Unwanted blood clots and excessive bleeding: balance is key

The physiological process of hemostasis protects the body by initiating clotting and stopping bleeding when vascular integrity is breached. Unwanted clot formation withinblood vessels results in a condition called thrombosis where the flow of blood is obstructed. This can be due to vascular injury, pre-existing conditions, or as a side effect of a therapeutic. Unwanted blood clot formation in both veins and arteries is the main cause for serious medical conditions such as pulmonary embolism, myocardial infarction and stroke. Together, these diseases are the leading causes of morbidity and mortality in the Western world.¹

Despite significant advances in antithrombotic and anticoagulant therapies, the problem persists. Current therapies are complicated by bleeding side effects as they interrupt key pathways involved in hemostasis. It is crucial to maintain a balance between thrombosis and haemostasis, particularly in the design of antithrombotic therapeutics.^2,3

Polyphosphates and the clotting cascade

One promising avenue in antithrombotic drug design is to target the contact pathway of coagulation,^4–6 since it is not required for hemostasis. Specifically, targeting recently identified biological negatively charged polymers (polyanions) that trigger the contact pathway of coagulation.^7–9 This pathway is also involved in the activation of inflammation and provides an important link between coagulation and immune-mediated reactions. This has been exemplified in the case of polyphosphate (polyP), a linear polymer of inorganic anionic phosphate.¹⁰ PolyP is a potent procoagulant and proinflammatory molecule^11–13 acting at multiple steps in the coagulation cascade.

In 2006, Morrissey and colleagues reported that polyphosphates play significant roles in modulating blood coagulation and fibrinolysis. As a potent accelerant of blood coagulation which is not involved in essential pathways of coagulation, polyP is a promising target for therapies to prevent thrombosis with minimal bleeding side effects.^11,14–17 The prevention of polyP’s procoagulant activity has been explored through the use of polycationic structures which bind polyP electrostatically and attenuate thrombosis in mouse models.^18,19 However, the concentrations required for such agents to provide thromboprotection were shown to be toxic,^20–22 precluding their clinical utility.

We present MPI: a potential therapeutic that can switch its charge upon binding to its target

With the objective of developing a selective and safe polyP inhibitor, we developed a drug design concept based on switchable protonation states, termed macromolecular polyanion inhibitors (MPIs). Beginning with design and synthesis utilizing a safe and biocompatible scaffold, we created a library of drug candidates in which the size of the macromolecules, the quantity of charges appended to the scaffold, the identity of the positively charged ligand, and the quantity of the ligands were systematically varied.

The structure of the binding ligands differs primarily in their ability, or likelihood, to carry additional charges when changes in their microenvironment provide a favorable energetic barrier to do so. This allowed us to develop positively charged drug candidates which have minimal charges at physiological pH, able to mitigate unwanted interactions with negatively charged blood proteins and cells. As the drug candidate approaches polyP in circulation, it acts as a charge switchable molecule and increases its positive charge, satisfying the requirements to electrostatically bind to the negatively charged polyP. This results in a highly stable complex and prevents unwanted blood coagulation initiation by polyP.

Optimization of size, charge, and ligands: screening of the MPI library and the importance of an interdisciplinary approach

Lead MPI candidates were thoroughly investigated through a series of biochemical, biophysical and biological assays to identify the optimal safe, selective and potent inhibitor to polyP. In highly involved titration experiments, each of the candidates were thoroughly characterized for the acidity (log K values) their conductivity (overall charge per macromolecule). Isothermal titration calorimetry experiments were also employed to evaluate the binding energies between polyP and lead candidates and used to show the recruitment of protons from the buffer upon binding to polyP. The biological activities of lead MPI candidates were initially assessed in vitro, and further evaluated for their antithrombotic activity in mice using various thrombosis models. MPI’s influence on bleeding and the dose tolerance in vivo were also investigated using mouse models.

 Our lead candidate, MPI 8, shows promise as an antithrombotic in mice   

MPI 8 was identified as the lead candidate, as it showed the most promise for antithrombotic activity while high concentrations did not interfere with human plasma and blood clotting as evaluated in biological assays. The antithrombotic activity of MPI 8 was evaluated in different models of thrombosis in mice. One experiment utilized a mouse model where the mice were administered fluorescent antibodies that tag fibrin and platelets (two key markers of a blood clot). The mice were separated into control groups (did not receive the drug, MPI 8) and experimental groups (received either a low dose or high dose of MPI 8). With a high-definition fluorescence microscope to visualize and record the cremaster arteriole, injury was applied to the arteriole via laser, and the accumulation of platelets and fibrin were visualized over time at the site of injury. This allowed us to demonstrate that when MPI 8 was administered, the time to produce a full blood clot was delayed compared to the group of mice that did not receive MPI 8.

In another thrombosis model, the flow of blood was monitored using a Doppler flow probe. MPI 8 demonstrated antithrombotic activity by delaying the time to occlusion (that is, the amount of time it took for the blood clot to form and close the site of injury). In both studies, MPI 8 demonstrated superior activity compared to controls. A third thrombosis model was employed, wherein the inferior vena cava (a large, thin-walled blood vessel) was constrained to induce blood clot formation. The blood flow was monitored, by ultrasound, and the final clots were assessed for their weight and density. Once again mice treated with MPI 8 produced clots that were significantly lighter in mass compared to untreated mice, showing the antithrombotic capability of MPI 8.

MPI 8 works as an antithrombotic. But is it safe?

In terms of investigating the safety of MPI 8, the focus was on the side effects of bleeding, the major setback of current antithrombotic medicines. One method used was the mouse tail bleeding model, commonly used to assess whether a drug compound would induce bleeding in mice. The advantage of a widely used model is the ability to compare to other drug candidates, including FDA approved drugs, such as unfractionated heparin (UFH). Mice were administered either a negative control (no drug), a dose of MPI 8, or a dose of UFH. After 10 minutes of circulation, the tails of mice were injured, the blood collected, and the bleeding time recorded. MPI 8 demonstrated no significantly prolonged bleeding time compared to the negative control. Mice administered with UFH demonstrated significantly longer bleeding times.

The acute and longer-term toxicity effects of MPI 8 in mice were investigated. Mice were administered very high doses of MPI 8, five times the efficacious concentration, or a negative saline control, and monitored for 15 days. No significant change in behaviour or body weight was observed, and upon assessment of the activity of biological markers of toxicity (LDH, ALT, AST), no significant changes were observed for mice administered with high doses of MPI 8. Stained images of organs such as the heart, lungs, liver, and kidneys all showed no signs of toxicity or tissue damage. The same assessments of enzymatic activity (LDH, AST, ALT) of mice administered high doses of MPI 8 were performed after 24 hours to confirm no acute toxicity was present.

Conclusion: MPI 8 is a viable antithrombotic therapeutic with unique advantages compared to current available medicine

 In conclusion, we present a tactical approach to design positively charged therapeutics which are safe in the blood stream. This platform could be tailored to other therapeutic applications or other negatively charged biological molecules. The significant advantages of our interdisciplinary approach are well evidenced by MPI 8’s ability to target polyP with high selectivity and deliver antithrombotic therapeutic effects, without the harmful side effects of bleeding that accompany current medicines.

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