Treatment of acute myocardial infarction is directed at the timely restoration of blood flow to reduce final infarct size and mortality. However, reperfusion may trigger injury beyond that induced by ischemia. This phenomenon, known as ischemia–reperfusion (IR) injury, can lead to myocardial stunning, myocyte cell death, microvascular dysfunction and reperfusion arrhythmias [1,2].

Prior exposure to sublethal ischemia, ischemic preconditioning (IPC) [3], may protect against prolonged ischemia and reperfusion [2,4–6]. IPC can be induced both locally and anatomically remote from the prolonged ischemia; a phenomenon known as remote IPC (RIPC) [4,7]. The clinical relevance of ischemic conditioning has recently been investigated, and a significant increase in myocardial salvage in patients with a first acute myocardial infarction was found [8].

Platelets appear to play an important role in the pathophysiology of IR injury and IPC [9,10]. In rodent hearts ex vivo, the extent of myocardial injury following IR injury is dependent on the activation status of platelets [9]. In humans, marked platelet activation has been demonstrated in patients presenting with acute coronary syndrome [11] or acute limb ischemia [12].

Attenuation of IR-induced platelet activation may be an important mechanism by which IPC attenuates IR injury [13,14].

Circulating platelet–monocyte aggregates (PMAs) are readily measured by flow cytometery, and have emerged as a highly sensitive marker of platelet activation in vivo [15,16]. Using measurements of PMAs, we sought to assess the effects of IR injury and IPC on platelet activation in vivo in humans.

Studies were performed with the approval of the Lothian research ethics committee and the informed consent of each subject, in accordance with the Declaration of Helsinki. Studies were performed in a temperature-controlled laboratory (22–24 °C).

We studied 36 healthy male volunteers in a single-blinded, randomized, crossover study with two different protocols. No subjects were excluded following screening or enrollment. All subjects were non-smoking, taking no regular medications and clinically well. Subjects abstained from vasoactive drugs for 7 days, and from caffeine-containing drinks and alcohol for 24 h, and fasted for at least 4 h, before each visit. Subjects attended for two study visits, at least 1 week apart. Subjects were rested supine for at least 15 min before study commencement and throughout the study.

Twelve subjects were randomized to IR injury (induced in the non-dominant arm by cuff inflation to 200 mmHg for 20 min) or sham IR (5 mmHg cuff inflation for 20 min) (protocol 1A). Twenty-four subjects were randomized to either RIPC or sham RIPC (protocol 1B) 30 min before IR injury was induced in all subjects. RIPC and sham RIPC were induced by cuff inflation around the dominant upper arm to 200 or 5 mmHg, respectively, for 5 min on three occasions, each inflation being separated by 5 min of reperfusion as previously described [16]. Although the study investigator was blinded to the study intervention, subjects clearly could not be, owing to the physical nature of the intervention. At time of the randomized intervention, study investigators left the laboratory. Randomization and intervention were performed by a clinical research nurse not involved in data collection or analysis. Randomization was performed by computer-generated sequence allocation. To maximize study power, data from subjects with IR injury alone (n = 12) and IR injury + sham RIPC (n = 24) were pooled. RIPC and IR injury were well tolerated by all subjects, without adverse events.

Venous blood samples were collected at baseline, and at 5 and 45 min following reperfusion, with a 17-gauge needle in the dominant arm to assess the systemic response to IR injury and RIPC of platelet activation. In a subgroup (n = 15), a further venous blood sample was collected immediately after RIPC or sham RIPC to assess the effect of RIPC and sham RIPC on PMAs. For each sample, care was taken to ensure that the blood was withdrawn smoothly. The first 5 mL of blood was discarded to minimize artefactual increases in PMA.

Three milliliters of venous blood was collected into tubes containing the direct thrombin inhibitor d-phenylalanyl-l-prolyl-l-arginine chloromethylketone (PPACK) (Cambridge Bioscience, Cambridge, UK). Monoclonal mouse anti-human CD14–phycoerythrin (Inverness Medical, Stockport, UK), mouse anti-human CD42a–fluorescein isothiocyanate (FITC) and mouse IgG1–FITC negative control (AbD Serotec, Oxford, UK) were used for immunolabeling. Aliquots of whole blood (60 μL) from the PPACK tube were incubated with 60 μL of either flow buffer (BD Biosciences, San Jose, CA, USA), IgG1/CD14 or CD14/CD42a for 20 min at 4 °C. After immunolabeling, samples were fixed and red cells were lysed by addition of 500 μL of FACS-Lyse solution (BD Biosciences). Samples were stored at 4 °C, and analyzed within 24 h of labeling with a Coulter EPICS XL flow cytometer equipped with a 488-nm-wavelength laser (Beckman Coulter, High Wycombe, UK). A minimum of 2500 cells were measured for each sample. PMAs were defined as monocytes positive for CD42a, and results are expressed as a percentage of PMAs. The mean interassay coefficient of variation for platelet–monocyte aggregation was 7.8% [17].

graphpad prism 5 was used for analysis (GraphPad Software, La Jolla, CA, USA). The study population was based on power calculations derived from preclinical studies to give a 90% power of detecting a 20% increase in PMA following IR injury at a significance level of 0.05 [18]. Data are expressed as means ± standard errors of the mean. Data were compared by use of one-way anova and two-tailed paired t-tests. Statistical significance was taken as a two-sided P-value of < 0.05.

There were no differences in baseline blood pressure, heart rate, hemoglobin level, platelet counts and leukocyte counts between visits, and no changes in heart rate or blood pressure throughout the studies (data on file). Mean baseline PMA was 23.4% in the sham IR group, 25.7% in the IR group, 31.9% in the RIPC group, and 24.6% in the sham RIPC group, and did not differ between groups (P = 0.10). As compared with baseline, PMA was increased at 5 and 45 min (32% at both time points; P = 0.04) following IR injury (Fig. 1A). RIPC abolished the increase in PMA associated with IR injury (Fig. 1B). As compared with baseline, neither sham IR (24.6%, P = 0.62) nor sham RIPC (25.3%, P = 0.21) had an effect on circulating PMAs.


Figure 1.  Percentage platelet–monocyte aggregates (% PMA) following ischemia–reperfusion (IR) injury with and without pretreatment with remote ischemic preconditioning (RIPC). (A) IR injury: *one-way anova, P = 0.04. Bars indicate t-test, P = 0.04 for both. (B) RIPC + IR injury: **one-way anova, P = 0.52. Bars indicate t-test, P > 0.05 for both. IR baseline vs. RIPC baseline, t-test = 0.10.

Download figure to PowerPoint

In a well-validated human model of IR injury [7,16], we have demonstrated, using a randomized blinded study design, that the increase in the circulating concentration of PMA associated with acute IR injury can be abolished by RIPC.

Our findings are consistent with preclinical data demonstrating activation of platelets following ischemia and reperfusion. In animal models of IR injury, systemic platelet activation was increased following IR injury [9,13]. In a similar human forearm model of IR injury, the level of circulating platelet–neutrophil complexes was increased[16]. We observed an increase in PMA levels 5 min following IR injury, and this increase persisted at 45 min following restoration of forearm blood flow. Similarly, platelet–neutrophil and platelet–leukocyte activation was increased following a treadmill test in patients with claudication; this increase was attenuated by a warm-up period prior to the treadmill test, a stimulus that could be viewed as equivalent to IPC [19].

In canine models of coronary IR injury, IPC attenuates platelet activation and aggregation [13] and platelet-mediated thrombus formation [14]. Consistently, we observed complete abrogation of the increase in platelet–monocyte aggregation associated with IR injury when preceded by RIPC. These findings are in contrast to those of a previous study, which failed to demonstrate any effect of IPC on circulating platelet–neutrophil complexes following IR injury in the human forearm [16]. These differences probably reflect the smaller sample size used in the earlier study and the lower sensitivity of platelet–neutrophil complexes than of PMAs as a marker of platelet activation [13].

IR injury induces impairment of endothelium-dependent vasomotor function in the downstream vascular bed [7]. Alterations in endothelial cell phenotype and the release of endothelium-derived factors favoring platelet activation may play an important role in the activation of platelets in the setting of acute atherothrombotic disease [20]. We have recently demonstrated a strong inverse correlation between platelet activation and endothelium-dependent vasodilatation in patients with coronary artery disease [21]. We propose that modulation of the complex dynamic interaction between endothelial cells and platelets plays a key role in the attenuation of platelet activation by RIPC and reduces tissue injury following IR.

We must acknowledge a number of limitations. In this hypothesis-generating study, we chose to focus on a single, well-validated measure of platelet activation status, PMA. Consequently, we cannot comment on the effects of IR injury or RIPC on other markers of platelet activation. Although it has been previously demonstrated that PMA is unaffected by menstrual cycle [22], we chose to perform this study in a homogeneous population of healthy male volunteers, to minimize potential confounding factors. Similarly, given the potential for interaction between platelet activation status, atherosclerosis and antiplatelet agents, we chose to examine the effect of IR and RIPC on PMA in the absence of any antiplatelet medication. Further work is required to determine the effects of IR and RIPC on platelet activation in patients with atherosclerotic vascular disease treated with cardiovascular medications.

In conclusion, we have demonstrated that systemic PMA increases following IR injury, and that this increase can be abolished by RIPC. Our findings provide support for a role for platelet activation in the pathophysiology of IR injury and the mechanism of RIPC in humans.


  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References

We would like to thank the staff of the Clinical Research Facility at the Royal Infirmary of Edinburgh, A. Ramasamy and S. Vun for their help with this project. Foundation Leducq and the British Heart Foundation (PG/08/093) supported this research. C. M. Pedersen has received funding from the Danish Agency for Science, Technology and Innovation, Det Classenske Fideicomis Jubilaeumsfond, Snedkermester Sophus Jacobsen og hustru Astrid Jacobsens Fond, Civilingeniør Stenild Hjorth’s Else Hjorth’s Fond, The A. P. Møller Foundation for the Advancement of Medical Science, the Institute of Clinical Medicin, Aarhus University, and Kirsten Antonius’ Mindelegat. R. K. Kharbanda is supported by the Oxford NIHR Biomedical Research Centre. Clinical Trial Registration Information: NCT00789451 and NCT00789243.

Disclosure of Conflict of Interests

  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References

The authors state that they have no conflict of interest.


  1. Top of page
  2. Acknowledgements
  3. Disclosure of Conflict of Interests
  4. References
  • 1
    Moens AL, Claeys MJ, Timmermans JP, Vrints CJ. Myocardial ischemia/reperfusion-injury, a clinical view on a complex pathophysiological process. Int J Cardiol 2005; 100: 17990.
  • 2
    Yellon DM, Hausenloy DJ. Realizing the clinical potential of ischemic preconditioning and postconditioning. Nat Clin Pract Cardiovasc Med 2005; 2: 56875.
  • 3
    Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986; 74: 112436.
  • 4
    Kharbanda RK, Li J, Konstantinov IE, Cheung MM, White PA, Frndova H, Stokoe J, Cox P, Vogel M, Van Arsdell G, MacAllister R, Redington AN. Remote ischaemic preconditioning protects against cardiopulmonary bypass-induced tissue injury: a preclinical study. Heart 2006; 92: 150611.
  • 5
    Cheung MM, Kharbanda RK, Konstantinov IE, Shimizu M, Frndova H, Li J, Holtby HM, Cox PN, Smallhorn JF, Van Arsdell GS, Redington AN. Randomized controlled trial of the effects of remote ischemic preconditioning on children undergoing cardiac surgery: first clinical application in humans. J Am Coll Cardiol 2006; 47: 227782.
  • 6
    Konstantinov IE, Li J, Cheung MM, Shimizu M, Stokoe J, Kharbanda RK, Redington AN. Remote ischemic preconditioning of the recipient reduces myocardial ischemia–reperfusion injury of the denervated donor heart via a Katp channel-dependent mechanism. Transplantation 2005; 79: 16915.
  • 7
    Kharbanda RK, Mortensen UM, White PA, Kristiansen SB, Schmidt MR, Hoschtitzky JA, Vogel M, Sorensen K, Redington AN, MacAllister R. Transient limb ischemia induces remote ischemic preconditioning in vivo. Circulation 2002; 106: 28813.
  • 8
    Botker HE, Kharbanda R, Schmidt MR, Bottcher M, Kaltoft AK, Terkelsen CJ, Munk K, Andersen NH, Hansen TM, Trautner S, Lassen JF, Christiansen EH, Krusell LR, Kristensen SD, Thuesen L, Nielsen SS, Rehling M, Sorensen HT, Redington AN, Nielsen TT. Remote ischaemic conditioning before hospital admission, as a complement to angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a randomised trial. Lancet 2010; 375: 72734.
  • 9
    Xu Y, Huo Y, Toufektsian MC, Ramos SI, Ma Y, Tejani AD, French BA, Yang Z. Activated platelets contribute importantly to myocardial reperfusion injury. Am J Physiol Heart Circ Physiol 2006; 290: H6929.
  • 10
    Mirabet M, Garcia-Dorado D, Inserte J, Barrabes JA, Lidon RM, Soriano B, Azevedo M, Padilla F, Agullo L, Ruiz-Meana M, Massaguer A, Pizcueta P, Soler-Soler J. Platelets activated by transient coronary occlusion exacerbate ischemia–reperfusion injury in rat hearts. Am J Physiol Heart Circ Physiol 2002; 283: H113441.
  • 11
    Sarma J, Laan CA, Alam S, Jha A, Fox KA, Dransfield I. Increased platelet binding to circulating monocytes in acute coronary syndromes. Circulation 2002; 105: 216671.
  • 12
    Burdess A, Nimmo AF, Campbell N, Harding SA, Garden OJ, Dawson AR, Newby DE. Perioperative platelet and monocyte activation in patients with critical limb ischemia. J Vasc Surg 2010; 52: 697703.
  • 13
    Linden MD, Whittaker P, Frelinger AL III, Barnard MR, Michelson AD, Przyklenk K. Preconditioning ischemia attenuates molecular indices of platelet activation–aggregation. J Thromb Haemost 2006; 4: 26707.
  • 14
    Hata K, Whittaker P, Kloner RA, Przyklenk K. Brief antecedent ischemia attenuates platelet-mediated thrombosis in damaged and stenotic canine coronary arteries: role of adenosine. Circulation 1998; 97: 692702.
  • 15
    Michelson AD, Barnard MR, Krueger LA, Valeri CR, Furman MI. Circulating monocyte–platelet aggregates are a more sensitive marker of in vivo platelet activation than platelet surface P-selectin: studies in baboons, human coronary intervention, and human acute myocardial infarction. Circulation 2001; 104: 15337.
  • 16
    Kharbanda RK, Peters M, Walton B, Kattenhorn M, Mullen M, Klein N, Vallance P, Deanfield J, MacAllister R. Ischemic preconditioning prevents endothelial injury and systemic neutrophil activation during ischemia–reperfusion in humans in vivo. Circulation 2001; 103: 162430.
  • 17
    Harding SA, Din JN, Sarma J, Jessop A, Weatherall M, Fox KA, Newby DE. Flow cytometric analysis of circulating platelet–monocyte aggregates in whole blood: methodological considerations. Thromb Haemost 2007; 98: 4516.
  • 18
    Ko W, Lang D, Hawes AS, Zelano JA, Isom OW, Krieger KH. Platelet-activating factor antagonism attenuates platelet and neutrophil activation and reduces myocardial injury during coronary reperfusion. J Surg Res 1993; 55: 50415.
  • 19
    Pasupathy S, Naseem KM, Homer-Vanniasinkam S. Effects of warm-up on exercise capacity, platelet activation and platelet–leucocyte aggregation in patients with claudication. Br J Surg 2005; 92: 505.
  • 20
    Rosenberg RD, Aird WC. Vascular-bed – specific hemostasis and hypercoagulable states. N Engl J Med 1999; 340: 155564.
  • 21
    Robinson SD, Harding SA, Cummins P, Din JN, Sarma J, Davidson I, Fox KA, Boon NA, Newby DE. Functional interplay between platelet activation and endothelial dysfunction in patients with coronary heart disease. Platelets 2006; 17: 15862.
  • 22
    Robb AO, Din JN, Mills NL, Smith IB, Blomberg A, Zikry MN, Raftis JB, Newby DE, Denison FC. The influence of the menstrual cycle, normal pregnancy and pre-eclampsia on platelet activation. Thromb Haemost 2010; 103: 3728.