) how sex affects platelet biology and response to antiplatelet agents, (ii) how sex and gender differences translate into clinical challenges and (iii) how the cardiological care in women might be improved.Finally, we highlight the challenges faced in clinical practice regarding the different needs and characteristics of female and male patients with CVD and address issues requiring further investigation.

Platelet reactivity
Numerous studies have identified differences between sexes in platelet reactivity.First, fibrinogen binding to platelets tends to be greater in women than in men due to higher density of fibrinogenbinding receptors [7] or increased expression of signaling cascade proteins in female platelets [8], leading to greater reactivity.In addition, female platelets exert more pronounced activation following interaction with adenosine 5'-diphosphate (ADP), collagen, and other mediators.Moreover, women exhibit greater baseline platelet count and higher levels of inflammatory markers, e.g., C-reactive protein, leukocyte count, and P-selectin expression.Additionally, women have greater concentrations of membrane microparticles that participate in inflammation.It is suggestive of more pronounced platelet reactivity in women, as activated platelets mediate inflammatory process [7][8][9].Conversely, increased reactivity in women seems to be independent of platelet size and expression of surface adhesion molecules [10,11].Receptors and ligands that may account for sex differences in platelet reactivity are shown in Figure 1.

Sex hormones and platelet reactivity
Differences between female and male platelets may arise due to the impact of sex hormones.In women, estradiol promotes the synthesis of an inhibitor of platelet aggregation, prostacyclin.Estrogen induces the release of vasodilatory nitric oxide, which suppresses the formation of platelet aggregates.Furthermore, progesterone was shown to suppress inflammatory process and stimulate nitric oxide synthesis after cerebral ischemia.The lowest levels of both sex hormones are observed during menstruation.Peak estrogen concentration occurs in proliferative phase, whereas progesterone reaches its maximum level in secretory phase.However, whether and how phases of menstrual cycle affect platelet properties remains unclear [8,12].Conversely, men exhibit higher platelet aggregation in response to thromboxane A2 (TXA2), which may be mediated by testosterone, the primary sex hormone in men [13].
Estrogens are believed to play a cardioprotective role, as CVD risk considerably increases in women after menopause or premature cessation of ovarian function.In the absence of estrogens, their receptors controlling platelet activation (Figure 1) become upregulated, thus platelets activate more easily, and the risk of ischemic events increases.Interestingly, exogenous estrogens may Figure 1.Receptors and ligands that may account for sex differences in platelet reactivity.Women exhibit: (i) greater ADP-and collagen-induced reactivity, (ii) higher density of surface GPIb-IX-V and integrin α IIb β 3 , (iii) high level of estrogens at premenopausal age, which is believed to play a cardioprotective role.ADP -adenosine 5'-diphosphate; GP -glycoprotein; VWF -von Willebrand factor.Created with BioRender.Com.
not mimic endogenous ones, as CVD risk cannot be diminished by means of hormone replacement therapy [14].

Cardiovascular diseases and platelet reactivity
Sex disparities in platelet reactivity observed in individuals with metabolic diseases and CVD may be explained by differences in CVD pathophysiology.Comorbidities, e.g., diabetes mellitus (DM), which promote platelet aggregation, are more often seen in women than in men with CVD, which may contribute to greater platelet reactivity in women [13,15].Despite a worse CVD risk profile, women present favorable atherosclerotic plaque characteristics, i.e., lower total plaque volume, plaque burden and fibrous tissue, fibro-fatty tissue, necrotic core and dense calcium volumes [16].Conversely, men more frequently develop vulnerable plaques and structural lesions in epicardial coronary arteries.Dysfunctional endothelium may overstimulate and consequently desensitize male platelets, leading to their lower reactivity [17].Additionally, women tend to present greater platelet reactivity while on treatment with antiplatelet agents.However, it remains unclear whether it results from the differences in baseline platelet reactivity or from the weaker response to the therapy [18].

Response to aspirin
Aspirin, also referred to as acetylsalicylic acid, suppresses conversion of arachidonic acid into T×A2 by irreversible inhibition of cyclooxygenase-1 (COX-1).As a result, aspirin impairs platelet aggregation, thus diminishing the risk of ischemic events [19] (Figure 2).However, aspirin increases the risk of major and gastrointestinal bleeding.Consequently, aspirin shall be considered as primary prevention only in individuals at high or very high CVD risk, when the benefits of ischemic protection outweigh the bleeding risk, regardless of sex.In contrast, it is used as secondary prevention in both women and men [1,2].
Ex vivo studies have shown higher platelet reactivity in women than in men on aspirin.Aspirin resistance has been primarily attributed to the diminished inhibition of indirect platelet activation pathways, e.g., the one induced by epinephrine [12].Further evidence associates female sex with increased concentrations of 11dehydrothromboxane B2.The presence of this T×A2 metabolite suggests inadequate inhibition of COX-1 and may explain higher arachidonic acid-induced platelet aggregation observed in women [13].
Some studies have reported that patients with high onaspirin platelet reactivity may be at greater CVD risk, and the assessment of arachidonic acid-induced platelet aggregation enabled to identify individuals at high risk of atherosclerotic events [20].However, other results from ex vivo analyses do not correspond to clinical outcomes, as similar ischemic event and bleeding rates have been reported in both sexes among patients undergoing PCI [18].Disparities occurred only when aspirin monotherapy was investigated as primary prevention.In this case, women were shown to benefit exclusively from the diminished stroke risk, whereas the advantage for men also included the reduction of myocardial infarction (MI) risk.Nonetheless, overall bleeding risk was similarly increased in both sexes, except for gastrointestinal bleeding, suggested to be more common in women [8,21].
Altogether, there is no sufficient evidence to support different approaches toward women and men in routine secondary prevention with aspirin.Conversely, sex should be considered if indications toward primary prevention with this antiplatelet agent occur, as it may not lower the risk of MI in women.
Figure 2. Action mechanism of aspirin and P2Y12 inhibitors.Aspirin irreversibly inhibits COX-1, thus suppresses TXA 2 synthesis and activation of TR receptor.Inactive clopidogrel is transformed to its active metabolite in two-step oxidation by CYP.Inactive prasugrel is transformed to its active metabolite in two-step process led by esterase and CYP.Ticagrelor is an active drug.They inhibit P2Y 12 receptors for ADP.As a result, inhibitory impact of G-protein's subunit α i on adenylyl cyclase is abolished.Increased level of cAMP leads to phosphorylation of VASP via PKA.VASP-P inhibits platelet activation.Furthermore, once P2Y 12 receptors are inhibited, G-protein's subunit β y cannot promote granule secretion, which normally leads to platelet activation.ADP -adenosine 5'-diphosphate; cAMP -cyclic adenosine 5'-monophosphate; COX-1 -cyclooxygenase-1; CYPcytochrome P; PGH 2 -prostaglandin H 2 ; PKA -protein kinase A; TR -thromboxane receptor; TXA 2 -thromboxane A 2 ; VASP -vasodilatorstimulated phosphoprotein.Created with BioRender.Com.

Response to P2Y 12 inhibitors
Inhibition of platelet aggregation may be achieved by antagonizing P2Y 12 receptor for ADP.Three P2Y 12 inhibitors are currently commonly used in clinical practice.Thienopyridines, clopidogrel and newer prasugrel, are orally administered as prodrugs.The former necessitates double oxidation by cytochrome P450.The latter undergoes hydrolysis led by carboxylesterases prior to single oxidation by cytochrome P450.Therefore, prasugrel is metabolized more efficiently than clopidogrel.In contrary, potent P2Y 12 inhibitor, ticagrelor, is an active agent which does not require metabolic conversion to exert antiplatelet effect [22,23] (Figure 2).Data derived from metaanalyses, randomized clinical trials, and registries investigating sex differences in the response to antiplatelet therapy with P2Y 12 inhibitors are summarized in Table I.

Clopidogrel
Ex vivo assessment revealed that women present higher onclopidogrel platelet reactivity, thus they are more likely than men to be hyporesponsive to treatment.The suboptimal laboratory results were not in line with clinical outcomes, as ischemic event and bleeding rates did not differ significantly between sexes [21].However, disparities have occurred in detailed analyses, e.g., clopidogrel was reported to diminish MI rates in women, whereas stroke and mortality risk were reduced only in men.Therapy outcomes may be influenced by mutations of CYP2C19, which is responsible for conversion of clopidogrel into active metabolite.As polymorphisms are distributed equally in women and men, no sex differences were identified in the investigation of CYP2C19 genotype-guided therapy [36].

Prasugrel and ticagrelor in cardiac ischemia
Trials comparing clopidogrel to prasugrel or ticagrelor as a part of DAPT in patients with ACS or chronic CAD showed similar results in both, women and men [27,37].However, another study identified novel P2Y 12 inhibitors as independent bleeding risk factor in women [26].Additionally, there is evidence that women may not benefit from prasugrel or ticagrelor to the same extent as men, as these potent P2Y 12 inhibitors failed to reduce ischemic event rates compared to clopidogrel, whereas the opposite was reported in men [34].Among DM patients undergoing PCI, women were shown to benefit less than men from novel P2Y 12 inhibitors, whereas no differences were reported for clopidogrel [38].Conversely, monotherapy with P2Y 12 inhibitor diminished the ischemic risk only in women and posed lower bleeding risk in both sexes, when compared to DAPT [33].

Dual antiplatelet therapy regimens
Sex-specific results from studies investigating different DAPT regimes are inconsistent.Some authors showed that DAPT continuation beyond 12 months may not further reduce CVD risk [30].Although men could benefit from prolonged DAPT, it significantly increases their bleeding risk [35].Conversely, prolonged DAPT may not necessarily be associated with excessive bleeding rates in women  [25].Therefore, the decision to continue DAPT, shall be made based on individual patient characteristics.Altogether, there is no sufficient evidence to support different approaches toward women and men in routine secondary prevention with clopidogrel, prasugrel, or ticagrelor.However, further studies are needed to tailor the treatment based on the individual ischemic and bleeding risk and sex.

Sex-specific antiplatelet challenges in clinical practice
Current guidelines do not advocate any sex-specific approach, thus shall be followed equally in treatment of both, women and men.However, multiple trials have shown disparities in diagnostic process, medicine prescription, therapy course and clinical outcomes, suggesting that women suffering from CVD may not receive equitable care, which would satisfy their needs.

Cardiovascular risk factors
Significant differences between sexes occur regarding baseline patient characteristics in cardiac patients.Women tend to experience the onset of CVD symptoms later in life, with their CVD risk considerably increasing after menopause.As a result, women seek medical attention at older age than men [39].Therefore, women treated because of CVD are more likely to suffer from various comorbidities.Multiple studies have consistently reported higher prevalence of arterial hypertension, dyslipidemia, or DM, including insulin-treated DM, in women than in men presenting with ACS [5,39,40].Moreover, pregnancy complications, e.g., placental, or hypertensive or diabetic disorders, may be identified as female-specific CVD risk factors [39].Interestingly, prolonged smoking [39] and DM [38] have been described as more hazardous to women.Furthermore, female gender is associated with higher prevalence of depression and greater mental stress, which predispose to CVDs [41].

Patient characteristics
Along with disparities in CVD risk factors, additional differences may also occur at the time of presentation.Women with ACS more frequently have a history of stroke, whereas men report previous MI and revascularizations.Women presenting with chest pain who undergo PCI may report worse quality of life than men [42].It has been suggested that women with ACS are more likely to show prodromal and atypical symptoms that physicians may underestimate and not associate with CVD, causing delay in treatment.Conversely, some authors found no discrepancies between sexes in the accuracy of symptom assessment, nor in duration of ischemic time [43,44].In contrast, other studies reported that women experienced significantly longer symptom-to-balloon time [45].Further disparities occur regarding ACS pathophysiology.Men more often present with ST-elevation MI and have underlying multivessel CAD.In contrary, women are more likely to be diagnosed with non-STelevation MI and MI with nonobstructive coronary arteries [46].

Patient management
Considering management strategies, women are likely to be treated less aggressively and to undergo less invasive procedures than men [47].Overall, women are prescribed antiplatelet along with hypolipemic and hypotensive agents less frequently than men [48,49].Considerable disparities occur regarding novel, potent P2Y 12 inhibitors.Women may more often receive DAPT with clopidogrel than with prasugrel or ticagrelor, which could be explained by the fear of increased bleeding risk [50,51].
Gender has been shown to influence the treatment course, as women are more likely to exhibit lower adherence to antiplatelet therapy.Female gender was identified as a predictor of discontinuation and disruption of DAPT, especially when it included novel P2Y 12 inhibitor.Women may resign from preventive measures due to family obligations, lack of social support, lower socioeconomic and education status [52].Another reason is that CVDs were long known as men's disease [5].However, social awareness of CVD risk in women is gradually being raised [4].

DAPT outcomes in patients after PCI
Considerable disparities may occur in treatment effects.Overall, higher rates of major adverse cardiac events, including ischemic incidences and death, have been reported in women than in men on DAPT after coronary interventions.Initial discrepancies usually disappeared after adjustment to baseline differences or procedure characteristics, suggesting that women and men benefit equally from the treatment [4,25,44,50,[53][54][55].Conversely, significant disparities in bleeding event rates, especially periprocedural bleeding rates, more often persisted after adjustment [24,50,56].Noteworthy, women undergoing PCI exhibit increased risk of access site bleeding [29], which may be explained by higher rates of femoral access in women [54] or inappropriate dosage of antiplatelet drugs [9,57].
Altogether, women and men have different CVD risk profiles and often present with dissimilar symptoms, comorbidities, and medical history.Women tend to be treated less invasively, to receive other medicines than men and to be less adherent to the therapy.Furthermore, response to antiplatelet therapy seems to be affected by sex.

Evidence gap
Trials investigating CVDs include the majority of men.Therefore, data on the efficacy and safety of antiplatelet therapy in women is limited.Due to the underrepresentation of women, the documented sex differences may not reach statistical significance [58].Women tend to participate in clinical trials less willingly, as they perceive greater risk of adverse events than men or have lower socioeconomic status.Moreover, researchers may be less likely to include women with CVD in their studies due to atypical presentation, comorbid conditions, or advanced age [3].However, there is lack of proper data regarding facilitators and barriers for women to participate in clinical trials [59].
Prior to the possible adjustment of therapeutic strategies according to sex-specific needs, better understanding of sex differences in platelet reactivity and in clinical outcomes in the response to antiplatelet agents must be obtained.For this purpose, future trials should be focused on women, or at least include greater representation of women.Therefore, interventions that will encourage women to participate in trials are needed.Researchers' awareness of sex and gender disparities and the importance of equal representation should be raised by measures undertaken at the institutional levels.
Furthermore, to assure that women and men receive antiplatelet therapy according to newest guidelines, healthcare professionals must be aware of sex disparities.Students should be taught about the different characteristics of women with CVD and receive updates on sex-related outcomes from clinical trials, which will enable them to provide the highest standard of care to both, women and men [41,[59][60][61].The key messages about antiplatelet therapy in women are summarized in Table II and Figure 3.

Summary
Altogether, the evidence from multiple trials suggest that women and men exhibit heterogenous baseline platelet reactivity and respond to antiplatelet therapy in a different way, which is reflected by laboratory results and clinical outcomes.Further investigations with appropriate representation of women are required to better understand the complex nature of sex-specific platelet response.As far as the guidelines recommend same treatment strategies in both sexes, there is a need to advocate the equality and equity in the management of women and men with CVD.This may be achieved by education of both women and healthcare professionals.
unusual and prodromal CVD symptoms in women Early detection of CVDs in women Consider sex by indications for primary CVD prevention with aspirin • Women benefit primarily from reduction of stroke risk Effective antiplatelet therapy in women Use secondary CVD prevention with aspirin in women and in men Choose P2Y 12 inhibitor based on individual patient characteristics • Use prasugrel or ticagrelor if indicated Choose DAPT duration based on individual patient characteristics • Follow the guidelines in women and in men Legend: CVD(s) -cardiovascular disease(s); DAPT -dual antiplatelet therapy.

Table I .
Sex differences in the response to antiplatelet therapy with P2Y 12 inhibitors.Data derived from meta-analyses, randomized clinical trials, and registries investigating P2Y 12 inhibitors with focus on sex differences, chosen based on title and abstract evaluation.Studies are presented in alphabetical order.

Table II .
Key messages about antiplatelet therapy in women.Action Benefit Raise women's awareness of CVD risk Improved antiplatelet therapy adherence and compliance among women Increased female enrollment and participation in CVD clinical trials Educate researchers on importance of appropriate female representation in clinical trials Increased inclusion and representation of women in CVD clinical trials More of statistically significant outcomes Encourage researchers to conduct sex-specific investigations Better understanding of pathophysiology of CVDs in women Relevant insight into benefits and adverse effects of antiplatelet therapy in women Chance to create sex-specific antiplatelet therapy guidelines Promote sex-specific research outcomes among healthcare professionals Effective patient management and treatment Treat comorbidities Effective CVD prevention and reduction of CVD risk Acknowledge