Remote ischaemic preconditioning and its effect on coronary physiology and platelet activation

Background Remote ischaemic preconditioning (RIPC) is the phenomenon where brief non-harmful ischaemia to a remote organ can confer protection to the heart against a future insult. Most commonly delivered with a sphygmomanometer on the arm, RIPC has been shown to confer protection when delivered prior to primary or elective percutaneous coronary intervention (PCI). The mechanism by which this occurs is not clearly defined. Coronary microcirculatory function is an important determinant of patient prognosis at the time of PCI. Platelets play an important role in the pathophysiology of coronary atherosclerosis related thrombotic events, with platelet inhibition an integral component of the clinical management of patients with coronary artery disease (CAD) including those undergoing PCI. Given the important role of both the microcirculation and platelet inhibition at the time of PCI, this project was designed to investigate the effect of RIPC on these two prognostically important factors. Methods and results This thesis has examined 1) the effect of RIPC on coronary artery physiology and 2) the effect of RIPC on platelet activation in response to platelet agonists and the interaction between these effects and antiplatelet medications. To study the effect of RIPC on coronary artery physiology and microcirculatory function, a randomised blinded placebo controlled trial was performed. Patients with a clinical indication for fractional flow reserve (FFR) measurement were randomised to receive RIPC or sham treatment while on the catheterisation table. A comprehensive coronary physiology study was performed with a temperature-pressure sensor coronary guidewire before and immediately after the allocated treatment. The index of microcirculatory resistance (IMR) and coronary flow reserve (CFR) were measured as markers of microcirculatory function. RIPC was associated with a significant reduction in the calculated IMR (median (interquartile range), 22.6 (17.9-25.6) vs 17.5 (14.5-21.3), P=0.007) and a significant increase in the CFR (mean ± standard deviation, 2.6 ± 0.9 vs 3.8 ± 1.7, P=0.001). These changes were associated with a reduction in hyperaemic transit time (0.33 (0.26-0.40) vs 0.25 (0.20-0.30), P=0.01) indicating an increase in coronary blood flow. There was no effect on FFR. Sham treatment had no effect on any coronary physiology parameter. Analysis of plasma stored from blood collected before and after RIPC/sham treatment found no changes in plasma nitrite, cGMP or adrenomedullin with RIPC. There was a reduction in plasma 6-keto-PGF1α (the metabolite of prostacyclin) with RIPC. The effect of RIPC on platelet activation was studied in a separate randomised controlled trial where patients referred for coronary angiography were randomised prior to their procedure to RIPC or sham treatment. Venous blood was collected from the contralateral arm before and immediately after their allocated treatment, prior to cardiac catheterisation. Platelets were stimulated in whole blood with adenosine diphosphate, protease activated receptor-1 agonist (SFLLRN), thrombin with and without collagen, and analysed by flow cytometry for expression of CD62P (marker of α-granule release), CD63 (marker of dense granule and lysosome release) and conformationally active glycoprotein IIb/IIIa (GPIIb/IIIa) (PAC-1 binding). RIPC was associated with decreased platelet PAC-1 binding in response to SFLLRN (50.4% (31.3-73.2) vs 49.3% (23.0-67.7), P=0.002) and thrombin and collagen (79.5 ± 11.9% vs 68.9 ± 22.5%, P<0.001). Similar effects were seen in patients on both aspirin and a P2Y12 receptor inhibitor. Despite their role in regulating GPIIb/IIIa activation, there was no effect of RIPC on plasma cAMP and cGMP levels or intra-platelet phosphorylated-VASP levels. RIPC was also assessed for its effects on the generation of procoagulant platelets, identified by the uptake of 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO, an intracellular cell death marker) and staining for CD62P (GSAO+/CD62P+). RIPC decreased circulating levels of procoagulant platelets in the circulation (2.0% (1.3-2.4) vs 1.3% (1.1-2.1), P=0.01), whereas sham had no effect. In the subgroup of patients on aspirin monotherapy, RIPC was associated with a reduction in procoagulant platelet formation in response to SFLLRN (11.4±5.2% vs 8.9±2.8%, P=0.04) and thrombin (12.9±5.9% vs 8.7±3.2%, P=0.04). In order to determine the mechanism behind the RIPC-mediated attenuation of procoagulant platelet formation, further patients were studied to assess for the effects of RIPC on platelet mitochondrial membrane potential using a fluorescent potential-sensitive probe (TMRE). RIPC was associated with a significant reduction in platelet mitochondrial membrane depolarisation in response to a range of thrombin (P=0.001) and thrombin and collagen (P=0.01) concentrations. To investigate whether RIPC modifies the plasma microRNA profile, plasma stored from blood collected before and after RIPC/sham in the platelet cohort was analysed. Plasma microRNA was extracted from paired plasma samples collected from 4 patients treated with RIPC. The plasma microRNA profile before and after RIPC was compared with a card based quantitative polymerase chain reaction (qPCR) array system and identified 6 candidate species which appeared to be increased with RIPC. A validation study was performed in the entire randomised platelet cohort (n=60) by extracting microRNA from paired plasma samples collected from patients before and after treatment with either RIPC or sham, and measuring the levels of the 6 candidate species with specific reverse transcription and qPCR reactions. This demonstrated no definite difference with RIPC over sham treatment in any of the 6 microRNA species. Conclusion RIPC leads to rapid improvements in coronary microcirculatory function as demonstrated by validated catheterisation based coronary physiology measurements. Additionally, RIPC attenuated platelet GPIIb/IIIa activation in response to potent platelet agonists which are present at the site of complicated atherosclerotic plaques. Attenuation of GPIIb/IIIa activation was evident in patients on contemporary antiplatelet therapy, suggesting benefit additional to these medications. RIPC also reduced the level of circulating procoagulant platelets. Reductions in procoagulant platelet activity appeared to be due to RIPC-mediated reduction in platelet mitochondrial membrane depolarisation. These novel actions of RIPC are likely to contribute to the beneficial effects of RIPC during elective and primary PCI