Aims:We report about a 35 year old male patient with severe and frequent epistaxis and hematoma since infancy. He presented with mild thrombocytopenia and increased mean platelet volume. Von Willebrand's disease and subhemophilia had been excluded. Previously, he was diagnosed with immune thrombocytopenic purpura. He never underwent elective surgery. His parents were asymptomatic. However, his young daughter also suffers from severe bleeding symptoms and was referred to our coagulation outpatient clinic to investigate the bleeding disorder.
Methods:Platelet function was assessed by light transmission-, lumi-aggregometry and flow cytometry. Lysates of gel-filtered platelets were analyzed for total granule P-selectin, CD63 and von Willebrand factor (VWF) content by Western blotting and for serotonin levels by ELISA, respectively. Molecular genetic analysis was performed using whole exome sequencing and pyrosequencing.
Results: Platelet aggregation in response to ADP was not inducible. Platelet aggregation in response to epinephrine resulted in monophasic curves. Platelet aggregation in response to TRAP-6 or collagen was inducible but impaired and accompanied by disaggregation. In contrast, arachidonic acid- and U46619-induced platelet aggregation as well as ristocetin-induced platelet agglutination/aggregation was within the normal range. Also platelet surface expression of GPIIb/IIIa, GPIb/V/IX, GPVI and CD29 was normal. Activation of GPIIb/IIIa in response to ADP, convulxin or TRAP-6 was inducible but diminished and in response to epinephrine or arachidonic acid normal. P-selectin (alpha-granule marker) and CD63 (dense body/lysosome marker) surface expression was not inducible by thrombin or convulxin and markedly reduced in response to TRAP-6 and arachidonic acid. Immunoblot analysis of isolated platelets demonstrated pronouncedly decreased content of total P-selectin, VWF and CD63. Fluorescent staining of adenine nucleotides by mepacrine in patient's platelet dense bodies and secretion of ATP induced by arachidonic acid (normal aggregation) were absent. However, patient's platelet serotonin levels were within the normal range. The molecular genetic diagnostic showed an undescribed mutation in the GATA1 gene (c.886A>C hemizygot, p.T296P). The bioinformatic check confirmed a pathogenetic importance of this mutation located in the domain I of the C-terminal zinc finger.
Conclusion: We describe the first case of a combined platelet alpha- and partial delta-storage pool disorder where the dense granules lack adenine nucleotides but contain normal levels of serotonin - causing a severe bleeding disorder.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.