Exploring the Function of Polyphosphate in the Contact Pathway of Blood Clotting and Developing Polyphosphate Probes for Enhanced Specificity

Abstract

The classic coagulation cascade has been well-studied for many years. In the traditional “waterfall” model, both the contact pathway and the tissue factor pathway can trigger blood clotting. The contact pathway is composed of two serine protease zymogens, factor XII and prekallikrein, together with a non-enzymatic co-factor, high-molecular-weight kininogen. Recent discoveries have implicated the contact pathway in thrombosis and inflammation, while it is clearly dispensable for hemostasis. Due to the complexity of the enzymatic reactions and molecular interactions in the contact pathway, the (patho)-physiological mechanisms of contact pathway activation remained unclear. Previous studies suggested several molecules that may modulate the contact pathway, including inorganic polyphosphate. Studying how polyphosphate modulates the contact pathway of coagulation holds promise for developing novel drug targets for treating thrombotic diseases without increasing the risk of bleeding. Despite being found in all kingdoms of life, polyphosphate polymer lengths are highly variable, with shorter polymers (approximately 60-100 phosphates) secreted from human platelets, and longer polymers (up to thousands of phosphates) in microbes. To reveal the molecular mechanisms of how polyphosphate regulates individual reactions in the contact pathway, we conducted in vitro measurements of enzyme kinetics to investigate the ability of varying polyphosphate sizes, together with high-molecular-weight kininogen and Zn2+, to mediate individual proteolytic reactions in the contact pathway and activation of the intrinsic pathway by coagulation factor XII. The results suggested that polyphosphate of different sizes, together with Zn2+ could activate subsets of the contact pathway reactions. The study also raised questions on whether the primary physiological role of coagulation factor XII is to trigger blood clotting. In addition, a protein engineering study has been conducted to develop a reliable polyphosphate probe with enhanced affinity and specificity. Currently, there is a lack of sensitive and specific method for polyphosphate visualization in mammalian cells and tissues. A recombinant protein from E. coli was used and random mutagenesis libraries for displaying on phage surfaces were generated for polyphosphate binding selection assays. Next-generation sequencing was used to detect mutant candidates with desired polyphosphate binding features. These candidates may be used as a functional polyphosphate inhibitor in vivo, to prevent its ability of accelerating unwanted coagulation (thrombosis). To interrogate the role of factor XII-mediated contact pathway in vivo, a new transgenic mouse model was generated using CRISPR/Cas knock-in technology, where a precise point mutation was introduced into coagulation factor XII, leading to the loss of enzymatic functions of factor XIIa. Different from currently available factor XII knock-out mice, these new transgenic mice possess intact zymogen factor XII, which should not interfere with neutrophil functions and hence should be a less biased model for coagulation or inflammation studies in the future.