Melissa A. Hayman

Attenuated exercise induced hyperaemia with age: mechanistic insight from passive limb movement

The influence of age on the central and peripheral contributors to exercise‐induced hyperaemia is unclear. Utilizing a reductionist approach, we compared the peripheral and central haemodynamic responses to passive limb movement (exercise without an increase in metabolism) in 11 old (71 ± 9 years of age s.d.) and 11 young (24 ± 2 years of age) healthy subjects. Cardiac output (CO), heart rate (HR), stroke volume (SV), mean arterial pressure (MAP), and femoral blood flow of the passively moved and control legs were evaluated second‐by‐second during 2 min of passive knee extension at a rate of 1 Hz. Compared to the young, the old group exhibited a significantly attenuated increase in HR (7 ± 4%vs. 13 ± 7%s.d.), CO (10 ± 6%vs. 18 ± 8%) and femoral blood flow in the passively moved (123 ± 55%vs. 194 ± 57%) and control legs (47 ± 43%vs. 77 ± 96%). In addition, the change in vascular conductance in the passively moving limb was also significantly attenuated in the old (2.4 ± 1.2 ml min−1 mmHg−1) compared to the young (4.3 ± 1.7 ml min−1 mmHg−1). In both groups all main central and peripheral changes that occurred at the onset of passive knee extension were transient, lasting only 45 s. In a paradigm where metabolism does not play a role, these data reveal that both central and peripheral haemodynamic mechanisms are likely to be responsible for the 30% reduction in exercise‐induced hyperaemia with age.

Central and peripheral contributors to skeletal muscle hyperemia: response to passive limb movement

The central and peripheral contributions to exercise-induced hyperemia are not well understood. Thus, utilizing a reductionist approach, we determined the sequential peripheral and central responses to passive exercise in nine healthy men (33 +/- 9 yr). Cardiac output, heart rate, stroke volume, mean arterial pressure, and femoral blood flow of the passively moved leg and stationary (control) leg were evaluated second by second during 3 min of passive knee extension with and without a thigh cuff that occluded leg blood flow. Without the thigh cuff, significant transient increases in cardiac output (1.0 +/- 0.6 l/min, Delta15%), heart rate (7 +/- 4 beats/min, Delta12%), stroke volume (7 +/- 5 ml, Delta7%), passive leg blood flow (411 +/- 146 ml/min, Delta151%), and control leg blood flow (125 +/- 68 ml/min, Delta43%) and a transient decrease in mean arterial pressure (3 +/- 3 mmHg, 4%) occurred shortly after the onset of limb movement. Although the rise and fall rates of these variables differed, they all returned to baseline values within 45 s; therefore, continued limb movement beyond 45 s does not maintain an increase in cardiac output or net blood flow. Similar changes in the central variables occurred when blood flow to the passively moving leg was occluded. These data confirm the role of peripheral factors and reveal an essential supportive role of cardiac output in the hyperemia at the onset of passive limb movement. This cardiac output response provides an important potential link between the physiology of active and passive exercise.

Understanding exercise-induced hyperemia: Central and peripheral hemodynamic responses to passive limb movement in heart transplant recipients

To better characterize the contribution of both central and peripheral mechanisms to passive limb movement-induced hyperemia, we studied nine recent (<2 yr) heart transplant (HTx) recipients (56 ± 4 yr) and nine healthy controls (58 ± 5 yr). Measurements of heart rate (HR), stroke volume (SV), cardiac output (CO), and femoral artery blood flow were recorded during passive knee extension. Peripheral vascular function was assessed using brachial artery flow-mediated dilation (FMD). During passive limb movement, the HTx recipients lacked an HR response (0 ± 0 beats/min, Δ0%) but displayed a significant increase in CO (0.4 ± 0.1 l/min, Δ5%) although attenuated compared with controls (1.0 ± 0.2 l/min, Δ18%). Therefore, the rise in CO in the HTx recipients was solely dependent on increased SV (5 ± 1 ml, Δ5%) in contrast with the controls who displayed significant increases in both HR (6 ± 2 beats/min, Δ11%) and SV (5 ± 2 ml, Δ7%). The transient increase in femoral blood volume entering the leg during the first 40 s of passive movement was attenuated in the HTx recipients (24 ± 8 ml) compared with controls (93 ± 7 ml), whereas peripheral vascular function (FMD) appeared similar between HTx recipients (8 ± 2%) and controls (6 ± 1%). These data reveal that the absence of an HR increase in HTx recipients significantly impacts the peripheral vascular response to passive movement in this population and supports the concept that an increase in CO is a major contributor to exercise-induced hyperemia.

158 The Anti-Platelet Effectiveness of P2Y12 Receptor Blockade is Strongly Influenced by the Endothelium-Derived Mediators Nitric Oxide and Prostacyclin

Introduction P2Y12 receptor antagonists that block the pro-aggregatory effects of ADP on platelets are commonly prescribed alongside aspirin, as dual antiplatelet therapy (DAPT) for secondary prevention of atherothrombotic events. It is emerging that these agents exert part of their effects through potentiation of the endogenous, endothelial-derived platelet inhibitors, nitric oxide (NO) and prostacyclin (PGI2), which constantly bathe circulating platelets reducing their reactivity through elevating levels of cyclic nucleotides. Thus, if the in vivo synergy between NO and PGI2 amplifies the anti-thrombotic effects of P2Y12 inhibitors, within the circulation variances in the levels of endothelial-derived mediators will be an important determinant of the efficacy of DAPT, suggesting that for the individual patient, endothelial function influences the therapeutic potential of these powerful agents. Here, we characterised the interplay of PGI2, NO and P2Y12 blockade on platelet function. Methods 8 healthy male volunteers received aspirin (75 mg) and prasugrel (10 mg) for 7 days. Platelet responses to TRAP-6 (25 ìM) or collagen (4 ìg/ml) in the presence of PGI2 (1 nM), NO (100 nM), PGI2+NO or vehicle were assessed by LTA and lumi-aggreggometry. Experiments were replicated in vitro (n = 4) utilising aspirin (30 ìM) and prasugrel active metabolite (PAM; 6, 3 or 1.5 ìM) representing maximal and partial P2Y12 blockade. Results Ex vivo, prior to DAPT, PGI2, NO, or PGI2+NO had little effect upon platelet aggregation. TRAP-6 (25 ìM): vehicle, 74 ± 3%; PGI2+NO, 66 ± 3%. Following DAPT, PGI2 or NO alone caused minor inhibition: vehicle, 57 ± 4%; PGI2, 47 ± 6%; NO 49 ± 6%. Conversely, the combination of PGI2+NO produced strong inhibitory effects, 19 ± 6% (p < 0.05). Lumi-aggregometry quantifying ADP+ATP, showed dense granule release was unaffected in individuals receiving DAPT (7.5 ± 1.7 to 7.7 ± 0.6 nmole) but was reduced with addition of DEA/NONOate+PGI2 (6.3 ± 1.9 to 3.7 ± 1.3 nmole, p < 0.05). In vitro, with maximal P2Y12 inhibition (PAM, 6 ìM) aggregation to TRAP-6 (25 ìM) was reduced: vehicle, 56 ± 5%; PGI2, 30 ± 12%; NO, 46 ± 7%; PGI2+NO, 15 ± 8% (p < 0.05). However, with partial inhibition (PAM, 3 ìM) only PGI2+NO inhibited aggregation: vehicle, 67 ± 6%; PGI2, 56 ± 9%; NO, 62 ± 5%; PGI2+NO, 33 ± 15%. Conclusion We demonstrate that PGI2 and NO synergise with P2Y12 blockade to produce the greatest platelet inhibition and that platelet reactivity, despite DAPT is powerfully influenced by the presence of NO and PGI2. This highlights that in vivo platelet reactivity will be a function of the level of P2Y12 receptor blockade and levels of extrinsic inhibitory endothelial mediators. This may be relevant to the efficacy of anti-platelet therapies in patients with endothelial dysfunction and introduces the concept of assessing endothelial function in patients undergoing ex vivo platelet function testing to help bridge the disconnect between results of platelet testing and thrombotic outcomes.

213 Synergy between Endothelial Mediators and P2Y12 Receptor Blockade as a Potential Determinant in Tailoring Anti-Platelet Therapy

Introduction Following ACS or PCI, P2Y12 receptor blockers such as clopidogrel or prasugrel are standardly prescribed alongside aspirin as dual anti-platelet therapy in a “one size fits all” approach, however recurrent thrombotic events occur. Despite the logical hypothesis that thrombotic risk should be associated with level of P2Y12 platelet inhibition, randomised control trials have repeatedly failed to show any clinical benefit of guiding anti-platelet therapy by platelet function tests (PFT). Whilst traditional PFT measure reactivity to ADP, they crucially ignore that P2Y12 receptor antagonists also produce powerful anti-thrombotic effects by potentiating the actions of the inhibitory endothelial mediators prostacyclin (PGI2) and nitric oxide (NO). Methods In vitro, IC50 curves produced from light transmission aggregation (LTA) traces for PGI2, NO and prasugrel active metabolite (PAM: 3 μM) were used to generate isobolograms. Ex vivo, healthy male volunteers (n = 8 each) received either prasugrel (10 mg) or aspirin (75 mg) for 7 days. Platelet-rich plasma (PRP) was obtained by centrifugation before and after treatment. Platelet responses to TRAP-6 amide (25 μM) or collagen (4 μg/ml) in the presence of PGI2 (1 nM) and/or the NO donor, DEA/NONOate (100 nM), or vehicle, were assessed by LTA, p-selectin binding by flow cytometry, and downstream cyclic nucleotide and VASP phosphorylation by immunoassay. Results Isobolographic analysis of in vitro studies showed P2Y12 blockade powerfully enhanced the synergy between NO and PGI2 (10 fold for TRAP-6). Ex-vivo platelet aggregation responses to TRAP-6 were unaffected by the addition of PGI2+NO prior to therapy. Aspirin monotherapy produced minimal inhibition of TRAP-6, though these became stronger in the presence of PGI2+NO (58%±8 to 28%±9; p < 0.05). Inhibitory effects were observed with prasugrel in the presence of PGI2 and NO individually (control, 63 ± 3%; PGI2, 43 ± 6%; NO, 50 ± 5%), and these became much greater with the combination (PGI2+NO, 7%±3). P-selectin expression was reduced from pre-treatment (vehicle, 32 ± 6%) to (PGI2+NO, 11 ± 4%) post aspirin, and from (vehicle, 25 ± 6%) to (PGI2+NO, 2 ± 4%) following prasugrel. cAMP levels increased with PGI2+NO in the aspirin group (0.97 ± 0.06 vs. 1.50 ± 0.19) compared to (1.41 ± 0.07 vs. 3.4 ± 0.58) in the prasugrel group. Conclusions PGI2 and NO synergise with P2Y12 blockade to cause powerful platelet inhibition suggesting that an individual’s endothelial function and production of NO and PGI2 are central determents of the anti-thrombotic protection from P2Y12 receptor blocker therapy. Whilst a therapeutic window of platelet reactivity is an attractive concept our research suggests that endothelial function testing alongside PFT would permit better risk stratification. Our research also suggests therapeutically that enhancing endothelial-derived mediators or optimising downstream cyclic nucleotide signalling could reduce both thrombotic and bleeding risk.