Transcriptomic Analysis of Extracellular Vesicles in the Search for Novel Plasma and Thrombus Biomarkers of Ischemic Stroke Etiologies.

Accurate etiologic diagnosis provides an appropriate secondary prevention and better prognosis in ischemic stroke (IS) patients; still, 45% of IS are cryptogenic, urging us to enhance diagnostic precision. We have studied the transcriptomic content of plasma extracellular vesicles (EVs) (n = 21) to identify potential biomarkers of IS etiologies. The proteins encoded by the selected genes were measured in the sera of IS patients (n = 114) and in hypertensive patients with (n = 78) and without atrial fibrillation (AF) (n = 20). IGFBP-2, the most promising candidate, was studied using immunohistochemistry in the IS thrombi (n = 23) and atrium of AF patients (n = 13). In vitro, the IGFBP-2 blockade was analyzed using thromboelastometry and endothelial cell cultures. We identified 745 differentially expressed genes among EVs of cardioembolic, atherothrombotic, and ESUS groups. From these, IGFBP-2 (cutoff > 247.6 ng/mL) emerged as a potential circulating biomarker of embolic IS [OR = 8.70 (1.84-41.13) p = 0.003], which was increased in patients with AF vs. controls (p < 0.001) and was augmented in cardioembolic vs. atherothrombotic thrombi (p < 0.01). Ex vivo, the blockage of IGFBP-2 reduced clot firmness (p < 0.01) and lysis time (p < 0.001) and in vitro, diminished endothelial permeability (p < 0.05) and transmigration (p = 0.06). IGFBP-2 could be a biomarker of embolic IS and a new therapeutic target involved in clot formation and endothelial dysfunction.

Role of factor XIII in ischemic stroke: a key molecule promoting thrombus stabilization and resistance to lysis.

BACKGROUND: Active coagulation factor XIII (FXIII) catalyzing crosslinking of fibrin and other hemostatic factors plays a key role in clot stability and lysis. OBJECTIVES: To evaluate the effect of FXIII inhibition in a mouse model of ischemic stroke (IS) and the role of activated FXIII (FXIIIa) in clot formation and lysis in patients with IS. METHODS: A ferric chloride IS murine model was performed before and after administration of a FXIIIa inhibitor (FXIIIinh). Thromboelastometry in human and mice blood was used to evaluate thrombus stiffness and lysis with FXIIIinh. FXIIIa-dependent fibrin crosslinking and lysis with fibrinolytic drugs (tissue plasminogen activator and tenecteplase) were studied on fibrin plates and on thrombi and clotted plasma of patients with IS. Finally, circulating and thrombus FXIIIa were measured in 85 patients with IS. RESULTS: FXIIIinh administration before stroke induction reduced infarct size, α2-antiplasmin (α2AP) crosslinking, and local microthrombosis, improving motor coordination and fibrinolysis without intracranial bleeds (24 hours). Interestingly, FXIII blockade after stroke also reduced brain damage and neurologic deficit. Thromboelastometry in human/mice blood with FXIIIinh showed delayed clot formation, reduced clot firmness, and shortened tissue plasminogen activator lysis time. FXIIIa fibrin crosslinking increased fibrin density and lysis resistance, which increased further after α2AP addition. FXIIIinh enhanced ex vivo lysis in stroke thrombi and fibrin plates. In patients with IS, thrombus FXIII and α2AP were associated with inflammatory and hemostatic components, and plasma FXIIIa correlated with thrombus α2AP and fibrin. CONCLUSION: Our results suggest a key role of FXIIIa in thrombus stabilization, α2AP crosslinking, and lysis resistance, with a protective effect of FXIIIinh in an IS experimental model.