Factor XI localization in human deep venous thrombus and function of activated factor XI on venous thrombus formation and hemostasis in rabbit

Background

Anticoagulant therapy is the first-line treatment for the prevention and management of venous thromboembolism (deep vein thrombosis and pulmonary embolism), as well as associated complications [1].

Currently available types of anticoagulant therapy operate through enzymatic inhibition. Heparins inhibit blood coagulation by enhancing the neutralizing effect of antithrombin on thrombin and factor Xa. Vitamin K antagonists bind to antithrombin, consequently inhibiting the synthesis of coagulation factors II, VII, IX, and X. In contrast, non-vitamin K oral anticoagulants (NOACs) directly inhibit thrombin or factor Xa.

These anticoagulant therapies all have a major downside however: they are associated with an increased risk of bleeding. This highlights the urgent need to explore new anticoagulant strategies in a variety of clinical contexts [2]. In this context, it is important to explore the relevance of factor XI (FXI) and its activated form, factor XIa (FXIa), as therapeutic targets and, moreover, whether targeting these factors could be a safer alternative to current anticoagulant therapies [3].

Key findings

Localization of FXI in human deep vein thrombosis.

First, the authors investigated the localization of FXI in human deep vein thrombosis (DVT). Fifteen thrombus samples were analyzed using immunofluorescence and immunohistochemistry techniques. The immunofluorescence images revealed that FXI was predominantly found in fibrin-rich areas, with less association to von Willebrand factor (VWF) or glycophorin A (a marker of erythrocytes). Immunohistochemical analysis distinguished “fresh” (non-organized) regions from “organized” areas based on the presence of CD34-positive cells (indicative of endothelialization). In these images, FXI was more abundant in the fresh areas, suggesting its role in initial thrombus formation, while its presence was reduced in organized regions, indicating potential degradation over time.

Oral administration of FXIa inhibitor prolongs aPTT rather than PT in rabbits, whereas oral administration of FXa inhibitor prolongs both PT and aPTT.

The authors compared the activated partial thromboplastin time (aPTT) and prothrombin time (PT) in rabbits after oral administration of ONO-1600586 (an FXIa inhibitor) and rivaroxaban (an FXa inhibitor). The results showed that ONO-1600586 prolonged aPTT without significantly affecting PT, in contrast to rivaroxaban, which increased both aPTT and PT. This suggests that ONO-1600586 specifically targets the intrinsic coagulation pathway related to FXIa.

Different contribution of activated FXIa and activated FXa on venous thrombus formation and skin bleeding in rabbits.

The authors observed differences in the size of venous thrombi in rabbits treated with ONO-1600586 and rivaroxaban compared to the control. Both inhibitors significantly reduced thrombus weight. Additionally, skin bleeding experiments indicated that the FXIa inhibitor did not increase bleeding time or volume, unlike the FXa inhibitor, which prolonged bleeding. This suggests that ONO-1600586 could be a safer clinical treatment option for human deep vein thrombosis, with a lower risk of hemorrhage.

Contribution of activated FXIa and activated FXa to ex vivo thrombus formation in a flow chamber system.

The authors next examined fibrin formation under low-shear blood flow conditions using a flow chamber system. The results showed that ONO-1600586 partially inhibited fibrin formation, whereas rivaroxaban more effectively reduced fibrin formation. This may explain why FXIa inhibitors have a lower impact on hemostasis compared to FXa inhibitors.

Effects of ONO-1600586 on human activated FXIa and in vitro thrombus formation.

Finally, the authors addressed the effects of ONO-1600586 on human blood coagulation. The inhibitor suppressed FXIa activity, reduced thrombin generation, and decreased fibrin formation during human blood perfusion. These findings suggest that FXIa inhibitors may prevent thrombus formation without significantly affecting coagulation under low-flow conditions.

Why I think this preprint is important:

The preprint’s findings have the potential to inspire future studies or clinical trials, reinforcing its relevance in the ever-evolving field of thrombosis research. By focusing on Factor XI as a therapeutic target, it addresses a crucial gap in current treatments, which frequently carry significant bleeding risks.

Suggestions and questions for the authors:

Q1): Would it be possible to include a more detailed description of the compound ONO-1600586 in the introduction, supported by specific references that validate its use as a selective FXIa inhibitor? Including this information would strengthen the introduction by offering a more comprehensive understanding of ONO-1600586’s role and supporting the interpretation of the experimental results discussed later in the text.

Q2): Considering the limitation related to the sample size, are you planning to increase the sample size beyond 15 individuals in a follow-up study? Alternatively, would you consider tempering the conclusions to reflect the current limitations? While the findings are promising, the restricted number of samples might constrain the broader applicability of the results. Increasing the sample size could significantly enhance the robustness and statistical significance of the data. Additionally, have you considered including patient data, such as the duration of hospital stay, which could potentially influence thrombus pathology?

Q3): A few specific changes to the data organization and formatting would really help the reader’s understanding and analysis of the results – my suggestions: i) organizing the lettering that labels the figures; ii) standardizing the position of the experimental ‘n’ values only in figure legends and not in the figure itself.

Q4): In the supplementary table, could you provide information on the number of female patients? It would perhaps be helpful to include a row for “Female sex, n (%)” or “Female sex (calculated)” to indicate how many women were included in the study. Additionally, to clarify the treatment distribution, you could perhaps include a row specifying the total number of patients who did not receive any medication or who received combinations of treatments. This would help provide a more comprehensive understanding of the treatment allocation.

Q5): Could you explain the differences between organized and non-organized areas within the thrombi in more detail (e.g., how these areas were defined, specific cellular characteristics, structural features, staining patterns). This could help the reader in understanding Figure 1.

Q6): For figures 1 and 3, could you please clarify the number of experimental samples (n) represented in the immunofluorescence and immunohistochemistry analyses?

Q7): For figure 3, could you please share details of the statistical analysis performed?

Q8): Your work demonstrates that FXI is localized in DVT and that FXIa inhibition reduces thrombus weight without increasing bleeding risk. Could you speculate on the mechanisms by which FXI inhibition affects thrombus organization and reduces fibrin formation?

References:

[1] Tracy A. DeWald, Jeffrey B. Washam, Richard C. Becker, Anticoagulants: Pharmacokinetics, Mechanisms of Action, and Indications, Neurosurgery Clinics of North America, Volume 29, Issue 4, 2018, Pages 503-515, ISSN 1042-3680, ISBN 9780323640916.

[2] Kluge KE, Seljeflot I, Arnesen H, Jensen T, Halvorsen S, Helseth R. Coagulation factors XI and XII as possible targets for anticoagulant therapy. Thromb Res. 2022 Jun;214:53-62. doi: 10.1016/j.thromres.2022.04.013. Epub 2022 Apr 25. PMID: 35490644.

[3] Gailani D, Gruber A. Targeting factor XI and factor XIa to prevent thrombosis. Blood. 2024 Apr 11;143(15):1465-1475. doi: 10.1182/blood.2023020722. PMID: 38142404; PMCID: PMC11033593.

Tags: coagulation factor x, coagulation factor xi, hemostasis, pathology, venous thromboembolism

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