Platelet and megakaryocyte abnormalities in the Gray platelet syndrome

DE CANDIA ERICA; LAROCCA LM; HELLER PAULA G; PODDA GIAN MARCO; PUJOL MOIX NURIA; GLEMBOTSKY ANA C; PECCI ALESSANDRO; ALBARELLI MA; CATTANEO M; BALDUINI CARLO L

Amsterdam

Congreso; XXIV Congress of the International Society on Thrombosis and Haemostasis; 2013

International Society on Thrombosis and Haemostasis

Background: The Gray Platelet Syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding symptoms, macrothrombocytopenia and gray appearance of peripheral platelets due to lack of a-granules. The genetic defect responsible for GPS was recently identified in biallelic mutations in the NBEAL2 gene. Aims: Although the GPS is considered a disease with a heterogenous phenotype, aim of the present study was the identification of bone marrow features and of platelet defects that could be considered specific for the disease. Methods: Five GPS patients from four unrelated families were recruited in four different institutions to study the platelet function and the bone marrow features. All subjects or their legal guardians gave written informed consent according to the Declaration of Helsinky. Protocols were approved by the Ethics review Board at the Institution that enrolled the patients. Four patients underwent extensive study of the platelet function and four patients underwent bone marrow (BM) biopsy. Patients underwent platelet studies at their Institutions. BM samples were obtained using standard procedures and embeded in paraffin. BM samples were shipped to the Catholic University of Rome for morphological and immunhistochemical analysis. Platelet studies included: (i) platelet aggregation according to standard Born method, (ii) flow cytometric analysis of platelet activation, by measurement of anti-P-selectin and PAC-1 antibodies binding to platelets activated by several agonists (iii) binding of FITC-fibrinogen to washed platelets activated by thrombin (iv) measurement of PAR1 expression on platelet membrane by flow cytometry and anti-PAR1 monoclonal antibodies (v) immunofluorescence quantitative analysis of the thrombospondin-1 content of alpha-granules on peripheral blood slides. BM studies included hematoxylin-eosin staining, reticulin staining and immunohystochemical analysis with the avidin-biotin-peroxidase complex method for the following proteins: (i) Platelet factor 4, (ii) P-selectin, (iii) CD61, (iv) c-MPL, (v) PAR1, (vi) PAR4. Results: All GPS patients who underwent platelet studies had a severe defect of platelet response to PAR1-activating peptide. Specifically, the PAR1-mediated platelet aggregation and activation, as measured by exposure of p-selectin and PAC1 binding sites, were severely affected in 4 GPS patients. The platelet response to other agonists was slightly or moderately affected in two patients, normal in one. Expression of PAR1 on platelet membrane was reduced in all four patients (50–70% of control values). The binding of fibrinogen to GPS platelets activated by thrombin was severely reduced. All four patients who underwent BM biopsy had marked fibrosis (grade 2-3) and marked emperipolesis. Fifthy to 80% of megakaryocytes contained up to four leukocytes engulfed within the cytoplasm. BM immunohistochemistry in two patients showed increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4, thrombospondin and c-MPL on MKs.Conclusion: Defective PAR1-mediated platelet responses and marked emperipolesis are typical features of GPS, as they were found in all patients studied. The defect of PAR1-mediated platelet responses can be used as a screening test for the diagnosis of the disease, which can be underdiagnosed. In fact, on the basis of the high number of compound heterozygous described so far, the disease might be more frequent than expected.