Platelets and cardiovascular risk
Barbara Renga, PhD; Franco Scavizzi, MD
Clinilab, P.S. Giovanni, Perugia, Italy.
Atherosclerosis and its late sequels are still the number one cause of death in Western societies. Platelets are a driving force not only during the genesis of atherosclerosis, but especially in its late stages, as evidenced by complications such as arterial thrombosis, myocardial infarction, and ischaemic stroke. Platelets are small, anucleate blood elements of critical importance in cardiovascular disease, a major cause of morbidity and mortality. Numerous risk scores exist to identify healthy individuals at increased risk of developing atherosclerosis and cardiovascular disease. However, markers of cardiovascular risk not routinely assessed (i.e. platelet activity, mean platelet volume and P-selectin) may also contribute to be useful in calculating cardiovascular risk. The present review and meta-analysis summarizes the evidence for measuring platelet function indices to identify patients at risk of developing cardiovascular events.
Keywords Atherosclerosis – cardiovascular risk – platelets – MPV – P-selectin.
Introduction on platelet function in atherosclerosis and cardiovascular disease
Despite remarkable progress in therapy, atherosclerosis and its associated complications, such as coronary heart disease, are still the leading cause of death in Western societies. Atherosclerosis is anticipated by morphologic and functional changes involving vessel wall and vascular endothelium, which in turn lead to endothelial dysfunction1. Growing evidence has linked the omega-3 intake with an improved endothelial function. The potential mechanisms for the enhanced endothelial function by omega-3 include: (i) increasing endothelium-dependent vasodilation; (ii) regulating endothelial cell turnover, and (iii) reducing both production of inflammatory cytokines and platelet activation2-4.
Platelets are small cell fragments of large importance in medicine. The classical function of platelets is coverage and closure of endothelial wounds, and contact between platelets and the subendothelial matrix triggers their activation and drives thrombus formation also during the early pathophysiological process of plaque formation (figure 1). Moreover, platelets have been shown to also interact with intact endothelium and recruit leukocytes even before an atherosclerotic plaque has formed5. Indeed, platelets of atherosclerotic apolipoprotein E deficient (ApoE–/–) mice were observed to adhere to intact endothelium via the von Willebrand factor receptor GPIbα and the fibrinogen receptor αIIbβ3 well before atherosclerotic plaques had formed5. Platelets express various inflammatory receptors, including fractalkine receptor (CX3CR1) which induces P-selectin on platelets upon binding to fractalkine (CX3CL1) expressed on inflamed endothelial cells. In turn, P-selectin exposure initiates local accumulation of leukocytes, driven by arterial shear forces6-7 (figure 1).
The importance of P-selectin in the genesis of atherosclerosis is underlined by the finding of increased intima-media-thickness in human subjects presenting with high levels of platelet P-selectin7. Indeed, both platelet and endothelial P-selectin contributed to lesion formation in a mouse model of atherosclerosis that was based on the adoptive transfer of P-selectin positive or negative platelets8.
Whether platelet-adhesion to the intima mediates direct damage to the endothelial lining remains unclear, but several studies found how platelets contribute to vascular inflammation via their interaction with leukocytes9. Activated platelets were shown to exacerbate atherosclerosis in ApoE deficient mice via the recruitment of monocytes and other leukocytes9. In turn, the formation of platelet- leukocyte aggregates (PLA) facilitated the deposition of inflammatory platelet mediators on endothelial cells10. Others found that the number of circulating PLAs was increased upon platelet activation11. As potential mechanisms underlying platelet-leukocyte crosstalk, several receptor/ligand pairs have been identified, including integrins or members of the JAM family of proteins9. A list of platelet expressed receptors with potential relevance for artherosclerosis is given in table 1. Finally, platelets may also contribute to vascular inflammation via release of active biomolecules from their granules12. As prominent examples, the release of chemokines such as CCL5 or CXCL4 contributes to atherosclerosis in a P-selectin-dependent manner13. Given the relevance of fatty acids in the genesis of artherosclerosis, it should be noted that also oxidized LDL, one of the major initiators and drivers of atherosclerosis, can be bound by platelets and interactions with lipoproteins can change platelet function14. In line with this, platelets of hypercholesterolaemic patients show hyper-aggregability in vitro and enhanced activity in vivo15. In conclusion, platelet activation seems to confer pro-atherosclerotic effects, as well as effects of atheromodulation and tissue/vascular remodelling.
Methods of measuring platelet function
The historical ‘‘gold standard’’platelet function test is turbidometric platelet aggregometry, which measures platelet coaggregation in platelet-rich plasma16. Samples are exposed to an agonist, such as adenosine diphosphate (ADP) or arachidonic acid, and the increase in light transmittance resulting from platelet-platelet aggregation is measured. Its advantages are that it can be used to monitor ASA, thienopyridines, and platelet glycoprotein αIIbβ3 inhibitor therapy17. Its disadvantages include the large sample volumes required, long processing times, and complex sample preparation. Impedance aggregometry is conceptually similar to turbidometric platelet aggregometry, but, rather than light transmission, it measures the increase in electrical impedance across two precious metal wires resulting from platelet coaggregation in response to an agonist16. It has the same disadvantages as turbidometric platelet aggregometry but it uses whole blood instead of platelet-rich plasma17. Other aggregation tests include the VerifyNow test (Acumetrics, San Diego, Calif., USA), a simple, rapid, point-of-care method that has several other advantages: required sample volumes are small, it uses whole blood, and no pipetting is required16. Verify Now has been used to monitor the pharmacodynamic effects of the 3 main classes of antiplatelet therapies: ASA, thienopyridines, and αIIbβ3 inhibitors.
Other methods assess activation-dependent changes on the platelet surface. These tests include measurement of levels of platelet surface P-selectin, activated αIIbβ3, and leukocyte-platelet aggregation. Their advantages include the small sample volumes required and the use of whole blood; disadvantages include complex sample preparation, the requirement for flow cytometry and experienced operators, and lack of commercial availability. They have been used to monitor the various classes of antiplatelet therapies. The thromboelastogram (TEG) Platelet Mapping System measures platelet contribution to clot strength16. It is a point-of-care method using whole blood to assess platelet clot formation and clot-lysis data. It is able to monitor all 3 classes of antiplatelet therapies. However, it requires pipetting and has undergone only limited study. The Impact cone and plate(let) analyzer is a simple, rapid, point-of care method assessing shear-induced platelet adhesion18. It uses whole blood, requires low-sample volumes, and needs no sample preparation. The drawbacks include the need for pipetting and lack of widespread availability. It is not recommended for monitoring of αIIbβ3 inhibitor therapy. The Platelet Function Analyzer (PFA)-100 measures in vitro the cessation of high-shear blood flow by the platelet plug. It is a simple, rapid, point-of-care, whole blood method that requires low-sample volumes and no sample preparation. Its disadvantages are that it is dependent on von Willebrand factor (vWF) and haematocrit levels and that it requires pipetting. It is not recommended for monitoring of thienopyridines18. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation measures activation-dependent platelet signalling. Its advantages include small required sample volumes, the use of whole blood, stability (allowing samples to be shipped to a remote laboratory), and dependency on the P2Y12 receptor, the site of action for thienopyridines. Its disadvantages are that it requires complex sample preparation, flow cytometry, and experienced technicians, and cannot be used to monitor ASA or αIIbβ3 inhibitor treatment18. The serum thromboxane B2 level reflects its activation-dependent release from platelets. Its chief advantage is that it is dependent on cyclooxygenase (COX)-1, the specific enzyme inhibited by ASA. However, thromboxane B2 levels may be influenced by prostaglandins produced by leukocyte-derived COX-2. Therefore, thromboxane B2 is not entirely COX-1- or platelet-specific. This indirect measure is not completely platelet-specific, however. These characteristics also apply to measurements of the ratio of the stable urinary metabolite of thromboxane B2, 11-dehydro-thromboxane B2 (UDTB) to creatinine. These tests cannot be used to monitor thienopyridines or αIIbβ3 inhibitor therapy.
In the HAPARG (haemostatic parameters as risk factors in healthy volunteers) study19 2,723 healthy volunteers were enrolled, their platelet function measured with light transmission aggregometry and they were followed for a period of 4-6 years for the development of fatal or non-fatal cardiovascular end points. Enhanced platelet aggregation increased with age and was more frequent in women. After multivariable adjustment, spontaneous platelet aggregation was modestly, albeit significantly, associated with incident vascular events19. A small prospective study of 150 apparently healthy men found that subjects with an ADP-induced platelet aggregation response above the median, as measured by rate of aggregation and lag time, had a significantly increased incidence of coronary heart disease mortality compared with those below the median20. However, no significant associations were seen with adrenaline or collagen-induced platelet aggregation20. Other studies were unable to demonstrate a significant association between platelet aggregation and cardiovascular events. Platelet aggregation was measured in a random sample of men participating in the Northwick Park Heart Study21. Among 1,369 apparently healthy men, there was no significant difference between ADP or adrenaline-induced aggregation in those who experienced ischaemic heart disease and those who remained event free24. In another study, the Caerphilly cohort measured ADP and thrombin-induced aggregation in more than 2,000 men aged 49-65 years. There was no significant association between aggregation induced by either thrombin or ADP and incident ischaemic heart disease over 5 years follow-up. However, men with a high secondary (irreversible) response versus a low secondary response to ADP were 20% more likely to develop disease and more likely to develop disease within 500 days (versus an event occurring 1,000 days); however, this finding was not statistically significant22. In longer follow-up from the Caerphilly cohort, no significant association was demonstrated between platelet aggregation and 10-year follow-up of myocardial infarction; however, a paradoxical association was noted between ADP-induced aggregation and subsequent stroke23. Using ADP-induced whole blood aggregometry, no difference was observed between subjects with and without ischaemic heart disease23. Overall, the data for platelet aggregometry and cardiovascular end points is far from conclusive.
Mean platelet volume
MPV and coronary artery disease. Mean platelet volume (MPV) is a useful biomarker of platelet activity24. Mean platelet volume is increased during acute myocardial infarction (AMI) and in the first subsequent weeks25. This finding is often associated with a transient decrease in platelet count25. Moreover, among patients with coronary artery disease, those with a higher platelet volume seem to have a greater risk of AMI than those with a lower MPV, regardless of the extent of the coronary lesions25, although some authors have reported contrasting data26. These findings have led to the conclusion that the increase in platelet size occurs before the acute event and may play an important role in the genesis of the disease. Kristensen et al. demonstrated that the bleeding time of patients with AMI is shorter than that in patients with unstable angina27. This effect seems to be mediated by thromboxane, which is produced in larger quantities in the presence of high MPV values. MPV also seems to be independent of the infarct size and site28, which reinforces the impression that this parameter is mainly determined before the acute event. However, large platelets may be released into the circulation, at least in part, also in response to myocardial ischaemia, as is suggested by the fact that MPV increases after exercise stress test in coronary patients29. Muscari et al. found a direct association between an MPV > 8.4 fl (the high tertile of its distribution) and ischaemic ECG changes30, while Pizzulli et al. found higher MPV values in patients with documented coronary artery disease than in controls, and in unstable angina more than in stable angina31.
Other authors investigated the association of MPV with the prognosis of acute coronary syndromes and with the outcome of percutaneous coronary interventions. For example, Martin et al. measured MPV 6 months after AMI in 1,716 patients, who were followed up for 2 years: the patients who had a second ischaemic event had higher baseline MPV values than the patients who had no further AMI; MPV values were also higher in those who subsequently died with respect to those who did not, showing that platelet size is a risk factor and a prognostic indicator in patients with AMI32. Mean platelet volume was even found to be an effective marker of impaired reperfusion in patients who underwent coronary angioplasty33. Some authors showed that the patients with a high MPV before the angioplasty had an increased risk of death and coronary restenosis in the following 4-8 months34.
MPV and cerebrovascular disease. Several studies have investigated the relationships of MPV with stroke and its prognosis. O’Malley et al. noticed an increase of MPV in all subtypes of ischaemic stroke35. This increase was already detectable in the acute phase (within 48 h of the onset of symptoms) and persisted long after the stroke (the second measurement was performed after 6 months). This finding led to the conclusion that increased MPV values were probably pre-existent to the acute event, and were potentially involved in the genesis of the disease, considering, in particular, that mean platelet life is only 8 days. Most studies found a greater MPV increase in cortical strokes than in lacunar strokes36. Importantly, in patients with previous cerebrovascular diseases, the PROGRESS study showed that MPV could predict the risk of a second stroke up to 4 years before the acute event, documenting an 11% increase of the relative risk of stroke for each femtoliter of MPV increment37. Mean platelet volume can even provide some information about the prognosis of stroke patients38.
P-selectin is an adhesion molecule found in platelets and endothelial cells. P-selectin expressed on activated platelets (pP-sel) plays an important role in the platelet-mononuclear cell interaction (a major role in platelet activation). Evidence supporting a role for P-selectin in atherogenesis is observed from several sources; p-selectin is found on human atherosclerotic plaques39, P-selectin has additional procoagulant and proinflammatory activities, P-selectin-deficient mice develop smaller atherosclerotic lesions, and anti P-selectin antibodies are able to inhibit platelet aggregation40. Soluble forms of P-selectin (sP-sel) can be measured in plasma and are derived from both the endothelium and platelet sources. Clinically, sP-sel may be a more reliable marker of platelet activation than pP-sel when assessing platelet activity by platelet aggregation. For example, sP-sel was found to be significantly associated with spontaneous platelet aggregation in those with peripheral artery disease, while pP-sel was not41. Also, sP-sel can be easily measured in vitro by an enzyme-linked immunosorbent assay (ELISA), whereas, pP-sel measurements require the more time and equipment intensive flow cytometry. For these reasons and others, sP-sel has been used more frequently as a marker of platelet activation. Numerous studies examine the association between sPsel levels and cardiovascular disease, but few have studied this marker prospectively. A cohort analysis from the Women’s Health Study of 345 apparently healthy women found that elevated baseline sP-sel values significantly correlated with future cardiovascular events during a 3.5-year follow-up. Women in the highest quartile group had a greater than 2-fold risk of developing a cardiovascular event than subjects in the lowest quartile (P = 0.01). For each quartile increase in sP-sel values, the risk of cardiovascular events increased by 28% (P = 0.03) 41. The Coronary Artery Risk Development in Young Adults (CARDIA) study examined the association between sP-sel levels and carotid intima-media thickness, a marker of subclinical atherosclerosis. In a group of 1,911 young men and women aged 33-45 years, sP-sel levels were significantly associated with carotid intima-media thickness measured 5 years later, most notably in European Americans42. A prospective case-control study with 16 years of follow-up also noted that sP-sel was associated with cardiovascular events in patients without baseline coronary heart disease; however, much of this association was attenuated after adjustment for clinical risk factors such as age, sex, smoking, and blood cholesterol levels and socio-economic status. Of note, this study measured sP-sel in the serum and therefore may prevent comparisons to other studies43.
Discussion and conclusions
Most researchers today consider that a high intake of saturated fat and elevated LDL cholesterol are the most important causes of atherosclerosis and coronary heart disease (CHD). Contrary to the widely held belief among doctors and researchers, there is little evidence that high cholesterol leads to cardiovascular disease44. High cholesterol was found to be a risk factor for CHD for the first time in the Framingham project45. However, at the 30-year follow-up, it appeared that high cholesterol was not a risk factor after age 47. Even more contradictory was that both coronary and total mortality was higher in those whose cholesterol had decreased during these years than in those whose cholesterol had increased. For each 1% mg/dl drop of cholesterol there was an 11 percent increase in coronary and total mortality45. Numerous studies have shown that for most populations high cholesterol is not a risk factor. They included Canadian men46, diabetic subjects47, patients with renal failure48, patients who already had CHD49, and almost all studies have found that it is not a risk factor for women or for old people either49. Indeed, old people with a high cholesterol live longer than old people with low cholesterol50. Thus, a possible cause of atherosclerosis and coronary heart disease may be due to defects in the haemostatic system and the reason why statin treatment is of benefit may therefore be its antithrombotic effect, not its effect on cholesterol. Supporting to this view came the observation that omega-3 fatty acids (i.e. n-3 fatty acids EPA and DHA from fish and fish oils) exert their antiatherosclerotic effects and provide cardiovascular protection particularly by acting as antithrombotic agents having anti-platelet, anti-inflammatory and anti-oxidative actions2-4.
In the present review we have illustrated many methods of platelet function testing. Platelet aggregometry is the most well-studied method of platelet function testing; however, the data for platelet aggregation and incident cardiovascular disease is conflicting. The inconsistency highlights the inherent limitations of turbidometry as a clinical test. Indeed, ex vivo manipulation of platelets is a delicate process, with the potential to significantly alter aggregation dynamics with even minor differences in procedure. The role of impedance aggregometry with whole blood remains an option that has yet to be fully tested.
MPV is another promising candidate. It is a commonly measured, standardized platelet parameter that has been studied extensively in the context of cardiovascular disease with clear associations with other markers of platelet activity and cardiovascular risk factors.
Plasma markers of platelet activation are also promising candidates. For example, P-selectin is easily measured with a standardized ELISA; however, timely and methodical handling of the specimens is crucial for proper interpretation. Although limited, there is some data to suggest a significant association between plasma markers of platelet activity and cardiovascular risk factors and incident cardiovascular disease.
In conclusion, only properly conducted studies will be able to fully evaluate the role of platelet function testing in predicting future cardiovascular events.
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Fig. 1. Upper part: model of platelet interaction with the damaged vessel wall: exposure of subendothelial matrix after endothelial lesion leads to platelet tethering, activation and accumulation to provide sealing of endothelial wound. Lower part: model of platelet interaction with the endothelium. Platelets interact with the endothelium via adhesion receptors such as GPIbα and PSGL-1 promoting rolling and subsequent firm adhesion via β3 integrins. Interaction with endothelial bound chemokines such as CX3CL1 (fraktaline) or CCL5 (Rantes) results in P-selectin-mediated recruitment of leukocytes to the vessel wall and subsequent transmigration. For both interaction with the subendothelial matrix and the endothelium, major adhesion receptors expressed on platelets are listed.