Diabetes and the platelet: Toward new therapeutic paradigms for diabetic atherothrombosis

Atherosclerosis 212 (2010) 367–376

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Review

Diabetes and the platelet: Toward new therapeutic paradigms for diabetic atherothrombosis Olivier Morel a,b,c,∗ , Laurence Kessler d , Patrick Ohlmann a , Pierre Bareiss a a Pôle d’activité médico-chirurgicale Cardiovasculaire des Hôpitaux Universitaires de Strasbourg, Nouvel Hôpital Civil, place de l’Hôpital, 67091 Strasbourg Cedex, France b Institut d’Hématologie et d’Immunologie, Université de Strasbourg, France c U.770 INSERM, Le Kremlin-Bicêtre, France d Pôle de Diabétologie et de Nutrition des Hôpitaux Universitaires de Strasbourg, Strasbourg, France

a r t i c l e

i n f o

Article history: Received 29 August 2009 Received in revised form 5 March 2010 Accepted 18 March 2010 Available online 25 March 2010 Keywords: Plaque Apoptosis Microparticles Thrombosis Thienopyridines Platelet Statins Acute coronary syndrome

a b s t r a c t Atherothrombosis – defined as atherosclerotic lesion disruption with superimposed thrombus formation – is a leading cause of death in patients with diabetes mellitus. Platelets play a pivotal role in atherothrombosis and platelets of diabetic patients are hyperreactive. Numerous studies have investigated the usefulness of antiplatelet therapy for primary and secondary prevention of atherothrombotic events in diabetic patients. However, there are limited evidences that aspirin may be effective in the reduction of atherothrombotic complication in this population. Additionally, dual antiplatelet therapy with aspirin and clopidogrel has been suggested to be harmful. In contrast, the role of antiplatelet therapy in secondary prevention after ischemic cardiac events is well established in diabetes. Glycoprotein IIb/IIIa receptor antagonists can reduce mortality in diabetic patients committed to undergo percutaneous coronary intervention (PCI). Upregulation of P2Y(12) signalling occurs in hyperglycemia, and the relevance of platelet P2Y(12) receptor inhibition with prasugrel in reducing adverse events following PCI has been recently suggested. Besides platelet activation, several other mechanisms may be involved in the pathophysiology of diabetic atherothrombosis. Tissue factor (TF)-bearing procoagulant microparticles (MPs) are a heterogeneous population of membrane-coated vesicles released by several cell lines upon activation or apoptosis. There is converging evidence that MPs and MP-associated TF activity are upregulated in patients with diabetes mellitus and can participate actively in promoting atherothrombotic complications. In this context, drugs that may reduce the release of microparticles and/or their thrombogenic capacity has the potential to improve upon current antiplatelet therapy, possibly resulting in lower adverse events rates in diabetic individuals. © 2010 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Platelet function in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microparticles in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hyporesponsiveness to antiplatelet therapy in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antiplatelet therapy for the primary prevention of atherothrombotic events in patients with diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antiplatelet therapy for the secondary prevention of atherothrombotic events in patients with diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antiplatelet therapy in diabetic patients undergoing percutaneous coronary intervention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Are platelets the key player in diabetic atherothrombosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ∗ Corresponding author at: Pôle d’activité médico-chirurgicale Cardiovasculaire des Hôpitaux Universitaires de Strasbourg, Nouvel Hôpital Civil, 1 place de l’hôpital, 67091 Strasbourg cedex, France. Tel.: +33 369550949; fax: +33 388127203. E-mail address: [email protected] (O. Morel). 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.03.019

Atherothrombosis – defined as atherosclerotic lesion disruption with superimposed thrombus formation – accounts for the majority of deaths in patients with diabetes mellitus (DM). Latest

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estimates predict that the global prevalence of DM will increase by 165% between 2000 and 2050. Therefore, approximately onethird of the population born in 2000 will develop DM, with the associated up to 30% reduction in life expectancy, mostly related to atherothrombosis, which accounts for up to 80% of all deaths among diabetic patients [1]. There is therefore a need for global efforts to reduce the increasing burden of atherothrombotic disease in diabetic patients. To this aim, a variety of therapeutic options – from tight glycemic control to a large number of pharmacologic agents – are currently available [2]. The usefulness of glycemic control, however, has recently been a matter of debate. For example, the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study was halted prematurely because of an excess mortality in the intensive glycemic control arm [3]. On the other hand, the Diabetes Control and Complications Trial (DCCT) [4] and the UK Prospective Diabetes Study (UKPDs) [5] have highlighted the importance of maintaining good glycemic control during the early stages of diabetes for achieving clinical benefit at follow-up. It seems thus likely that pharmacological interventions will remain an important means for reducing atherothrombotic cardiovascular events in patients with diabetes. A better comprehension of the pathophysiology of diabetic atherothrombosis holds the promise of improving drug therapy by optimizing efficacy and reducing toxicity. In this context, excess risk of atherothrombosis in diabetic patients is associated with a variety of factors, including endothelial damage, plaque neovascularization and inflammation, increased expression of matrix metalloproteinases, and the shedding of tissue factor (TF)-bearing procoagulant microparticles (MPs) from stimulated platelets and apoptotic cells [6,7] (Fig. 1). Erosion or rupture of a vulnerable, lipid-rich, inflammatory atherosclerotic plaque with superimposed thrombosis is the key

factor in the development of acute cardiovascular events. Diabetes has been viewed as a coronary heart disease risk equivalent owing to its association with a hypercoagulable state and elevated concentrations of procoagulant factors including TF, fibrinogen, von Willebrand factor, and factor VII [8,9]. Blood with an increased level or activity of circulating thrombogenic factors is referred to as vulnerable [10], and this seems to be a typical feature of diabetic patients (Fig. 1). This review summarizes the evidence for blood thrombogenicity in patients with diabetes, and discuss the implications of therapy with antiplatelet agents in this setting. We will also highlight the potential role played by TF-bearing procoagulant microparticles in diabetic atherothrombosis, which may in the future prove suitable as a novel therapeutic target. 2. Platelet function in diabetes Growing evidence suggests that platelet of diabetic patients is larger and hyperreactive, showing increased adhesion and aggregation, and increased platelet-dependent thrombin generation [11]. Several mechanisms may account for the increased platelet reactivity in patients with diabetes. Nonenzymatic glycation of proteins is one of the key mechanisms in the pathogenesis of diabetic complications and may be significant in diabetic atherothrombosis [12]. There is an enhanced glycation of platelet membrane protein in diabetes, and most of the glycated platelets can be incorporated into thrombi [13]. In the hyperglycemic milieu, there is an increased platelet surface expression of glycoprotein Ib (GP Ib), which mediates binding to von Willebrand factor, and GP IIb/IIIa, which mediates platelet-fibrin interaction and represents the final common pathway of platelet activation, leading to platelet

Fig. 1. Possible pathways involved in the thrombotic state observed in T2DM. Numerous factors may contribute to the occurrence of a vulnerable blood in patients with diabetes. These include, but are not limited to, endothelial damage, monocyte activation, plaque neovascularization and inflammation, and the shedding of procoagulant microparticles by stimulated or apoptotic cells. Within the atherosclerotic plaque, stimulated smooth muscle cells and macrophages are the main contributor to tissue factor activity through the shedding of procoagulant microparticles. Microparticles sequestered at sites of vascular injury represent a reservoir of highly thrombogenic material that may be released in the bloodstream following plaque rupture. In addition, microparticles released from the plaque may promote angiogenesis, one of the mechanisms involved in intraplaque hemorrhage and plaque rupture among diabetic patients. Blood-borne tissue factor mainly harboured by MPs (that are typically increased in patients with diabetes) can be promptly recruited at sites of endothelial injury, ultimately leading to the triggering of the coagulation cascade.

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aggregation [14]. Platelets from diabetic subjects show low intracellular levels of glutathione and other antioxidants, and this in turn has been linked to an increased production of thromboxane A2 (TxA2) [15]. Of note, tight metabolic control has been shown to result in a significant reduction in TxA2 biosynthesis in platelets of diabetic individuals [16]. Nitric oxide (NO) produced by platelet nitric oxide synthase (NOS) inhibits platelet activation and adhesion to the vascular endothelium by increased cytoplasmic cGMP levels. The generation of NO mediated by ␤-adrenoceptors is impaired in platelets from patients with type 2 diabetes mellitus, and shows a significant negative relation with glycated hemoglobin [17]. Additionally, growing evidence suggests an altered calcium hemostasis in platelets of diabetic patients. Since intraplatelet calcium regulates platelet shape change, TxA2 formation, and platelet aggregation, disordered calcium regulation may contribute significantly to abnormal platelet activity in diabetes [18]. In this pletora of platelet abnormalities that have been suggested to play a role in the thrombogenesis of diabetes, recent research has focused on the potential role of platelet ADP receptors. ADP is released from platelet dense granules upon platelet activation by numerous agonists and thereby amplifies platelet responses regardless of the initial stimulus. Platelets possess two receptors for ADP, P2Y(1) and P2Y(12) [19]. The P2Y(1) receptor is one of many platelet receptors coupled to Gq and initiates ADP-induced activation. The P2Y(12) receptor is linked to Gi and plays a key role in the amplification of platelet activation triggered by other pathways. The P2Y(12) receptor plays a key role and is strikingly upregulated in diabetic platelets compared to control platelets. It has been also suggested that microaggregates of platelets via P2Y(12) receptors could play a key role in the hyperreactivity of platelets in diabetic patients [20].

3. Microparticles in diabetes Other cellular components beside of platelets may be involved in diabetic atherothrombosis. Microparticles (MPs) are a heterogeneous population of submicron membrane-coated vesicles that emerge by budding from their parental cells upon apoptosis or activation [21]. They retain at least some functions of their cell of origin, which can include platelets, endothelial cells, and various leukocytes. Microparticles have the ability to induce coagulation activation with consequent thrombosis of vascular beds. Platelet-derived MPs have been extensively investigated for their participation in thrombosis because they expose negatively charged phospholipids, which provide binding sites for activated coagulation factors [22]. Platelet MPs can also bind to the exposed subendothelial matrix thereby providing a substrate for further platelet adhesion via GPIIb/IIIa fibrinogen bridging [23]. Quiescent platelets express pre-mRNA for tissue factor and, in response to activation, splice this intronic-rich message into mature mRNA [24]. This capacity may allow platelets to synthesize active TF for propagation and stabilization of the thrombus. In an amplification loop, monocyte-derived MPs exposing TF may bind, fuse and transfer proteins and lipids to stimulated platelets through interactions between P-selectin glycoprotein ligand-1 on MPs and platelet P-selectin [23]. Additionally, circulating MPs represent a phospholipase A2 substrate and lead to the production of lysophosphatidic acid, a strong platelet agonist [25]. Other possible noxious pathways regulated by MPs include endothelial and leukocyte activation, the recruitment of monocytes within the plaque, the stimulation of neoangiogenesis, the induction of apoptosis in endothelial or smooth muscle cells, and the promotion of TxA2 release which causes blood vessels to constrict [26]. There is now emerging evidence that microparticles and related TF activities participate actively in the atherothrombotic process in diabetic patients. Increased levels of platelet-derived MPs have

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been reported in diabetic patients with macrovascular complications and have been suggested to play a role in these complications [27]. Interestingly, elevated levels of TF-bearing MPs correlate with the components of the metabolic syndrome in uncomplicated patients with type 2 diabetes [28,29]. 4. Hyporesponsiveness to antiplatelet therapy in diabetes Antiplatelet therapy, with either aspirin or the newer platelet aggregation inhibitors, has been shown to be safe and costeffective for reducing the risk of recurrent atherothrombotic events in nondiabetic patients. Antiplatelet therapy in diabetes, however, is still a matter of intense debate due to a high prevalence of hyporesponsiveness to aspirin and clopidogrel [30]. Platelet hyporesponsiveness – defined as a lower than expected platelet response to a platelet function test – may be a major contributor to poorer outcomes among diabetic patients and may be attributed to several different causes (hyperglycemia and poor metabolic control, increased oxidative stress, reduced sensitivity to nitric oxide, occurrence of a proinflammatory and/or prothrombotic status). For example, it has been recently reported that platelet reactivity is tightly associated with glycemic control in patients with type 2 diabetes on dual antiplatelet therapy [31]. These data suggest that diabetic patients with poor metabolic control and the highest degrees of platelet reactivity may benefit from aggressive antiplatelet strategies. The occurrence of aspirin hyporesponsiveness assessed by an in vitro platelet aggregation test has been documented in almost 20% of diabetic patients, more in those with type 2 than in those with type 1 diabetes [30,32]. Reduced susceptibility of various platelet proteins and receptors on blood platelet membranes to acetylation, due to protein glycation, can be a determinant of platelet hyporesponsiveness to aspirin in diabetic patients [30]. Because higher concentrations of aspirin can counteract the effects of hyperglycemia, one simple mean to overcome aspirin hyporesponsivenes might be to increase the daily dosage of aspirin from 100 to 300 mg among diabetic patients [32]. Patients with type 2 diabetes seem to benefit most from clopidogrel, a P2Y(12) receptor antagonist, compared with aspirin. However, diabetic patients may still have a reduced responsiveness to clopidogrel compared with nondiabetic individuals [33]. The alteration of the P2Y(12)-dependent pathway of platelet reactivity in diabetic patients can partially explain the negative findings of the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA) trial [34]. In this study, 42% of the 15,603 randomized patients had diabetes (17% insulin-treated). The clopidogrel plus aspirin combination did not result in a better cardiovascular protection compared with aspirin alone. Whether more potent P2Y(12) antagonism using a higher maintenance dose of clopidogrel or novel antagonists (cilostazol, prasugrel) will be able to inhibit more efficiently the upregulated P2Y(12) pathway in platelets of patients with type 2 diabetes deserves future studies. In this regard, promising results have been recently obtained with cilostazol in type 2 diabetic patients on standard dual antiplatelet therapy [35–37]. 5. Antiplatelet therapy for the primary prevention of atherothrombotic events in patients with diabetes In contrast with prevailing beliefs, existing data suggest that the clinical efficacy of low-dose aspirin in patients with diabetes is substantially lower than in individuals without diabetes (Table 1). The Physicians’ Health Study included 22,071 male physicians, 40–84 years of age at entry in 1982, who were randomized to receive 325 mg/day of aspirin or placebo [38]. Results showed a signifi-

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Table 1 Clinical studies investigating the potential benefits of antiplatelet therapy in primary prevention for patients with diabetes. Number of diabetics

Antiplatelet therapy

Clinical effects

Follow-up (years)

Reference

Physicians’ Health Study

533 (all males)

Aspirin 325 mg

5

[38]

HOT (Hypertension Optimal Treatment)

1501

Aspirin 75 mg/day

3.8

[39]

ETDRS (Early treatment Diabetic retinopathy Study)

3711

Aspirin 2× 325 mg

5

[42]

POPADAD (Prevention of progression of arterial disease and diabetes

1276

Aspirin 100 mg/day

6.7

[43]

PPP (Primary prevention Project)

1031

Aspirin 100 mg/day

3.7

[40]

JPAD (Japanese Primary Prevention of Atherosclerosis With Aspirin for Diabetes)

2539

Aspirin low dose (81–100 mg/day)

4.37

[44]

CHARISMA (Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance)

2655

Aspirin (75–162 mg/day) + clopidogrel (75 mg/day)

Significant reduction of myocardial infarction (4 versus 10.1%; relative risk 0.39) in the whole population (22,071). Non-significant reduction of myocardial infarction in the diabetic subgroup (11/275 in the aspirin group and 26/258 in the placebo group) Aspirin significantly reduced cardiovascular events by 15% and myocardial infarction by 36% in the entire study cohort. Subgroup analysis in diabetic patients was not provided. Increase of nonfatal bleeding (RR 1.8; P < 0.001) in the whole cohort Significant 28% reduction in the risk for myocardial infarction. Lack of prevention of retinopathy progression. Non-significant reduction in the composite endpoints (death from coronary heart disease or stroke, non-fatal myocardial infarction or stroke, or above-the-ankle amputation for critical limb ischemia [relative risk: 0.98 (95% CI: 0.76–1.26); P = 0.86] Non-significant reduction in the main end point (cardiovascular deaths, nonfatal myocardial infarction, and nonfatal stroke [relative risk 0.90, 95% CI: 0.50–1.62] and in total cardiovascular events (0.89, 0.62–1.26) and with a non significant increase in cardiovascular deaths (1.23, 0.69–2.19). Non-significant 20% reduction in atherosclerotic events [hazard ratio (HR) 0.80 (0.58–1.10); P = 0.16] (Primary endpoint). Significant reduction of coronary and cerebrovascular mortality [HR 0.10 (0.01–0.79); P = 0.0037] In the primary prevention subgroup (3284 patients, 80.8% diabetics), non-significant increase in the primary endpoint (myocardial infarction, stroke and cardiovascular death) for patients on dual antiplatelet therapy (6.6% versus 5.5%; P = 0.020). Significant increase of cardiac vascular death (3.9 versus 2.2%; P = 0.01) and death from all causes (5.4% versus 3.8%; P = 0.04) in patients on dual antiplatelet therapy

2.33

[34]

cant reduction in myocardial infarction in the entire study cohort. However, in the subgroup of patients with diabetes, no significant differences were seen between the two arms (11/275 in the aspirin group and 26/258 in the placebo group) probably due to the small sample size. The Hypertension Optimal Treatment (HOT) trial examined the effects of 75 mg/day of aspirin versus placebo in 18,790 hypertensive patients who were randomized to achieve diastolic blood pressure goals of 90, 85, or 80 mmHg [39]. Aspirin significantly reduced cardiovascular events by 15% and myocardial infarction by 36% in the entire study cohort. Unfortunately, a subgroup analysis of patients with diabetes was not performed. In the 1031 diabetic patients of the Primary Prevention Project (PPP), aspirin therapy was associated with a nonsignificant 10% decrease of cardiovascular death, stroke or myocardial infarction [40]. This finding may be due to the small number of diabetic patients included in this study. Of note, the authors required a sample of at least 4000 diabetic patients to ensure an adequate power. However, study recruitment was stopped prematurely (n = 1031 patients) due to a statistically significant result from the scheduled interim evaluation in the entire study cohort. Thus, the study was not adequately powered for assessing the effect of aspirin on vascular events in patients with diabetes [41]. The Early Treatment Diabetic Retinopathy Study (ETDRS) was designed to assess the possibility that aspirin may retard the progression of diabetic retinopathy. Diabetic patients (n = 3711) with known retinopathy were randomly assigned to aspirin (325 mg twice a day) or placebo. A history of cardiovascular disease was

present in 49% of the study participants. All-cause mortality was specified as the primary outcome measure for assessing the systemic effects of aspirin. Other outcome variables included causespecific mortality and cardiovascular events. In the entire study period, the relative risk for all-cause mortality for aspirin-treated patients compared with placebo-treated patients was 0.91 (99% confidence interval: 0.75–1.11). Macrovascular events were a secondary endpoint and were not significantly decreased on aspirin [42]. However, in the first five years of follow-up, the relative risk for myocardial infraction was significantly reduced in the aspirin arm (RR 0.72 99% CI 0.55–0.95; P < 0.01). The Prevention Of Progression of Arterial Disease And Diabetes (POPADAD) trial looked at the efficacy and safety of aspirin plus antioxidant compared with aspirin alone, antioxidant alone and placebo in patients with diabetes and asymptomatic peripheral arterial disease [43]. All participants (n = 1276) were aged 40 years or older and had type 1 or type 2 diabetes and an ankle brachial pressure index ≤ 0.99 but no symptomatic cardiovascular disease. The study produced no evidence to support the use of either aspirin or antioxidants in the primary prevention of cardiovascular events and mortality in individuals with diabetes. Finally, the Japanese Primary Prevention of Atherosclerosis With Aspirin for Diabetes (JPAD) trial demonstrated that low-dose aspirin use was associated with a nonstatistically significant 20% reduction in atherosclerotic events among Japanese adults with type 2 diabetes [44]. In this context, the current evidence suggests that diabetes should be considered as a separate entity, not just one of the many subgroups at high

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atherothrombotic risk, at least with regard to the potential benefits associated with aspirin in primary prevention. Another trial, A Study of Cardiovascular Events in Diabetes (ASCEND, clinicaltrials.gov: NCT00135226), is recruiting at least 10,000 patients with diabetes (either type 1 or type 2) without overt vascular disease. ASCEND participants are randomly allocated to take 100 mg aspirin daily or placebo in a double-blind design. Hopefully, the ASCEND trial will provide the most reliable evidence about the effects of aspirin for primary prevention in patients with diabetes [41]. The usefulness of clopidogrel for primary prevention of atherothrombotic events is diabetes has not been properly evaluated. The Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA) trial has compared clopidogrel (75 mg/day) plus low-dose aspirin (75–162 mg/day) to placebo plus low-dose aspirin in 15,603 high risk patients, either for primary or secondary prevention, with a median follow-up of 28 months [34]. The rates of major vascular events were not significantly different between the two randomized treatment arms. Subgroup analysis showed that among the 3284 patients in primary prevention with multiple risk factors, including diabetes in 80.8%, the rate of the primary end point was 6.6% with clopidogrel plus aspirin and 5.5% with aspirin plus placebo (P = 0.20). There was a trend toward higher risk of severe bleeding in the primary prevention group compared with the secondary prevention group. Of note, subgroup analysis of the CHARISMA trial has clearly shown that dual therapy with clopidogrel and aspirin in asymptomatic patients is associated with excess cardiovascular mortality compared with subjects treated with aspirin alone [34]. Based on the data from 3284 primary prevention patients (of whom 80.8% were diabetics), there was a significant (P = 0.01) increase of cardiovascular death from 2.2% in the aspirin-only group up to 3.9% in patients treated with clopidogrel and aspirin combination. Likewise, in this subgroup there was a significant increase of all-cause mortality in patients treated by dual antiplatelet therapy (5.4% versus 3.8%; P = 0.04) [45]. There is no obvious explanation for the excess mortality in the dual therapy arm, but an increased incidence of intraplaque hemorrhage may play a role. In asymptomatic patients, the rates of severe and moderate bleeding were 2.0% and 2.2%, respectively, in the clopidogrel group, and 1.2% and 1.4% in the placebo group (P = 0.07 and P = 0.08). There is evidence to suggest that atherosclerotic plaque in diabetic patients are characterised by increased vasa vasorum neovascularization [46]. This pathological finding can be associated with a higher risk of intraplaque hemorrhage with consequent rupture or thrombosis. In the light of this evidence, the use of dual antiplatelet therapy cannot be recommended in diabetic patients without overt cardiovascular disease.

6. Antiplatelet therapy for the secondary prevention of atherothrombotic events in patients with diabetes Diabetic patients with a history of atherothrombotic events are at a high risk for recurrences and, in the absence of any absolute contraindication, should be treated with antiplatelet therapy. In over 4500 diabetic patients enrolled in the Antithrombotic Trialists’ Collaboration (ATC), the incidence of vascular events was reduced from 23.5% in the control group to 19.3% in the subjects receiving antiplatelet therapy, mainly aspirin at doses ranging from 75 to 325 mg/day (P < 0.01) (Antiplatelet Trialists’ Collaboration). Collaborative overview of randomized trials of antiplatelet therapy. I. Prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients [41]. The Clopidogrel versus Aspirin at Risk of Ischemic Events (CAPRIE) study has demonstrated that clopidogrel (75 mg) is slightly more effective than aspirin (325 mg) in reducing the com-

371

bined risk of stroke, myocardial infarction, or vascular death in 19,185 patients with atherosclerotic vascular disease [47]. A subgroup analysis in diabetic patients identified 3866 patients from the CAPRIE cohort [48]. There was an event rate per year of 17.7% in the diabetic patients who received aspirin, and a significantly lower (2.1%, P = 0.042) event rate of 15.6% in those on clopidogrel. The Clopidogrel in Unstable angina to prevent Recurrent Events (CURE) trial examined whether the addition of clopidogrel to aspirin therapy in the context of acute coronary syndrome (ACS) improved outcome [49]. In contrast to CAPRIE [47] which performed a headto-head comparison of the two agents, CURE examined the role of synergistic antiplatelet therapy in 12,562 patients with non-ST elevation ACS. The primary composite outcome of death, nonfatal myocardial infarction or stroke occurred in 9.3% of the aspirin plus clopidogrel group and in 11.4% of the aspirin alone group (P < 0.001). In the CURE study, 2840 patients had diabetes. As in CAPRIE, the diabetic subgroup showed a higher vascular event rate compared with their nondiabetic counterparts. When an endpoint of cardiovascular death, nonfatal MI or stroke was considered, 14.2% of those on combined therapy suffered such an event. Of note, 16.7% of the aspirin-only group suffered from a vascular event. The relative benefit of combined therapy over aspirin alone in diabetes narrowly failed to achieve statistical significance.

7. Antiplatelet therapy in diabetic patients undergoing percutaneous coronary intervention Percutaneous coronary intervention (PCI) with stenting is known to enhance platelet aggregation. In this context, optimization of antiplatelet therapy has a crucial role in patients undergoing percutaneous revascularization and even more aggressive antiplatelet therapies have been employed to prevent postprocedural thrombotic complications. Although diabetic patients undergoing PCI show similar short-term outcomes compared with nondiabetic patients, they show an increased risk of subsequent myocardial infarction, restenosis, and mortality [50]. Notably, diabetic patients have trends toward increased rates of stent thrombosis [51]. Growing evidence has accrued that platelet glycoprotein GPIIb/IIIa inhibitors have an enhanced benefit in diabetic patients undergoing PCI for acute coronary syndromes. Data from a pooled analysis from three trials of abciximab in diabetics have suggested that abciximab may be associated with a 1-year survival advantage in this patient group [52] (Table 2). The Intracoronary Stenting and Antithrombotic Regimen: Is Abciximab a Superior Way to Eliminate Elevated Thrombotic Risk in Diabetics? (ISARSWEET) study investigated 701 patients with diabetes who were pretreated with a 600-mg dose of clopidogrel at least 2 h before PCI. Patients were subsequently randomized to receive either abciximab or placebo during PCI, followed by at least 6 months of clopidogrel after PCI. In the entire study population and in the subgroup of diabetic patients, abciximab was no better than placebo in reducing the primary composite endpoint of death and myocardial infarction at 1-year follow-up [53]. In the OPTIMIZE-it trial, a high-dose bolus of tirofiban in stable diabetic patients undergoing elective PCI, along with double antiplatelet therapy, was associated with a significant further inhibition of platelet aggregation which, however, did not translate in a lower incidence of postPCI distal embolization [54]. Another group of antiplatelet agents that have been shown to reduce periprocedural complications are thienopyridines. In the subgroup of patients undergoing PCI in the CURE trial (PCI-CURE study), pretreatment with clopidogrel for a median of 6 days followed by long-term therapy was associated with lower rates of cardiovascular death, myocardial infarction, or any revascularization (P = 0.03) and of cardiovascular death or myocardial infarction (P = 0.047). This strategy may also be benefi-

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Table 2 Clinical studies investigating the potential benefits of antiplatelet therapy for patients with diabetes undergoing PCI. Number of diabetics PCI-Cure (substudy)

504

OPTIMIZE-IT

46

Bhatt et al. (meta-analysis) TRITON-TIMI 38 (substudy)

1462 3146

Antiplatelet therapy

Clinical effects

Reference

Clopidogrel 300 mg loading dose before PCI and aspirin 75–325 mg. After PCI, most patients (>80%) in both groups received open-label thienopyridine for about 4 weeks, after which study drug was restarted for a mean of 8 months. High-dose bolus (25 ␮g/kg per 3 min) of tirofiban versus placebo in patients undergoing elective PCI along with dual antiplatelet therapy. Abciximab versus placebo in patients undergoing PCI. Aspirin in all patients. Prasugrel (60/10 mg) versus clopidogrel (300/75 mg). Aspirin in all patients

Non-significant reduction of cardiovascular death, myocardial infarction, or urgent target-vessel revascularization within 30 days of PCI (16.5–12.9%) (relative risk: 0.77; 95% CI: 0.48–1.22).

[55]

Significant further inhibition of platelet aggregation which, however, did not translate in a lower incidence of post-PCI distal embolization.

[54]

Significant reduction of 1 year mortality in diabetic patients treated by abciximab (4.5–2.5%, P = 0.031) Significant reduction of cardiovascular death, nonfatal MI, or nonfatal stoke in diabetics patients treated by prasugrel (12.2% versus 17.0%; hazard ratio, 0.70; P < 0.001)

[52]

cial to patients with diabetes, who experienced a reduction from 16.5% to 12.9% in cardiovascular death or myocardial infarction [55]. This difference, however, did not reach statistical significance. Research has been carried out to investigate the clinical impact of more profound inhibition of the platelet surface-membrane P2Y(12) receptor with prasugrel in patients with diabetes. In a subanalysis of the TRITON-TIMI 38 trial, dual antiplatelet therapy has been assessed in patients with diabetes mellitus [56]. The novel thienopyridine prasugrel was superior to clopidogrel in reducing primary efficacy outcomes in patients with diabetes mellitus without increasing the risk of bleeding (12.2% versus 17.0%; HR, 0.70; 95% CI, 0.58–0.85; P < 0.001). The benefit of prasugrel was observed regardless of the concomitant use of GPIIb/IIIa antagonists. Future studies are needed to assess whether triple antiplatelet therapy (aspirin, clopidogrel, and cilostazol) [57] may be used clinically to prevent thrombotic complications after PCI in patients with diabetes [36,37]. There is an ongoing debate as to the optimal duration of antiplatelet therapy in patients with diabetes undergoing PCI. Several reports have shown that withdrawal of oral antiplatelet agents may be associated with an increased risk of death and myocardial infarction, probably due to a rebound in platelet reactivity [58]. Current recommendations limit the use of a dual antiplatelet regimen to 1 year, following which patients withdraw clopidogrel and indefinitely maintain treatment with aspirin. Of note, diabetic patients on long-term dual antiplatelet therapy experience proinflammatory and prothrombotic effects following clopidogrel withdrawal. Current evidences, however, display a worrisome paucity of data. There is thus an urgent need to develop evidence-based practices as to the optimal duration of antiplatelet therapy in patients with diabetes. 8. Are platelets the key player in diabetic atherothrombosis? Although there is no doubt about the role played by platelet activation following PCI in diabetes, recent data have questioned the supposed pivotal role of the platelet in diabetic atherothrombosis. Accordingly, long-term outcomes of antiplatelet therapies in diabetes have been frequently disappointing. It is thus possible to postulate that, beyond platelets, other players may be involved in diabetic atherothrombosis. Microparticles, shed by platelets or stimulated cells, represent another important reservoir of bioactive vascular effectors involved in thrombotic response, vascular wall inflammation and remodelling. Circulating MPs provide an additional procoagulant phospholipidic surface, probably enabling the assembly of clotting enzyme complexes and subsequent thrombin

[56]

generation. The catalytic properties of MPs rely on a procoagulant anionic phospholipid, phosphatidylserine, made accessible at the outer leaflet of the membrane following plasma membrane remodelling and on the eventual presence of tissue factor, the main activator of blood coagulation [59]. Microparticles constitute the main reservoir of blood-borne TF and there is evidence indicating that acute thrombosis may be triggered by TF disseminated by MPs in the peripheral circulation [59]. This suggests that MPs not only can serve as a reliable biomarker of vascular damage but may actually play an active detrimental role in the disruption of vascular homeostasis. Using real-time intravital microscopy thrombosis models, it is been previously shown that swift accumulation of leukocyte-derived MPs harbouring TF activity seems to be mandatory for thrombus growth [60,61]. In addition, soluble P-selectin (sP-selectin) has been shown to promote the shedding of leukocytederived TF-bearing MPs, resulting in a full hemostasis correction in a mouse model of hemophilia A [62]. To shed more light on the role of circulating or sequestered TF-bearing MPs in thrombus development, reciprocal bone marrow transplants between wild-type and engineered mice expressing minimal levels of TF were performed [63]. The study showed that arterial vessel wall TF is involved in the initiation of platelet activation, while blood-borne TF spread by MPs mediates thrombus propagation [63]. In an experimental model of venous thrombosis, leukocyte and platelet-derived MPs were found to be significantly associated with thrombus weight and tissue factor activity. Of note, reinjections of MPs resulted in an increased thrombus weight [64]. Taken together, this evidence clearly indicates that TF-positive MPs are key players during thrombus formation. Recent research has highlighted the crucial role played by TFbearing MPs in the atherothrombotic complications of diabetes [65]. Of note, advanced glycation end products (AGEs), one of the most potent inducers of vascular damage in type 2 diabetes, have been shown to induce a time- and concentration-dependent increase in TF activity that is enhanced by proinflammatory molecules (e.g., tumor necrosis factor-alpha) and inhibited by antioxidants such as N-acetyl cysteine [66]. Interestingly, TF expression in monocytes is significantly higher in patients with type 2 diabetes than in normoglycemic subjects [67]. Similarly, the induction of hyperglycemia and hyperinsulinemia in normal subjects has been shown to induce platelet activation and monocyte TF expression resulting in promoting a procoagulant state that may contribute to atherothrombosis [68]. In diabetes, a wide panel of blood and vascular cells – including platelets, endothelial cells, monocytes, and islets of Langerhans – release MPs [69,70]. Shedding of MPs is triggered by different stimuli, including proinflammatory cytokines (tumor necrosis

O. Morel et al. / Atherosclerosis 212 (2010) 367–376

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Fig. 2. Possible antithrombotic effects of statins in T2DM. Statin therapy reduces the expression and the release of tissue factor from activated monocytes. In turn, this phenomenon leads to reduced platelet stimulation and aggregation. In patients with type 2 diabetes, pravastatin reduces the expression of GPIIb-IIIa on platelet-derived MPs, leading to blunted platelet activation and aggregation. Combination of pitavastatin with eicosapentaenoic acid may exert synergistic effects in reducing platelet-derived MPs and sCD40 levels in diabetic patients with hyperlipidemia. Statins can also exert potential benefits by facilitating the detachment of activated endothelial and the release of endothelial-derived MPs cells at sites of endothelial injury. This mechanism could contribute to the improvement of the overall condition of the remaining vascular endothelium. In addition, statins may reduce the expression of active tissue factor in the damaged endothelium.

factor-alpha, interleukin1-beta), soluble CD40L, advanced glycation endproducts, oxidative stress, and hyperglycemia. Other studies in the hyperglycemic milieu have suggested that TF-bearing microparticles may exert proinflammatory effects and promote neoangiogenesis, thereby being involved in the development of diabetic vasculopathy [71]. Of note, the phenotype and procoagulant potential of MPs may vary according to the type of diabetes or glycemic control [69]. In this regard, high levels of procoagulant activity borne by circulating MPs have been detected in patients with poorly controlled HbA1c levels [69]. Similarly, diabetic patients with other common cardiovascular risk factors (such as hypertension) show raised levels of endothelial- and platelet-derived MPs [72]. Interestingly, MPs have emerged as a reliable biomarker of diabetic vascular dysfunction [25]. Experimental animal studies have recently shown that diabetic mice show reduced levels of circulating endothelial progenitor cells (EPCs) and increased plasma endothelial microparticles compared with euglycemic animals [73]. Similar findings have been reported in human studies. Of note, Pirro and coworkers have reported that low levels of EPCs and raised EPC-derived MPs are associated with the aortic stiffness ratio independently of the Framingham risk score [74]. In addition, raised levels of endothelial-derived MPs have been associated with increased brachial-ankle pulse wave velocity and diminished endothelium-dependent flow-mediated dilation of the brachial artery [75]. In line with these findings, a significant inverse association between circulating levels of endothelial-derived MPs and endothelial-dependent vasodilation has been reported in diabetic patients with coronary artery disease. In this study, elevated endothelial-derived MPs emerged as the most significant predictor CAD, with a higher predictive value compared with traditional risk factors [6]. In patients with type 2 diabetes and acute coronary syndromes, elevated endothelial-derived MPs have been linked to non-calcified atheromatous lesions as detected by multidetector

computed tomography. In this report, there were no associations between platelet-derived MPs levels and either acute coronary syndromes or the presence of non-calcified atheromatous lesions [76]. The importance of endothelial-derived MPs for predicting cardiovascular events has been recently demonstrated in a study of 387 stable patients at high risk for coronary heart disease (of whom 41.5% were diabetics) [77]. Among a wide variety of agonists that can promote the release of MPs, the binding of oxidized low-density lipoproteins (oxLDL) to their receptor has been shown to promote the release of circulating MPs from endothelial cells in diabetic patients, an effect inhibited by the sulfonylurea glyclazide [78]. Insulin-resistant macrophages entrapped within the atheromatous plaque may also promote the shedding of TF-bearing MPs [79]. Hyperglycemia may thus promote a prothrombotic state through their effects on tissue factor and circulating microparticles. Both endothelial erosion and plaque rupture may recruit MPs from the bloodstream, resulting in an increased coronary accumulation of TF ultimately leading to arterial thrombosis (Fig. 1) [25]. After plaque disruption, circulating MPs may also support a cellular cross-talk leading to vascular inflammation and tissue remodelling, endothelial dysfunction, and leukocyte adhesion [26]. Blood-borne TF mainly harboured by circulating MPs may act synergistically as potent triggers of blood coagulation at sites of plaque rupture [80]. Recent data from our group have supported this possibility. Procoagulant MPs were measured within the occluded artery at the vicinity of the coronary thrombus during myocardial infarction [81]. Although there was no significant increase in platelet-derived MPs, both endothelial and leukocyte-derived TF-bearing MPs were found to be increased. These findings suggest that endothelial and leukocyte activation might play a significant role in coronary atherothrombosis through the shedding of procoagulant MPs. Of note, it has been recently confirmed that TF activity mainly harboured by MPs predicts cardiovascular mortality in patients with acute myocardial infarction [82].

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In this context, pharmacological approaches that enable the modulation of tissue activity or the release of procoagulant MPs within the vasculature hold promise for reducing the burden of atherothrombosis. Recent evidence suggests that insulin reduces the expression of TF in monocytes and monocyte-derived microparticles [83]. In addition, statins – probably the most efficient drugs for reducing atherothrombotic complications in patients with diabetes – may target MPs (Fig. 2). An in vitro study has shown that fluvastatin may inhibit shedding of microparticles from human coronary artery endothelial cells partly through its action on Rho family proteins [84]. Of interest, statins have been shown to reduce the expression of fibrinogen receptor GPIIb/IIIa on platelet-derived MPs, a mechanism by which pravastatin could blunt platelet activation and aggregation [85]. Combination of pitavastatin with eicosapentaenoic acid has been suggested to reduce both platelet-derived MPs and CD40 levels in hyperlipidemic patients with diabetes [86]. In patients with type 2 diabetes, the combination of simvastatin and ramipril has been shown to reduce plasma TF activity and prothrombin fragment F1/2 to a greater extent than in those receiving monotherapy with either agent (Fig. 2) [87]. Statins can also exert potential benefits by promoting the detachment of activated endothelial cells and the release of endothelial-derived MPs at sites of endothelial injury. The shedding of these MPs could be viewed beneficial as these microparticles contribute to the sorting of several deleterious proapoptotic factors such as caspase-3, thus preventing endothelial cell apoptosis [88]. Pharmacological approaches to modulate oxidative stress may also influence the release of MPs. Interestingly, the administration of vitamin C in diabetic patients with a history of myocardial infarction has been shown to have reduce the levels of platelet-derived MPs [89]. Additionally, eicosapentaenoic acid administration may reduce the number of endothelial-derived MPs levels in patients with type 2 diabetes and high angiopoietin2 concentrations [90]. Taken together, these results suggest that the pharmacological modulation of microparticle release appears a promising approach for reducing the burden of atherosclerotic complications in patients with diabetes. Future trials are needed to assess prospectively the value of this approach on cardiovascular events in diabetic patients.

9. Concluding remarks There is limited evidence that aspirin may be effective and safe for primary cardiovascular prevention in diabetic patients [40,42–44]. Additionally, the CHARISMA trial has indicated that the benefit/risk ratio of dual antiplatelet therapy may be harmful in diabetics [34]. In contrast, the role of antiplatelet therapy in secondary prevention after ischemic cardiac events is well established in diabetes. Glycoprotein IIb/IIIa receptor antagonists can reduce mortality in diabetic patients committed to undergo PCI [52]. Upregulation of P2Y(12) signalling occurs in hyperglycemia [20], and the relevance of platelet P2Y(12) receptor inhibition with prasugrel in reducing adverse events following PCI has been recently suggested [56]. Nonetheless, new therapeutic paradigms must be created for bridging the gap between the large body of evidence implicating the platelet in the initiation and progression of experimental atherothrombosis, and the relatively minor role play by platelets in the macrovascular complications of diabetes. Converging evidence indicates that MPs are not only a reliable marker of vascular damage but can also participate actively in promoting atherothrombotic complications. In this context, drugs that may reduce the release of microparticles and/or their thrombogenic capacity may have the potential to improve upon current antiplatelet therapy, possibly resulting in lower adverse events rates in diabetic individuals.

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