Alexandra Cranston, Abigail Hall, Anushri Umesh, Nathan J. Alves

Exploring the Impact of Plasmin Inhibitors on Clotting Characteristics as a Novel Therapeutic for Thromboembolic Events

Introduction

Exploring the impact of plasmin inhibitors on clotting characteristics as a novel therapeutic for thromboembolic events. Explore novel plasmin inhibitor therapeutics for thromboembolic events. Our research optimizes safer clot-digesting strategies using plasmin and pentamidine, minimizing bleeding risks.

Abstract

Background:Thromboembolisms are common, life-threatening emergencies. Systemic hemorrhaging post-thrombolytic treatment is an adverse reaction, highlighting the need for safer clot-digesting therapeutics. Our research focuses on an endogenous fibrinolytic enzyme, plasmin. We investigated how co-delivery of plasmin and pentamidine, a reversible bivalent plasmin inhibitor, impacts clotting and thrombolysis. Plasmin and pentamidine delivery were tested across ex-vivo stagnant viscoelastic assays (thromboelastography, TEG) and shear-based clot formation and digestion assays (Chandler Loop). This research aims to optimize safer clot-digesting therapeutics that minimize adverse bleeding. Methods:Consenting healthy volunteers (n=13) donated whole blood into citrated tubes using an approved IRB protocol. Whole blood was analyzed via Chandler Loop (20 RPM at 37C for 60min) and TEG (37C for 90min). Pentamidine (0-800μM) and plasmin (0-0.1mg/mL) were tested independently and mixed measuring: clot mass, clot strength (MA), R-time, angle, and K. Results:At increasing pentamidine concentrations, R-time and K increased while MA, angle, clot weight, and clot length decreased. At increasing plasmin concentrations, R-time, K, and angle remained unchanged while MA, clot weight, and clot length decreased. Concentrations >700μM pentamidine and >0.1mg/mL plasmin inhibited clotting. In the presence of fixed pentamidine (50μM in TEG, 200μM in Chandler), the initial impact to clot formation resembled 50μM of pentamidine alone followed by limited impact at increasing plasmin concentrations with a flat decrease in clot MA (18.3%), clot length (19.5%), and clot weight (18.9%) and increase in R-time (43.4%) and K (50.6%). Conclusion/Impact:Increasing plasmin concentrations results in increased clot digestion while increasing pentamidine concentrations functions more akin to an anticoagulant preventing clot formation. Co-administered plasmin with pentamidine demonstrates how an inhibitor can be used to deliver an active clot digesting enzyme. Leveraging the results presented herein, and the principles of multivalency, plasmin inhibitory molecules can be developed to create safer and more effective direct fibrinolytics for clinical use. FUNDING:This research is supported by the NIH NHLBI Award #R01HL167877 and T35 HL110854.

Review

This study introduces a compelling and potentially safer approach to treating thromboembolic events by investigating the co-delivery of plasmin and its reversible inhibitor, pentamidine. The background clearly articulates the critical need for novel thrombolytic agents that mitigate the risk of systemic hemorrhaging, a significant adverse effect of current treatments. By focusing on plasmin, an endogenous fibrinolytic enzyme, and exploring the controlled interaction with a bivalent inhibitor, the research proposes an innovative strategy to achieve targeted clot digestion while minimizing unintended systemic effects, thus addressing a major clinical challenge in thrombosis management. The methodology employed ex-vivo stagnant viscoelastic assays (thromboelastography, TEG) and shear-based clot formation/digestion assays (Chandler Loop) using whole blood from healthy volunteers. The independent testing revealed that increasing pentamidine concentrations functioned akin to an anticoagulant, significantly increasing clot formation time (R-time, K) and decreasing clot strength (MA, angle), weight, and length. In contrast, increasing plasmin concentrations primarily led to clot digestion, evidenced by decreased clot strength, weight, and length, with minimal impact on initial clot formation parameters. Critically, the co-administration experiments, using fixed pentamidine concentrations, showed an initial anticoagulant-like effect similar to pentamidine alone, followed by a limited, flat decrease in clot parameters (MA, length, weight) and an increase in R-time and K, demonstrating a controlled modulatory effect on clotting and lysis. The conclusions drawn from this research are significant, highlighting the distinct mechanisms of action for pentamidine as an anticoagulant and plasmin as a clot digester. The most impactful finding is the demonstration that an inhibitor like pentamidine can be effectively leveraged to deliver and potentially modulate the activity of an active clot-digesting enzyme like plasmin. This proof-of-concept suggests a promising pathway for developing more precise and safer direct fibrinolytics. The authors propose that by applying these principles and incorporating multivalency, future plasmin inhibitory molecules can be designed to create highly optimized therapeutic agents for clinical use, marking a substantial step forward in the quest for safer and more effective treatments for thromboembolic disorders.

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