PO:33:201 | Metabolic and functional state of circulating platelets in patients with systemic sclerosis

Annalisa Villa¹, Simona Truglia¹, Francesca Maiorca², Annamaria Sabetta², Ludovica Lombardi², Silvia D'Orso², Lucia Stefanini², Fabrizio Conti¹, Marcella Visentini², Valeria Riccieri¹ | ¹Dipartimento di Scienze Cliniche, Internistiche, Anestesiologiche e Cardiovascolari, Università degli Studi di Roma La Sapienza; ²Dipartimento di Medicina Traslazionale e di Precisione, Università degli Studi di Roma La Sapienza

Background. Systemic Sclerosis (SSc) is a systemic autoimmune disease characterized by vasculopathy, inflammation and fibrosis. Microangiopathy manifests with Raynaud’s phenomenon and digital ulcers, but also visceral involvement. Endothelial dysfunction represents an early event, associated with imbalance in vasoconstriction–vasodilation, chronic hypoxia and altered angiogenesis. Platelets, besides their known role in hemostasis, recruit immune cells and release pro-fibrotic mediators. Recent studies show increased levels of platelet-derived products in SSc, suggesting higher platelet activity, although the use of antiplatelet therapy in this field remains controversial. Evidence on a complete phenotypic analysis of platelets in SSc is still limited. This preliminary study aims to investigate platelet functionality in SSc.

Materials and Methods. This single-center study involved patients diagnosed with SSc according to the 2013 ACR/EULAR criteria, adults, not on antiplatelet therapy, in outpatient follow-up. As healthy controls, age- and sex-matched subjects without autoimmune diseases and not on antiplatelet therapy were enrolled. Blood samples were analyzed within 30 minutes after collection. Platelet activation and reactivity were evaluated by labeling diluted blood with Tyrode buffer, CD62P-PE and PAC1-FITC, in the absence or presence of agonists (ADP, convulxin, TRAP-6). To identify procoagulant platelets, staining with CD41-APC and Annexin V-PE was used, in buffer with Ca² and fibrin inhibitor. Mitochondrial functionality was studied with MitoTracker Green FM and MitoTracker CMXRos on washed platelets, with or without stimulation with agonists. Analysis was performed using BD Accuri C6 Plus flow cytometer and FlowJo™ and GraphPad Prism software version 10.4.2.

Results. Eleven patients with SSc (mean age 58.4 ±11.7, F 11) and six healthy controls (age 54.7 ±13.6, F 6) were enrolled. Circulating platelets showed greater mass and mitochondrial membrane potential. Basal platelet activation was significantly higher in SSc patients, with greater expression of PAC1+ CD62P+ (4.4±9.3% vs 0.4±0.9%, p<0.05) and mean fluorescence intensity for PAC1 (243.8±330.0 vs 61.3±57.5, p<0.05) compared with controls. Moreover, platelets from SSc patients showed higher responsiveness following stimulation with ADP (a weak agonist), but not in response to stronger agonists mimicking thrombin or collagen. No significant differences emerged in the percentage of procoagulant or apoptotic platelets.

Conclusions. This study shows increased basal platelet activation in SSc patients, with an altered response pattern to stimuli. Mitochondrial integrity suggests elevated platelet activity, implying their role in vascular damage and the possible reconsideration of antiplatelet therapy in SSc. Further studies on larger cohorts and better-characterized patients by disease form, duration, and activity will be needed to clarify platelet function in SSc pathophysiology.