peripheral arterial disease: Implications for
clinical management
Sriram Rajagopalan, MRCS,aIan Mckay, MRCS,aIsobel Ford, PhD,cPaul Bachoo, MSc, FRCS,b Michael Greaves, MD, FRCP, FRCpath,cand Julie Brittenden, MD, FRCS,aForesterhill, United Kingdom
Introduction: Patients with peripheral arterial disease (PAD) have increased mortality from cardiovascular events compared with age and sex matched controls Platelets play a major role in atherosclerosis and thrombotic vascular events. Platelet reactivity is increased in patients with PAD compared with healthy controls. We aimed to determine the relationship, if any, between platelet activation and severity of disease.
Methods and Results:One hundred eighty-two patients with intermittent claudication (IC) or subcritical limb ischemia (SLI), defined as the presence of rest pain or ulceration, had the following investigations performed: platelet P-selectin expression and bound fibrinogen by flow cytometric analysis and platelet aggregation using the rapid platelet function assay with arachidonic acid (AA) and thrombin receptor activation peptide (TRAP) as agonists. Patients with SLI compared with IC had significantly enhanced ADP stimulated P-selectin expression (median 42.45% [inter-quartile range 33.32% to 58.5%] vs 35.2% [26.07% to 46.32%], Pⴝ .002) and bound fibrinogen (73.7% [54.3% to 83.2%] vs 63.7%
[43.8% to 76.5%], Pⴝ .001). TRAP stimulated aggregation was higher (207 [153 to 238] PAU vs 183[155 to 199]
PAU, Pⴝ .04) but AA mediated aggregation was not significantly different. An ankle-brachial pressure index (ABPI) of
less than 0.6 was associated with increased ADP stimulated P-selectin and bound fibrinogen (P < .05). ABPI correlated inversely with ADP stimulated P-selectin expression (rⴝ ⴚ0.228, P ⴝ .003), ADP stimulated fibrinogen binding (r ⴝ ⴚ0.156, P ⴝ .043) and TRAP stimulated aggregation (r ⴝ ⴚ0.179, P ⴝ .04).
Conclusion:We have demonstrated for the first time that progression of severity of PAD is not only reflected by symptoms, signs, and ABPI but also by increased platelet activity as assessed by both flow cytometry and aggregation. As patients with more severe PAD have increased cardiovascular mortality, our findings suggest that new strategies for platelet inhibitory therapy are indicated in these patients. ( J Vasc Surg 2007;46:485-90.)
Peripheral arterial disease (PAD) is common, with a prevalence of both symptomatic and asymptomatic disease estimated at 16% in adults over the age of 55.1,2 Patients
diagnosed as having PAD have a two- to three-fold in-creased risk of cardiovascular and cerebrovascular mortality when compared with an age and sex matched group with-out PAD.3,4 Thus, the first line treatment of patients with
PAD is directed at cardiovascular risk factor manage-ment.5,6 The major risk factors for both cardiovascular
disease and peripheral vascular disease are well established and include cigarette smoking, dyslipidaemia, hyperten-sion, and diabetes. In addition to management of these, it is recommended that all patients are prescribed anti-platelet therapy.
Abnormal platelet function has been implicated in the development and progression of atherosclerosis, as well as in the pathogenesis of acute cardiac ischemic events.7
Plate-let activation is increased in patients with lower limb isch-emia compared with healthy controls suggesting an under-lying prothrombotic state.8-11 The antithrombotic trialist
collaboration meta-analysis has shown that anti-platelet therapy reduces serious adverse vascular events by 23% in patients with peripheral arterial disease (PAD).12 PAD
exists as a spectrum of disease ranging from asymptomatic to severe limb ischemia. The severity of disease is related to overall survival.
Patients with more severe PAD, as assessed by symp-toms or the ankle brachial pressure index (ABPI) have reduced survival. In the Edinburgh Artery Study, almost 20% of patients with intermittent claudication had died within 5 years, 13.7% from cardiovascular causes.13 With
more severe limb ischemia, patients may present with pain at rest, ulceration, or gangrene. The present of these symp-toms is associated with an increased cardiovascular mortal-ity rate.14
In addition to utility in the diagnosis of PAD, the ABPI has been shown in many studies to be a good predictor of future cardiovascular events.15-18 This association is
inde-pendent of the metabolic syndrome and other major car-diovascular risk factors.19 Significant linear trends have
been shown across ABPI categories and cardiovascular outcome.15,17,18 In particular, an ABPI less than 0.6 has
consistently been shown to be associated with increased mortality.
From the Vascular Unit, University of Aberdeen,athe Aberdeen Royal Infirmary, NHS Grampian,band Medicine and Therapeutics Unit, Uni-versity of Aberdeen.c
Competition of interest: Julie Brittenden, MD, FRCS, and Paul Bachoo, MSc, FRCS, have been paid consulting fees and speaker fees by Sanoffi-aventis.
Reprint requests: Julie Brittenden, Senior Lecturer and Consultant Vascular Surgeon, Vascular Unit, Aberdeen Royal Infirmary, Foresterhill AB25 2ZN UK (e-mail: [email protected]).
0741-5214/$32.00
Copyright © 2007 by The Society for Vascular Surgery. doi:10.1016/j.jvs.2007.05.039
We hypothesised that platelet reactivity would be in-creased in patients with more severe peripheral arterial disease. The aim of this study was to determine the rela-tionship between platelet activation in vivo and reactivity ex vivo as assessed by flow cytometry markers of activation and aggregation and severity of PAD in terms of symptoms and the ankle brachial pressure index.
MATERIALS AND METHODS
Consecutive consenting patients who had intermittent claudication (n ⫽ 90) or severe limb ischemia (n ⫽ 92) characterized by the presence of rest pain or ulceration were recruited from the claudication clinic and the vascular unit, respectively. Patients with claudication had chronic, stable intermittent claudication for at least 6 months and had not undergone any prior intervention. The study protocol was approved by the relevant research ethics committee.
Risk factors were managed as recommended by the Scottish Intercollegiate Guideline Network (SIGN) guide-lines.5 All patients were already prescribed 75 mg daily of
aspirin and statin therapy of at least 6 weeks duration. Statin therapy consisted of 40 mg of simvastatin or pravastatin and 10 mg of atorvastin, depending on individual general prac-titioners prescribing practice. Comorbidities, smoking his-tory, and ankle brachial pressure indices were documented. Exclusion criteria. Patients with diabetes, acute limb ischemia, active infection, those on clopidogrel, warfarin, nonsteroidal anti-inflammatory drugs, and those unable to give informed consent were excluded.
Study design. Blood samples were taken by a 21-gauge needle from the ante-cubital without the use of tourniquet from fully rested patients. Blood samples were collected in 3.2% citrate vacutainers for flow cytometry and platelet aggregation. Ultegra rapid platelet function anal-yser (RPFA) was performed within 15 minutes of blood drawing. The following analyses were performed:
Whole blood flow cytometry (Coulter Epics XL-MCL Flow Cytometer (Beckman Coulter Inc, Calif ): The per-centage of platelets expressing P-selectin (CD62P) and bound fibrinogen on platelets with and without ex vivo stimulation with ADP (1⫻10⫺5mol/L) in diluted whole
blood samples was measured, as previously described by our laboratory.9,21,22
Platelet aggregation was assessed using the RPFA (Ac-cumetrics, San Diego, Calif). This is a well validated, point of care; whole blood cartridge based turbidimetric optical detection system, which assesses the ability of activated platelets to bind fibrinogen coated micro-beads contained within the cartridge test wells. Fibrinogen-coated micro-beads agglutinate in whole blood in proportion to the number of available GPIIb/IIIa receptors, causing an in-crease in light transmittance.23,24 The test has been shown
to correlate well with conventional platelet aggregom-etry.25 The agonist in the RPFA-TRAP assay is thrombin
receptor activating peptide (TRAP) and in the RPFA-ASA assay is arachidonic acid (AA). The analyser measures the change in the optical signal and reports it as platelet aggre-gation units (PAU) for the RPFA-TRAP assay and aspirin reaction units (ARU) for the RPFA-ASA. An ARU of greater than or equal to 550 indicates a nonresponse to aspirin. Inter-assay variation was 5.8% for RPFA-ASA and 5.0% for RPFA-TRAP.26,27
Statistical analysis. Based on a 10% anticipated in-crease in platelet aggregation (TRAP) in patients with subcritical limb ischemia compared with claudication, at 90% power, 0.05 significance, 90 patients per group were required.26
Calculations were performed with SPSS for Windows version 13.0 statistical software (SPSS, Chicago, Ill). The results of platelet activity markers and aggregation between the two cohorts were compared using the Mann Whitney U test. Spearmans correlation coefficient was used to deter-mine the relationship between ABPI and platelet activity.
RESULTS
Patient characteristics. One hundred eighty-two patients were recruited for the study. Clinical character-istics of the study population are summarized in Table I. There was no difference between the two groups. The ABPI s of the patients included in this study ranged from 0.34 to 0.88.
Table I. Patient demographics
Intermittent claudication N⫽ 90 Severe limb ischemia N⫽ 92 P
Median age : 71.5 69 .28
(range) 43-97 48-88
Male:female 64:26 57:35 .26
Hypertension (%)* 61 (68%) 56.7 (51%) .12
Coronary artery disease (%)† 27 (30.3%) 28.3 (26%) .75
Body mass index (median, interquartile range) 27 (25-30) 25 (21-29) ⬎.05
Current (%) 41 (47%) 48 (53%) .35
Reformed smoker (%): (1 year abstinence) 43 (48%) 35 (38%)
Beta-blockers 15 (16%) 23 (25%) ⬎.05
Ca channel blockers 26 (28%) 28 (30%)
ACE inhibitors 19 (21%) 27 (29%)
*Hypertension was defined as a systolic blood pressure greater than 140 mm Hg and/or diastolic greater than 90 mm Hg. †Angina, myocardial infarction, previous revascularisation.
Flow cytometry detected markers. Unstimulated P-selectin expression and bound fibrinogen were similar in patients with intermittent claudication and subcritical limb ischemia. The ABPI did not correlate with unstimulated P-selectin (r⫽ ⫺0.117, P ⫽ .132) or fibrinogen binding (r ⫽ 0.71,⫺0.236). ADP stimulated platelet P-selectin expression and fibrinogen binding were significantly increased in patients with subcritical limb ischemia compared to patients with in-termittent claudication (Table II) (P ⬍ .01).
The ABPI correlated with ADP stimulated P-selectin expression (r⫽ ⫺0.228, P ⫽ .003) and ADP stimulated fibrinogen binding ( r ⫽ ⫺0.156, P ⫽ .043) (Figs 1 and 2). ADP stimulated P-selectin expression was significantly in-creased in patients with an ABPI less than compared with greater than 0.6. (34.2 [27 to 40.4] vs 42.3 [29.7 to 56.25] P ⫽ .003). Similarly, ADP stimulated bound fibrinogen was increased in patients with an ABPI ⬍0.6 compared with those with ABPI⬎0.6 9 (63.9 [41.9 to 76.9] vs 73.0 [55.05 to 82.45], P⫽ .02).
Platelet aggregation. AA mediated platelet aggrega-tion was not statistically different between the two groups and did not correlate with the ABPI (r⫽ 0.059, P ⫽ .51) (Fig 3). TRAP stimulated platelet aggregation was
sig-nificantly increased in patients with subcritical limb isch-emia (Fig 4, P ⬍ .05). Overall, 11% of the patients had a ARU greater than 550, suggesting an inadequate response to aspirin. Patients with severe limb ischemia were over-represented in this group (14% vs 5%, P⬎ .05).
The ABPI showed an inverse correlation with TRAP mediated aggregation (r ⫽ ⫺0.179, P ⫽ .04) (Fig 5).There was no difference in either AA or TRAP mediated aggrega-tion between patients with an ABPI less than or greater than 0.6.
Correlation between platelet assays. ADP stimu-lated P-selectin expression and bound fibrinogen were cor-related (r⫽ 0.61, P ⬍ .001) as were AA mediated aggre-gation and ADP stimulated P-selectin expression (r⫽ 0.22, P⫽ .009). There was no difference in the flowcytometry results between patients who were aspirin responders and nonresponders.
DISCUSSION
This is the first study to show that platelet activation as assessed by aggregation and flow cytometry is significantly enhanced in patients with subcritical limb ischemia com-pared with patients with intermittent claudication, despite Table II. Ankle brachial pressure index (ABPI) and platelet function tests in patients with intermittent claudication and severe limb ischemia
Intermittent claudication (N⫽ 90) Severe limb ischemia (N⫽ 92) P†
ABPI 0.61 (0.55-0.74) 0.40 (0.34-0.50) ⬍.001
P-selectin* 0.61(0.38-1.01) 0.70 (.36-1.28) .78
P-selectin (⫹ADP)* 35.2 (26.07-46.32) 45.05 (34.12-58.8) .001
Fibrinogen binding* 1.68 (.58-4.47) 1.68 (.58-4.47) .42
Fibrinogen binding* (⫹ ADP) 63.7 (43.8-76.5) 74.55 (59.2-85.35) ⬍.001
Results are expressed as medians and interquartile ranges.
*P-selectin and fibrinogen binding are expressed as percentage platelet expression. †P value: Mann-Whitney U test.
0.80 0.60 0.40 0.20 80 60 40 20 0 % Platelets ABPI
Fig 1. Spearmans correlation coefficient between ADP-stimulate P-selectin expression and the ankle brachial pressure index (ABPI).
0.80 0.60 0.40 0.20 100 80 60 40 20 0 % Platelets ABPI
Fig 2. Spearmans correlation coefficient between ADP-stimulate fibrinogen binding and the ankle brachial pressure index (ABPI).
the use of aspirin and statin therapy. Previous studies have shown that platelet activation is increased in patients with PAD compared with healthy controls.7-9,11 Furthermore,
we showed previously a tendency for increased P-selectin expression but no change in fibrinogen binding in patients with more severe arterial disease, although that study was not powered to assess differences in platelet function ac-cording to the severity of disease and did not assess platelet aggregation.9 To assess platelet function comprehensively
both flow cytometric parameters and aggregation should be measured, in view of the range of pathways involved in platelet activation.8,28,29 The current study supports the
use of such an approach.
Platelet aggregation as assessed by TRAP as agonist significantly increased in patients with subcritical limb isch-emia compared with those with intermittent claudication. There was a tendency for increased AA mediated aggrega-tion and also a higher proporaggrega-tion of nonresponders to aspirin in the severe limb ischemia cohort (5% vs 14%).
We have previously shown that unstimulated platelet P-selectin expression and bound fibrinogen are increased in patients with PAD compared with healthy controls. In a small cohort of 20 patients with subcritical limb ischemia and tissue loss, we found that ADP stimulated fibrinogen binding but not unstimulated fibrinogen binding was sig-nificantly reduced compared with healthy controls.9 Many
of these patients had diabetes, and it was felt that the presence of infection, poor diabetic control, and specific diabetic therapy may have accounted for this finding. Thus, we actively excluded diabetic patients from this current study. A control group was not included in this study due to the inability to standardize for variables, which may affect platelet function such as statin and aspirin therapy and smoking. The primary aim of the current study was not to compare PAD and controls, for which we already have published data, but to look for a relationship between platelet activation measures and severity of vascular dis-ease. Now, we demonstrate significantly increased ADP-stimulated P-selectin expression and bound fibrinogen in patients with more severe ischemia but no difference when platelets are studied in the unstimulated state. Our findings are consistent with those of Tan et al, who found significantly increased platelet microparticles in patients with severe limb ischemia compared with those with intermittent claudication.30
This increase in platelet reactivity is consistent with the hypothesis that circulating platelets are more reactive in patients with more severe PAD despite anti-platelet therapy Severe limb ischemia
Intermittent claudication 700 600 500 400 300 200 100 0 ARU
*
Fig 3. Arachadoinc acid (AA) mediated platelet aggregation. Data presented as box-plots-line in center of box represents the median value; the box represents the inter-quartile range and the whiskers represent the range.*P⬎ .05, Mann-Whitney U test.
Severe limb ischemia Intermittent claudication 400 350 300 250 200 150 100 50 0 PAU
**
Fig 4. Thrombin receptor activating peptide (TRAP) stimulated platelet aggregation. Data presented as box-plots-line in center of box represents the median value; the box represents the inter-quartile range and the whiskers represent the range.**P⬍ .05, Mann-Whitney U test. 0.80 0.60 0.40 0.20 400 350 300 250 200 150 100 50 PAU ABPI
Fig 5. Spearmans correlation coefficient between thrombin re-ceptor activating peptide (TRAP) stimulated platelet aggregation and ankle brachial pressure index (ABPI).
with aspirin. Consequently, there may be an increased risk of thrombus formation in response to an in vivo stimulus, typically as plaque rupture.
The patient cohorts were well matched apart from the severity of PAD as reflected by the significantly lower ABPI in patients with subcritical limb ischemia. It is known that, overall, patients with an ABPI less than 0.6 have reduced survival.18 In this study, 37 patients in the claudication
group had an ABPI less than 0.6. We have shown now that ex vivo ADP stimulated P-selectin expression and bound fibrinogen are increased in patients with an ABPI less than 0.6. Significant inverse correlations were observed across the spectrum of ABPIs included in this study, ADP stimu-lated platelet expression, and TRAP mediated aggregation. This is consistent with the observation that the presence of small platelet aggregates correlates with the ABPI in pa-tients with PAD and known coronary artery disease.30 We
have shown, as have previous studies, wide variation in the degree of platelet activation amongst individual patients and the lack of a clear discrimination between patients with subcritical limb ischemia, intermittent claudication, and healthy controls. There appears be no clear “cut-off” value for P-selectin, fibrinogen binding, or TRAP mediated ag-gregation, which would indicate the need for alternative or additional anti-platelet therapy to aspirin 75 mg daily. However, our findings support the use of more aggressive platelet suppressive, regimens, such as the addition of clo-pidogrel, in patients with more advanced PAD. These patients are known to have reduced survival due to an increased incidence of thrombotic events such as myocar-dial infarction and stroke. While the anti-platelet triallest collaboration has shown the benefit of anti-platelet therapy in patients with PAD, it should be noted that in many of the studies patients were prescribed a variety of anti-platelet agents. The Clopidogrel versus Aspirin in Patients at Risk of Ischaemic Events (CAPRIE) trial has shown that clopi-dogrel is more effective than aspirin in reducing serious adverse events. In the patients with PAD, clopidogrel achieved a 23.8% relative risk reduction compared with aspirin.32 Clopidogrel has been shown to be a more
effec-tive at inhibiting ADP induced platelet activation and flow cytometry markers of platelet activation.21,33,34 In the
United Kingdom, the National Institute for Clinical Excel-lence has stated that the cost-effectiveness for clopidogrel in patients with PAD is not proven.35 Targeting the use of
clopidogrel to patients with more severe disease who have reduced survival may make this therapy cost-effective.
CONCLUSION
This study has shown that progression of severity of PAD is not only reflected by symptoms, signs, and ABPI but also by increased platelet reactivity. This has implica-tions for therapy. Clinical trials of more aggressive anti-platelet therapy are indicated in patients with severe PAD as manifested by the presence of rest pain or ulceration or an ABPI less than 0.6.20, 31
The research fellow Sriram Rajagopalan was sponsored by a grant from the Scottish Chief Scientist Office. Ian McKay was sponsored by a BHF project grant. The statis-tical advice was given by Mr G. Prescott and Mr E. Amalraj (Department of Public Health, University of Aberdeen). AUTHOR CONTRIBUTIONS
Conception and design: IF, PB, MG, JB Analysis and interpretation: SR, IM, PB, JB Data collection: SR, IM, PB, JB
Writing the article: SR
Critical revision of the article: JB, IF, PB Final approval of the article: JB, MG Statistical analysis: SR, IM
Obtained funding: JB, IF, MG, PB Overall responsibility: JB, MG
REFERENCES
1. Belch JJF, Topol EH, Agnelli C, Bertrand M, Califf RM, Clement DL, et al. Critical issues in peripheral arterial disease detection and manage-ment: a call to action. Arch Intern Med 2003;163:884-92.
2. Hirsch AT, Criqui MH, Treat-Jacobson D, Regensteiner JG, Creager MA, Olin JW, et al. Peripheral arterial disease detection, awareness, and treatment in primary care. JAMA 2001;286:1317-24.
3. Fowkes FG. Epidemiology of atherosclerotic arterial disease in the lower limbs. Eur J Vasc Surg 1988;2:283-91.
4. Dormandy J, Heeck L, Vig S. The natural history of claudication: risk to life and limb. Semin Vasc Surg 1999;12:123-37.
5. SIGN Scottish Intercollegiate Guidelines Network (SIGN). Diagnosis and management of peripheral artery disease-A national clinical guide-line. October 2006. www.sign.ac.uk
6. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. TASC II Working Group. Inter-Society Consensus for the Man-agement of Peripheral Arterial Disease (TASC II). [Consensus Devel-opment Conference Journal Article]. J Vasc Surg 2007;45(Suppl S):S5-S67.
7. Cassar K, Bachoo P, Brittenden J. The role of platelets in peripheral vascular disease. Eur J Vasc Endovasc Surg 2003;15:6-15.
8. Robless PA, Okonko D, Lintott P, Mansfield AO, Mikhailidis DP, Stansby GP. Increased platelet aggregation and activation in peripheral arterial disease. Eur J Vasc Endovasc Surg 2003;25:16-22.
9. Cassar K, Bachoo P, Ford I, Greaves M, Brittenden J. Platelet activation is increased in peripheral arterial disease. J Vasc Surg 2003;38:99-103. 10. Koksch M, Zeiger F, Wittig K, Siegemund A, Reininger CB, Pfeiffer D, et al. Coagulation, fibrinolysis and platelet P-selectin expression in peripheral vascular disease. Eur J Vasc Endovasc Surg 2001;21:147-54. 11. Galt SW, McDaniel MD, Ault KA, Mitchell J, Cronenwett JL. Flow cytometric assessment of platelet function in patients with peripheral arterial occlusive disease. J Vasc Surg 1991;14:747-55; discussion 755-6.
12. Antithrombosis Trialists Collaboration 2002. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002;324:76-81.
13. Leng GC, Lee AJ, Fowkes FGR, Whiteman M, Dunbar J, Housley E, et al. Incidence, Natural history and cardiovascular events in symptom-atic and asymptomsymptom-atic peripheral arterial disease in the general popula-tion. Int J Epid 1996;25:1172-81.
14. Criqui MH, Fronek A, Barrett-Connor E, Klauber MR, Gabriel S, Goodman D. The prevalence of peripheral arterial disease in a defined population. Circulation 1985;71:510-5.
15. Lee AJ, Price JF, Russell MJ, Smith FB, van Wijk MC, Fowkes FG. Improved prediction of fatal myocardial infarction using the ankle brachial index in addition to conventional risk factors: the Edinburgh Artery Study. Circulation 2004;110:3075-80.
16. Abbott RD, Petrovitch H, Rodriguez BL, Yano K, Schatz IJ, Popper JS, et al. Ankle-brachial blood pressure in men⬎70 years of age and the risk of coronary heart disease. Am J Cardiol 2000;86:280-4.
17. Heald CL, Lee AJ, Murray GD, Fowkes FGR. Ankle-brachial index as an independent indicator of mortality in fifteen international population cohort studies. Circulation 2005;112(Suppl II):798.
18. O’Hare AM, Katz R, Shlipak MG, Cushman M, Newman AB. Mortality and cardiovascular risk across the ankle-arm index spectrum: results from the Cardiovascular Health Study. Circulation 2004;113:388-93. 19. Wild SH, Byrne CD, Smith FB, Lee AJ, Fowkes FG. Low ankle-brachial pressure index predicts increased risk of cardiovascular disease indepen-dent of the metabolic syndrome and conventional cardiovascular risk factors in the Edinburgh Artery Study. Diabetes Care 2006;29:637-42. 20. Zheng ZJ, Sharrett AR, Chambless LE, Rosamond WD, Nieto FJ, Sheps DS, et al. Associations of ankle-brachial index with clinical coronary heart disease, stroke and preclinical carotid and popliteal atherosclerosis: the Atherosclerosis Risk in Communities (ARIC) Study. Atherosclerosis 1997;131:115-25.
21. Cassar K, Bachoo P, Ford I, Greaves M, Brittenden J. Randomized controlled trial of the antiplatelet effects aspirin-clopidogrel combina-tion vs aspirin alone at angioplasty. Br J Surg 2005;92:166-70. 22. Methods in Molecular Biology 272 Platelets and Megakaryocytes
Vol 1: Functional assays ISBN: 1-58829-101-4 E-ISBN: 1-59259-782-3 ISSN: 1064-3745, humanapress.com, Humana Press Inc., Totowa, NJ, 2004. Volume editors: Gibbins JM and Mahaut-Smith MP. Chapter 19: Flow-cytometric analysis of platelet-membrane glycoprotein expression and platelet activation. Chapter authors: Goodall AH and Appleby J. Pages 225-253A.
23. Smith JW, Steinhubl SR, Lincoff AM, Coleman JC, Lee TT, Hillman RS, et al. Rapid platelet-function assay: an automated and quantitative cartridge-based method. Circulation 1999;99:620-5.
24. Harrison P. Platelet function analysis. Blood Rev 2005;19:111-23. 25. Wheeler GL, Braden GA, Steinhubl SR, Kereiakes DJ,
Kottke-Marchant K, Michelson AD, et al. The Ultegra rapid platelet-function assay: comparison to standard platelet platelet-function assays in patients undergoing percutaneous coronary intervention with abcix-imab therapy. Am Heart J 2002;143:602-11.
26. Collins P, Ford I, Ball D, Macaulay E, Greaves M, Brittenden J. A preliminary study on the effects of exercising to maximum walking
distance on platelet and endothelial function in patients with intermittent claudication. Eur J Vasc Endovasc Surg 2006;31:266-73. 27. Collins P, Ford I, Greaves M, Macaulay E, Brittenden J. Surgical revascularisation in patients with severe limb ischemia induces a pro-thrombotic state. Platelets 2006;17:311-17.
28. Hezard N, Metz D, Nazeyrollas P, Droulle C, Potron G, Nguyen P. PFA-100 and flow cytometry: can they challenge aggregometry to assess antiplatelet agents, other than GPIIbIIIa blockers, in coronary angioplasty? Thromb Res 2002;108:43-7.
29. Matzdorff AC, Kuhnel G, Kemkes-Matthes B, Voss R. Comparison of GP IIB/IIIA inhibitors and their activity as measured by aggregometry, flow cytometry, single platelet counting, and the rapid platelet function analyzer. J Thromb Thrombolysis 2001;12:129-39.
30. Tan KT, Tayebjee MH, Lynd C, Blann AD, Lip GY. Platelet micropar-ticles and soluble P-selectin expression in peripheral artery disease: relationship to extent of disease and platelet activation markers, vol 16. Ann Med 2005;3761-6.
31. Kudoh T, Sakamoto T, Miyamoto S, Matsui K, Kojima S, Sugiyama S, et al. Relation between platelet microaggregates and ankle brachial index in patients with peripheral arterial disease. Thromb Res 2006; 117:263-9.
32. A randomised, blinded, trial of clopidogrel vs aspirin in patients at risk of ischemic events (CAPRIE). CAPRIE Steering Committee. Lancet 1996;348:1329-39.
33. Jagroop IA, Matsagas MI, Geroulakos G, Mikhailidis DP. The effect of clopidogrel, aspirin and both antiplatelet drugs on platelet function in patients with peripheral arterial disease. Platelets 2004;15:117-25. 34. Klinkhardt U, Bauersachs R, Adams J, Graff J, Lindhoff-Last E, Harder
S. Clopidogrel but not aspirin reduces P-selectin expression and forma-tion of platelet-leukocyte aggregates in patients with atherosclerotic vascular disease. Clin Pharmacol Ther 2003;73:232-41.
35. Jones L, Griffin S, Palmer S, Main C, Orton V, Sculpher M, et al. Clinical effectiveness and cost-effectiveness of clopidogrel and modified-release dipyridamole in the secondary prevention of occlusive vascular events: a systematic review and economic evaluation. Health Technol Assess 2004; 8:1-196.
