Background Endovascularly retrieved clots are a potential resource for diagnosing stroke etiology, which may influence secondary prevention treatment. In this study we measured thrombin activity eluted by serially washing clots.
Methods Clots were retrieved from 68 patients with acute ischemic stroke, freshly frozen and classified by standard criteria into proven atrial fibrillation (AF, 18 patients), atherosclerotic origin (AS, 15 patients), cryptogenic stroke (Cr, 17 patients) and other known causes (18 patients). Thawed clot samples were washed by transferring them into 1 mL buffer in seven hourly cycles and a fluorescent substrate assay was used to measure secreted thrombin activity. The clots were also examined histologically. Artificial fibrin and red blood cell-rich clots were similarly assayed for wash-eluted thrombin activity as an external control.
Results Thrombin activity eluted from clots of AF origin decreased significantly with time in contrast to steady levels eluted from AS origin thrombi (P<0.0001 by repeated measures ANOVA). The Cr stroke group was indistinguishable from the AF group and differed statistically from the AS group (P=0.017 by repeated measures ANOVA). In artificial clots we found a biphasic activity pattern, with initially decreasing levels of eluted thrombin (AF pattern) and then, with continuing washes, steady eluted thrombin levels (AS pattern).
Conclusions An assay measuring the change in thrombin in clots retrieved during acute stroke endovascular thrombectomy procedures may serve as a diagnostic marker of the origin of the clot. The suggested mechanism for these differences may be the clot location before its retrieval, with high blood flow causing thrombin washout in atherosclerotic clots, in contrast to atrium appendage low blood flow retaining high thrombin levels.
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Z'eIH and EAB contributed equally.
ESS and DO contributed equally.
Contributors ZIH, EAB, NS, MN: performed sample collection, data aquisition and analysis. ZIH: planned the experiment, devised the final draft and conducted the patient clinical part of the study. MB, GY, BK, SB: sample acquisition and patient management. TD, SM, AG, YS: patient management and requirement, clinical data analysis. AA: scientific revision. JC: major scientific revision. DO, ESS: equal scientitfic supervision. ESS: planning and supervision of bench experiments.
Funding This study was funded in part by a generous donation from the Budai family.
Competing interests US provisional patent application has been filed according to the results of this study (Application No. 62/880,681).This study was performed as part of the requirements for EAB's MD thesis.
Patient consent for publication Not required.
Ethics approval All the experiments were approved by the institutional ethical committee detailed in the Methods section.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information. All the data including de-identified patient data are included in the submission files.
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