In studies on human platelets, nitroprusside (NP) alone at 1–10 µmol/l increased platelet cyclic AMP (cAMP) by 40–70%, whereas increases in cyclic GMP (cGMP) were much larger in percentage though not ...in concentration terms. Collagen enhanced these increases in cAMP up to fourfold, without affecting cGMP. This effect was partly prevented by indomethacin or aspirin, indicating that platelet cyclo‐oxygenase products acted synergistically with NP to increase cAMP. ADP released from the platelets by collagen tended to restrict this cAMP accumulation. Addition of 2′,5′‐dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase, decreased both the inhibition of collagen‐induced platelet aggregation by NP and the associated accumulation of cAMP without affecting cGMP, indicating that cAMP mediates part of the inhibitory effect of NP. Unlike DDA, 1H‐1,2,4oxadiazolo4,3‐aquinoxalin‐1‐one (ODQ), an inhibitor of guanylyl cyclase, blocked all increases in both cGMP and cAMP caused by NP, as well as the inhibition of platelet aggregation, suggesting that cAMP accumulation was secondary to that of cGMP. Human platelet cGMP‐dependent protein kinase (PKG) coelectrophoresed with the purified bovine type Iβ isoenzyme. An inhibitor of this enzyme (Rp)‐β‐phenyl‐1,N2‐etheno‐8‐bromoguanosine 3′,5′‐cyclic‐monophosphorothioate, diminished the inhibition of collagen‐induced platelet aggregation by NP, but had little additional effect when DDA was present. This showed that both PKG and cAMP participate in the inhibition of collagen‐induced platelet aggregation by NP. Moreover, selective activators of PKG and cAMP‐dependent protein kinases had supra‐additive inhibitory effects, suggesting that an optimal inhibitory effect of NP requires simultaneous activation of both enzymes.
The mutant gunmetal mouse exhibits reduced rates of platelet synthesis, abnormalities of platelet alpha and dense granules and hypopigmentation. Several of these features resemble those of human ...alpha/delta platelet storage pool disease, grey platelet syndrome and Hermansky–Pudlak syndrome. Gunmetal mice have reduced levels of Rab geranylgeranyltransferase (RabGGTase), which adds lipophilic prenyl groups to the carboxyl terminus of Rab proteins. The degree of prenylation and the subcellular distribution of several Rab proteins were evaluated in mutant platelets, melanocytes and other tissues. Significant deficits in prenylation and membrane binding of most Rabs were observed in platelets and melanocytes. In contrast, minimal alterations in Rab prenylation were apparent in several other gunmetal tissues despite the fact that RabGGTase activity was equally diminished in these tissues. The mutant tissue‐specific effects are probably due to increased concentrations of Rab proteins in platelets and melanocytes. These experiments show that Rab proteins are differentially sensitive to levels of RabGGTase activity and that normal platelet synthesis, platelet organelle function and normal pigmentation are highly sensitive to the degree of prenylation and membrane association of Rab proteins. Further, the tissue‐specific effects of the gunmetal mutation suggest that RabGGTase is a potential target for therapy of thrombocytosis.
cGMP enhances cAMP accumulation in platelets via cGMP-inhibited phosphodiesterase (PDE3) Maurice and Haslam (1990) Mol. Pharmacol. 37, 671-681. However, cGMP might also limit cAMP accumulation by ...activating cGMP-stimulated phosphodiesterase (PDE2). We therefore evaluated the role of PDE2 in human platelets by using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to inhibit this enzyme selectively. IC50 values for the inhibition of platelet PDE2 by EHNA, with 10 microM cAMP as substrate in the absence and in the presence of 1 microM cGMP, were 15 and 3 microM respectively. Changes in platelet cyclic 3Hnucleotides were measured after prelabelling with 3Hadenine and 3Hguanine. Nitroprusside (NP) caused concentration-dependent increases in 3HcGMP and a biphasic increase in 3HcAMP, which was maximal at 10 microM (49+/-6%) and smaller at 100 microM (32+/-6%) (means+/-S.E.). In the presence of EHNA (20 microM), which had no effects alone, NP caused much larger increases in platelet 3HcAMP (125+/-14% at 100 microM). EHNA also enhanced 3HcGMP accumulation at high NP concentrations. In accord with these results, EHNA markedly potentiated the inhibition of thrombin-induced platelet aggregation by NP. The roles of cAMP and cGMP in this effect were investigated by using 2', 5'-dideoxyadenosine to inhibit adenylate cyclase. This compound decreased the accumulation of 3HcAMP but not that of 3HcGMP, and diminished the inhibition of platelet aggregation by NP with EHNA. We conclude that much of the effect of NP with EHNA is mediated by cAMP. Lixazinone (1 microM), a selective inhibitor of PDE3, increased platelet 3HcAMP by 177+/-15%. This increase in 3HcAMP was markedly inhibited by NP; EHNA blocked this effect of NP. Parallel studies showed that NP suppressed the inhibition of platelet aggregation by lixazinone. EHNA enhanced the large increases in 3HcAMP seen with 20 nM prostacyclin (PGI2), but had no effect with 1 nM PGI2. NP and 1 nM PGI2 acted synergistically to increase 3HcAMP, an effect attributable to the inhibition of PDE3 by cGMP; EHNA greatly potentiated this synergism. In contrast, NP decreased the 3HcAMP accumulation seen with 20 nM PGI2, an effect that was blocked by EHNA. The results show that, provided that cGMP is present, PDE2 plays a major role in the hydrolysis of low cAMP concentrations and restricts any increases in cAMP concentration and decreases in platelet aggregation caused by the inhibition of PDE3. At high cAMP, PDE2 plays the major role in cAMP breakdown, whether cGMP is present or not.
GTP‐binding proteins of the Rab family were cloned from human platelets using RT‐PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in ...platelets previously, were isolated. The coding sequence of Rab31 (GenBank accession no. U59877) corresponded to a 194 amino‐acid protein of 21.6 kDa. The Rab32 sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but the 5′ coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino‐acid protein of 25.0 kDa with the unusual GTP‐binding sequence DIAGQE in place of DTAGQE. Northern blots for Rab31 and Rab32 identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti‐peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 µg·mg of platelet protein−1) and contained substantial amounts of Rab32 (0.11 µg·mg protein−1). Little Rab31 was present (0.005 µg·mg protein−1). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28‐kDa Rab32 was replaced by a 52‐kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione S‐transferase (GST)‐fusion proteins, did not bind α‐32PGTP on nitrocellulose blots but did bind 35SGTPS in a Mg2+‐dependent manner. Binding of 35SGTPS was optimal with 5 µm Mg2+free and was markedly inhibited by higher Mg2+ concentrations in the case of GST–Rab31 but not GST–Rab32. Both proteins displayed low steady‐state GTPase activities, which were not inhibited by mutations (Rab31Q64L and Rab32Q85L) that abolish the GTPase activities of most low‐Mr GTP‐binding proteins.
We have used reverse transcriptase PCR, platelet mRNA and degenerate primers based on platelet peptide sequences, to amplify a fragment of platelet cGMP-inhibited phosphodiesterase (cGI-PDE; PDE3). ...Sequence analysis of this clone established that both the platelet and the cardiac forms of PDE3 were derived from the same gene (PDE3A). A RT-PCR product representing the C-terminal half of platelet PDE3 cDNA and corresponding to amino acid residues 560-1141 of the cardiac enzyme, was cloned and expressed in Escherichia coli cGI-PDEDelta1. Further deletion mutants were constructed by removing either an additional 100 amino acids from the N-terminus (cGI-PDEDelta2) or the 44-amino-acid insert characteristic of the PDE3 family, from the catalytic domain (cGI-PDEDelta1Deltai). In addition, site-directed mutagenesis was performed to explore the function of the 44-amino-acid insert. All mutants were evaluated for their ability to hydrolyse cAMP and cGMP, their ability to be photolabelled by 32PcGMP and for the effects of PDE3 inhibitors. The Km values for hydrolysis of cAMP and cGMP by immunoprecipitates of cGI-PDEDelta1 (182+/-12 nM and 153+/-12 nM respectively) and cGI-PDEDelta2 (131+/-17 nM and 99+/-1 nM respectively) were significantly lower than those for immunoprecipitates of intact platelet PDE3 (398+/-50 nM and 252+/-16 nM respectively). Moreover, N-terminal truncations of platelet enzyme increased the ratio of Vmax for cGMP/Vmax for cAMP from 0.16+/-0.01 in intact platelet enzyme, to 0.37+/-0.05 in cGI-PDEDelta1 and to 0.49+/-0.04 in cGI-PDEDelta2. Thus deletion of the N-terminus enhanced hydrolysis of cGMP relative to cAMP, suggesting that N-terminal sequences may exert selective effects on enzyme activity. Removal of the 44-amino-acid insert generated a mutant with a catalytic domain closely resembling those of other PDE gene families but despite a limited ability to be photolabelled by 32PcGMP, no cyclic nucleotide hydrolytic activities of the mutant were detectable. Mutation of amino acid residues in putative beta-turns at the beginning and end of the 44-amino-acid insert to alanine residues markedly reduced the ability of the enzyme to hydrolyse cyclic nucleotides. The PDE3 inhibitor, lixazinone, retained the ability to inhibit cAMP hydrolysis and 32PcGMP binding by the N-terminal deletion mutants and the site-directed mutants, suggesting that PDE3 inhibitors may interact exclusively with the catalytic domain of the enzyme.
Hermansky Pudlak syndrome (HPS) is an autosomal recessive inherited disorder characterized by defects in synthesis and/or secretion of three related subcellular organelles: melanosomes, ...platelet-dense granules, and lysosomes. In the mouse, mutant forms of any of 14 separate genes result in an HPS-like phenotype. The mouse pearl and mocha genes encode subunits of the AP3 adaptor protein complex, confirming that HPS mutations involve proteins regulating intracellular vesicular trafficking. Therefore, expression of several additional proteins involved in vesicular transport was examined by immunoblotting of platelet extracts from HPS mutant and control mice. Platelet levels of SCAMPS (secretory carrier membrane proteins), Rab11, Rab31, NSF (N-ethylmaleimide-sensitive fusion protein), syntaxin 2, syntaxin 4, munc18c, and p115/TAP (p115/transcytosis-associated protein) were not significantly altered in several different HPS mutants. However, gunmetal (gm/gm) platelets contained decreased amounts of SNAP-23. The Snap23 gene was mapped to mouse chromosome 5, demonstrating it cannot encode the gm gene, which maps to chromosome 14. It is likely therefore that the gm gene functions upstream of SNAP-23 in vesicular trafficking.
Ultraviolet irradiation of human platelet cytosol in the presence of 32P-labelled cyclic GMP (cGMP) can specifically label 110, 80, 55, 49 and 38 kDa proteins; the 110 kDa species is the subunit of ...cGMP-inhibited phosphodiesterase (PDE III) and the 80 kDa species that of cGMP-dependent protein kinase (Tang et al., 1993, Biochem. J. 294, 329). We have now shown that although photolabelling of platelet PDE III was inhibited by unlabelled cGMP, 8-bromo-cGMP and cyclic AMP (cAMP), it was not affected by phosphorothioate analogues of these cyclic nucleotides. Specific concentration-dependent inhibitions of the photolabelling of PDE III were observed with the following PDE inhibitors: 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). Thus, measurements of the inhibitory effects of compounds on the photolabelling of platelet PDE III provide a simple quantitative means of investigating their actions at a molecular level that avoids the need to purify the enzyme. Photolabelling of rat platelet lysate or rat heart homogenate by 32PcGMP showed that the 110 kDa PDE III present in human material was replaced by a 115 kDa protein, labelling of which was also blocked by PDE III inhibitors. Heart and other rat tissues contained much less of this putative 115 kDa PDE III than rat platelets. In contrast, the 80 kDa protein was labelled much less in platelets than in many other rat tissue homogenates (e.g., heart, aorta, uterus and lung). Thus, comparison of the relative amounts of specific photolabelled proteins in different cells may provide an indication of different patterns of cyclic nucleotide action. We compared the abilities of phosphodiesterase inhibitors to block the photolabelling of PDE III in human platelet cytosol and to increase the iloprost-stimulated accumulation of cAMP in intact platelets. Whereas trequinsin (EC50 = 19 +/- 3 nM), lixazinone (EC50 = 122 +/- 8 nM), milrinone (EC50 = 5320 +/- 970 nM) and siguazodan (EC50 = 18880 +/- 3110 nM) all increased platelet cAMP to the same maximum extent, cilostamide and IBMX increased cAMP further, indicating that they inhibited a PDE isozyme in addition to PDE III.
Atriopeptin II and isoproterenol acted synergistically to inhibit the phenylephrine-induced contraction of aortic smooth muscle from Wistar-Kyoto (WKY) rats. Thus, a weakly inhibitory concentration ...of atriopeptin II (10 nM) caused a 5-fold decrease in the IC50 of isoproterenol from 169 nM to 32 nM, whereas a low concentration of isoproterenol (100 nM) increased the maximum inhibition attributable to atriopeptin II from 43% to 74%. Atriopeptin II (10 nM) increased the cGMP found in aortic smooth muscle and approximately doubled the accumulation of cAMP caused by isoproterenol. The results suggest that cGMP, formed by the action of atriopeptin II on receptor guanylyl cyclase (GC-A), may inhibit aortic cyclic nucleotide phosphodiesterase type III (PDE III) and that an increased accumulation of cAMP then mediates the observed synergism.
GTP‐binding proteins of the Rab family were cloned from human platelets using RT‐PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in ...platelets previously, were isolated. The coding sequence of
Rab31
(GenBank accession no.
U59877
) corresponded to a 194 amino‐acid protein of 21.6 kDa. The
Rab32
sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no.
U59878
) but the 5′ coding sequence was only completed later by others (GenBank accession no.
U71127
). Human
Rab32
cDNA encodes a 225 amino‐acid protein of 25.0 kDa with the unusual GTP‐binding sequence DIAGQE in place of DTAGQE. Northern blots for
Rab31
and
Rab32
identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti‐peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 µg·mg of platelet protein
−1
) and contained substantial amounts of Rab32 (0.11 µg·mg protein
−1
). Little Rab31 was present (0.005 µg·mg protein
−1
). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28‐kDa Rab32 was replaced by a 52‐kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione
S
‐transferase (GST)‐fusion proteins, did not bind α‐
32
PGTP on nitrocellulose blots but did bind
35
SGTPS in a Mg
2+
‐dependent manner. Binding of
35
SGTPS was optimal with 5 µ
m
Mg
2+
free
and was markedly inhibited by higher Mg
2+
concentrations in the case of GST–Rab31 but not GST–Rab32. Both proteins displayed low steady‐state GTPase activities, which were not inhibited by mutations (Rab31
Q64L
and Rab32
Q85L
) that abolish the GTPase activities of most low‐
M
r
GTP‐binding proteins.
PDF
