A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

Abstract

The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′, 5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K I = 43.4 ± 7.2 μM and k cart = 0.007 ± 0.0006 min −1 ). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K d = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). NAD + , neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.

Increased adhesive properties of platelets in sickle cell disease: roles for αIIbβ3‐mediated ligand binding, diminished cAMP signalling and increased phosphodiesterase 3A activity

“…NO is a key regular of platelet function, inhibiting platelet adhesive, secretion and aggregation properties, in a mechanism that occurs principally via the formation of the second messenger, cGMP (Oberprieler et al , 2007a; Naseem & Riba, 2008). cGMP, in turn, regulates the platelet cAMP pathway by competitively inhibiting PDE3A, permitting cAMP accumulation (Maurice & Haslam, 1990; Hung et al , 2008; Naseem & Riba, 2008). Determination of intracellular cGMP demonstrated similar levels of this cyclic nucleotide in control and SCD platelets, suggesting that platelet cGMP‐dependent signalling is not significantly affected by diminished NO bioavailability in SCD individuals.…”