Hemostatic agents of broad applicability produced by selective tuning of factor Xa zymogenicity

Abstract

Key Points Modulation of FX(a) zymogenicity yields proteins with a broad range of half-lives and biologic function yet potent in vivo efficacy. Zymogen-like variants are attractive molecules for alleviating bleeding in different clinical scenarios such as hemophilia.

Rendering factor Xa zymogen-like as a therapeutic strategy to treat bleeding

“…In vitro and ex vivo studies of increasing complexity showed that FXa I16L , a prototypical zymogen-like FXa species, was highly effective in promoting thrombin generation in hemophilic plasma with or without inhibitors and in hemophilic blood [25, 26, 33]*. As expected from previous biochemical studies, these effects were FVa-dependent.…”

“…AT and TFPI) and, consequently a prolonged half-life in plasma. Interestingly, variants with lowest catalytic activity (the most “zymogen-like”) were the most resistant to inhibitors [24, 25]*. Importantly, since the zymogen-like state has plasticity and is highly dynamic, ligands that engage the enzyme can readily alter its conformational equilibrium.…”

“…Despite its zymogen-like character, FXa I16L has a relatively short half-life in mouse plasma (<10 min), limiting its use as a prophylactic agent [25, 26]. However, it may be ideal for acute bleeding, where the desired effect is to stop blood loss quickly without prolonged activation of coagulation.…”

“…FXa I16T ; >1.5 hr) were most effective when given prior to injury (a prophylaxis strategy), whereas short-to-intermediate half-life variants (e.g. FXa I16L; <10 min; Table 1) were best when given after injury (an on-demand strategy) [25]*. Together these studies underscore that the tunability of the FX/Xa zymogen-to-protease transition allows for the development of therapeutics with broad applicability, and prompted us to explore other clinical applications outside of hemophilia.…”

Assessing Blood Clotting and Coagulation Factors in Mice

“…In some variations of the method, the tail is cut at 1 to 5 mm from the tip (Maroney et al, 2012;Sambrano, Weiss, Zheng, Huang, & Coughlin, 2001;Tranholm et al, 2003), while in others the tail is amputated at 1 to 4 mm tail diameter (Holmberg, Lauritzen, Tranholm, & Ezban, 2009;Ivanciu & Camire, 2015). The measurable parameters are: bleeding time, measured as cessation of blood flow due to hemostatic plug formation, and total blood loss, measured as the hemoglobin content of blood.…”