Prostaglandin F(2alpha) receptor-dependent regulation of prostaglandin transport.

Prostaglandin (PG) F(2alpha) may act on its G protein-coupled receptor (FP) or be imported intracellularly via a transporter, which has high affinity for PGF(2alpha) and PGE(2), but not prostacyclin (PGI(2)). In cells overexpressing the epitope-tagged FP together with the human prostaglandin transporter (hPGT), stimulation of the FP with PGF(2alpha) (1 nM-1 microM), or the less potent FP agonist, the isoprostane 8,12-iso-iPF(2alpha)-III, inhibited prostaglandin uptake via the hPGT. This effect was abolished by pretreatment of the cells with cholera toxin, but not with pertussis toxin. Furthermore, two dominant negative constructs directed against Galpha(s) partially blocked FP-mediated regulation of hPGT function, also suggesting Galpha(s) involvement in this phenomenon. Surprisingly, neither an activator (dibutyryl cyclic AMP) nor an inhibitor (H89) of cyclic AMP-dependent protein kinase had any effect on FP-mediated inhibition of hPGT activity. Furthermore, although PGF(2alpha) increases intracellular cyclic AMP via Galpha(s) activation, it does not induce phosphorylation of the transporter, excluding a role of cyclic AMP-dependent protein kinase in hPGT regulation. Activation of the PGI(2) receptor, which is also coupled to Galpha(s), does not regulate hPGT activity, despite markedly augmenting adenylate cyclase activation. In conclusion, activation of the FP reduces intracellular import of prostaglandins for metabolic inactivation, increasing prostanoid availability for membrane receptor activation. This effect seems to be mediated via Galpha(s), independent of adenylate cyclase and cyclic AMP-dependent protein kinase activation.

Directed vascular expression of the thromboxane A2 receptor results in intrauterine growth retardation.

Thromboxane (Tx) A2 is a platelet agonist, smooth muscle cell constrictor, and mitogen. Urinary Tx metabolite (Tx-M) excretion is increased in syndromes of platelet activation and early in both normal pregnancies and in pregnancy-induced hypertension. A further increment occurs in patients presenting with severe preeclampsia, in whom Tx-M correlates with other indices of disease severity. TxA2 exerts its effects through a membrane receptor (TP), of which two isoforms (alpha and beta; refs. 5,6) have been cloned. Overexpression of TP in the vasculature under the control of the pre-proendothelin-1 promoter results in a murine model of intrauterine growth retardation (IUGR), which is rescued by timed suppression of Tx synthesis with indomethacin. IUGR is commonly associated with maternal diabetes or cigarette smoking, both conditions associated with increased TxA2 biosynthesis.

Differential signaling by the thromboxane receptor isoforms via the novel GTP-binding protein, Gh.

Thromboxane A2 acts via G protein-coupled receptors; two splice variants of the thromboxane A2 receptor (TPalpha and TPbeta) have been cloned. It is unknown whether they differ in their capacity to activate intracellular signaling pathways. Recently, a high molecular weight G protein, Gh, that can also function as a tissue transglutaminase, has been described. We investigated whether Gh functions as a signaling protein in association with thromboxane receptors. First, we sought Gh expression in cells known to express TPs. Reverse transcription-polymerase chain reaction and immunoblotting demonstrated Gh expression in platelets, megakaryocytic cell lines, and endothelial and vascular smooth muscle cells. Second, immunoprecipitation of both TPalpha and TPbeta in transfected COS-7 cells resulted in the co-immunoprecipitation of Gh, indicating that TPs may associate Gh in vivo. Finally, agonist activation of TPalpha, but not of TPbeta, resulted in stimulation of phospholipase C-mediated inositol phosphate production in cells cotransfected with Gh. By contrast, agonist activation of both TP isoforms resulted in Gq-mediated inositol phosphate signaling. Gh is expressed in platelets and vascular cells and may associate with both TP isoforms. However, stimulation of TP isoforms results in differential activation of downstream signaling pathways via this novel G protein.

Rapid, agonist-dependent phosphorylation in vivo of human thromboxane receptor isoforms. Minimal involvement of protein kinase C.

Thromboxane A2 (TxA2) is a potent vasoconstrictor and platelet agonist. Its biological function is tightly regulated. G protein-coupled membrane receptors transduce the effects of TxA2. However, although a single thromboxane receptor (TP) gene has been identified, two splice variants have been cloned from human placenta and megakaryocytic lines (TPalpha) and from human endothelial cells (TPbeta). These differ in the length of their carboxyl-terminal extensions (15 versus 79 residues), which contain multiple potential sites for receptor phosphorylation. Given that TP agonists activate protein kinase C (PKC), it would seem possible that PKC-dependent phosphorylation of TPs might play a central role in homologous desensitization of these receptors. To determine if the TP isoforms were differentially phosphorylated in response to agonist in vivo, human embryonic kidney (HEK) 293 cells were stably transfected with TPalpha and TPbeta. Isoform-specific anti-peptide antibodies were developed and used to immunoprecipitate the phosphorylated receptors. U46619, a PGH2/TxA2 mimetic, induced specific phosphorylation of both isoforms. Phosphorylation of the two isoforms was similar in dose and time dependence, reaching a plateau at around 100 nM U46619. Inhibition of PKC with either GF 109203X (5 microM) or RO 31-8220 (5 microM) or of protein kinase A with H-89 (50 microM) marginally influenced agonist-dependent phosphorylation of either isoform and failed to modulate homologous desensitization of agonist-induced stimulation of inositol phosphate formation. Similar results were obtained when PKC was down-regulated by long term incubation with the phorbol ester, phorbol myristate acetate. Although short term stimulation with phorbol myristate acetate caused PKC-dependent phosphorylation of TPs in vivo, thrombin stimulation of the TP-transfected HEK cells in vivo failed to phosphorylate either of the TP isoforms. Thus, despite the capacity of PKC to phosphorylate TPs in HEK 293 cells and the likely activation of PKC by TP stimulation, this enzyme, like protein kinase A, contributes marginally to rapid, agonist-induced phosphorylation of either TP isoform.

Antivasoconstrictor and antiaggregatory activities of picotamide unrelated to thromboxane A2 antagonism.

Picotamide is a dual thromboxane (Tx) A2 receptor antagonist/Tx synthase inhibitor although some observations suggest an anti-vasoconstrictor effect independent of TxA2 inhibition/antagonism. The aim of our study was to assess whether picotamide antagonises vascular contractions induced by different vasoactive substances in vitro. Picotamide inhibited competitively the contraction of rabbit aortic rings induced by the TxA2 mimetic U46619 (pA2 = 3.59) but also the contractions induced by phenylephrine (pA2 = 3.93) and serotonin (5-HT) (pA2 = 5.81) although in a not competitive way. Picotamide did not inhibit potassium-induced contractions, thus excluding aspecific effects on vascular smooth muscle. Picotamide inhibited 5-HT-induced platelet aggregation in vitro with an IC50 (212 microM) similar to that found when other aggregating stimuli are used, but it did not affect shape change (IC50 > 1 mM) suggesting that the effects of picotamide can not be ascribed to 5-HT2-receptor antagonism; in the same experimental conditions neither a Tx-receptor antagonist (BM13.177) nor a dual Tx-receptor antagonist/synthase inhibitor (ridogrel) affected 5-HT-induced platelet responses. Our studies demonstrate that picotamide exerts antivasoconstrictor and platelet inhibitory effects unrelated to TxA2 antagonism. This activity may contribute to the anti-thrombotic/anti-ischaemic effects of the drug in vivo.

Regulation of cyclooxygenases by protein kinase C. Evidence against the importance of direct enzyme phosphorylation.

Cyclooxygenases (COXs) are key prostaglandin biosynthetic enzymes. While COX-1 expression is largely constitutive, COX-2 is highly regulated by cytokines, growth factors, and tumor promoters, such as the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA). While phosphorylation of transcription factors may regulate COX transcription, the existence of PKC consensus sequences suggests that direct enzyme phosphorylation might also regulate differential expression of the enzymes. Nevertheless, phosphorylation of both human recombinant COX-1 and COX-2 by rat brain PKC in vitro was minimal, as was phosphorylation of peptides based on PKC consensus sequences in COX-1 (less than 4% of the phosphorylation of the PKC-alpha pseudosubstrate peptide). Similarly, phosphorylation of the corresponding COX-2 peptides was not observed using either the phosphocellulose paper absorption method or electrospray mass spectrometry. MEG-01 and NIH 3T3 cells were labeled with [32P]orthophosphate to investigate COX phosphorylation in vivo. COX-2 synthesis was induced by PMA (100 nM) or serum stimulation in NIH 3T3 cells. COX-1 was expressed constitutively in MEG-01 cells. Specific polyclonal antibodies raised against sequences of human COX-1 (Ala24-Cys35) and COX-2 (Asn580-Lys598) were used for immunoprecipitation. Neither COX-1 nor COX-2 was phosphorylated in vivo, irrespective of the presence of a phosphatase inhibitor (1 microM okadaic acid). Although COX-1 and COX-2 are differentially regulated, no differences were observed in terms of susceptibility to phosphorylation by PKC either in vitro or in vivo. Despite regulated expression of COX-2 by PMA and the existence of consensus sequences for PKC phosphorylation, it appears that it is an unfavorable substrate for this enzyme.

Protein kinase C inhibitors enhance G-protein induced phospholipase A2 activation in intact human platelets.

Washed intact human platelets were prelabelled with [3H]arachidonic acid ([3H]AA) and stimulated with thrombin or with AlF4-, a known unspecific activator of G-proteins. Both stimuli induced the liberation of [3H]AA, the release of beta-thromboglobulin (beta-TG) and platelet aggregation. PMA did not induce liberation of [3H]AA although it induced beta-TG release and aggregation; preincubation with PMA did not modify significantly the amounts of [3H]AA and beta-TG released by thrombin or AlF4-. Different inhibitors of PKC (staurosporine, H-7 and calphostin C) increased the release of [3H]AA and inhibited beta-TG release and aggregation induced by AlF4- but they had no effect when platelets were stimulated with thrombin (0.5 U/ml). Calphostin C was able to release [3H]AA by itself without inducing aggregation of beta-TG release. Okadaic acid (a serine/threonine phosphoprotein phosphatase inhibitor) greatly inhibited the release of [3H]AA, beta-TG and aggregation in AlF4--stimulated platelets. These results indicate the presence of a G-protein mediated mechanism for the activation of a platelet phospholipase A2 which is negatively affected by a protein kinase, sensible to putative inhibitors of protein kinase C, and it is activated by a protein phosphatase, sensible to okadaic acid.

Thromboxane synthase inhibitors suppress more effectively the aggregation of thromboxane receptor-desensitized than that of normal platelets: role of adenylylcyclase up-regulation.

Exposure of human platelets to U46619, a thromboxane (Tx) A2 mimetic, desensitizes the TxA2/prostaglandin (PG) H2 receptor and sensitizes adenylylcyclase to stimuli, such as PGI2 or PGD2. This phenomenon may occur in vivo in conditions associated with platelet activation. Tx synthase inhibitors produce a rediversion of arachidonic acid metabolism toward the adenylylcyclase stimulators PGD2 and PGI2. We assessed whether the desensitization of the platelet TxA2 receptor affects the antiplatelet activity of drugs acting on the arachidonic acid metabolic cascade. A Tx synthase inhibitor (OKY046), a PGH2/TxA2 receptor antagonist (BM13.505), their combination, two dual Tx synthase inhibitors/receptor antagonists (picotamide and ridogrel) or the cyclooxygenase inhibitor aspirin were studied. OKY046 alone or combined with BM13.505, picotamide and ridogrel, as well as PGD2, but not BM13.505 or aspirin, caused a stronger inhibition of platelet aggregation with desensitized platelets; this effect was potentiated by the phosphodiesterase inhibitor HL725 and was almost abolished by the adenylylcyclase inhibitor SQ22,536. A larger increase in cAMP synthesis was observed in desensitized as compared with control platelets with a Tx synthase inhibitor or with dual Tx synthase inhibition/receptor antagonism. No differences were observed in the degree of TxA2 suppression. Our observations showed that Tx synthase inhibitors exerted a stronger antiaggregatory effect in TxA2 receptor-desensitized platelets due to a stimulation of adenylylcyclase. This can be of relevance in the treatment of thrombotic disorders in which an in vivo desensitization of platelet TxA2 receptors takes place.

Thrombocytopenia in HIV infected patients. Prevalence and clinical spectrum.

We studied the prevalence, clinical spectrum and epidemiologic features of thrombocytopenia among 442 (333 male, 109 female) HIV infected patients. Thrombocytopenia was defined as a platelet count < 100,000/mmc and severe if platelet count was < or = 30,000/mmc. Intravenous drug abusers were 83% (369/442). At the first clinical evaluation according to Walter-Reed (WR) classification, 90% (396/442) of patients were in stage 1-5 and 10% (45/442) in stage 6. Severe thrombocytopenia (platelet count < or = 30,000/mmc) was present in 24% (11/45) of the entire thrombocytopenic population. Forty percent (18/45) of the thrombocytopenic patients were positive to: HBV (6), HCV (7), HBV+HCV (5). Mild bleeding was present in 16% (7/45) of the patients but one case, with severe thrombocytopenia, died of intracranial hemorrhage. Major hemorrhagic sequelae with even fatal events are possible, especially when a low platelet count is associated with other hemostatic abnormalities (e.g. haemophilia, liver disease, disseminated intravascular coagulation). Zidovudine therapy (range 500-1250 mg/day) is effective in normalizing the platelet count (platelets > 100,000/mmc) only in 29% (9/31) of the patients.

Platelet glycohydrolase activities: characterization and release.

Granules containing acid hydrolases have been detected in human platelets but have not been thoroughly characterized. We have studied the activity and characteristics of glycohydrolases present in normal human platelets, evaluated their release upon stimulation with thrombin, and assessed the contribution of platelet - released lysosomal contents to the glycohydrolase activity present in normal serum. Platelets contained a remarkable glycohydrolase activity with a prevalence of beta - N-acetylhexosaminidase. All glycohydrolases were released to some extent upon stimulation with thrombin and contributed to the glycohydrolase activity found in human serum. alpha-Mannosidase and alpha-galactosidase were partially inactivated after release by a mechanism as yet undefined. In addition, thrombin stimulation affects the intraplatelet isoenzyme pattern of beta-N-acetylhexosaminidase by producing the appearance of a new form.

Interferon-alpha is effective in the treatment of HIV-1-related, severe, zidovudine-resistant thrombocytopenia. A prospective, placebo-controlled, double-blind trial.

OBJECTIVE: To determine the effect of interferon-alpha for severe, zidovudine-resistant, HIV-1-related thrombocytopenia. DESIGN: Prospective, randomized, double-blind, placebo-controlled, multicenter, crossover trial. SETTING: Outpatient clinics in Central Northern Italy. PATIENTS: 15 sequential patients positive for HIV-1 with platelet counts less than 25 x 10(9)/L who were refractory to 1 month of full-dose (1000 mg/d) zidovudine. INTERVENTION: Interferon-alpha (3 million units) or placebo (1 mL saline) three times a week subcutaneously for 4 weeks, followed by a 4-week washout period. Patients were then switched to the alternative treatment for the next 4 weeks, followed by another 4 weeks of washout, and they were randomly assigned to either sequence of treatment. Patients received zidovudine (200 mg three times daily) throughout the study. MEASUREMENTS: The primary end point was the platelet count (measured weekly). Secondary end points were qualitative assessment of the platelet response; bleeding time; p24 antigen in serum; CD4/CD8 counts; beta 2-microglobulin in serum; and platelet-associated IgG. RESULTS: Interferon-alpha significantly increased platelet counts in the 12 patients who completed the study (baseline level, 15.6 +/- 7.1 x 10(9)/L; after 4 weeks of interferon-alpha therapy, 82.2 +/- 52.2 x 10(9)/L). The estimated increase in the platelet count after interferon-alpha compared with placebo was 60.0 x 10(9)/L (95% CI, 23.2 to 96.8 x 10(9)/L). The increase was already statistically significant after 3 weeks (66.6 +/- 49.7 x 10(9)/L) and remained significantly increased 1 week after discontinuing interferon-alpha therapy (58.2 +/- 45.0 x 10(9)/L). Placebo did not modify the platelet count. The bleeding time was significantly shortened by interferon-alpha. Four of 12 patients who had more serious alterations of some measures reflecting disease severity did not respond to interferon-alpha. No relevant side effects were observed. CONCLUSIONS: Interferon-alpha is a safe and effective treatment for zidovudine-resistant, HIV-related thrombocytopenia.

[Pregnancy and labor in a primigravida with Glanzmann's thrombasthenia]. / Gravidanza e parto in primigravida affetta da tromboastenia di Glanzmann.

Glanzmann's thrombasthenia is an autosomal, recessive, inherited haemorrhagic disorder due to the lack of the glycoprotein IIb/IIIa (GP IIb/IIIa) complex of the platelet membrane. The disease is characterized by a prolonged bleeding time and by a severe haemorrhagic mucocutaneous diathesis. The only possible antihaemorrhagic therapeutic measure is the infusion of platelet concentrates, but this is almost constantly associated with the production of antibodies against the GP IIb/IIIa complex that lead to the rapid removal of the infused platelets from the circulation. For this reason, platelet transfusions are usually without efficacy and must be limited to severe, profuse haemorrhages. We report the case of a primigravidae with Glanzmann's thrombasthenia in which a spontaneous delivery was made possible by a series of measures taken during the parturition to avoid haemorrhagic complications, without the use of platelet transfusions.

Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C.

Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P-labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

Platelet thromboxane A2 receptor number and function in normal and hypertensive pregnancy.

PROBLEM: Our study evaluated the number and function of platelets for thromboxane A2/prostaglandin H2 (TxA2/PGH2) and the platelet response to TxA2 receptor agonists in normal and hypertensive pregnancy. In addition, correlations between platelet membrane lipid composition and TxA2 receptor number and function were evaluated. METHODS: Ten normotensive healthy pregnant women (GC), 10 hypertensive pregnant women (PIH), and 10 normotensive nonpregnant healthy women (C) were examined. Radioligand binding and aggregation studies were performed. Lipid composition of platelet membrane was assessed as cholesterol to phospholipids ratio (CH/PL) and normalized to protein content. RESULTS: TxA2/PGH2 receptor binding sites and affinity did not differ among the groups. An enhanced response to TxA2 receptor agonist U46619 in GC and in PIH compared to C was observed, while platelet response to thrombin was not different among the groups. CH/PL was altered in PIH respect to GC and C. No significant correlation was found between CH/PL and number and function of platelet TxA2 receptors in PIH. CONCLUSIONS: In PIH there is no alteration of number or function of TxA2 receptors on platelet membrane. However, the enhanced TxA2 production associated with the increased sensitivity of platelet to TxA2 in pregnancy may contribute to the microcirculatory thrombotic changes of PIH.