Investigating Platelet Alpha Degranulation Defect in Patients with Gaucher Disease Using Immunofluorescence-Microscopy on Peripheral Blood Smears

Background: Gaucher disease (GD) is caused by mutations in the GBA1 gene, leading to a dysfunctional glucocerebrosidase (GCase) enzyme. This enzyme's inability to hydrolyze its substrate, glucocerebroside, results in its accumulation in lysosomes, primarily within macrophages. Bleeding tendency is a prominent feature in patients with GD, arising from thrombocytopenia, abnormal coagulation, and platelet dysfunction. In a previous study, we demonstrated that the main platelet dysfunction associated with the bleeding phenotype in GD was platelet α-degranulation.

Study aim: To determine if the platelet α-degranulation defect is associated with α-granule deficiency, evaluated through peripheral blood smears by immunofluorescence microscopy.

Methods: Blood smears were prepared from EDTA blood on SuperFrost slides, dried overnight at room temperature (RT), fixed in ice-cooled acetone at -30°C for 2 minutes, dried for 2-4 hours at RT, and stored at -30°C. On the day of testing, slides were warmed to RT, blocked with 10% normal goat serum for 30 minutes, and incubated with markers of α-granules, i.e., von Willebrand factor (rabbit anti-human von Willebrand factor) and P-selectin (mouse anti-human CD62P) for 1 hour in the dark. After washing, slides were incubated with secondary antibodies for 1 hour in the dark, washed, coated with glycerine, and stored at 2-4°C overnight. Fluorescence microscopy was performed using Alexa Fluor 488 and 568 for green and red emissions, respectively. All participants signed informed consent for biobanking.

Results: We included 37 patients with GD at a median age of 30 years (range: 4-70 years), of whom 23 received GD-specific therapy. The median (range) platelet count was 184 x10⁹/L (65-430 x10⁹/L). Of these, 17 patients (45%) exhibited a low P-selectin response to thrombin receptor activating peptide (TRAP) and collagen, indicating an α-degranulation defect. In six of these patients, blood smears showed low α-granule markers compared to controls, four of whom were bleeders. The remaining 11 patients with low activity showed normal staining of α-granule markers, with only one being a bleeder. All patients with a normal response to P-selectin had normal staining of α-granule markers. The α-degranulation defect and α-granule deficiency were not related to age, platelet count, or GD-treatment status.

Conclusions: Our data indicate that the platelet α-degranulation defect in patients with GD can be partially explained by α-granule deficiency. However, a low P-selectin response was also observed in the absence of α-granule deficiency, suggesting a defect in α-granule release. Further studies are needed to explore whether the accumulation of α-synuclein secondary to mutant GCase in patients with GD contributes to the abnormal α-granule release, as was shown in vitro. Understanding the cause of platelet dysfunction may help in managing the bleeding risks and in developing targeted treatments for GD.

Revel-Vilk:Takeda: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding. Zimran:Takeda: Consultancy, Honoraria; Pfizer: Honoraria; Bio-Events: Honoraria; Agyany Pharma Ltd: Current equity holder in private company.