Platelet dysfunction in Ehlers–Danlos syndrome is associated with low GPVI and PAR1 expression on platelets

Abstract

Abstract Introduction: People with Ehlers–Danlos syndrome (EDS) are at an increased risk for hemorrhagic events, which can range from minor bruising to life-threatening bleeding episodes. Although prior research has suggested the presence of platelet dysfunction in EDS populations, the underlying mechanisms remain unclear. We systematically evaluated platelet function in human patients with EDS and a murine model of classical EDS. Methods: Blood samples were obtained from 44 people with various EDS types—hypermobile (40%), classical (32%), classical-like (23%), and vascular (5%)—and compared with 44 age- and gender-matched healthy controls in accordance with the Declaration of Helsinki. The Col5a1+/- mouse model was employed for the classical type of EDS. The function of human and mouse platelets was evaluated using standardized agonist-induced in vitro assays. Quantification of plasma inflammatory markers and soluble GPVI (sGPVI) was done using ELISA. H&E staining, transmission electron microscopy (TEM), and flow cytometry assay were used to characterize murine megakaryocytes. Results: The mean age of the study population was 34.5 (range 16-69) years. Platelets from people with EDS exhibited reduced GPVI and PAR1 levels in resting platelets compared to healthy controls. Evaluation of EDS platelet function demonstrated significantly reduced aggregation and αIIbβ3 activation in response to collagen, collagen-related peptides, and thrombin receptor activator peptide-6, despite normal surface expression of αIIbβ3. Furthermore, EDS platelets showed impaired platelet spreading on fibrinogen- and collagen-coated surfaces. Platelet functional deficits in people with EDS were associated with decreased phosphorylation of LAT at Y212, PLCγ2 at Y1217 in collagen-stimulated platelets, and decreased total tyrosine and Srс phosphorylation at Y416 in thrombin-stimulated platelets. Phosphorylation of talin-1 at S425 was reduced in collagen- or thrombin-stimulated EDS platelets. Platelet granule secretion levels were similar in both groups. Plasma analysis revealed elevated TNFα levels, but not IL6 levels, in EDS with no correlation to platelet aggregation. Human plasma sGPVI levels were comparable between groups. Col5a1+/- mice exhibited a similar platelet function phenotype, with defects in aggregation, αIIbβ3 activation, platelet signaling, and spreading on fibrinogen compared to wild-type mice. As observed in humans, platelet function defects in Col5a1+/- mice were associated with decreased GPVI levels. TEM analysis revealed the absence of organized collagen fibrils around vessels in Col5a1+/- mice. While megakaryocyte number, morphology and proplatelet structure were comparable, flow cytometry analysis of murine bone-marrow-derived megakaryocytes from Col5a1+/- mice revealed decreased GPVI levels compared to littermate wild-type mice. Conclusions: We provide evidence for the first time that platelet dysfunction in EDS is linked to reduced GPVI and PAR1 levels in quiescent platelets concomitant with defects in integrin αIIbβ3 signaling. Findings from the EDS mouse model suggest that platelet dysfunction in EDS may be partially attributed to low GPVI expression in megakaryocytes. Currently, we are elucidating the underlying mechanism by which disorganized collagen fibrils in EDS reduce GPVI levels in megakaryocytes.