Date of Award
Doctor of Philosophy (PhD)
Dr. Richard J. Haslam
The first objective of this thesis was to investigate the targets of cyclic GMP (cGMP) action in platelets. Proteins that bind cGMP were first detected by photoaffinity labelling with [³²P]cGMP and subsequently identified by molecular, pharmacological and immunological criteria. Since cGMP was already known to exert major effects in platelets through the cGMP-inhibited phosphodiesterase family (PDE3) (Maurice and Haslam, 1990a), an additional objective was to explore the molecular basis of the unique properties of this enzyme by cloning and mutagenesis studies. A photolabelling technique using [³²P]cGMP was modified to permit the rapid detection of cGMP-binding proteins in crude platelet extracts. Five labelled proteins (110, 80, 55, 49 and 38 kDa) were detected in platelet supernatant and four (80, 65, 49 and 38 kDa) in platelet membranes. The sensitivity of photolabelling to PDE3 inhibitors and specific immunoprecipitation established that the 110 kDa photolabelled species was a product of the PDE3 gene family. In addition, the 80 kDa species was identified as cGMP-dependent protein kinase (PKG) by similar methods. Interestingly, cyclic AMP (cAMP) greatly enhanced the labelling of the 80 kDa protein, suggesting the existence of a novel co-operative interaction between cAMP and cGMP. This study also detected a previously unknown 65 kDa cGMP-binding protein in platelet membranes. Since both cAMP and cGMP inhibited labelling of this protein, it may represent a novel target for both cyclic nucleotides in platelets, possibly a subunit of a cyclic nucleotide-gated (CNG) ion channel. The inhibitory effects of variouus compounds on the photolabelling of PDE3 were quantitated by [³²P]cGMP. Thus, concentration-dependent inhibition of photolabelling of PDE3 was observed with trequinsin (IC₅₀ = 13 ± 2 nM), lixazinone (IC₅₀ = 22 ± 4 nM), milrinone (IC₅₀ = 56 ± 12 nM), cilostamide (IC₅₀ = 70 ± 9 nM), siguazodan (IC₅₀ 117 ± 29 nM) and 3-isobutyl 1-methylxanthine (IBMX) (IC₅₀ = 3950 ± 22 nM). The effects of these phosphodiesterase inhibitors on iloproststimulated cAMP accumulation in intact platelets were also investigated. Discrepancies between the abilities of these compounds to inhibit photolabelling of PDE3 and to increase platelet cAMP accumulation are probably related to differences in the rates of entry of the individual inhibitors into the intact platelet. The general applicability of this photolabelling technique and its value in the detection of novel cGMP-binding proteins in crude cell extracts was demonstrated in rat tissues. Distinctive photolabelling patterns were observed in different rat tissues and the 110 kDa protein found in human platelets was replaced by a 115 kDa species in rat platelets. To clarify the molecular mechanisms by which cGMP and inhibitory drugs modulate PDE3 activity, an attempt was made to define the roles of different PDE3 domains in the action of the enzyme. To that end, the C-terminal half of platelet PDE3 was cloned, identified as a product of the PDE3A gene and expressed as an active enzyme in E.coli. Further deletion mutants were generated by removing either an additional 100 amino acids from the N-terminus or the 44 amino acid insert, characteristic of members of the PDE3 family, from the catalytic domain. Site-directed mutagenesis of the 44 amino acid insert was also conducted to explore the function of this region. Kinetic analysis of these mutant enzymes demonstrated that the deletion of N-terminal sequences from PDE3 was accompanied by progressively lower Km values and by an increased Vmax for cGMP relative to that for cAMP. Thus, N-terminal sequences exert modulatory effects on cGMP hydrolysis. Deletion of the 44 amino acid insert abolished enzyme activity, as did site-directed mutagenesis of putative β-turns located at the N- and C-termini of the insert. Mutation of a cluster of negatively charged residues in the insert did not have major effects on the hydrolysis of cAMP or cGMP. The results suggest that this insert is required to preserve an effective catalytic domain structure in PDE3. In conclusion, the major targets of cGMP in platelets were shown to include not only PKG, but also PDE3A and an unidentified membrane protein, possibly a CNG ion channel. This thesis has also identified some of the functionally and structurally important domains within PDE3A.
Tang, Katherine Mary, "Targets of cyclic GMP in Blood Platelets: Photolabelling, mutagenesis and pharmacological analysis of the cyclic GMP-inhibited phosphodiesterase" (1997). Open Access Dissertations and Theses. Paper 1236.