Severe early-onset manifestations of pseudoxanthoma elasticum resulting from the cumulative effects of several deleterious mutations in ENPP1, ABCC6 and HBB: transient improvement in ectopic calcification with sodium thiosulfate.

Pseudoxanthoma elasticum (PXE) is a rare disorder characterized by fragmentation and progressive calcification of elastic fibres in connective tissues. Overlap has been reported between the inherited PXE phenotype associated with ENPP1, ABCC6 or NT5E mutations and acquired PXE clinical manifestations associated with haemoglobinopathies induced by HBB mutations. No treatment is currently available for PXE. A young boy presented with severe early-onset systemic calcifications occurring in the skin as elastosis perforans serpiginosa (EPS) and in the arteries, causing mesenteric and limb ischaemia. Analyses revealed deleterious ABCC6, ENPP1 and HBB mutations. The diagnosis of severe PXE was retained and we have coined the term 'PXE+ syndrome' to describe the cumulative effects of the various mutations in this uncommon phenotype. Given the severity, rapid progression and a potentially fatal prognosis, intravenous sodium thiosulfate (STS) was initiated at 25 g three times weekly for 6 months. Numerous side-effects prompted dosage adjustment to 10 g intravenously daily. Treatment efficacy was evaluated at 6 months. Asthaenia, anorexia and pre-/postprandial pain had subsided, entailing weight gain. Abdominal EPS had diminished. Calcific stenosis of the coeliac and mesenteric arteries was no longer detectable on arterial ultrasonography. Follow-up revealed only transient efficacy of STS. Discontinuation of treatment to evaluate the persistence of effects resulted in relapse of the initial symptomatology after 4 months. STS efficacy is conceivably due to strong antioxidant properties and chelation of calcium to form soluble calcium thiosulfate complexes. This case is suggestive of PXE+ syndrome for which STS may represent potential treatment in severe cases. What's already known about this topic? Generalized arterial calcification of infancy may occur in association with ABCC6 mutations and pseudoxanthoma elasticum (PXE) can be linked to ENPP1 mutations. A PXE-like phenotype has also been reported in a subset of patients with inherited haemoglobinopathies, namely sickle cell disease or ß-thalassaemia, related to HBB mutations. To date, there is still no cure for PXE. What does this study add? We report a severe case of PXE resulting from the cumulative effects of several deleterious mutations in ENPP1, ABCC6 and HBB. We suggest the term 'PXE+ syndrome' to describe such patients. Sodium thiosulfate therapy could represent a potential option in severe cases of PXE+ syndrome.

Adenine triphosphate nucleotides are antagonists at the P2Y receptor.

The aim of the present study was to characterize the pharmacological profile of the P2Y(12) receptor for several adenine triphosphate nucleotides in view of their possible roles as partial agonists or true antagonists. Two distinct cellular systems were used: P2Y(1) receptor deficient mouse platelets ( platelets) previously shown to express a native and functional P2Y(12) receptor and 1321 N1 astrocytoma cells stably expressing the human P2Y(12) receptor (1321 N1 P2Y(12)). ADP and its structural analogues inhibited cAMP accumulation in a dose-dependent manner in both platelets and 1321 N1 P2Y(12) cells with a similar rank order of potency, 2 methylthio-ADP (2MeSADP) >>ADP - Adenosine 5'-(betathio) diphosphate (AlphaDPbetaS). Commercial ATP, 2 chloro; ATP (2ClATP) and 2 methylthio-ATP (2MeSATP) also inhibited cAMP accumulation in both cell systems. In contrast, after creatine phosphate (CP)/creatine phosphokinase (CPK) regeneration, adenine triphosphate nucleotides lost their agonistic effect on platelets and behaved as antagonists of ADP (0.5 microm)-induced adenylyl cyclase inhibition with IC(50) of 13.5 +/- 4.8, 838 +/- 610, 1280 +/- 1246 microm for 2MeSATP, ATP and 2ClATP, respectively. In 1321 N1 P2Y(12) cells, CP/CPK regenerated ATP and 2ClATP lost their agonistic effect only when CP/CPK was maintained during the cAMP assay. The stable ATP analogue ATPgammaS antagonized ADPbetaS-induced inhibition of cAMP accumulation in both platelets and 1321 N1 P2Y(12) cells. Thus, ATP and its triphosphate analogues are not agonists but rather antagonists at the P2Y(12) receptor expressed in platelets or transfected cells, provided care is taken to remove diphosphate contaminants and to prevent the generation of diphosphate nucleotide derivatives by cell ectonucleotidases.

The P2Y(12) receptor induces platelet aggregation through weak activation of the alpha(IIb)beta(3) integrin--a phosphoinositide 3-kinase-dependent mechanism.

High concentrations of adenosine-5'-diphosphate ADP are able to induce partial aggregation without shape change of P2Y(1) receptor-deficient mouse platelets through activation of the P2Y(12) receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizes activated mouse alpha(IIb)beta(3) integrin, revealed a low level activation of alpha(IIb)beta(3) in P2Y(1) receptor-deficient platelets in response to 100 microM ADP or 1 microM 2MeS-ADP. Adrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of (32)P-labeled platelets showed that P2Y(12)-mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P(20)) or pleckstrin (P(47)) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y(12) receptor is able to trigger a P2Y(1) receptor-independent inside-out signal leading to alpha(IIb)beta(3) integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the alpha(IIb)beta(3) integrin, and (iii) the transduction pathway triggered by the P2Y(12) receptor is independent of PKC but dependent on phosphoinositide 3-kinase.