Beta-blockade does not reduce the neuroendocrine stress response, suggesting that this mechanism is not responsible for the previously reported improved cardiovascular outcome. However, it confers several advantages, including decreased analgesic requirements, faster recovery from anesthesia, and improved hemodynamic stability. The release of cardiac troponin I suggests the occurrence of perioperative myocardial damage in this elderly population, which appears to be independent of the neuroendocrine stress response.

The isoform(s) of adenylyl cyclase (AC) present in human platelets has not been identified, and evidence supporting a role for AC in platelet aggregation is equivocal. We recently characterized deaggregation as an active component of the platelet aggregation response that may be an important determinant of the extent and duration of aggregation. G(i)-coupled receptors are linked to the inhibition of AC and are targets of antiplatelet drugs. They also affect platelet aggregation by modulating deaggregation, suggesting a role for AC in modulating this response. The purpose of this study was to identify the AC isoform(s) present in human platelets and to identify its physiological modulators. RT-PCR screening of platelet, buffy coat layer cell and bone marrow megakaryocyte cDNA, and Western blot analysis with AC type III (AC-III) antibodies identified AC-III in platelets and in megakaryocytes. Human platelet AC-III was cloned and expressed in HEK293 cells and its characteristics compared to native platelet AC. Both platelet AC and cloned AC-III required Mg(2+) for activity, were insensitive to Ca(2+) and were G(s)- and G(i)-coupled. Zn(2+) and SQ22536 inhibited platelet AC activity. The affinity of SQ22536 was increased with Mg(2+)-related stimulation of AC, while that of Zn(2+) was unchanged, which is consistent with a non-competitive interaction between the two metal ions on AC. The Zn(2+) chelator TPEN reversed the inhibitory effects of Zn(2+). This study identified AC-III as the predominant AC isoform in human platelets, the activity of which may affect the extent and duration of the net aggregation response by modulating deaggregation.

Deaggregation, the partial reversal of the initial aggregation of platelets is observed following low, but not higher, micromolar ADP concentrations. This study tested the hypothesis that deaggregation results from a balance between concurrent, opposing, aggregation and deaggregation processes which are ADP (adenosine 59 -diphosphate) receptor occupancy-dependent. Aggregation of human platelet-rich plasma (PRP) prepared in r-hirudin was assayed in a 96-well plate reader over 20 min by measurement of the optical density (OD) at 580 nm. Aggregation and the time to reach peak aggregation were directly proportional to ADP receptor occupancy. The magnitude and time course of the response to ADP were comparable to those previously reported with standard aggregometry. The rate constant of platelet deaggregation, as assessed by a fourcompartment kinetic model, was inversely proportional to agonist concentration. The ratio of the rate constants of aggregation and deaggregation was receptor occupancy-dependent and directly proportional to aggregation. Consequently, platelet aggregation was proportional, and deaggregation inversely proportional, to ADP receptor occupancy. We propose that the response of PRP to ADP and to 2-MeS-ADP (2-methylthioadenosinediphosphate), in vitro, consists of at least two active, concurrent processes, aggregation and deaggregation. Incremental occupancy of the P 2T ADP receptor subtype attenuates deaggregation and governs the balance between these two processes.

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Single-receptor pharmacology does not satisfactorily explain the physiology of the ADP-induced platelet aggregation response. It has been shown that, in addition to Gq-coupled receptor activation, one Gi-coupled receptor, either the ADP P2T or the alpha2-adrenoceptor, is required for elicitation of aggregation. The underlying mechanism of this action, however, has not been elucidated. By systematically assaying the entire time course of the aggregation and its fade using two methods of aggregometry, we have investigated the role of graded activation of these two Gi-coupled receptors. We demonstrate that constant activation of either of two Gq-coupled receptors, the ADP P2Y1 or the 5-HT2A, and incremental activation of either of the two Gi-coupled receptors, tightly regulates the aggregation response in vitro, through the apparent release of a tonic inhibition of platelet aggregation. This tightly regulated release of inhibition, which appears analogous to the phenomena of disinhibition observed in the central nervous system, may be instrumental for the continuous adaptation of the aggregation response to variable physiological conditions.