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

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 Gsαdependent 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. Pseudohypoparathyroidism (PHP) characterises a heterogeneous group of disorders, of which the common feature is an end-organ resistance to parathyroid hormone. Patients diagnosed with pseudohypoparathyroidism

Assessment of a complete and classified platelet proteome from genome-wide transcripts of human platelets and megakaryocytes covering platelet functions

“…Earlier analyses indicated that the platelet proteome from healthy subjects is quite stable with < 15% of changes 51 . Similarly, the global platelet proteomes from the few patients, extensively studied so far—such as Albright hereditary osteodystrophy, Glanzmann or Scott syndrome patients—showed only minor changes compared to that of control subjects 27 , 28 , 48 . the technical abilities to study this in the future is made in the revised discussion (page 16).…”

“…https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054341/ b. Supplemental Table 1 and 2 : identified phosphopeptides and proteins c. ProteomeXchange repositories PXD002883 and 10.6019/PXD002883 Cohort 5 28 a. https://www.nature.com/articles/s41598-020-68379-3#Sec25 b. Datafile S1 and Datafile S2 : identified phosphopeptides and proteins c. ProteomeXchange repository PXD016534 Cohort 6 29 a.…”

“…Since still little is known of many of the proteins, the list of 20 k transcripts reveals a wealth of novel information on proteins that will influence platelet structure and function. Knowledge for understanding disease processes is still limited, as prior work from our and other labs describe only small-size alteration in platelet (phospho)proteomes of patients with Scott ( ANO6 ) 27 or Glanzmann ( ITGA2B ) 48 disorders or with pseudohypoparathyroidism ( GNAS ) 28 . Altogether, this underscores that our approach to define a complete platelet proteome provides a valuable scaffold for further exploring and understanding platelet traits in and beyond thrombosis and hemostasis.…”

The role of phospho-tyrosine signaling in platelet biology and hemostasis

“…Phosphotyrosine profiling shows that total cellular tyrosine phosphorylation increases upon stimulation with ATP, and is near completely abolished by concurrent treatment of cells with PKAactivating prostacyclin analogue iloprost [48]. This suggests that kinase activity does not indiscriminately result in enhancement of platelet functions, as is often thought.…”

Long-term platelet priming after glycoprotein VI stimulation in comparison to Protease-Activating Receptor (PAR) stimulation

“…The more limited ability of iloprost to revert the CRP-XL induced aggregation (in comparison to TRAP6) can be explained by the longer Ca 2+ signal with this GPVI agonist, implying a lower ability of PKA to overrule this signal. On the other hand, our laboratories have described that iloprost and other cAMP-elevating agents, as a pretreatement, are able to affect the collagen-induced platelet adhesion under stasis and flow conditions [ 28 , 29 ].…”