Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS

Frauke Swieringa,Paola E J Van Der Meijden,Barbara Zieger,Nadine J A Mattheij,Connie T R M Stumpel,Joachim Pohlenz,Jörg Faber,René P Zahedi,Oliver Pagel,Johan W M Heemskerk,Jingnan Huang,Fiorella A Solari,Albert Sickmann,Alexandra Russo,Dirk E Schrander,Florian Beck,Marion A H Feijge,Irene M L W Körver-Keularts,Kerstin Jurk

doi:10.1038/s41598-020-68379-3

Abstract

Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gsα. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gsα induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gsα-dependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gsα-PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHO, the changes in platelet phosphoproteome were reversed. This combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia.

Highlights

Pseudohypoparathyroidism (PHP) characterises a heterogeneous group of disorders, of which the common feature is an end-organ resistance to parathyroid hormone
Some patients have mental retardation. These characteristics are classified as Albright hereditary osteodystrophy (AHO) syndrome
In order to assess abnormalities of the Gsα-adenylyl cyclase (AC)-protein kinase A (PKA) pathway, we analysed the platelets from seven rare patients with confirmed or suspected pseudohypoparathyroidism type Ia (PHP Ia) (Table 1)

Summary

Introduction

Pseudohypoparathyroidism (PHP) characterises a heterogeneous group of disorders, of which the common feature is an end-organ resistance to parathyroid hormone. Consistent among patients with established PHP (including AHO) is impairment in the Gsα-mediated signalling effects of tissues in response to multiple h ormones[2]. This impairment is linked to dysfunctional mutations in the GNAS complex locus, encoding for the Gsα protein. The other high-affinity receptor for prostacyclin, EP1, is not expressed on platelets[14] Both iloprost and PGE1 elevate platelet cAMP levels via Gsα, activate PKA and establish suppression of key signalling events including C a2+ fluxes and integrin αIIbβ3 activation[15,16]. Earlier studies describe that in patients with PHP Ia (AHO) the Gsα functional defect is linked to lower platelet responses to both iloprost and PGE117

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Conclusion