Emerging Clinical Problem of Resistance to Antiplatelet Therapy in Primary Prevention and Treatment of Cardiovascular Events in People Living with HIV: Conundrum despite Effective cART

Abstract

Despite the extensive use of combined antiretroviral therapy (cART) for effective human immunodeficiency viral (HIV) suppression, people living with HIV have an increased risk of cardiovascular events compared to the general population. Antiplatelet agents are recommended for primary prevention and treatment of individuals at risk of ischaemic stroke and heart attack. However, these guidelines and recommendations are hinged on data from non-HIV populations. Accumulating evidence has revealed that response to antiplatelet agents varies in people living with HIV compared to non-HIV individuals. The variability may be attributed to consequences of HIV infection, metabolic derangements, and effects of cART and other drug interactions. Given that interventions employed in primary and secondary prevention of cardiovascular events heavily rely on guidelines developed for the general population that emphasize on identification, optimization and stratification of traditional risk factors, there is need to tailor these interventions with knowledge of HIV status and co-administration of cART. This chapter will synthesize the current topic regarding antiplatelet agents in people living with HIV. Specifically, we will critically examine the effects of individual antiplatelet agents on platelet function tests, drug interactions with cart and clinical data on the reduction of cardiovascular events.

1. Introduction

Global statistics indicate that 28.7 of the 38.4 million people were living with HIV (PLWH) infection in 2022, 650,000 were on antiretroviral medications and 650,000 deaths were recorded [1]. The availability of effective combined antiretroviral therapy (cART) for HIV infection has transformed the disease from an infectious to a noncommunicable disease (NCD) including cardiovascular events, consisting of aggregate of interrelated chronic conditions, such as chronic inflammation, immune activation and metabolic syndrome (MetS).

The prevalence of MetS among PLWH is high, ranging from 11 to 45% [2] plausibly linked to some cART regimens containing zidovudine, some non-nucleoside reverse transcriptase inhibitor drugs (NNRTIs) (e.g. efavirenz) and protease inhibitors (PIs) (e.g. indinavir) [3, 4]. MetS is a risk factor and precursor for the development of cardiovascular disease [5], in the background of chronic inflammatory processes linked to cardiovascular events [6]. Collectively, these factors combine to aggravate an already existing or accelerate the development of cardiometabolic complications such as intravascular thrombosis.

Platelet hyperactivity links HIV, MetS and cardiovascular diseases [7], and antiplatelet medications are indicated depending on risk stratification in order to prevent cardiovascular complications. However, in the general population, the protective effect of aspirin is variable, with some individuals being resistant [8]. Emerging evidence indicates that the suboptimal response may be greater in PLWH. It is now acknowledged that the risk factors for the development of MetS also modulate the efficacy of antiplatelet agents, resulting in abrogated protection against cardiovascular complications, a phenomenon known as treatment failure or drug resistance.

2. Cardiovascular events in HIV

2.1 Epidemiology of cardiovascular events in HIV

People living with HIV have 50% increased risk of cardiovascular events than non-HIV patients [9] or twofold risk than the general population [10]. Although cardiovascular diseases account for 8–22% deaths among HIV-infected patients, the rates are expected to increase with the aging HIV-infected population on effective cART [11]. Whereas the substantial risk of death associated with HIV infection increases with the use of cART [12], HIV and cART independently exhibit hyperactive platelets in spite of viral suppression [13].

2.2 Ischaemic stroke in HIV

The incidence of ischaemic stroke in HIV is one and half times greater than in non-HIV-positive patients, a situation that persists independently after controlling for other traditional risk factors [14]. The rates are higher across gender and age groups, although younger age groups are more affected in HIV than in non-HIV-infected controls [15]. Hyperactive platelets have been implicated in the independent causal relationship between ischaemic stroke, HIV infection and cART treatment [16]. Surprisingly, the incidence and risk factors are not reduced by cART [17].

3. Antiplatelet therapy in HIV

3.1 Available antiplatelet agents

Available antiplatelet agents fall into various chemical groups acting on different pharmacological targets. Aspirin or acetylsalicylic acid (ASA) at low doses (30–150 mg) irreversibly blocks constitutive isoenzyme cyclooxygenase-1 (COX-1) in platelets preventing the generation of Thromboxane A2 (TXA2) from membrane arachidonic acid (AA), thus attenuating amplification pathways in platelet activation and aggregation [18]. Given that aspirin acts exclusively via arachidonic acid pathway, other signaling pathways stimulated by adenosine diphosphate (ADP), thrombin, collagen, adrenaline and thrombin receptor activating peptide (TRAP) are unaffected. Clopidogrel is an oral thienopyrimidine prodrug that undergoes a series of biotransformation in the hepatic circulation to the active form. It binds selectively and irreversibly to the purinergic receptor P2Y12 on the platelet membrane, blocking access to the platelet agonist ADP [18]. The glycoprotein IIbIIIa (GPIIbIIIa) agents are only available as intravenous (iv) formulations for peri-procedural usage and thereafter replaced by orally available agents for continued long-term antiplatelet therapy (Table 1, Antiplatelet agents adapted from [19, 20]).

Agent	Drug class	Molecular target	Route	Time to onset of action	Time to platelet function recovery after stopping
Aspirin	Acetylsalicylic acid	Irreversible cyclooxygenase 1 (COX-1) inhibition	Oral	20 min	5–7 days
Clopidogrel; Ticlopidine; Prasugrel	Thienopyridines	Irreversible P2Y12 receptor inhibition	Oral	2–6 h; 30 min; 30 min	7 days; 7–10 days
Cangrelor	Nucleoside/Nucleotide analogues-Adenosine triphosphate (ATP) analogue	Reversible P2Y12 ADP receptor inhibition	IV	<5 min	30–50 min
Ticagrelor	Nucleoside/Nucleotide analogues-Cyclopentyl-triazolo-pyrimidine		Oral		3–5 days
Abciximab; Eptifibatide; Tirofiban	Monoclonal antibody; Synthetic RGD analogues	GPIIbIIIa receptor inhibitors	IV	10–20 min	4–8 h
Vorapaxar	Tricyclic himbacine derivative	PAR 1 receptor inhibitor	Oral		4–8 weeks
Cilostazol; Dipiridamole	2-Oxoquinoline; pyrimidopyridine	PDE-enzyme inhibitor	Oral		12–16 h
Iloprost; Beraprost; Treprostinil; prostacyclin		Prostaglandin (PGI2) analogues	IV/Inhalation		2 h

Table 1.

Classification of antiplatelet agents, route of administration and time to platelet recovery after cessation.

3.2 Antiplatelet therapy rationale in HIV

People living with HIV (PLWH) have 1.5 times increased risk of cardiovascular events [21] associated with platelet activation exhibiting increased membrane expression of P-selectin [22, 23, 24], integrin GPIIbIIIa (PAC-1) [24], microparticles [22] and platelet-leucocyte aggregates [25]. Increased platelet activation and aggregation parameters do not improve with cART treatment [26, 27]. These parameters have been reported as definitive contributors to microvascular thrombosis in heart attack and ischaemic stroke [28] that necessitate antiplatelet therapy [9], especially in sub-Saharan Africa where HIV is most prevalent [29]. The criteria for initiation of antiplatelet therapy in primary prevention of cardiovascular diseases are based on the Framingham risk score [30] that incorporates age (>45 year males, >50 year females) and MetS features [31]. PLWH meet criteria for antiplatelet therapy, though at a much younger age compared to non-HIV [32].

3.3 Antiplatelet therapy and platelet function in HIV

Although aspirin is the most popular and widely used antiplatelet agent, in cART-stabilized HIV patients, an aspirin loading dose of 325 mg oral aspirin followed by a daily oral dose of 81 mg for 5 days failed to suppress urinary and serum thromboxane levels and arachidonic acid (AA)-stimulated Light Transmission Aggregometry (LTA) compared to controls [33]. However, in the same patients, there was a decrease in response to other agonists [33]. The lack of response to aspirin on AA stimulation is consistent with the development of acquired aspirin resistance [34]. This finding contrasts with that of another study where the subjects who received 100 mg oral aspirin for 2 weeks had suppressed urinary and serum thromboxane B2 (TxB2) levels and showed a decrease in AA-stimulated platelets on LTA, surface expression of biomarkers and prolongation of platelet function analyzer-100 (PFA-100) closure time [35], suggesting that the resistance could be overcome by dose increment.

In the Evaluation of Residual Platelet Reactivity After Acute Coronary Syndrome (ST+/ST−) in HIV (EVERE₂ST-HIV) study, a comparison of the platelet function in acute coronary syndrome patients on dual antiplatelet therapy (a combination of aspirin and P2Y12 inhibitors) found that the proportion of patients with residual platelet reactivity for both drugs was higher in HIV + ve patients compared to non-HIV, as assessed by residual platelet aggregation (RPA) on LTA, platelet reaction units (PRUs) on VerifyNow and platelet reaction index (PRI) on vasodilator-associated protein (VASP) [13]. In the same study, the resistance showed to antiplatelet remedies was consistent across the drugs and the methods of assay, correlated with HIV status but was greater with protease inhibitors more than NNRT [13].

The EVERE₂ST-HIV study findings demonstrated that platelet reactivity is high among patients with recurrent acute coronary syndrome and that antiplatelet therapy was associated with: (i) high residual platelet reactivity on a number of laboratory tests, (ii) resistance displayed to aspirin, clopidogrel, prasugrel and ticagrel, (iii) resistance was correlated with HIV RNA viral load, cluster of differentiation 4 (CD4) count and cART, and (v) genetic polymorphism was uniformly distributed across all study groups and therefore could not account for drug responses [13]. The observed high value on treatment with the antiplatelet agents could provide an explanation for recurrent ischaemic events in PLWH.

Another study that evaluated aspirin, clopidogrel and placebo on platelets in PLWH reported interesting findings summarized as follows: (i) diminished platelet aggregation upon aspirin administration on LTA stimulated by arachidonic acid and ADP, even though the platelet receptor expression evaluated by flow cytometry remained unaffected; (ii) clopidogrel lowered ADP (but not AA)-induced platelet aggregation on LTA and showed greater suppression of platelet activation biomarkers (P-selectin, PAC-1) and (iii) clopidogrel, but not aspirin, decreased endothelial and inflammatory biomarkers after 2 weeks of treatment compared to aspirin or placebo [36]. The results of this study suggested that suboptimal response may be limited to aspirin, and clopidogrel may provide a better profile and therefore may be the preferred therapy in PLWH. The uncoupling of responses between aggregation and activation and differences in drug responses indicate that aspirin inhibition of COX-1 does not protect against platelet hyperactivity from HIV-associated immune activation consistent with other studies [37].

Overall, the studies on PLWH suggest that platelet resistance to aspirin is higher compared to clopidogrel and potentially illustrates that platelet activation is not solely dependent on COX-1. Although data point to aspirin resistance in HIV, it must be noted that PLWH present with preexisting platelet hyperactivity prior to initiation of antiplatelet therapy [33]. Aspirin inhibition of platelet functions is selective for arachidonic acid-stimulated pathway, but insensitive to other agonists [38]. Consequently, this suggests that the application of cut-off levels to test results may be inappropriate to categorize patients, given the variability to aspirin response in normal individuals with pre-existing platelet hyperactivity [39].

3.4 Clinical thrombosis and antiplatelet therapy in HIV

Despite adequate antiplatelet therapy and other interventions during the first episode of acute coronary thrombosis, high recurrence rates still continue to be reported in HIV patients [40, 41]. This observation therefore indicates clinical resistance to antiplatelet medication in PLWH. However, the clinical spectrum and mechanisms have not been well defined.

3.5 Other outcomes of antiplatelet therapy in HIV

Aspirin’s potential to dampen the inflammatory response has been employed for the treatment of an array of inflammatory conditions over the years [42, 43]. On this basis, a clinical trial pilot study in stable cART-treated patients found that the administration of a 325 mg oral loading dose of aspirin followed by 81 mg daily decreased T-cell activation (cluster of differentiation 38 (CD38) and human leucocyte antigen-DR (HLA-DR)) and monocyte activation (soluble cluster of differentiation 13 (CD13)), but enhanced leucocyte response to toll-like receptor (TLR) stimulation [33], suggestive of attenuation of immune responses. Similarly, in another study, 81 mg aspirin reduced T-cell activation and genital mucosal inflammation in PLWH [44]. However, a clinical trial by the same investigators comparing 300 mg and 100 mg aspirin with placebo over a 12-week period did not reveal any difference in inflammatory markers such as soluble cluster of differentiation 14 (CD14), interleukin 6 (IL-6), cluster of differentiation 163 (CD163), T-cell activation or any other inflammatory markers [37], indicating that cardioprotective doses of aspirin have no effect on inflammation. Consistent with other studies, administration of low-dose aspirin (81 mg) in premature coronary artery disease (CAD) over 14 days failed to lower elevated inflammatory markers such as high-sensitivity C-reactive protein (hs-CRP) and IL-6 whose levels correlated with urinary 11-dehydrothromboxane B2 (uTxM), biomarker of aspirin resistance [45]. Moreover, low-dose aspirin in HIV patients does not attenuate pro-inflammatory lipid mediators of inflammation [46].

4. Concept and mechanism of antiplatelet resistance

4.1 Concept of antiplatelet resistance

The concept of antiplatelet resistance encompasses a variety of broad phenomena such as suboptimal, poor or nonresponse, treatment failure or variability. In the narrowest sense, it refers specifically to endogenous mechanism in certain individuals, which prevents the drug from exerting its full antithrombotic effect when applied at therapeutic doses compared to expectations in normal individuals [47, 48, 49, 50]. It is multifactorial and includes both clinical and laboratory evidence of deficiency in activity, despite adequate therapy and compliance. The incidence varies widely in studies depending on the antiplatelet agent, methodology of assessment and cut-off levels.

Clinical ‘resistance’ to antiplatelet drugs has been defined to occur with the onset of new, or worsening of ischaemic cardiovascular events in particular in patients while on recommended appropriate doses of antiplatelet agents. It is broad and encompasses treatment failure, interindividual variability or failure to protect from thrombotic ischaemic vascular events. Since this terminology does not imply the cause-effect relationship between the presence of drug and consequent events, its use is discouraged [18].

Chemical or true resistance specifically refers to de-acetylation of cyclooxygenase 1 at serine 529 (COX-1 Ser529) in platelets by aspirin. There are no chemical definitions of Thienpyrimidines or GPIIbIIIa blockers’ resistance. Individuals resistant to clopidogrel are grouped into intermediate or poor metabolizers. Intermediate metabolizers process some clopidogrel, so they receive partially benefit from the treatment but are not protected from developing a harmful blood clot. Poor metabolizers process little or no clopidogrel, so they benefit minimally from the treatment and are at risk of forming a harmful blood clot.

Laboratory ‘resistance’ to antiplatelet drugs refers to lack of, poor response, nonresponsiveness or deficiency of platelet reactivity on in vitro tests, despite the use of oral antiplatelet drugs when evaluated on a predetermined cut-off level of inhibition [18]. This pharmacological definition refers to the lack of effect at the target site of action upon stimulation by agonist specific to each drug on the various platelet function tests. It may, or may not be specific to inhibition of a COX-1 system. Based on pharmacokinetic and pharmacodynamic characteristics, laboratory resistance has been stratified into three levels [51, 52], namely:

1. Responders: Normal platelet aggregation and TxB2 levels >95.2 ng/ml. Therapeutic oral aspirin blocks platelet aggregation and reduces TxB2 to <0.5 ng/ml.

2. Type I resistance: Pharmacokinetic, reduced bioavailability at sites of action. Ex vivo platelet aggregation is not inhibited and TxB2 levels ≤82.9 ng/ml. TxB2 levels and platelet aggregation are successfully inhibited by in vitro-added antiplatelet agents.

3. Type II resistance: Pharmacodynamic, normal bioavailability at sites of action. Platelet aggregation is present, but TxB2 is partially reduced to ≤32–17 ng/ml by oral aspirin. Persistent TxA2 is present in serum and urine due to altered sites of action. Thromboxane A2 and platelet aggregation takes place when antiplatelet is added in vitro. Accelerated platelet turnover takes place, with introduction into bloodstream of newly formed, drug unaffected platelets. It is suggestive of COX-1 inhibition with persistent platelet activation, possibly from inducible cyclooxygenase 2 (COX-2) production of prostaglandin H2 (PGH2) conversion to TXA2, increased sensitivity to other agonists such as ADP, epinephrine, collagen and thrombin.

4. Type III resistance: pseudo-resistance or treatment failure. Oral aspirin does not inhibit platelet aggregation but Thromboxane A2 is successfully inhibited by both in vivo and in vitro added antiplatelet agents. Platelet aggregation can cause to be inhibited from non-TXA2-mediated pathways, such as due to smoking, hypercholesterolaemia, exercise or stress.

Because the different antiplatelet drugs block different pharmacological targets, it has been proposed that aspirin resistance be restricted to COX-1-dependent TXA-2 pathways, while that of thienopyrimidine (clopidogrel) be limited to the inability to inhibit purinergic P2Y12 receptor pathways [48]. There is evidence to suggest that resistance occurs to direct GPIIbIIIa inhibitors [53] but data are limited.

4.2 Laboratory measurement of antiplatelet resistance

The laboratory methods, in definition of antiplatelet resistance, involve either biochemical or functional assays. Due to the complexity in relating clinical outcomes to laboratory tests, and the performance and interpretation of test results, a consensus statement that guides the testing of aspirin resistance has been compiled by a group of experts [54]. In summary, they recommend as prerequisites that include the following points: the patient needs to be on stable antiplatelet therapy for at least 5 days and there should be confirmation of compliance and method for establishing drug bioavailability either in plasma or urine.

The recommended tests are a combination of biochemical (urinary or serum TXA2) and functional (platelet aggregation-LTA) assays. The assays should include in vitro addition of COX-1 inhibitor (aspirin) and COX-2 inhibitor if necessary to further elucidate the mechanism. Analysis of membrane surface receptor expression may be performed to characterize polymorphisms.

4.2.1 Bleeding time (BT)

This is the oldest method for the assessment of primary hemostasis that demonstrated the prolongation of bleeding time (BT) with ingestion of aspirin [55, 56]. In a clinical trial of aspirin, a subset of healthy volunteers (40%) and patients undergoing coronary artery bypass graft (CABG) (42%) were nonresponders as therapeutic doses of aspirin failed to prolong bleeding time [57]. In other studies, bleeding time of nonresponders [58] correlated with alternative in vitro platelet function test [59]. However, there is great unpredictability in responses ranging from exaggerated (increased bleeding time) response [59] to paradoxical prothrombotic (decreased) bleeding time in some individuals [60] or lack of correlation with other laboratory tests [61].

4.2.2 The biochemical assays

4.2.2.1 COX-1-dependent assay

During platelet activation, arachidonic acid is metabolized to intermediate prostaglandins in reactions catalyzed by COX-1 mainly, and COX-2 under special conditions. The terminal end products, thromboxane metabolites, are stable and appear in urine and serum. Thus, the measurement of TxA2 metabolite (serum thromboxane-A2) and urinary 11-dehydrothromboxane-B2 (11dhTxB2) [62] reveals activation status. Low-dose aspirin (30–300 mg) specifically blocks the COX-1 pathway leading to low activation sates (low serum/urine thromboxane metabolites). As urine levels of TxB2 usually vary depending on the rate and volume of urine collected, no normal values have been agreed upon [63]. Owing to this, it is recommended that the urinary TXB2 be correlated with creatinine levels, such that nonresponders have TxB2 > 33.8 ng/mmol cr while levels in partial responders are 15.1 to 33.8 ng/mmol cr [64]. The results are influenced by non-COX-1 sources of TxA2 that include thromboxane synthases in monocytes, macrophages and endothelial cells. Furthermore, COX-2 can also produce TxA2 independent of COX-1 [62]. Although aspirin consistently suppresses platelet COX-1 production of thromboxanes in platelets, there is lack of uniformity upon evaluation by functional assays [65].

4.2.2.2 Intraplatelet VASP assay

Vasodilator-stimulated phosphoprotein (VASP) is an intracellular protein whose activity links purinergic P2Y12 receptor occupancy to intracellular signaling leading to GPIIbIIIa activation with attendant fibrinogen binding [66]. During platelet activation, ADP occupancy of platelet P2Y12 receptors leads to VASP dephosphorylation, GPIIbIIIa activation and fibrinogen binding. Conversely, clopidogrel displacement of ADP at P2Y12 receptors triggers VASP phosphorylation (VASP-P) and stabilizes GPIIbIIIa in an inactive resting state that cannot bind fibrinogen [67, 68]. Thus, assays of intracellular VASP-P (flow cytometry or enzyme-linked immunosorbent assay (ELISA)) can estimate the level of platelet inhibition by thienopyrimidine (such as clopidogrel). Platelet VAS-P assay results are presented as platelet reactivity index (PRI) [69]. Since high levels of VASP-P correspond to low PRI [67, 70], there is an almost linear inverse relationship between PRI and level of P2Y12 blockade [68]. Because of the latter relationship, VASP is considered a specific biomarker for thienopyrimidine (e.g. clopidogrel) on purinergic P2Y12-mediated pathways, although it does not indicate the extent of receptor activation or aggregation.

4.2.3 Platelet functional assays

The commonly used clinical platelet functional assays include:

4.2.3.1 Platelet aggregometry

1. Light transmission aggregometry (LTA): It is considered the gold standard reference for platelet function testing stratifying aspirin responders as Aspirin sensitive (AS) or Aspirin resistant (AR). Of the various agonists used, AA has high specificity as it amplifies COX-1 synthesis of TxA2 targeted by aspirin. The cut-off for AR is agonist and dose dependent, but consensus considers the AA concentration at 1.6 mg/mL as appropriate for the investigation of AR [62].

2. Platelet function analyzer-100 (PFA-100™): It consists of passage of whole blood through capillary apertures coated with a combination of either collagen or ADP (Coll/ADP) or collagen and epinephrine (Coll/Epi). When platelets are stimulated with either epinephrine or ADP, they plug the aperture and time to closure (CT) is recorded. Aspirin prolongs the closure time beyond 300 s. It is considered specific for aspirin.

3. VerifyNow™ system: It is a whole blood-based turbidometric method where blood is passed through cartridges coated with a combination of fibrinogen and agonists for pathways to be t-tested for each drug, as in AA for aspirin, TRAP for GPIIbIIIa inhibitors and ADP or PGE1 to be tested for each drug, such that AA for aspirin, TRAP for GPIIbIIIa inhibitors and ADP or PGE1 for purinergic P2Y12 receptor blockers. In this assay, changes in light transmittance correspond to level of platelet aggregation. Platelet adhesion occurs in proportion to available fibrinogen receptors and results reported in aspirin response (aspirin reaction unit (ARU)), the normal value being >550 [64]. It was developed to monitor aspirin effects on platelets.

4. Impedance aggregometry (Multiple electrical aggregometry-MEA™/Multiplate): Impedance aggregometry (Multiple electrical aggregometry-MEA™/Multiplate) uses whole blood sample to measure change in electrical resistance as platelets are activated by agonists. The results are expressed in ohms (Ω) or Area under the curve (AUC) calculated from aggregation units (AUs) after subtracting baseline levels obtained with 0.9% saline (Table 2).

Test	Agonist	Cut-off	ASA cut-off [71]	Clopidogrel cut-off [71]
VerifyNow [62]	ADP, AA, TRAP	PRU > 208	ARU > 550	>240 clopidogrel reaction units
Biochemical serum and urine TxA2 [62]	—	AR urine >1500 pg./mg cr Serum >3.1 ng/mg cr	Urine 11dhTxB2 > 67.9 ng/mmol cr
MEA [62]	ADP, AA, TRAP, collagen	AU > 46; AR > 30%
TEG platelet mapping	ADP	Ma > 47 mm
VASP	PGE1 and ADP	PRI > 50%		N > 70 PRI; Clo > 50%PRI
RPFA [64]	Cationic propyl thiogallate	ARU > 550
PFA-100 [62, 64]	Coll/ADP (CADP); Coll/Epinephrine (CEPI)	CT > (N < 163–190 s); CADP <(69–140 s); CEPI< (80–200 s)	CT(N < 193)
LTA [62, 72]	AA (0.5–1 mg/mL); ADP (10 μmol/L)	≥20% AA; ≥70ADP; ≥70%collagen	>20%	<10% reduction
TEG [72]	Platelet mapping	≥50%AA
P-selectin [73]	Flow cytometry	+ve cells>5%
GPIIbIIIa [74]	Flow cytometry	Log MFI > 220

Table 2.

Laboratory tests in the evaluation of antiplatelet resistance and their cut-off levels.

4.2.4 Flow cytometry

The method uses monoclonal antibodies to identify and quantify platelets’ membrane receptor expression of P-selectin (alpha granule secretion), GPIIbIIIa or fibrinogen binding (PAC-1), glycoprotein Ib-V-IX (GPIb-V-IX) and CD40 ligand (CD40L). These antibodies are used to characterize the extent of pharmacogenetic influence on antiplatelet resistance in combination with gene profiling [75]. Notably, P-selectin expression is less sensitive to aspirin effects in patients [76], as opposed to healthy volunteers [77]. More importantly, patients who require antiplatelet agents, such as for acute coronary syndrome and ischaemic stroke, already have preexisting increased platelet expression that is not abrogated by aspirin or clopidogrel [78]; thus, it is a less sensitive indicator of antiplatelet resistance.

4.2.5 Global coagulation assays

Thromboelastography (TEG) estimates platelet functions in whole blood in the presence of other cellular elements including red blood cells (RBCs) and leucocytes thus considered more physiological. A modified technique, platelet mapping, defines the contribution of platelets to clot strength using AA or ADP. In comparison with other tests, it is less precise [79] and overestimates platelet resistance [80].

4.2.6 Comparison of methods

Generally, near-patient testing methods, such as PFA-100, VerifyNow™ and Impedance Aggregometry, are preferred for clinical testing [81]. Since antiplatelet drugs block diverse signaling pathways, the recommended agonists for pathways testing include AA for aspirin and ADP for thienopyrimidine (e.g. clopidogrel) [82]. While it has been observed that platelets are resistant to aspirin, they exhibit hypersensitivity to low dose or submaximal doses of agonists on a dose-response curve [83].

While it is generally agreed that some individuals exhibit suboptimal response to antiplatelet medication, there is no consensus on laboratory investigation since various methods have been employed. Similarly, there is disagreement on the interpretation of laboratory test results that report increased platelet activation during treatment and linkage to clinical events for clinical decision-making is still debatable [84]. Many clinicians prefer the term ‘high on treatment platelet reactivity’ [85], equivalent to decreased effectiveness [86]. High on treatment platelet reactivity (HTPR) is a screening tool to estimate platelet functions while on treatment. However, the various methods give different incidence/prevalence values of aspirin resistance. Furthermore, when compared to each other, there is wide heterogeneity and lack of concordance with biochemical tests and therefore are not interchangeable [87]). Notably, optical aggregometry (LTA), VerifyNow and PFA-100 each gives different values for the prevalence of aspirin resistance in patients with ischaemic stroke [88]. However, they neither showed reproducibility nor correlation/agreement with one another [79]. Overall, the available evidence shows that TEG-PM is least suitable [80], whereas Multiplatelet Impedance method serves as the most reproducible, acceptable reliable method among healthy donors and patients for monitoring antiplatelet medications [80]. Moreover, test results vary on a temporal scale depending on the timing of blood collection from the onset of treatment [80]. Given that drug responses follow a bell-shaped curve, the interindividual responses on the platelet function tests could plausibly be a reflection of normal biological distribution on either side of the curve but not true resistance [80]. The differences in responses could arise from alternative activation pathways not targeted by the drugs tested [80].

4.3 Mechanisms of antiplatelet resistance

Factors contributing to the resistance mechanisms arise from changes at the clinical, cellular and genetic levels [89]. In the case for aspirin, the mechanisms are characterized as Thromboxane dependent or independent, while the diverse clopidogrel mechanisms encompass genetic polymorphism of purinergic receptors. The modulating factors act at the level of pharmacokinetic/pharmacodynamic resistance, platelet properties, postreceptor signaling and metabolic conditions such as diabetes mellitus, insulin sensitivity and obesity [64].

Pharmacokinetic resistance occurs when, despite ingestion of adequate doses of aspirin, there is failure of inhibition of COX-1 owing to inadequate plasma levels, malabsorption or genetic polymorphism [90]. Pharmacodynamic resistance is experienced when thromboxane production continues despite adequate COX-1 inhibition, due to COX-2 from other sources [90]. Pseudo-resistance is characterized by the total inhibition of thromboxane but platelet activation still occurs through thromboxane-independent pathways such as ADP, epinephrine or thrombin [90].

The proposed biological basis for aspirin resistance [91] includes the following points: (i) aspirin-insensitive TxA2 biosynthetic pathways via inducible COX-2 or regenerated COX-1 from other cells such as macrophages, monocytes and endothelium; (ii) alternate platelet activation pathways arising from increased sensitivity to collagen, catecholamine surges (exercise, stress), non-TxA2-mediated (e.g. ADP, thrombin and platelet activating factor (PAF)); (iii) prostaglandin-like compounds (lipid peroxidation); and (iv) vascular inflammation due to increased expression of CD40L.

4.4 Management of antiplatelet resistance

Although there is a growing body of evidence regarding resistance to antiplatelet agents in the general population and in PLWH in particular, no consensus has been formed on the appropriate laboratory method to detect it, or whether laboratory evaluation should be routinely performed in clinical practice. Furthermore, it is still unclear whether treatment failures are due to drug resistance, or how the drug resistance results translate to clinical outcomes. Queries regarding PLWH on antiplatelet therapy have been raised (on whether the right agents are being used either singly or in combination) [92]. Nevertheless, the general practice is to increase the dosage, while balancing against the risk of adverse drug effects, especially gastrointestinal (GI) bleeding. Alternatively, adding another antiplatelet (dual therapy), which acts through a different pathway, may be an option [93]. Alternatively, a switch to another agent with less resistance, or adding a third antiplatelet agent, may be considered.

5. Risk factors for antiplatelet resistance in HIV

5.1 cART interactions as contributors to antiplatelet resistance in HIV

Human immunodeficiency viral infection and some cART contribute to platelet hyperactivity [94]. In the EVERE₂ST-HIV study, patients receiving cART with PIs and other combinations had increased platelet reactivity to P2Y12 inhibitors and higher prevalence of HPR, consistent across all functional tests (RPA, VASP-PRI and PRU). Conversely, patients treated with NNRTIs had consistently decreased platelet reactivity and lower prevalence of HPR [95]. According to this study, resistance was consistent for aspirin across all cART, but variable for P2Y12 inhibitors.

In other studies (e.g. ATP-binding cassette (ABC)-treated patients) though, aspirin reduced urinary and serum Thromboxane A2 and AA induced platelet aggregation on LTA, indicating treatment compliance. Other biomarkers remain above cut-off levels of reactivity and did not normalize compared to healthy uninfected controls [35], indicating suboptimal response or resistance to therapy.

The mechanism of cART-induced platelet hyperreactivity includes induction of MetS [96] and modulation of extra-platelet enzymatic degradation of nucleotides promoting feedback activation by ADP [97]. cART also contributes to suboptimal antiplatelet effects through pharmacokinetic drug interactions. PIs and NNRTIs are strong inhibitors of hepatic cytochrome P450 3A (CYP3A) enzymes, diminish clopidogrel and prasugrel bioactivation and decrease the bioavailability of the active drug metabolite, thereby decreasing antiplatelet effects [92, 98].

5.2 Other concomitant drug (proton pump inhibitor (PPI), statins, antifungals and over-the-counter nonsteroidal anti-inflammatory drugs (OTC NSAIDs) interactions and antiplatelet resistance in HIV

There is potential for drug interactions in PLWH, owing to the intake of multiple drugs.

Many PLWH are on proton pump inhibitors (PPIs) for bleeding prophylaxis or to counteract the effects of antiplatelet or nonsteroidal anti-inflammatory drugs (NSAIDs). Coronary artery disease (CAD) patients treated with daily oral 75 mg aspirin together with PPI exhibit increased platelet aggregation than those not on PPI [99], indicating suboptimal response to aspirin. Similarly, omeprazole decreases antiplatelet effects of clopidogrel as evaluated by VASP PRI [100]. Clinically, patients on aspirin or clopidogrel plus PPI have increased incidence of major adverse cardiovascular events [101, 102]. By acting on platelet cyclooxygenase, over-the-counter treatment (OTC) NSAIDs displace aspirin from its active site, thus decreasing its effectiveness [103].

5.3 Concomitant infections and antiplatelet resistance in HIV

Concomitant bacterial, viral, fungal and mycobacterial (e.g. tuberculosis) infections are common in PLWH. Early in sepsis, platelet activation and aggregation are enhanced compared to healthy controls with variation in levels observed between Gram-positive and Gram-negative bacteria [104, 105]. Whereas agonist-induced aggregation deteriorates with progression of sepsis severity [106, 107, 108, 109], activation biomarkers as demonstrated by the expression of adhesion molecules (cluster of differentiation 42a (CD42a), cluster of differentiation 42b (CD42b), cluster of differentiation 36 (CD36), cluster of differentiation 29 (CD29) and PAR-1) [110] and alpha granule secretion (P-selectin) [105, 111] remain unchanged, or increased [112]. However, when platelet count is taken into account, aggregation and activation status correlate [113] with the overall phenomenon of increase in platelet reactivity with sepsis severity [114]. Microorganisms and their products such as lipopolysaccharides interact with platelets directly through TLR, and indirectly via bridging proteins that include fibrinogen, fibronectin, von Willebrandt factor (vWF) and thrombospondin through platelet receptors such as GPIIbIIIa and GPIa-IX-V [115].

5.4 Metabolic syndrome/hyperlipidaemia complications and antiplatelet resistance in HIV

Metabolic syndrome (MetS), characterized by insulin resistance, visceral adiposity, atherogenic dyslipidaemia and endothelial dysfunction [31], occurs in PLWH partly due to HIV, and due to some cART [2, 116]. The prevalence of MetS in PLWH could be as high as 21% [3]. MetS is associated with exaggerated platelet function [117] and the suboptimal response to aspirin in up to 69% patients [118, 119, 120]. The decreased response to aspirin in MetS may be due to hyperlipidaemia, inflammation (high hs-CRP) [121] and increased platelet turnover that releases large reticulocytes into circulation [122] with enhanced expression of COX-2 [123] being less sensitive to aspirin [124, 125]. Furthermore, apoptotic platelet changes induced by HIV [126], hyperlipidaemia [127] and aspirin [128] mimic laboratory characteristics of antiplatelet resistance. The independent factor contributing to platelet hyperactivity in MetS appears to have increased hs-CRP, a marker of inflammation [129].

5.5 Deficiencies of vitamin D and other micronutrients as risk factors for antiplatelet resistance in HIV

Vitamin D deficiency is a common finding in HIV infection [130, 131]. The said deficiency is associated with platelet activation [132] and antiplatelet resistance [133]. In vitamin D deficiency, platelet hyperactivity is triggered by oxidative stress [134] and immune activation [135, 136]. These factors increase MetS-linked endothelial activation [137] and cause platelet-endothelial adhesive protein vWF release [138] leading to activation via GPIb-IX-V receptors by bypassing the aspirin-inhibited pathway, thus enhancing the risk of thrombosis.

Magnesium contributes to antiplatelet effects [139, 140] by: (i) interfering with fibrinogen binding of GPIIbIIIa due to induced membrane alterations, (ii) competing with membrane transporters of Ca++, and (iii) intracellular signal transduction pathways that promote Ca++ influx and mobilization from stores [140, 141] (e) decreasing thromboxane biosynthesis [142, 143]. Also, hypomagnesaemia produces oxidative stress and increased risk of cardiovascular events [144].

Although diet has not been specifically investigated in relation to antiplatelet resistance in HIV, there is strong justification for its contribution as a risk factor. Diets rich in micronutrients and vitamins, such as practiced among Mediterranean population, are reputed to lower cardiovascular risk factors due to antiplatelet effects [145]. It is speculated that individuals who do not partake these diets may be at risk of micronutrient deficiencies and rebound platelet hyperactivity.

5.6 Inflammation, megakaryopoiesis/reticulated platelets in HIV

Human immunodeficiency viral (HIV) infection is characterized by high levels of inflammation. Elevated inflammatory biomarkers, such as hs-CRP, tumor necrosis factor α (TNF-α) and phospholipase 2 (PLA2), are associated with platelet hyperactivity and aspirin resistance [146, 147, 148]. Inflammation contributes to aspirin resistance through several mechanisms including initiation of COX-1-independent platelet activation mechanisms, acceleration of platelet turnover and thrombopoiesis, generation of reticulated platelets rich in COX-2, generation of reactive oxygen species (ROS) and promotion of the expression of surface adhesion molecules, such as P-selectin, GPIIbIIIa and CD40L [89]. vWF, a platelet-vascular adhesive molecule released during inflammation [149], is a biomarker elevated in HIV [150, 151]. It contributes to aspirin resistance [146] through platelet activation via glycoprotein Ib (GPIb) receptor [152]. The signaling pathways are independent of COX-1-mediated thromboxane. Immune complexes and complements bind to platelets and activate platelets through immunoreceptor tyrosine-based activation motif (ITAM) signaling pathways [153, 154].

People living with HIV have enhanced megakaryopoiesis characterized by increased immature platelet reticulocytes in circulation (up to 10%) compared to control non-HIV [155, 156]. Although immature platelets display increased agonist-stimulated aggregation and membrane activation receptor expression [157], these responses persist, despite antiplatelet therapy [125], consistent with resistance to medication. Antiplatelet resistance arises due to increased expression of COX-2 during maturation [158] that is neither inhibited by aspirin nor inhibited by clopidogrel [124].

5.7 Gut microbial translocation and platelet hyperactivity in HIV

HIV virus is highly predisposed to localize in the gut epithelial lining, and CD4 and CD8 (cluster of differentiation 8) lymphocytes in underlying Peyer’s patches and lymph nodes. The subsequent epithelial destruction leads to loss of barrier functions, resulting in ‘leaky gut’ and passage of gut microbiota into the systemic circulation, a process termed ‘gut microbial translocation’ [159]. The translocated bacteria, and their products, such as lipopolysaccharides (LPS) and Trimethylamine N -oxide (TMAO), interact with platelet Toll-like receptor-4 (TLR-4) contributing to platelet hyperactivity [160]. Although it has been demonstrated that in non-HIV, acute coronary patients with translocated gut microbiota have platelet hyperactivity despite being on antiplatelet agent ticagrelor [161], no data on antiplatelet resistance are available for PLWH.

5.8 Racial and genetic polymorphisms in antiplatelet resistance and HIV

People of African descent have higher rates of vascular ischaemic events compared to other racial groups, a phenomenon that persists even after the administration of antiplatelet medications [162]. Genetic polymorphism disaggregated based on race arises in response to antiplatelet medications [163]. African Americans exhibit clopidogrel HTPR as assessed on VerifyNow (PRU) and VASP (PRI) compared to Caucasians, which corresponds to a higher prevalence of CYP2C19*2 allele carrier status [164]. The response is also reflected in LTA and VerifyNow testing patients on aspirin and clopidogrel at baseline and on stimulation with other agonists at different concentrations [165]. The differences could be traced to genetic polymorphism to GPIIbIIIa and P2Y12 receptors [166]. Plausibly, genetic polymorphism could explain the antiplatelet resistance reported in a study of PLWH where a majority of them were people of black ancestry [36]. However, it must be emphasized that although data suggest that the people of African descent are likely more resistant to antiplatelets compared to other races, the reality is that less than one fifth of those who qualify for and could potentially benefit actually are prescribed for and take antiplatelets [167].

6. Guidelines for antiplatelet therapy

6.1 Guidelines for antiplatelet therapy in HIV

Globally, incidences of noncommunicable diseases (NCDs) are on the rise [168], partly due to HIV infection [169]. For the primary prevention of cardiovascular events, the US Preventive Task Force (USPTF) recommends the calculation of risk assessment and initiation of antiplatelet therapy to those who meet the criteria [170]. Despite the unique risk factors for cardiovascular events in HIV [32], no specific guidelines exist for antiplatelet therapy for HIV-specific cardiovascular preventive strategies, despite the concerns raised [171]. Unfortunately, many guidelines have not incorporated focused antiplatelet therapy for at-risk PLWH, despite thrombosis being reported in many case series in patients not on aspirin [172]. Despite the known cardiovascular risks in HIV, antiplatelets are grossly underutilized in PLWH [173, 174, 175]. Even in centres where antiplatelets are prescribed, disparities in prescription and quality of care are rampant [176], resulting in high rates of adverse cardiovascular events among PLWH [177]. Whereas the potential for drug-to-drug interactions between cART and antiplatelets is real, a study found aspirin and clopidogrel dual therapy continues to be prescribed alongside PI and NNRTI in coronary artery disease, resulting in 100% adverse interactions [92].

6.2 Guidelines for laboratory testing of antiplatelet resistance

Although it is clear that antiplatelet resistance is glaring, most professional societies’ guidelines do not consider that there is sufficient evidence to routinely perform the laboratory evaluation of antiplatelet resistance to justify altering prescribing practice based on platelet function testing results [62]. The role of routine aspirin or any other antiplatelet therapy in primary prevention of cardiovascular events is increasingly being questioned, since it only targets 25% of cardiovascular events, the rest being from other causes [178]. Thus, the emergence of drug resistance or suboptimal response introduces a new dimension in the conundrum of care for cardiovascular diseases in general, and HIV in particular. Given the escalating burden of cardiovascular diseases, and the role of platelets in pathophysiology, there is a burgeoning paradigm shift away from the traditional approach of risk stratification, antiplatelet therapy and platelet function testing for research purposes. The current direction is a personalized approach, whereby individual platelet phenotypes are evaluated by alternative means and targeting therapy [179]. Currently, it is increasingly becoming clear that there is a lack of equipoise in testing and therapeutic practice, instead, further studies are recommended to guide practice.

7. Conclusion

The incidence of major cardiovascular events, including acute coronary syndromes and ischaemic stroke, is elevated in PLWH and hyperactive platelets are implicated. The risk factors for hyperactive platelets include HIV, cART and the attendant metabolic syndrome. Although guidelines recommend antiplatelets such as aspirin, the problem of their resistance in HIV is greater than in the general population, which predisposes to greater morbidity and mortality risk. Identification of drug resistance in laboratory functional assays is challenging, owing to the different definitions of the phenomenon and the limitations of correlating it with clinical observations. It is emerging that the contributing factors to platelet hyperactivity and related antiplatelet drug (e.g. aspirin) resistance are the interplay of concurrent inflammation, micronutrient deficiencies and drug interactions acting at both pharmacokinetic and pharmacodynamic levels. Although many guidelines have been developed for antiplatelet medications in cardiovascular disease management, none is specific for HIV. Despite effective uptake of cART globally, the issue of antiplatelet medication in PLWH is a conundrum. The population most affected by HIV and concurrent MetS who meet the criteria for antiplatelet medications are not getting it. The reasons are unclear, but may include the lack of awareness and screening for MetS, not following the guidelines with regard to problems in polypharmacy. Even in cases where antiplatelets are prescribed, the emergence of antiplatelet resistance hampers effective primary prevention or treatment of cardiovascular events in PLWH.