R8 - The Impact of Hemostatic Clots on Endothelial Dynamics in Wound Healing

Introduction: : Hemostasis is a crucial phase of wound healing, essential for restoring tissue integrity and function following vascular injury. By preventing excessive blood loss and facilitating the re-establishment of normal blood flow, hemostasis plays a key role in the healing process. This is achieved through the formation of a stable blood clots that are able to withstand external mechanical and hydrodynamic forces. Stabilization of the blood clot is aided through platelet-driven contraction resulting in its densification and prepares it for the following stages of wound healing. Moreover, endothelial cells (ECs) contribute to blood clot resolution through their release of pro-fibrinolytic agents such as tissue-type plasminogen activator (tPA). While the hemostatic clot has been extensively studied both in vivo and in vitro, its role in wound healing, particularly the impact of clot composition on tissue repair, remains underexplored. In particular, the influence of fibrin on EC behavior—such as cell migration and sprouting phenotypes—warrants further investigation. This study aims to examine the dynamic interplay between plasma clot formation, subsequent lysis, and the spatial-temporal changes in ECs through developing injury models based on wound healing assays with hemostatic clots, providing insights into the mechanisms that drive wound healing.

Materials and

Methods: : Human umbilical vein endothelial cells (HUVECs) at no later than passage 5 were seeded into double-chamber culture inserts on collagen-coated tissue-culture treated dishware. Culture inserts were used for wound creation (~0.5mm). Platelet-rich plasma (PRP) was obtained by centrifuged citrated human whole blood from pooled donors at 200×g at 25°C for 15 min. Platelet-poor plasma (PPP) was obtained from spinning down PRP at 1600xg at 25°C for 15 min. To visualize fibrin in fluorescence confocal microscopy, Alexa-Fluor 647-labeled fibrinogen (40 μg/mL) was added to aliquoted plasma. Following culture insert removal, CaCl2 (25 mM) and thrombin (1 U/mL or 0.3 U/mL) were added to plasma, which was subsequently applied at the center of the gap on top of cells. The clots were allowed to stabilize for 10 mins prior to adding cell culture media to dishware. HUVECs and platelets in PRP clots were stained with Calcein AM for visualization beneath the plasma clots. External fibrinolysis was induced by adding tissue plasminogen activator (tPA) at 1 ug/ml in cell culture media in incubator conditions to dishes with PPP clots. Cleaved fibrin was washed away by regularly replacing cell culture media. Confocal microscopy of cells and fibrin was performed using Zeiss LSM 880 confocal laser microscope. Fibrin fiber and network characteristics were measured with the open-source image software Fiji manually and using ‘OrientationJ’ plugin. Wound closure and cell morphology were also manually analyzed in Fiji.

Results, Conclusions, and Discussions:: Quantitative analysis of confocal microscopy images of wound healing assays clots acquired over the course of wound closure reveal that the PRP and PPP clots significantly delay or prevent wound closure. In agreement with experiments utilizing purified PPP, wound closure was inhibited with applied PPP clots. In contrast, PRP clots allowed wound closure, though, at a slower rate than in the absence of clots. In both conditions, EC sprouting morphology was observed. Similarly, low density fibrin network through low thrombin concentration found slower rates of wound closure. Fibrinolysis experiments demonstrated that the dissolution of the clots restored both normal ECs morphology and migration rates. These findings suggest that the presence of fibrin fibers mechanically prevent cell migration while platelets promote ECs movement throughout the clot leading to wound closure. It is also observed that the alteration of the fibrin structure promotes wound closure observed by low density fibrin networks.

Acknowledgements and/or References (Optional):: This project is supported by the National Institute of Health grant (R21DE030294) and American Society of Hematology Junior Faculty Scholar Award.