How Clot Composition Influences Fibrinolysis in the Acute Phase of Stroke: a Proteomic Study of Cerebral Thrombi

Background: The dramatic clinical improvement offered by mechanical thrombectomy (MT) raised questions about the relevance of prior intravenous thrombolysis (IVT) in large vessel occlusion strokes. Hence, studying IVT susceptibility and its dependence on thrombus composition is crucial. We used observational proteomic study of whole thrombi retrieved by MT to identify factors associated with fibrin content and fibrinolytic activity (FA). Methods: In 104 stroke patients, the thrombi proteome was established by mass spectrometry coupled to liquid chromatography. FA were estimated in clots both outside (FAout) by measuring D-dimer levels at the blood-thrombus interface and inside (FAin) by evaluating the ratio of fibrinogen ⍺ to its plasmin-cleaved forms using proteomics coupled with protein electrophoresis. The factors associated with fibrin content, FAin and FAout were determined by IVT-adjusted linear regression. Results: FAout (p<0.0001) and FAin (p=0.0147) were driven by rt-PA administration (47/104) and thrombus composition. Indeed, FAout was greater with fibrin-rich than erythrocyte-rich thrombi, presumably because of more (r)t-PA substrates. Thus, FAout was increased with cardioembolic thrombi (72/104), which are rich in fibrin (p=0.0300). Opposite results were found inside thrombus, suggesting that (r)t-PA penetrability was hampered by the density of the fibrinous cap. Moreover, blood cells had a strong impact on thrombus structure and susceptibility to (r)t-PA. Indeed, fibrin content was negatively associated with erythrocyte-specific proteins in the thrombus, admission hematocrit (p=0.0139) and hemoglobin level (p=0.0080), which underlines the key role of erythrocytes in thrombus composition. Also, an increased number of neutrophils impaired FAout (p=0.0225), which suggests that their aggregation around the thrombus prevented the (r)t-PA attack. Only FAout was significantly associated with reduced thrombus weight (p=0.0310), increased recanalization rate (p=0.0150), good clinical outcome (p=0.0480) and reduced mortality (p=0.0080). Conclusions: Proteomics can offer new insights into the close relation between thrombus composition and susceptibility to fibrinolysis, paving the way for new adjuvant therapies.