Impaired Platelet Function in Ehlers-Danlos Syndrome: Insights from Human Subjects and a Murine Model

Background: Bleeding complications remain a serious problem in people with Ehlers-Danlos syndrome (EDS). Hemorrhage can be observed across multiple types of EDS. Bleeding in people with EDS is heterogeneous and may be multifactorial. Several prior studies have found platelet function abnormalities in people with EDS. Although the association of bleeding with EDS has been long-term acknowledged, the contributing mechanisms remain poorly understood.

Objective: To characterize platelet function in a cohort of EDS subjects and a murine EDS model.

Methods: In this study, we included a cohort of 53 people with the hypermobile (49%), classical (26%), classical-like (21%), or vascular (4%) types of EDS and a group of 53 age- and gender-matched healthy control subjects in accordance with an Institutional Review Board-approved protocol. The median ages of the EDS study group and healthy controls were 35 (range 16 - 69) and 36 (range 16 - 61), respectively. As no single mutation has been identified for the hypermobile type of EDS, the most prevalent type in our EDS cohort, we used COL5A1^+/− mice as a murine model of the classical type, the second most prevalent EDS type in the cohort. The function of human and mouse platelets was evaluated using standardized agonist-induced in vitro assays. To study the in vivo interaction of platelets and arterial extracellular matrix, we utilized a FeCl₃ (5%) injury-induced model of the common carotid artery. Statistical analysis included the Mann-Whitney U test and the two-way ANOVA to compare the cohorts for continuous variables, and the Fisher's exact test for categorical variables.

Results: The mean ISTH-BAT score was significantly higher in EDS subjects compared to healthy controls (9.2 vs. 0.1, p < 0.001). Evaluation of platelet function revealed reduced GPVI and PAR1 levels in resting platelets from EDS subjects compared to healthy controls (p < 0.01). Platelets from EDS subjects exhibited reduced platelet aggregation and αIIbβ3 activation (with normal αIIbβ3 surface expression) in response to stimulation with collagen, collagen-related peptide, or thrombin receptor activator peptide-6 compared to healthy controls (p < 0.01). Reduced platelet aggregation and αIIbβ3 activation were associated with the downregulation of LAT (Y212), PLCγ2 (Y1217), and talin-1 (S425) phosphorylation in EDS patients (p < 0.05 vs. healthy controls). We also observed reduced platelet spreading on the fibrinogen-coated surface (p < 0.01 vs. healthy controls). Platelet granule secretion was comparable between both study cohorts. COL5A1^+/− mice demonstrated a bleeding tendency characterized by prolonged tail-bleeding time (p < 0.05 vs. wild-type control, WT), which was concomitant with reduced platelet aggregation and αIIbβ3 activation, similar to findings observed in EDS subjects. In a FeCl₃ injury-induced carotid artery thrombosis model, COL5A1^+/− male and female mice were less susceptible to thrombosis (p < 0.05 vs. WT), with 41% of COL5A1^+/− mice having no occlusion within 40 min of observation time.

Conclusions: For the first time, we demonstrate that EDS platelets are characterized by reduced baseline GPVI and PAR1 expression, which is associated with defects in integrin αIIbβ3 inside-out and outside-in signaling. The data from the EDS murine model suggest that EDS platelet dysfunction results in impaired arterial thrombosis. We conclude that while platelet dysfunction in people with EDS may protect them from cardiovascular disorders, it may also contribute to post-traumatic or post-surgical hemorrhagic complications.

Staber:BioMarin: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Lentz:Novo Nordisk: Consultancy; Sanofi: Consultancy.