Molecular and biochemical evidence for the presence of Type III adenylyl cyclase in human platelets

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. Gi-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 Mg2þ for activity, were insensitive to Ca2þ and were Gsand Gi-coupled. Zn2þ and SQ22536 inhibited platelet AC activity. The affinity of SQ22536 was increased with Mg2þ-related stimulation of AC, while that of Zn2þ was unchanged, which is consistent with a non-competitive interaction between the two metal ions on AC. The Zn2þ chelator TPEN reversed the inhibitory effects of Zn2þ. 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.