Smart thrombosis inhibitors without bleeding side effects via charge tunable ligand design

Venous and arterial thromboembolic disorders (pulmonary embolism, myocardial infarction, stroke) are major causes of morbidity and mortality. While antithrombotic and anticoagulant therapies have advanced, bleeding side effects remain a significant problem due to disruption of key hemostasis processes. A balance between thrombosis and hemostasis is crucial in antithrombotic drug design. The contact pathway of coagulation, not essential for hemostasis, presents a promising target. Polyphosphate (polyP), a linear polymer of inorganic anionic phosphate, is a potent procoagulant and proinflammatory molecule involved in the contact pathway. PolyP triggers clotting, accelerates factor V activation, enhances fibrin clot structure, and accelerates factor XI back-activation by thrombin, depending on its chain length. Long-chain polyP from bacteria is a potent contact pathway activator, while platelet-released short-chain polyP forms nanoparticles on cell surfaces, activating contact pathways and inhibiting fibrinolysis. Patients with polyP production defects show protection against thrombosis. As a potent coagulation accelerant not involved in essential clotting pathways, polyP is a promising target for thrombosis prevention with minimal bleeding side effects. Previous approaches, enzyme-mediated cleavage and use of cationic structures (PEI, PAMAM), either showed slow hydrolysis, removal of phosphates from other molecules, or toxicity. Previous work on universal heparin reversal agents (UHRAs) as polyP inhibitors showed some bleeding effects at therapeutic doses. This research explores a novel inhibitor design concept based on switchable protonation states for selective polyP inhibition, focusing on macromolecular polyanion inhibitors (MPIs).

The literature extensively documents the challenges associated with current antithrombotic therapies, highlighting the persistent risk of bleeding as a major limitation. The role of the contact pathway and polyphosphate (polyP) in thrombosis has been increasingly recognized as a key area of research. Studies have shown polyP's involvement in various stages of coagulation and its contribution to thrombotic events. Different approaches to inhibiting polyP's procoagulant activity have been explored including enzyme-mediated cleavage (e.g., using alkaline phosphatase or *Escherichia coli* exopolyphosphatase), and the use of cationic compounds (PEI, PAMAM). However, these methods have limitations, including slow reaction rates, off-target effects, and toxicity. Therefore, the need for a novel approach with enhanced selectivity and biocompatibility was identified. This research draws upon previous work on universal heparin reversal agents (UHRAs), while aiming to improve upon their limitations and address the issue of bleeding side effects. The authors cite relevant literature to support the development of their proposed macromolecular polyanion inhibitor (MPI) design concept.

The study employed a rational design approach to develop MPIs. A library of MPIs was generated using a biocompatible hyperbranched polyglycerol (HPG)-polyethylene glycol (PEG) copolymer scaffold, functionalized with different cationic binding groups (CBGs) at varying densities. The CBGs were designed to exhibit switchable protonation behavior, with low cationic charge at physiological pH but increased charge upon polyP binding. The selection of CBGs was based on their ability to efficiently bind polyP while maintaining biocompatibility. Two CBGs (CBG I and CBG II) were selected based on their pKa values and the effects of amine spacing on charge switching. The properties and performance of these CBGs were evaluated at different densities and on different HPG-PEG scaffold sizes (10 and 20 kDa). Potentiometric and conductometric titrations were performed to determine the average protonation strength of each MPI and the number of CBGs per molecule. The binding affinities of MPIs to short-chain (SC) and long-chain (LC) polyP were determined using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). ITC experiments also probed the change in CBG protonation states during polyP binding. The biological activities of the MPIs were evaluated using plasma clotting and thrombin generation assays in human plasma with LC and SC polyP. The hemocompatibility of MPIs was assessed by measuring clotting time and thrombin generation in tissue factor (TF)-initiated plasma clotting in FXII-deficient plasma. Three lead MPI candidates were selected based on their in vitro activity and hemocompatibility profiles for further in vivo studies. The in vivo antithrombotic activities of the lead candidates were evaluated using three mouse models: a laser-induced cremaster arteriole thrombosis model (intravital microscopy), a FeCl3-induced carotid artery injury model, and an inferior vena cava (IVC) stenosis ligation model. The safety of the lead MPI candidates was investigated in mouse models assessing bleeding time (tail bleeding, saphenous vein hemostasis) and acute/chronic toxicity. Various techniques were used for characterization, including NMR, GPC, SPR, ITC, ROTEM, SEM, and standard biochemical and hematological assays. Statistical analyses were performed using appropriate methods, and p-values < 0.05 were considered statistically significant.

A library of MPIs with varying CBG structures, densities, and scaffold sizes was synthesized and characterized. Potentiometric titrations and ITC confirmed the switchable protonation behavior of the MPIs, with significantly increased positive charge upon polyP binding. SPR and ITC analyses revealed that MPIs bound polyP with high affinity (sub-micromolar Kd) despite having lower charge density at physiological pH compared to UHRA controls. In vitro studies demonstrated that MPIs effectively inhibited the procoagulant activity of both LC and SC polyP in human plasma, as evidenced by normalized plasma clotting times and thrombin generation parameters (lag time, peak thrombin, endogenous thrombin potential). The hemocompatibility assays showed that MPIs did not cause significant changes to clotting times or thrombin generation in TF-initiated clotting systems, in contrast to UHRA controls which exhibited off-target effects. Three lead MPIs (MPI 1, MPI 6, and MPI 8) were selected for further in vivo studies. In vivo studies using mouse thrombosis models showed that lead MPIs effectively reduced thrombosis without increasing bleeding risk. MPI 8 showed superior antithrombotic activity compared to UHRA-10 in the carotid artery thrombosis model and the IVC stenosis model, significantly reducing thrombus formation. In the mouse tail bleeding and saphenous vein hemostasis models, MPI 8 did not prolong bleeding time or cause excess blood loss, even at high doses (300 mg/kg). Acute and chronic toxicity studies in mice showed that MPI 8 was well-tolerated, even at a very high dose (500 mg/kg). Histological analysis revealed no organ damage. The data suggest that MPI 8 inhibits polyP selectively without interfering with other coagulation factors or small phosphate-containing molecules such as ADP.

The results demonstrate the successful development of a novel class of polyP inhibitors (MPIs) that effectively prevent thrombosis without the increased bleeding risk associated with current therapies. The key to their efficacy lies in the design concept of switchable protonation, which allows for high selectivity and biocompatibility. Unlike previous polycationic approaches which suffer from toxicity, the MPIs' low charge density at physiological pH, coupled with increased charge upon polyP binding, limits off-target interactions and enhances specificity. The in vitro and in vivo data strongly support the hypothesis that this charge-switching mechanism enables targeted polyP inhibition with minimal interference with hemostasis. The lead candidate, MPI 8, exhibits superior antithrombotic efficacy and safety profile compared to previous generation polyP inhibitors and has a substantially larger therapeutic window. The modular design platform employed allows for flexibility in optimizing MPI properties, paving the way for further development of even more effective and safer antithrombotic agents.

This study successfully demonstrates the concept of charge-tunable ligand design for the creation of selective and biocompatible polyP inhibitors. The lead compound, MPI 8, exhibits potent antithrombotic activity in multiple mouse models without inducing bleeding or toxicity, addressing a significant unmet clinical need. The modular nature of the MPI design platform allows for further optimization of structure and activity, and the findings offer a promising new therapeutic avenue for thrombosis prevention.

The study was conducted primarily in mouse models, and further investigation is needed to confirm the efficacy and safety of MPIs in humans. While the in vitro studies demonstrated selectivity, the potential for off-target effects in a complex biological system like the human body remains a possibility. The long-term effects of MPI treatment also require further investigation. More detailed mechanistic studies of the interactions between MPIs and polyP and their influence on coagulation pathways would also enhance the understanding of the therapeutic action.