Increased platelet purinergic sensitivity in peripheral arterial disease--a pilot study

Peripheral arterial disease (PAD) is associated with platelet hyperaggregability as well as an increase in morbidity and mortality from myocardial infarction (MI) and stroke. Purinergic signaling has been shown, both experimentally and clinically, to play an important role in the activation of platelets. Platelets express three different purinergic receptors: P2Y1, P2Y12 and P2X1. We assessed the hypothesis that the hyperaggregability associated with PAD is partly due to an increased P2 receptor expression at the transcriptional and/or translational level. Patients with PAD (n=8) and controls (n=8) were studied. Using a high-resolution channelyzer, platelet shape change (PSC) was assessed by measuring the median platelet volume (MPV). The fall in free platelet count following the addition of ADP was also assessed. Real-time PCR was used to quantify the mRNA expression and Western blots to quantify the protein expression of P2 receptors in platelets. The median (and range) fall in free platelet count after adding ADP was significantly (P=0.02) greater for patients [11% (5-24); n=8] than for controls [0.5% (0-10); n=8] despite using a lower concentration of ADP for the patient samples. The MPV did not differ significantly. The mRNA levels for the three P2 receptors were similar in PAD patients and controls. Western blot detected no significant differences in protein expression between these groups. Thus, platelets from PAD patients show an increased activation after stimulation with ADP (even though all patients were on aspirin). This hyperactivity was neither due to an obvious up-regulation of the mRNA levels nor to altered protein levels of P2 receptors in the platelets. It is suggested that the increased sensitivity to ADP in PAD is caused by post-receptor mechanisms.