Variability in Individual Responsiveness to Aspirin - Ingenta Connect

Cardiovascular & Haematological Disorders-Drug Targets, 2007, 7, 274-287

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Variability in Individual Responsiveness to Aspirin: Clinical Implications and Treatment Isabel Coma-Canella* and Amelia Velasco Department of Cardiology and Cardiovascular Surgery, University Hospital of Navarra, School of Medicine, University of Navarra, Pamplona, Spain Abstract: Aspirin protects from cardiovascular events because of its antiaggregant effect. The occurrence of new events in patients who take aspirin has been called clinical aspirin resistance. Many authors believe that aspirin resistance must be detected by biochemical tests, although there is no agreement on which is the best. Nor is there agreement on the term aspirin resistance. Tests used in research laboratories are aggregometry (turbidometric and impedance), tests based on activation-dependent changes in platelet surface, and tests based on activation-dependent release from platelets. Point-ofcare tests are PFA-100, IMPACT and VerifyNow, which can detect platelet dysfunction that may be due to aspirin effect, but their use for this purpose is not yet recommended. Aspirin response may be modified by different factors: patient’s compliance, dose, smoking, hyperlipidemia, hyperglucemia, acute coronary syndrome, percutaneous revascularization, recent stroke, extracorporeal circulation, heart failure, exercise, circadian rhythm, absorption, concomitant medications, polymorphisms. Patients with aspirin resistance may have an increased risk of cardiovascular events, and possible therapeutic options are to increase the dosage, to replace aspirin with another antiaggregant drug or to add another drug. In conclusion, there are many reasons that explain the variability in individual responsiveness to aspirin. The term resistance is probably not exact in describing this phenomenon.

Key Words: Aspirin, cyclooxigenase, thromboxane A2, drug resistance. INTRODUCTION

Platelet Adhesion

Acetylsalicylic acid or aspirin is probably the simplest drug available in Cardiology. It can be given orally, once a day, is effective and cheap, and so, very cost-effective. Aspirin is considered the gold standard of antithrombotic therapy in the prevention and treatment of cardiovascular diseases. The use of aspirin is associated with a 22% reduction in myocardial infarction, stroke, and vascular death [1]. However, some patients do not respond adequately, and they are considered to be “resistant” to the antiaggregant effect of aspirin. The purpose of the present review is to analyze the concept of aspirin resistance or the variability in individual responsiveness to aspirin. The first part, written for clinicians, is a simple explanation of platelet function and the mechanism of aspirin-action. This may facilitate the understanding of the different reasons why aspirin may not work properly. The main causes of ineffectiveness are reviewed, as well as alternative treatments for those patients who do not respond adequately to the antithrombotic effect of aspirin.

The first step in the formation of a plug or thrombus is platelet adhesion which occurs when the endothelium is disrupted. At the site of vascular lesions, circulating von Willebrand factor (vWF) binds to the exposed collagen, which will subsequently bind to the glycoprotein (GP) Ib/IX/V receptor on the platelet membrane [2]. (Fig. 1) Under pathological conditions and in response to changes in shear stress, vWF can be secreted from the storage organelles in platelets or endothelial cells, reinforcing the activation process [5].

PLATELET FUNCTION

Fig. (1). in normal conditions the endothelium is formed by a layer of cells, and the platelets circulate freely in the blood as discoid cells. In the case of endothelial disruption (mainly due to atherosclerosis), the platelets become adhered to the subendothelial collagen by means of glycoprotein (GP) Ia receptors and to von Willebrand Factor (vWF) by GP Ib/IX/V receptors.

The physiological function of platelets is the formation of the haemostatic plug. Platelets play a critical role not only in the physiological haemostasis, but also in the vascular repairing process after endothelial dysfunction. After activation, platelets adhere to the areas of endothelial dysfunction to form a plug, and at times form a luminal thrombus, a pathological process [2-4]. *Address correspondence to this author at the Departamento de Cardiología, Clínica Universitaria de Navarra, Avenida de Pio XII 36, 31008 Pamplona, Spain; Tel: 948 296387; Fax: 948 296500; E-mail: [email protected] 1871-529X/07 $50.00+.00

PLATELET

vWF vWF

endothelium VESSEL WALL

collagen

GP Ib/IX/V

Platelet Activation Platelets can be activated by adhesion to the arterial wall or by interacting with circulating agents such as epinephrine, thrombin, serotonin, thromboxane (TX)A2 and adenosine diphosphate (ADP), via specific platelet surface receptors. When they are activated there is a change in platelet shape © 2007 Bentham Science Publishers Ltd.

Variability in Individual Responsiveness to Aspirin

Cardiovascular & Haematological Disorders-Drug Targets, 2007, Vol. 7, No. 4 275

(Fig. 2) and calcium translocation within the platelet. Platelet activation induces phospholipase A2 activation that triggers TXA2

ADP

thrombin

plasmin epinephrine

serotonin Inactivated I ti t d platelet

collagen shear forces

fibrinogen platelet activating factor degranulation

eration. TXA2 is a very potent platelet-aggregating agent located in the alpha granules of the platelet, which increases the expression of fibrinogen receptors on the platelet membrane and is released into the circulation. TXA2 binds to TX receptors on the surface of adjacent platelets to trigger their activation (Fig. 4), and also acts synergistically with other products released by activated platelets (such as ADP, fibrinogen, and factor V) to further enhance platelet aggregation. When calcium is increased, there is a conformational change of the GP IIb/IIIa receptors on the platelet surface (Fig. 4).

F

ADP FV TXA2 FR

FR

Activated platelets

Fig. (2). Platelets may be activated not only by collagen but also by other factors as depicted in the figure. Once activated, there is a conformational shape change that allows a great enlargement of the membrane surface. TXA2: thromboxane A2. ADP: adenosine diphosphate.

the conversion of membrane-bound phospholipids to arachidonic acid (Fig. 3). Platelet cyclooxigenase (COX) enzymes catalyze the conversion of arachidonic acid to prostaglandin

PLATELET ACTIVATION Phospholipase A2 activation Membrane Phospholipids

COX-1 (pl+EC)) (p

COX-2 (EC) ( )

PGI Synthase (EC+GM) (EC GM) peroxidase PGG2

PGH2

GP IIb/IIIa R

Fig. (4). After activation, platelet alpha granules liberate thromboxane (TX)A2, adenosine diphosphate (ADP), fibrinogen, factor V, etc. TXA2 increases the expression of fibrinogen receptors (FR) on the platelet membrane and binds to TX receptors (TXR) on the surface of adjacent platelets to trigger their activation. There is also external expression of glycoprotein (GP) IIb/IIIa receptors (R) on the platelet surface.

Platelet Aggregation

Arachidonic Acid

Cyclooxigenase or PGG/H Synthase

TXR

GP IIb/IIIa receptors bind to adhesive proteins, mainly fibrinogen (and also circulating vWF), promoting plateletplatelet interaction or aggregation (Fig. 5).

PGI2 TXA2

TX Synthase (pl) TXB 2

Fig. (3). When the platelet is activated there is activation of the enzyme phospholipase A2. This enzyme catalyzes the conversion of membrane phospholipids to arachidonic acid. The enzyme cyclooxigenase (COX) or PGG/H synthase has two isoforms: COX-1, usually present in platelets (pl) and endothelial cells (EC) and COX-2, usually absent in pl but present in EC. COX allows the conversion of arachidonic ancid into prostaglandin (PG)G2 and the enzyme peroxidase catalyzes the conversion of PGG2 into PGH2. The enzyme PGI syntase, usually present in EC and gastric mucosa (GM) catalyzes the conversion of PGH2 into PGI2 or prostacyclin. The enzyme thromboxane (TX) synthase, present in platelets, catalyzes the conversion of PGH2 into TXA2. The active metabolite of TXA2 in blood is TXB2.

(PG) G2/H2. Thromboxane synthase enzyme catalyzes the conversion of PGH2 to TXA2, which plays a role in platelet aggregation, vasoconstriction and smooth muscle cell prolif-

GP IIb/IIIa R

F F vWF

Fig. (5). Fibrinogen and von Willebrand Factor (vWF) links activated platelets through glycoprotein (GP) IIb/IIIa receptors (R), causing platelet aggregation.

THE MECHANISM OF ACTION OF ASPIRIN Inhibition of TXA2 Aspirin works by acetylating the serine moiety at position 529 of COX-1 and thereby irreversibly inhibits the key

276 Cardiovascular & Haematological Disorders-Drug Targets, 2007, Vol. 7, No. 4

enzyme required for the production of TXA2 in platelets (Fig. 3). The aspirin-induced inhibition of platelet COX-1 acts by blocking the access of arachidonic acid through a narrow hydrophobic channel to the catalytic site in the core of the enzyme molecule [6]. Because platelets do not have nuclei, they cannot synthesize new COX-1 and are, therefore, permanently inhibited by aspirin. The inhibition lasts for the lifetime of the platelet, approximately 7 days [7]. The COX-1 isoform, present in all tissues, represents the constitutive form of the enzyme, and is expressed in inflammatory states in response to inflammatory stimuli [8], oxygen reactive species, endotoxins [9], cytokines, or growth factors [10]. COX-2 can be found in human atherosclerotic plaques [11] and also in small amounts in newly formed platelets [12-13]. The number of COX-2–expressing platelets may increase in conditions of high platelet regeneration [14]. This enzyme is also responsible for generation of PGI2 in the endothelium, but not in platelets [15] (Fig. 6). Platelet COX-1 Permanent inhibition Transient inhibition inhibition

AA ii i Aspirin EC COX-1

TXA2

platelets

no inhibition EC COX-2

vessels

PGI PGI 2 PGI2 is produced at daily low doses of aspirin

Fig. (6). When aspirin is given in daily low doses, it produces permanent inhibition of COX-1 in the platelets (because they have no nuclei) and therefore there is no synthesis of (TX)A2. However, the endothelial cells (EC) are nucleated and can resynthesize COX-1 after inhibition of this enzyme by aspirin. In addition, endothelial COX-2 is insensitive to low doses of aspirin. Therefore, PGI2 is produced in spite of aspirin.

COX-1 is responsible for the production of prostacyclin (PGI2) in the vessel wall and the gastrointestinal tract. Aspirin also blocks the production of PGI2 in the vessel wall [16], where it acts as antithrombotic and vasodilator, and in the gastric mucosa, where is responsible for maintaining mucosal integrity. However, inhibition of COX in these tissues is reversible. In vessels, the antithrombotic effects of TX inhibition predominate over the possible prothrombotic effects of PGI2 inhibition [17]. TXA2 is largely a COX-1-derived product (mostly from platelets). Therefore TXA2 is highly sensitive to aspirin inhibition under physiologic conditions. Vascular PGI2 can derive from both COX-1 (sensitive to transient aspirin inhibition) and to a greater extent from COX-2 [18] (largely insensitive to aspirin inhibition at conventional antiplatelet doses). This may account for the substantial PGI2 biosynthesis in vivo at daily low doses of aspirin, despite transient suppression of COX-1-dependent PGI2 release [19] (Fig.6).

Coma-Canella and Velasco

Additional Antithrombotic Effects of Aspirin • Reduction of thrombin generation with the subsequent attenuation of thrombin-mediated coagulant reactions such as factor XIII activation [20]. • Acetylation of lysine residues in fibrinogen, resulting in increased fibrin clot permeability and enhanced clot lysis, as well as directly promoting fibrinolysis at high-doses [20]. • Inhibition of platelet aggregation by neutrophils and antioxidant effects [21-23]. • Inhibition of inflammation and thrombosis through blockade of sCD-40L release [24]. Aspirin-treated platelets can still aggregate in response to potent agonists such as collagen or thrombin, at the site of vascular injury [2,4]. Pharmacokinetics of Aspirin Aspirin is a weakly acidic drug (optimum absorption in the human stomach occurs in the pH range of 2.15 to 4.10) that crosses the mucosa of the stomach and upper intestine in its lipophilic state. Aspirin achieves peak blood concentrations within 30–40 minutes after ingestion of a soluble formulation. Part of the drug is hydrolysed by abundant mucosal esterases (in the stomach and intestine) to salicylic acid, its inactive form [25]. Aspirin has a short half-life (15 to 20 min) in human circulation and is approximately 50-fold to 100-fold more potent in inhibiting COX-1 than COX-2 [26]. Once-a-day dosing will maintain virtually complete inhibition of platelet TXA2 production (Fig. 6). In contrast, the inhibition of COX2-dependent pathophysiologic processes (i.e., hyperalgesia and inflammation) requires larger doses of aspirin and a much shorter dosing interval because nucleated cells rapidly resynthesize the enzyme. Thus, there is an approximately 100-fold variation in the daily doses of aspirin when used as an anti-inflammatory rather than as an antiplatelet agent [27]. After a single 325mg dose of aspirin, platelet COX-1 activity recovers by about 10% per day due to new platelet formation. Production of TXA2 is completely inhibited by daily doses of aspirin as low as 30 mg [28]. This explains why regular low doses of aspirin suppress more than 95% of TXA2 generation after several days’ medication. Although it may take 10 days for the total platelet population to be renewed, hemostasis can be restored when 20% of the platelets have normal COX activity. ASPIRIN RESISTANCE This concept was proposed in the 1990s [29] because some patients suffered untoward cardiovascular events while taking aspirin. To date there is no agreement on its definition, but one possible definition is the occurrence of serious vascular events despite the use of recommended doses of aspirin [30], which could be termed clinical resistance. Another definition is the inability of aspirin to inhibit platelet thromboxane formation [31], or to produce an anticipated effect on one or more platelet function tests [32]. This could



be termed biochemical or laboratory resistance. Sometimes there is apparent resistance, due to lack of patient’s compliance, which can lead to an overestimation of the problem. Clinical Resistance Clinical resistance could also be called treatment failure. Just as with any other drug, patients differ in their clinical response to aspirin. The drug only blocks one of the multiple pathways involved in platelet activation so platelets can still become activated by other mechanisms. Considering that the nature of atherothrombosis is multifactorial, platelet-mediated thrombosis may not be responsible for all vascular events. It is not surprising that only a fraction of all vascular complications can be prevented by a single drug. If the term “clinical resistance” to aspirin were correct, the same term could also be applied to any drug that fails to achieve its clinical goal. For instance, it could be applied to betablocking drugs and nitrates when they fail to avoid angina. Biochemical Resistance The effects of some drugs, such as statins can be measured objectively through analytical tests. If a patient has the same levels of cholesterol before and after taking a statin for a given time, we could speak of statin resistance, or lack of effect of the drug. However, if a patient has a new cardiovascular event after reaching a very low cholesterol level with a statin, we cannot blame statin resistance as the reason for the event, because the drug is working properly. Some patients have multiple cardiovascular events despite very low cholesterol level, because cholesterol is not the only risk factor involved in the atherothrombotic process. In the same way, we could speak of aspirin resistance only when a proper test detects a lack of aspirin-antiaggregant effect. Therefore, the term aspirin resistance, if accepted, should be a biochemical concept, not a clinical one. Although the term aspirin resistance is debatable and some people criticize it [33], hundreds of articles have been published on this topic during the last decade. The majority of authors agree on the need for further research to clarify this matter. The nomenclature should probably be changed on the basis of new knowledge. However the most important matter is to understand the mechanisms of ineffectiveness, the best way of measuring aspirin effect, and the adequate blood levels for improved clinical results. It is also important to know in what ways therapy should be altered in patients with insufficient response. There is need for a suitable test to know the antiaggregant effect of aspirin in a given patient. Point-of-care assays of platelet function may improve the ability of clinicians to modify therapy according to the response. A possible mistake with the tests available is to categorize aspirin resistance as a dichotomous response, when it is more likely to be a continuous variable, similar to blood pressure or cholesterol level. Prevalence of Aspirin Resistance The prevalence of aspirin resistance is estimated to range from 5.2% to 60%, depending on the population studied and

platelet-function assay used. A frequency of 5.2% was calculated in patients with cardiovascular disease but without a recent clinical event [34], and about 13% in the Antithrombotic Trialists' Collaborative study [1]. Another study reported the incidence of aspirin resistance as 60% in patients submitted to peripheral arterial angioplasty [35]. Many other studies have found an intermediate proportion between these extreme figures [36]. Mechanism of Aspirin Resistance The mechanism of aspirin resistance remains uncertain and is probably multifactorial. Proposed mechanisms of “true aspirin resistance” [37] are increased isoprostane activity (derived from the free radical oxidation of arachidonic acid), platelet hypersensitivity to agonists, increased COX-2 activity in newly formed platelets [12,14] and the presence of different polymorphisms. In cases of increased platelet turnover, the proportion of newly formed platelets is augmented and COX-2 activity is higher than normal. Another possible mechanism is transcellular formation of TXA2 from PGH2 released by other blood cells or vascular cells (monocytes and macrophages) [38]. Although TXA2 may be fully inhibited by aspirin, platelets can still become activated by COX-independent mechanisms, such as ADP, collagen and thrombin [39,40]. In addition, cell-cell contact between activated platelets and erythrocytes or neutrophils may modulate the effect of aspirin on platelets [41,42]. This explains why total inhibition of TXA2 by aspirin is compatible with normal platelet aggregation. Some causes of “clinical aspirin resistance” are not due to “true resistance” but to decreased bioavailability of aspirin. This can be due to competition of aspirin with other nonsteroidal anti-inflammatory drugs (NSAIDs) [43,44], poor patient compliance, or low aspirin absorption. In case of noncompliance we can speak of pseudo-resistance, whereas in the other cases the term ineffectiveness could be more appropriate. If the lack of biochemical response to aspirin correlates with unfavorable clinical outcome, testing the biochemical effect of aspirin would be clinically meaningful. Although more research is necessary to recommend this practice, it is interesting to be acquainted with the available tests for quantification of the antiaggregant effect of aspirin. LABORATORY TESTS TO DETECT ASPIRIN RESISTANCE An official communication of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis lists the different tests available to measure aspirin resistance [45]: 1. Bleeding time consists of in vivo cessation of blood flow by a platelet plug. It is a physiological test but insensitive and with high inter-operator coefficient of variation. 2. PFA-100 is based on in vitro cessation of high shear blood flow by a platelet plug. The test is simple and quick, needs a low sample volume, no sample preparation and uses whole blood at high shear.


3. IMPACT (cone and platelet analyzer) is based on shearinduced platelet adhesion. It has the same advantages as the PFA-100 but the instrument is not yet widely available. 4. Aggregometry in response to arachidonic acid and ADP (turbidometric) is based on platelet-to-platelet aggregation and is widely available. It needs high sample volume, sample preparation and intensive labor. 5. Aggregometry in response to ADP and collagen (impedance) is also based on platelet-to platelet aggregation. It is a whole blood assay, needs a high sample volume and is time-consuming. 6. VerifyNow (Ultegra RPFA), with arachidonic acid or propyl gallate cartridge is based on platelet-to-platelet aggregation. The test is simple and quick, needs a low sample volume (whole blood) and does not need sample preparation. 7. Tests based on activation-dependent changes in platelet surface: platelet surface P-selectin, platelet surface activated GPIIb/IIIa or leukocyte-platelet aggregates in response to arachidonic acid (flow cytometry). They need a low sample volume of whole blood, sample preparation, flow cytometer and an experienced operator. 8. Tests based on activation-dependent release from platelets: serum TXB2, arachidonic acid or collagen-induced platelet TXA2 production, as measured by TXB2 or urinary 11-dehydro TXB2. All three are directly dependent on aspirin’s target COX-1 and reflect in vivo TX production. They need sample preparation and are time-consuming. Urinary 11-dehydro TXB2 depends on renal function and there is potential contribution by cells other than platelets. The majority of these tests are operator-dependent and time-consuming, so cannot be used in clinical practice. Point-of-care tests are much simpler and have been approved by the FDA to measure the antiaggregant effect of aspirin. However, the International Community still does not recommend the routine measurement of aspirin effect until further research clearly demonstrates its usefulness [27,45]. Platelet Function Analyzer (PFA-100) PFA-100 was originally developed by Kratzer & Born to measure platelet adhesion, activation and aggregation under conditions of high shear [46]. The prototype instrument (Thrombostat-4000) has been replaced by the commercially available PFA-100 (Dade-Behring, Marburg, Germany). The FDA has approved PFA-100 to detect platelet dysfunction, von Willebrand disease, and aspirin-induced platelet inhibition. A citrated whole-blood sample is applied to a disposable cartridge containing a membrane coated with either collagen/epinephrine (CEPI) or collagen/ADP (CADP) with a microscopic (147m) aperture. Under high shear rates (50006000 s1), contact of blood with the membrane causes platelets to aggregate and occlude the aperture. The time taken to occlude the aperture or closure time is the end-point which is


recorded in a similar manner to the bleeding time. The maximal closure time that can be recorded is 300 sec. The technique is simple and quick, but sensitive to a number of variables including platelet number, hematocrit, ABO blood group and plasma vWF levels. Aspirin increases the closure time. As a normal result has a high negative predictive value, it is becoming more popular as a screening test for platelet dysfunction [47]. Closure time is abnormal in some congenital and acquired platelet function defects, but is not prolonged by coagulation factor deficiencies [48]. There are limitations to the PFA-100, among them, an incorrect citrate concentration. Closure times are significantly lower for samples collected in 3.2% compared with 3.8% citrate. This concentration of citrate increases the collagen/epinephrine (CEPI) sensitivity to aspirin [49]. Therefore, 3.8% concentrations are recommended. Also, the cut-off value to determine aspirin sensitivity is poorly defined. The manufacturer advises that each laboratory should establish its own PFA-100 closure time reference ranges. In addition, the manufacturer's sample stability time of 4 hours may be too long; samples can show deterioration after only 2 hours. Verify Now Aspirin Assay This test was previously marketed as the Ultegra Rapid Function Platelet Assay (RPFA) and is FDA-approved "to aid in the detection of platelet dysfunction due to aspirin ingestion" [50]. It is an in vitro semiquantitative measurement of aspirin-dependent aggregation that requires whole blood collected in 3.2% sodium citrate. The cartridge may contain propyl gallate or arachidonic acid. The instrument tests aggregation of activated platelets through binding to human fibrinogen-coated beads [51]. The numerical result is reported in aspirin-response units (ARU) with the aspirin resistance cut-off defined as 550 ARU (aspirin-related platelet dysfunction not detected) [50]. Its sensitivity as a screen for aspirin-induced platelet dysfunction is approximately 91%, according to the manufacturer's product information [50]. A limitation is that VerifyNow cannot be used in patients with inherited platelet defects or in those receiving other anti-platelet drugs. However, the principal limitation is that diagnostic criterion for aspirin resistance is based on a cut-off that was established by comparison with light or optical platelet aggregation in response to adrenaline after a single 325-mg dose of aspirin [52,53]. Therefore, the biochemical response to lower doses of aspirin could be not well assessed with this method. Other Methods for Detection of Aspirin Effect PlateletWorks is FDA-approved to detect platelet dysfunction due to inhibition secondary to diet, aspirin, and/or other drugs. It is an in vitro quantitative measurement of platelet activation that requires whole blood collected in 3.2% sodium citrate [54]. This assay uses separate tubes containing collagen and ADP (adenosine diphosphate) and traditional cell counting principles (electronic impedance). Both pre- and post-activation platelet counts are obtained and the numerical result represents percent inhibition. There is a very short time allowed (10 minutes) between sample collection



and assay. Dietary substances such as garlic, ginger, Vitamin E, dark chocolate and beer may give false positive results. It is possible that these substances produce platelet inhibition detectable by PlateletWorks. Comparison of Different Tests Light transmission aggregometry (LTA) is considered the gold standard to measure the antiaggregant effect of aspirin. However it is poorly standardized, presents a high variability and requires a specialized laboratory. For all these reasons LTA is unlikely to be used in clinical practice. Therefore, there is need for other tests that can be used in everyday practice. Harrison et al. [55] have compared LTA with the two point-of-care tests approved by the FDA: PFA-100 and VerifyNow. The agreement among the tests was poor and very few patients were aspirin non-responsive on all three tests. The PFA-100 detects more aspirin non-responsiveness than LTA [56,57]. Because the PFA-100 is a high-shear system, it may be more physiologically relevant than the others [55]. Platelet hyperfunctional response and high vWF levels could both contribute to the occurrence of normal closure times in aspirin non-responders [58-60]. A recent study [61] has measured platelet function with PFA-100, platelet aggregation with LTA and platelet activation with flow cytometry in the same group of patients. Patients with aspirin resistance, determined by PFA-100, also had increased platelet aggregation and activation with the other tests. The optimal platelet function test for assessment of platelet inhibition by aspirin should be reproducible, easy to perform, quick and cheap. The best test and criteria for the diagnosis of aspirin resistance are not clear at present. Some authors have chosen criteria combining several tests [62], but this option is valid only for research. SITUATIONS THAT CAN MODIFY ASPIRIN RESPONSE Changes in Aspirin Resistance with Time An important consideration is that aspirin resistance may be temporary [63]. Most studies have been performed with a single determination. However, some [35, 64] show changes in the antiaggregant effect of aspirin with time. In addition, there is considerable movement between aspirin-sensitive and -resistant groups [35]. The reason for this phenomenon is unknown. It could indicate the dynamic nature of aspirin response; however, variability in the platelet aggregation assay may be a contributing factor. In healthy controls complete suppression of platelet COX-1 by aspirin is maintained during the first month of treatment [7], but a loss of effect has been reported in some patients during long-term treatment [64,65]. Different Doses Guidelines on acute myocardial infarction recommend aspirin for secondary prevention. “A daily dose of aspirin 75 to 162 mg orally should be given indefinitely to patients recovering from ST elevation myocardial infarction (Level of

Evidence: A)” [66]. The dose of aspirin needed to alter platelet function tests is not related to body surface [36], thus the same dose can be recommended for everyone. However, the dose required to inhibit platelet aggregation may differ among patients according to their particular situation. When platelet function tests have been repeated in the same patients with different doses, there is an increasing antiaggregant effect and less resistance with higher doses [59,64,67,68,69]. In a study on aspirin resistance with VerifyNow, low aspirin dose was associated with a higher incidence of resistance [70]. Other authors [71] found significantly lower platelet activation in a small group of patients who received high vs. low dose of aspirin. However, PFA100 was unable to detect these differences [71]. Cardiovascular Risk Factors Aspirin resistance has been found in a high proportion of patients with cardiovascular risk factors such as smoking [36,72], hyperlipidemia [73] and diabetes [74]. We have found more aspirin resistance in smokers and patients with dyslipemia, but not in diabetics [36]. Other authors also found the same response in patients with and without diabetes [65]. Smoking A recent report [72] shows a major association between aspirin resistance (measured with VerifyNow) and smoking (risk ratio 11,47 vs. no smoking). The association suggests that antiplatelet therapy resistance may be one mechanism by which smoking generates risk of thrombotic events [72]. This has also been found in previous studies [75]. Hyperlipidemia Platelets taken from hyperlipidemic patients are highly thrombogenic, due to an increase in reactive oxidant species [76,77]. Other factors, such as upregulation of COX-2 expression, may contribute to poor platelet responsiveness to aspirin [12]. Diabetes A recent experimental study finds that aspirin ineffectiveness is related to poor metabolic control in diabetes [78]. Hyperglycemia induces platelet activation and monocyte tissue factor expression, promoting a procoagulant and proinflammatory state that may contribute to acute vascular events and atherogenesis [79]. However, platelet responsiveness to activation with ADP or thrombin appears not to be altered by hyperglycemia-hyperinsulinemia [79]. According to our results [36], platelet responsiveness to activation with epinephrine is not altered either. Other authors [74] have reported that high concentrations of glucose in the medium appear to affect not only platelet and endothelial functioning per se, but also the action of aspirin. Aspirin resistance (measured by PFA-100) has been associated with high plasmatic levels of glucose [80]. It is possible that only bad management of diabetes favors aspirin resistance, whereas wellcontrolled diabetics present the same proportion of aspirin resistance as non-diabetics.


Gender and Age The relationship between aspirin resistance and gender or age differs in different publications [36,81]. Its relevance is still uncertain. Coronary Artery Disease In atherosclerotic vascular disease, especially in unstable coronary syndromes, TXA2 synthesis is only partially suppressed by aspirin [26,38]. This is evidenced by relatively large amounts of TX metabolites in urine, despite inhibition of platelet TXA2 production [31]. Aspirin resistance is common in patients with coronary artery disease [36,57], mainly in unstable situations [26,38]. The occurrence of acute coronary syndrome in patients taking aspirin in the previous 7 days is not infrequent, and is considered a factor for negative prognosis, included in the calculation of TIMI risk score in non-ST-elevation acute coronary syndrome [82]. In a recent report [83] previous aspirin use was found to increase the odds of in-hospital mortality during acute myocardial infarction. Another article [63] reports a reduction in the proportion of patients resistant to aspirin several months after myocardial infarction. However no prognostic significance was found after one year of follow-up [63]. Percutaneous Revascularization This treatment is frequent in coronary artery disease. Some authors [69] have found that a high aspirin dose is necessary to inhibit platelet activation in these patients. A high incidence of myonecrosis has been reported in patients with aspirin resistance after non-urgent percutaneous coronary intervention [84]. Patients with stent thrombosis have an impaired response to antiplatelet therapy with aspirin [85,86], compared with controls and volunteers [85]. Wenaweser et al. found that additional treatment with clopidogrel did not overcome these differences in platelet aggregation [85]. They conclude that aspirin, but not clopidogrel resistance, appears to be associated with stent thrombosis. Recent Stroke Some patients who have suffered strokes have increased platelet reactivity [59,67,87]. Those who respond to aspirin have a significantly better prognosis than the patients with high platelet reactivity despite the use of aspirin [64,67]. The main factors that may trigger a cardiovascular event are vulnerable plaque, vulnerable blood and a vulnerable patient (with some inflammatory process). The inflammatory cells can release proteolytic enzymes capable of degrading collagen and other structurally important constituents of the plaque's fibrous cap. Thus, when there is inflammation in the intima, the collagen responsible for the integrity of the plaque's fibrous cap is under double attack, subject to both decreased synthesis and increased degradation, which sets the stage for plaque disruption. The inflammatory cells also are responsible for signaling and producing increased quantities of tissue factor, a potent procoagulant deemed responsible for thrombosis of ruptured plaques [88]. Thrombin itself


is a strong stimulus of platelet aggregation. Another factor involved in this process may be COX-2. Upregulation of COX-2 has been demonstrated in atherosclerotic tissue [89] and may be associated with greater synthesis and transfer of prostaglandin H2 to platelets, thus bypassing platelet cyclooxygenase-1 and leading to aspirin-insensitive thromboxane biosynthesis in these patients. In the coronary arteries the vulnerable plaque (with high lipidic content and a thin fibrous cap) is the most important factor in triggering a cardiovascular event. However the thrombogenic factor may be of greater importance in the carotid arteries. Extracorporeal Circulation This situation produces an important decrease in platelet count and augmented platelet turnover. Aspirin may be unable to inhibit the activation of the new platelets that enter the circulation with a functioning COX system. However, this does not sufficiently explain aspirin resistance. Zimmermann et al. [13] found that platelet counts increased to almost 80% above control levels within 10 days after CABG and platelet COX-2 was increased. Therefore, aspirin resistance may be caused by COX-2 in platelets by generating critical amounts of TX despite aspirin treatment. Aspirin resistance can probably be overcome in this case by prolonged dispensation, i.e. repeated daily doses [13]. Heart Failure Congestive heart failure has been found to be associated with increased degrees of platelet activation [90]. Not only severe, but also even mild to moderate heart failure is associated with increasing risk for thromboembolic events. This has been attributed to a hypercoagulable state including formation of intraventricular thrombi [91]. The pathophysiology of thrombogenesis in heart failure could well be explained in the context of Virchow's original triad. In addition to "abnormal flow" through low cardiac output, dilated cardiac chambers and poor contractility, patients with heart failure also demonstrate abnormalities of hemostasis and platelets (that is "abnormal blood constituents") and endothelial dysfunction ("vessel wall abnormalities") [92]. Reduced formation of the platelet inhibitor nitric oxide apparently contributes to platelet activation in heart failure [91]. Physical Exercise Exercise can produce aspirin resistance by increasing the level of cathecolamines [93] and platelet activation [94]. Norepinephrine infusion, at plasma levels easily attained during exercise, enhances platelet aggregability and platelet secretion in vivo in healthy humans [95]. Aspirin may be less effective as an antithrombotic during sympathoadrenal activation in humans [95]. Aspirin resistance has been detected only immediately after an exercise stress test in some patients who had a normal response before exercise [96-98]. Moderate physical exercise provokes 12% increase in aspirin resistance occurrence [99], connected with a concentration increase in the sCD40L. Some authors [100] suggest that catecholamines probably do not induce platelet aggregation



per se. This may be induced secondarily through potentiation of agonists such as ADP or thrombin. Circadian Variation Circadian variation may also be involved in the response to aspirin. There is a surge of cathecolamines soon after getting up in the morning, which can produce an increase in platelet activation [101,102]. Plasma levels of beta-thromboglobulin and platelet factor 4 are lowest with patients who are supine and resting at 7 and 8 A.M., and they increase with activity [103]. There is a trend toward circadian variation in “in vitro” platelet aggregability to epinephrine. These data indicate that changes in platelet function have a diurnal occurrence. In the Physicians’ Health Study, the aspirin treated patients showed a 59.3% reduction in the incidence of infarction during the morning compared with a 34.1% reduction for the remaining hours of the day [104]. According to this study, aspirin counteracted the adverse effect in aggregation of the morning surge of cathecolamines. We have studied the response to aspirin at different times of day and the highest proportion of resistance was found in the morning [105]. Similar results have been reported by others [106]. Influence of Intestinal Absorption

aspirin [110]. However, a report from the United Kingdom supports the concept of an increased risk of myocardial infarction with both conventional NSAIDs and coxibs [115]. These authors found no significant interaction between any NSAID and aspirin, indicating that the risk of myocardial infarction for each NSAID does not vary with the prescription of aspirin. In January 2007 an extensive review on Coxibs and heart disease was published [116]. This review shows unfavorable cardiovascular outcomes with celecoxib, rofexoxib, valdecoxib and parecoxib. Ibuprofen and naproxen had fewer untoward effects than selective COX-2 inhibitors according to previous trials. The Prospective Randomized Evaluation of Celecoxib Integrated Safety vs. Ibuprofen or Naproxen (PRECISION) trial has already been launched. This trial with an approximate sample of 20,000 patients with osteoarthritis or rheumatoid arthritis, both with cardiovascular disease or at high risk of cardiovascular disease, will randomize patients to ibuprofen vs. celecoxib vs. naproxen and will examine the rate of cardiovascular death, myocardial infarction, or stroke. Both concomitant aspirin-users and non-users will be included. In addition, gastrointestinal and renal safety, as well as arthritis efficacy, will be evaluated [117].

Enteric-coated formulations take longer to achieve peak concentrations in blood than soluble formulations. In addition, some studies have found that uncoated aspirin prolongs the closure time more than enteric-coated aspirin [59]. Acid suppression with proton pump inhibitors can increase the potential for mucosal esterases to hydrolyze aspirin to its inactive form, and thereby reduce enteral absorption of acetylsalicylic acid. At present there is controversy on this topic [107,108].

As this topic is so controversial, the Food and Drug Administration has published a scientific paper [118] with the following main points:

Influence of Concomitant Medications

• The clinical implication of this interaction may be important because the cardioprotective effect of aspirin, when used for secondary prevention of myocardial infarction, could be attenuated.

The PFA-100 CEPI closure time has found to be prolonged approximately 30% above baseline in subjects taking the serotonin-reuptake inhibitor paroxetine [109]. Aspirin and other non-steroidal anti-inflammatory drugs [NSAIDs] prolong the PAF-100 CEPI closure time without altering the CADP closure time. When NSAIDs are discontinued, closure time abnormalities revert by 6 days with aspirin [110] and by 24 hours with ibuprofen [111]. Theoretically the NSAIDs may reduce the risk of myocardial infarction but may also interfere with aspirin's cardioprotective effect [112]. Some of these drugs compete with aspirin for a common site within the COX channel [i.e., arginine-120], which aspirin binds to with weak affinity prior to the irreversible acetylation of serine-529. Concomitant administration of reversible COX-1 inhibitors, such as ibuprofen [113] and naproxen [114] might prevent the irreversible acetylation of platelet COX-1 by low-dose aspirin. This pharmacodynamic interaction does not occur with rofecoxib or diclofenac, which have variable COX-2 selectivity [113]. Diclofenac even decreases platelet CD 62 (P selectin) expression, and therefore can favor the antiaggregant effect of

• Existing data using platelet function tests suggest there is a pharmacodynamic interaction between 400 mg ibuprofen and low dose aspirin when they are dosed concomitantly. The FDA is unaware of data addressing whether taking less than 400 mg of ibuprofen interferes with the antiplatelet effect of low dose aspirin.

• For single doses of ibuprofen, the pharmacodynamic interaction can be minimized if ibuprofen is given at least 8 hours before or at least 30 minutes after immediate- release aspirin (81mg; not enteric-coated). • The clinical implication of the interaction has not been evaluated in clinical endpoint studies. • There is no clear data regarding the potential effect of chronic ibuprofen dosing of over 400 mg on the antiplatelet effect of aspirin. • The timing of administration of ibuprofen and low-dose aspirin is important for preserving the cardioprotective effect of aspirin. Polymorphisms Single nucleotide polymorphisms can modify the antiplatelet effect of aspirin [119-123]. Aspirin resistance may be associated with different polymorphisms, mainly affect-


ing COX-1 [119,121] and the platelet glycoprotein IIIa [124]. However, both increased [119] and diminished [121] responses to aspirin have been associated with the same COX-1 haplotype. Collagen receptor and vWF receptor polymorphisms may also be involved in aspirin resistance [124,125], as well as genetic variation of the platelet surface adenosine 5-diphosphate receptor gene P2Y1 [126]. Other factors Blood group, food, platelet count [67], hematocrit value [71], levels of vWF [127,128] and of C reactive protein [129] have been reported as altering the effect of aspirin, when measured by PFA-100. As it is for the bleeding time, the closure time is prolonged by significant reductions in the platelet count or hematocrit [130,131]. The PFA-100 test is highly dependent on vWF binding to the platelet membrane GP receptors Ib/IX/V and integrin aII/b3 [IIb/IIIa] under high shear [132-134]. The closure time shows an inversely proportional relationship to plasma vWF levels. Increased levels of plasma vWF may be an associated factor leading to the observed in vitro aspirin resistance. Its detection, revealed by PFA-100 closure time prolongation failure, may be a good tool for the assessment of clinical thrombotic risk under aspirin treatment [127]. Closure time is longer in patients with group O, probably because of their lower plasma vWF [135]. Consumption of flavonoid rich foods (e.g. red wine, cocoa and chocolate) can prolong the CEPI closure time [136]. In the same way, the results obtained with PlateletWorks are also modified by food. Fig. 7 summarizes the main factors that can influence on aspirin response.


periodically. Although the term “aspirin resistance” is probably not accurate, it is possible that the aspirin effect needs to be tested regularly in order to give the adequate dose in each circumstance. RELATIONSHIP BETWEEN ASPIRIN RESISTANCE AND FUTURE CARDIOVASCULAR EVENTS The following tests have been studied in relation with future cardiovascular events: PFA-100 [81,137-142]; aggregometry in response to arachidonic acid and ADP (turbidometric) [56]; aggregometry in response to ADP and collagen (impedance) [35]; VerifyNow [84,127]; urinary 11dehydro thromboxane B2 in urine [31]. Some studies have demonstrated a relationship between aspirin resistance and negative prognosis: Gum et al. [56] found that patients with aspirin resistance (measured by turbidometric aggregometry) had a worse prognosis than those with a normal response. The same authors found a lack of correlation between aspirin resistance measured by PFA-100 and prognosis. Andersen and associates [139] noted a trend of increased vascular events in patients with aspirin resistance (measured with PFA-100), although the results were not statistically significant. Recent studies [81,140-142] have found an association between increased platelet aggregation measured by PFA-100 and future cardiovascular events in patients who take aspirin. Eikelboom et al. [31] measured aspirin resistance by means of urinary 11-dehydro TXB2. Patients without this metabolite in urine had better clinical prognosis than those with normal levels, because of aspirin resistance. The prognosis was related to the different quartiles of TXB2. Grotemeyer et al. [67] carried out a two-year follow-up of 181 patients after cerebrovascular accidents. They found a significantly greater number of events in those with increased platelet reactivity in spite of aspirin treatment. There are still few studies with VerifyNow to assess prognosis. In the study by Chen et al. [84], aspirin resistance was correlated with increased markers of myonecrosis after percutaneous revascularization, but there was no follow-up of these patients. In a study that did attempt to correlate test results with clinical outcomes, there were confounding variables, such as other anti-platelet drugs [128]. POSSIBLE THERAPEUTIC OPTIONS IN CASE OF ASPIRIN INEFFECTIVENESS

Fig. (7). The main factors of variability in the responsiveness to aspirin are showed. ACS: acute coronary syndrome, PCI: percutaneus coronary intervention, vWF: von Willebrand factor.

USEFULNESS OF LABORATORY TESTS Despite the many factors that can change the response to the above-mentioned laboratory tests, testing platelet inhibition is not necessarily a waste of time. A patient with a temporary condition that modifies either platelet function or aspirin response may need higher or lower aspirin doses to be properly antiaggregated. In the same way a patient who takes warfarin undergoes many changes in protrombin time. In fact, according to the INR, the warfarin dose must be changed

If a test can prove that a patient has normal platelet function despite aspirin, and this lack of antiaggregant effect has clinical consequences, there are several therapeutic options. a) Increase in the dose of aspirin; b) Use of another antiplatelet drug, or c) Addition of another antiplatelet drug to aspirin. Before taking one of these options, it is necessary to treat any possible cause: lack of compliance, smoking, bad glycemic control, hyperlipidemia, as well as eliminating drugs or food that can interfere with aspirin absorption or action. a) Increase in the Dose Although theoretically a very low dose of aspirin can completely inhibit the synthesis of TXA2, in a significant number of patients the doses of aspirin currently used for



antithrombotic treatment are insufficient to block platelet reactivity [143] as promoted by erythrocytes [144]. In addition, many other factors already mentioned can alter the effect of aspirin, therefore, these patients may need higher doses. In a meta-analysis by the Antithrombotic Trialists, aspirin reduced cardiovascular events by 22% [1]. However, when stratified by dose, trials that used doses of aspirin of 75 mg/day provided significant reduction in cardiovascular events, whereas the three trials using doses