An architecturally rational hemostat for rapid stopping of massive bleeding on anticoagulation therapy

Introduction: Approximately 35% of hemorrhagic deaths occur before hospital arrival, exacerbated by anticoagulant therapies that complicate hemostasis and increase severe bleeding risks. Uncontrolled hemorrhage is a significant contributor to global trauma-related mortality, causing over five million deaths annually. In the U.S., over eleven million people on anticoagulants face elevated emergency visits due to bleeding complications.
Current hemostatic dressings often fail to provide immediate and effective hemostasis in patients on anticoagulants and do not maintain hemostasis long enough for clinical treatment. Ideal hemostatic agents should rapidly form clots, sustain hemostasis, and be removable without causing further tissue damage. They should also be long-lasting, easy to store and use, and biocompatible, specifically targeting patients on anticoagulant therapy—a growing patient population unmet by existing solutions.
Rapid absorption is crucial for hemostatic agents as it allows swift concentration of clotting components at bleeding sites and creates high shear zones that accelerate platelet activation and aggregation. This rapid and voluminous blood absorption has been achieved primarily by introducing new materials or modifying the chemical properties of existing materials. Another promising approach is altering material microstructures, which can impact liquid absorption speed and capacity.
Our research introduces a next-generation chitosan hemostat, designed with optimized pore size and connectivity. This hemostat enables rapid blood absorption and concentration of clotting components, effectively counteracting anticoagulants. Contrary to the belief that chitosan does not participate in physiological clotting, our findings reveal it activates platelets via Toll-like receptor 2 (TLR-2), initiating the coagulation cascade even in the presence of anticoagulants.

Materials and

Methods: The study involved engineering single-phase chitosan scaffolds with various porous structures, including spherical and oblate pores. Computational simulations were performed to predict fluid dynamics and particle accumulation within different pore geometries. We observed geometrical differences between commercially available hemostats and our optimized chitosan hemostat using microCT and Field Emission Scanning Electron Microscopy (FESEM). After clotting, each hemostat was fixed and cryosectioned to record the final location of fibrin and platelet accumulations. The molecular mechanism of chitosan-induced platelet activation via TLR-2 was investigated using flow cytometry with activated platelet labeling and the incorporation of various inhibitors. Further, we tested the water and blood absorption capacities and measured the blood clotting index of each hemostatic material. We also developed ex vivo bleeding models with different bleeding rates (2.5 and 35-50 mL/min) to create Grade 1: Mild Bleeding and Grade 3: Severe Bleeding according to the Validated Intraoperative Bleeding Scale (VIBe SCALE). In vivo studies evaluated the hemostatic performance of the engineered chitosan pads in murine and swine liver injury models, swine carotid artery dissection, and a clinical study involving human radial artery bleeding under anticoagulant therapy.

Results, Conclusions, and Discussions: In computational simulations, we observed an increased flow rate and decreased platelet accumulation as pore size increased (Figure 1a). To validate our models, we prepared porous structured hemostats with varying pore sphericities. Notably, more spherical pores facilitated rapid blood absorption and concentrated platelets and fibrin microthrombi within localized regions, consistent with our predictions. Compared to commercially available hemostats, ours showed larger, more spherical structures that support efficient accumulation of platelets and fibrin for rapid clotting (Figure 1b).
To understand the interaction mechanism between chitosan and platelets at a molecular level, we validated whether chitosan can directly activate platelets through receptor binding. Using pharmacological inhibitors to block various platelet receptors, we found that chitosan activated platelets even when different receptors were inhibited, suggesting a distinct receptor's involvement. Blocking TLR1/2 with inhibitors abrogated chitosan-mediated PtSer exposure, indicating that chitosan stimulates platelets via TLR (Figure 1c-d).
The engineered chitosan hemostat effectively stopped bleeding from various vascular injuries, even under anticoagulant treatment, with significantly reduced blood loss and hemostasis time compared to other hemostats. Ex vivo bleeding models with different bleeding rates demonstrated the superior performance of the porous chitosan pad (Figure 1e). In vivo studies on murine and swine models, including liver injury and carotid artery dissection, confirmed its hemostatic efficacy (Figure 1f). A clinical study involving human radial artery bleeding further validated its effectiveness, achieving hemostasis within minutes across various patient conditions, including those on high doses of heparin (Figure 1h-i).
Overall, the optimized chitosan hemostat demonstrated superior performance, rapid hemostasis, and effective platelet activation, making it a valuable tool for managing severe bleeding in clinical settings. This hemostat addresses the need for effective hemorrhage control in trauma and surgical settings and advances the management of bleeding in anticoagulated patients. By merging advanced engineering design with molecular insights, our hemostat offers a practical solution for emergency care, potentially reducing mortality rates associated with severe bleeding and anticoagulation therapy.

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