Remote ischemic preconditioning prevents systemic platelet activation associated with ischemia–reperfusion injury in humans

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 Correspondence: Christian M. Pedersen, Aarhus University Hospital Skejby, B-forskning, Brendstrupgaardsvej 100, 8200 Arhus N, Denmark. Tel. : +45 89 49 62 32; fax: +45 89 49 60 09. E-mail: Christian.M. Pedersen@ki.au. dk.