Diabetes and Aspirin Resistance

3.1. Aspirin as a primary prevention strategy in diabetes mellitus

The Japanese Primary Prevention of Atherosclerosis With Aspirin for Diabetes (JPAD) trial (Ogawa, Nakayama et al. 2008) was the first prospectively designed trial to evaluate the use of aspirin (81 mg or 100 mg) in the primary prevention of cardiovascular events in patients with type 2 diabetes (n = 2,539) aged 30–85 years in Japan, and reported that aspirin use was associated with a 32% reduction in the risk of the primary end point at 4.7 years of follow up. The ongoing trials that will provide insights into the appropriateness of aspirin usage in diabetic patients include the Aspirin and Simvastatin Combination for Cardiovascular Events Prevention Trial in Diabetes (ACCEPT-D).(De Berardis, Sacco et al. 2007) Results of the Prevention Of Progression of Arterial Disease And Diabetes (POPADAD) trial have been reported, with no benefit with aspirin or antioxidants in primary prevention of cardiovascular events.(Belch, MacCuish et al. 2008) ADA recommends enteric-coated aspirin at a dosage of 81–325 mg to be used as a preventive strategy in high-risk diabetic individuals.(Pignone, Alberts et al. 2010) An important consideration is that patients may acquire additional risk factors over time, which would necessitate a reassessment of their overall risk profile. Several meta-analyses have explored the benefit of aspirin therapy in the primary prevention of major adverse cardiovascular events (MACE) among patients with diabetes.(Baigent, Blackwell et al. 2009; Calvin, Aggarwal et al. 2009; De Berardis, Sacco et al. 2009; Pignone, Alberts et al. 2010; Younis, Williams et al. 2010; Zhang, Sun et al. 2010) There are several tools to calculate the risk. Tools that can be used in patients with diabetes are available from several sources, for example: UKPDS Risk Engine:http://www.dtu.ox.ac.uk/riskengine/index.php; ARIC CHD Risk Calculator:http://www.aricnews.net/riskcalc/html/RC1.html; American Diabetes Association Risk Assessment Tool, Diabetes PHD:http://www.diabetes.org/phd. The AHA has issued similar guidelines and recommends 75–160 mg/day of aspirin as a primary prevention strategy in high-risk individuals, defined as those with a 10-year risk of coronary artery disease (CAD) over 10%.

3.2. Aspirin as a secondary prevention strategy in diabetes mellitus

Two large meta-analyses of major secondary prevention trials by the Antithrombotic Trialists' Collaboration (ATC) showed oral aspirin to be protective in patients at high risk for CVD, including those with diabetes (1994; 2002). The meta-analyses included 287 secondary prevention trials involving 212,000 high-risk patients with acute or prior vascular disease or another condition that increased their risk of vascular disease. Of note, a low dose of aspirin (75–150 mg/day) was found to be at least as effective as higher daily doses. In more than 4,500 diabetic patients studied in the ATC, the incidence of vascular events was also reduced from 23.5% in the control group to 19.3% in the group treated with antiplatelet therapy (P< 0.01) and from 17.2% to 13.7% in the ~42,000 nondiabetic patients (P< 0.00001).The ADA recommends the use of aspirin (81–325 mg/day) as a secondary prevention measure in diabetic patients with atherosclerotic disease. (Pignone, Alberts et al. 2010)

3.3. Aspirin resistance

Aspirin resistance, defined as failure of suppression of thromboxane generation, increases the risk of cardiovascular events in a high-risk population.(Eikelboom, Hirsh et al. 2002)Causes of aspirin resistance include concurrent use of nonsteroidal anti-inflammatory drugs such as ibuprofen that may compete with aspirin at the COX-1 receptor site,(Catella-Lawson, Reilly et al. 2001)polymorphisms in the COX-1 gene,(Eikelboom, Hirsh et al. 2002; Halushka and Halushka 2002) poor glucose control, body weight, and conditions associated with a high platelet turnover.(Zimmermann, Wenk et al. 2003; Zimmermann, Kurt et al. 2005; Guthikonda, Lev et al. 2007; Modica, Karlsson et al. 2007)

3.4. Terminology (Ben-Dor, Kleiman et al. 2009)

The lack of agreement on a standardized definition for “aspirin resistance” has contributed to the disparity in reports of its incidence among different studies. Whereas some use the term “aspirin treatment failure,” others like to call it “aspirin non responsiveness.” The term aspirin resistance has been used to describe the occurrenceof cardiovascular events despite regular aspirin intake at recommendeddoses.

3.5. Diabetes and aspirin resistance

The benefit of aspirin in diabetic patients has been consistently documented in several trials. Aspirin is recommended for primary and secondary prevention in DM. Yet, in the meta-analysis of the ATC, the event rate of DM patients on treatment was similar to that of non-DM patients off treatment.(2002)In the Primary Prevention Project Trial, aspirin treatment reduced cardiovascular events and deaths in high-risk non-diabetic patients, but not in patients with type 2 DM (T2DM).Furthermore, in the recent Japanese Primary Prevention of Atherosclerosis with Aspirin for Diabetes Study,(Ogawa, Nakayama et al. 2008)a low dose of aspirin in primary prevention did not reduce the risk of cardiovascular events at 4 years in diabetic patients. Other studies in secondary prevention similarly suggested that aspirin might be less effective in T2DM, especially in patients with poor metabolic control, than in non-DM patients, the underlying mechanism being still debated. Since platelets play a key role in the development of atherothrombotic events, the dysfunctional status of platelets in DM patients may contribute to the enhanced atherothrombotic risk of these patients. It has been proposed that reduced sensitivity to aspirin in diabetic patients might be owing to accelerated thrombopoiesis, (Di Minno, Silver et al. 1986) or to reduced platelet permeability to aspirin caused by membrane glycosylation.(Winocour, Watala et al. 1992)

The mechanisms that lead to increased platelet reactivity observed in patients with DM can be grouped together into the following aetiopathogenic categories: a) hyperglycaemia, b) insulin deficiency and resistance, c) associated metabolic conditions, and d) other cellular abnormalities (as shown in Figure 2). Poor glucose control and body weight are also proposed to contribute to aspirin resistance. (Watala, Golanski et al. 2004; Singla, Antonino et al. 2009) Poorly controlled patients with diabetes have the greatest platelet reactivity. High platelet reactivity was defined as >46% for 5 micromol/L ADP-induced and >59% for 20 micromol/L ADP-induced platelet activity and may require alternative antiplatelet strategies, and further clinical investigations are warranted.(Singla, Antonino et al. 2009)

Platelets: Platelets play a key role in the development of atherothrombotic events. Platelets are essential for primary hemostasis and repair of the endothelium, but they also play a key role in the development of acute coronary syndromes and contribute to cerebrovascular events. Platelet adhesion is an essential function in response to vascular injury and is generally viewed as the first step during which single platelets bind through specific membrane receptors to cellular and extracellular matrix constituents of the vessel wall and tissues. Beyond acute activation as a consequence of vascular injury, circulating platelets are actively involved in all phases of the atherogenetic process, from atherosclerotic plaque formation to plaque inflammation and rupture. (Davi and Patrono 2007; Ruggeri and Mendolicchio 2007; Langer and Gawaz 2008)

Mechanism of aspirin resistance in diabetes: Increased platelet reactivity observed in patients with DM may be secondary to several factors. Acute hyperglycemia as well as poor control of diabetes is associated with increased platelet reactivity. Comparative studies of patients with good glycemic control show they have better response to aspirin compared to the patients with poor glycemic control. Although this might implicate that better glucose control leads to less incidence of aspirin non-responsiveness, the clinical significance of such findings should be carefully inspected, since in 2 of the largest trials assessing the role of aspirin on primary prevention of cardiovascular events in patients with type 2 diabetes, low-dose aspirin did not decrease the risk of cardiovascular events when compared to placebo. Insulin resistance, TNF-alfa and IL-6 are shown to affect platelet reactivity. The clinical determinants that help identify aspirin resistance in diabetes are suggested to be CVD, microalbuminuria, poor diabetes control, and increased waist circumference.(Yassine, Davis-Gorman et al. 2010)

Platelet abnormalities in Type 2 diabetes: The abnormalities described in patients with diabetes are listed here.

1. Increased production of thromboxane A2 from arachidonic acid. (Halushka, Mayfield et al. 1981; Halushka, Rogers et al. 1981)

2. Increase in platelet-dependent thrombin generation.

3. Increased expression of platelet surface adhesion molecules such as CD31, CD49b, CD62P, and CD63, leading to increased platelet activation.

4. Increased platelet surface receptors such as P-selectin, GP Ib, and GP IIb/IIIa. (Gresele, Guglielmini et al. 2003)

5. Reduced vascular synthesis of the anti-aggregants PGI2 and NO, shifting balance towards aggregation and vasoconstriction.

6. Disordered calcium homeostasis that affects platelet shape change, secretion, aggregation, and thromboxane formation (Halushka, Mayfield et al. 1981; Halushka, Rogers et al. 1981).

7. Decreased platelet insulin receptor number and affinity and failure to reduce platelet responses to the agonists ADP, collagen, thrombin, arachidonate, and PAF.

8. Glycation of circulating LDL rendering platelets hypersensitive. Glycated LDL causes an increase in intracellular calcium concentration and platelet NO production, as well as inhibition of the platelet membrane Na+/K+-ATPase activity.

Key: GP = glycoprotein; PGI2 = prostacyclin; NO = nitric oxide; ADP = adenosine diphosphate; PAF = platelet-activating factor; LDL= low-density lipoprotein; Na+/K+-ATPase = Na+/K+-adenosine triphosphatase

An accelerated platelet turnover represented by the presence of a higher number of reticulated platelets has been observed in patients with DM.(Guthikonda, Alviar et al. 2008) The dysfunctional status of platelets in patients with DM may contribute to the enhanced atherothrombotic risk of these patients. Platelets obtained from diabetic patients show increased adhesiveness, hyperfunction both spontaneous as well as in response to agonists. These observed hyperfunctions are attributed to increased expression, activation or abundance of surface membrane receptors for agonists as well as cell matrix components, increased binding of fibrinogen, altered membrane fluidity, changes in activation mechanisms and signaling pathways. Changes in platelets in diabetes include enhanced GP receptor binding of agonists and adhesive proteins; decreased membrane fluidity; enhanced activation of the arachidonic acid pathway resulting in increased TxA₂ formation; altered PI turnover leading to changes in diacylglycerol and inositol triphosphate production, calcium mobilization, and protein phosphorylation; impaired responses to antiaggregants resulting in decreased PGI₂ receptor binding, cyclic nucleotide production and cyclic nucleotide–dependent protein phosphorylation; and reduced sensitivity to the inhibitory actions of insulin. These changes translate to impaired PGI₂ stimulation of cAMP and blindness to the inhibitory actions of both PGI₂ and NO. Platelet dysfunction coupled with decreased endothelial production of these antiaggregatory agents conspire to amplify the risk of CVD in patients with type 2 diabetes.(Vinik, Erbas et al. 2001)

Metabolic control and platelet reactivity

In the early 1960s Bridges et al showed that both in vitro as well as in vivo administration of glucose increased platelet stickiness.(Bridges, Dalby et al. 1965)Combined hyperinsulinemia and hyperglycemia in healthy volunteers increased circulating tissue factor, plasma thrombin generation, and coagulation factors VII and VIII activities, suggesting that the coagulation system had been activated.(Boden and Rao 2007)Chronic hyperglycemia has been identified as a causal factor for in vivo platelet activation and platelet hyperreactivity in DM patients as evidenced by enhanced TXA2 biosynthesis.(Davi, Gresele et al. 1997; Davi, Ciabattoni et al. 1999) Of note, T2DM platelets are characterized by enhanced thromboxane biosynthesis and tight metabolic control, shown to lead to a reduction of thromboxane levels.(Davi, Catalano et al. 1990)Acute, short-term hyperglycemia induces an increased activation of platelets exposed to high shear stress conditions in vitro (filtration method) or in vivo (bleeding time). In vivo platelet activation is reflected by an increased urinary excretion of 11-dehydro-TxB (2).(Gresele, Guglielmini et al. 2003)This acute hyperglycemia-induced enhancement of platelet activation is resistant to aspirin: an NO -donating agent suppresses it.(Gresele, Marzotti et al. 2010)LDL, a circulating complex of lipids and proteins that is increased in hypercholesterolemia, enhances platelet function and sensitizes platelets via binding of apoB-100 to a receptor on the platelet membrane and via transfer of lipids to the platelet membrane.(Relou, Hackeng et al. 2003) Hyperglycemia also induces an increase in nonenzymatic glycation of LDL (glycLDL), which renders them more susceptible to oxidative stress.(Angiolillo 2007)

Insulin and platelet reactivity

The T2DM patients had platelet aggregation and shear-induced platelet function significantly increased compared to nondiabetic patients using all assays. Platelet aggregation was increased in ITDM (n = 68) compared with NITDM (n = 133) patients after P2Y12-specific stimuli. Insulin treatment was the strongest predictor of ADP-induced aggregation. Platelet function profiles were similar between ITDM and NITDM using assays non-specific to the P2Y12 pathway. Platelet dysfunction was independent of glycemic control and inflammatory status.(Angiolillo, Bernardo et al. 2006)

NF-kB is a transcription factor that stimulates numerous genes and activates inflammatory responses related to insulin resistance. Salicylates inhibit NFkB activation. This inhibition was shown to be associated with a significant decrease in IL-6 and TNF-α release, mediated through inhibition of IKKβ activity.

Platelet activity measures

A major urinary metabolite of thromboxane A2 synthesized from extra renal sources is 11-dehydro thromboxane B2. A major portion of this metabolite is believed to come from the platelet, but there are additional cellular sources. In the Heart Outcomes Prevention Evaluation (HOPE) trial, patients whose urinary 11-dehydro thromboxane B2 levels were in the highest quartile had an odds ratio of 2 for having a myocardial infarction and an odds ratio of 3.5 for a risk of having a cardiovascular-related death compared to those patients in the lowest quartile. Serum aspirin esterase (AE) activity may account for part of aspirin pharmacokinetics and has been proposed as one source of variation in aspirin effectiveness.(Adebayo, Williams et al. 2007)Elevated MPV values are associated with a shortened bleeding time and increased thromboxane B2 plasma levels. Thus, MPV could be considered an indicator of platelet function.(Vizioli, Muscari et al. 2009)Diabetic patients with coronary heart disease have significantly higher MPV values compared to control patients.(Tavil, Sen et al. 2010)

Methods that directly measure the capacity of platelets to synthesize TxA₂ are certainly preferable. Of these, the urinary levels of the TxB₂ metabolite, 11-dehydrothromboxane B₂, represent a time-integrated index of TxA₂ biosynthesis in vivo.(Patrono, Ciabattoni et al. 1986) 11-dehydro-TxB₂ is the most abundant urinary metabolite of TxB_2. Detection in the urine of this metabolite, which is not formed in the kidney, reflects systemic TxA₂ formation, which largely, albeit not exclusively, occurs in the platelets. It has been calculated that about 30% of the urinary metabolite derives from extra-platelet sources, as in inflammatory diseases, the contribution of extra-platelet sources may increase in atherosclerosis and inflammatory conditions.(Catella and FitzGerald 1987) Test measures consider the end products of the TxA₂pathway such as serum TxB₂, or urine 11-dehydro-TxB₂, for assessing aspirin activity.(Eikelboom, Hirsh et al. 2002) In fact, these 2 tests may better reflect the amount of TxA₂ derived from sources other than platelets such as macrophages and monocytes, and on the COX-2 linked pathway of arachidonic acid, which is blocked by aspirin at very high doses (1200 mg/ day) only.(Bucchi, Bodzenta et al. 1986) Urinary 11-dehydro- TxB₂ concentration is affected by renal production of this substance. However, measurement of this metabolite is still commonly used in trials assessing aspirin resistance, due to its low cost and ease of measurement.(Eikelboom, Hirsh et al. 2002)

In aspirin-treated patients, elevated urinary 11-dehydro thromboxane B₂levels identify patients who are relatively resistant to aspirinand who may benefit from additional antiplatelet therapies ortreatments that more effectively block in vivo thromboxane productionor activity.(Eikelboom, Hirsh et al. 2002)

Clinical implications

The clinical implication of aspirin resistance as measured in vitro by the inability of aspirin to reduce platelet activation and aggregation by failure to suppress the platelet production of TXA has not yet been elucidated via prospective trials that have controlled for confounders, such as hypertension, and dyslipidemia. Large meta-analyses have found low-dose aspirin to be as effective as high-dose aspirin in preventing vascular events, making a dose-dependent improvement in laboratory response clinically irrelevant. Causes of aspirin resistance include poor compliance, inadequate dose, drug interactions, genetic polymorphisms of cyclooxygenase-1, increased platelet turnover, and upregulation of nonplatelet pathways of thromboxane production. At present, there is no standardized approach to the diagnosis and no proven effective treatment for aspirin resistance. Further research exploring the mechanisms of aspirin resistance is needed in order to better define aspirin resistance, as well as to develop a standardized laboratory test that is specific and reliable, and can correlate with the clinical risk of vascular events.

Management

Factors that need to be considered in the approach to patients with suspected treatment failure include: compliance with aspirin use, ensure the optimal dose and drug form (avoid use of enteric-coated aspirin formulations), evaluate concomitant infections or inflammatory conditions, and assess possible drug–drug interactions. Several approaches have been evaluated for treatment failure and some of these approaches are based on laboratory testing for evidence of resistance. The role of testing in directing management is still controversial. Management strategies are currently limited to dosing alteration and introduction of other anti platelet agents. However, these measures have not met the expected efficacy or safety.

Increased aspirin doses:The idea of increasing aspirin dose has been assayed in many studies, as there is some evidence that response to aspirin may be dose dependent.(ten Berg, Gerritsen et al. 2002)Because patients with diabetes exhibit a higher prevalence of aspirin resistance on standard aspirin doses (81 mg/day) and have significantly higher ADP- and collagen-induced platelet aggregation, 11-dehydro- TxB₂ levels and the aspirin resistance may be partially overcome by higher aspirin doses. (Di Chiara, Bliden et al. 2007)Laboratory and genetic inconsistency, as well as dose dependence, is seen when agonists other than arachidonic acid (the most specific in assessing aspirin resistance), such as ADP, collagen, and epinephrine, are used for in vitro assessment of platelet inhibition by aspirin.(McCabe, Harrison et al. 2005; Assadian, Lax et al. 2007; Gurbel, Bliden et al. 2007). The Clopidogrel vs Aspirin in Patients at Risk of Ischaemic Events (CAPRIE) trial compared the efficacy for secondary prevention of clopidogrel (75 mg daily) versus aspirin (325 mg daily) in a high-risk population (n = 19,185) consisting of patients with a history of recent MI, recent ischaemic stroke, or established peripheral artery disease.(Diener, Bogousslavsky et al. 2004) The benefit of clopidogrel therapy was higher (15.6% vs.17.7%; P = 0.042) in DM patients, despite the increased incidence of ischaemic outcomes in this subgroup. The absolute reduction in events was highest among diabetic patients requiring insulin therapy.(Diener, Bogousslavsky et al. 2004)In large, well-designed multicentre trials, such as CURE, COMMIT, and CLARITY-TIMI 28, the addition of clopidogrel to aspirin therapy improved outcomes in patients with acute coronary syndromes.(Plosker and Lyseng-Williamson 2007)

Addition of other antiplatelet agents: Dual antiplatelet therapy using acetylsalicylic acid and clopidogrel is of great importance following coronary stenting. Clopidogrel, a thienopyridine, works by irreversibly inhibiting ADP binding to the P2Y12 receptors on the platelet surface, and ultimately interfering with platelet-fibrinogen binding. The PLATO (PLATelet Inhibition and Patient Outcomes) study has shown that ticagrelor, an agent with a similar mechanism of action to clopidogrel and still in phase III trials, showed a significant reduction of a combined endpoint of cardiovascular death, myocardial infarction, or stroke as compared with clopidogrel (hazard ratio 0.84; 95%CI 0.75, 0.94; P = 0.0025).(Cannon, Harrington et al. 2010) In the subgroup of patients undergoing coronary artery bypass graft (CABG) within 7 days after the last study drug intake, ticagrelor compared with clopidogrel was associated with a substantial reduction in total and cardiovascular mortality without excess risk of CABG-related bleeding.(Held, Asenblad et al. 2011) Addition of dipyridamole to aspirin can lead to significant platelet inhibition in aspirin-resistant patients. The addition of dipyridamole to aspirin appears to be more effective than aspirin alone in the prevention of secondary vascular events in stroke patients and does not cause an increase in haemorrhagic events compared to aspirin alone in an identical dose.(Diener, Darius et al. 2001; Diener, Darius et al. 2001) There is also a suggestion that dipyridamole may partially compensate for aspirin resistance in patients with ischaemic stroke via an alternative antithrombotic mechanism.(Serebruany, Malinin et al. 2005; Serebruany, Malinin et al. 2006) In a study to determine whether treatment with dipyridamole or clopidogrel, in addition to aspirin, is more effective at reducing embolization and transient ischemic attacks, King and associates have shown that both dipyridamole and clopidogrel reduced embolization to a similar extent.(King, Bath et al. 2011)Other antiplatelet agents that may be more potent via alternative pathways are under investigation.

Statins to improve aspirin resistance: There is evidence that statins may be useful in treating aspirin resistance. Tirnaksiz and associates reported that in a study of patients with stable coronary artery disease, 11.2% were found to be aspirin resistant as measured by PFA-100, with a closure time of <186 seconds with collagen/adrenaline cartridges.(Tirnaksiz, Pamukcu et al. 2009) After 3 months of statin therapy (atorvastatin 10 mg/day), 65% of the aspirin-resistant patients became aspirin sensitive by PFA-100 measurements (P< 0.0001).(Tirnaksiz, Pamukcu et al. 2009) Tekten and his colleagues have shown that statins reduced platelet aggregation.(Tekten, Ceyhan et al. 2004)

Another recommendation is that because saturated fat ingestion increases in vivo thromboxane production despite aspirin therapy, diabetic patients on ASA therapy should have low dietary saturated fat intake and aggressive lipid management.(Yassine, Davis-Gorman et al. 2010)