A Zymogen-like Factor Xa Improves Hemostasis in a Murine Bleeding Model

In many clinical indications, effective control of bleeding is needed. Factor Xa (FXa) is a vitamin K-dependent trypsin-like serine protease that interacts with non-enzymatic coagulation factor Va (FVa) on negatively charged membrane surfaces to generate thrombin during hemostasis. Based on its central role in the coagulation cascade at the intersection of both intrinsic and extrinsic pathways, direct administration of FXa is an attractive approach to restoring hemostasis in bleeding disorders by leading to direct thrombin generation and fibrin formation. However, the short plasma half-life of the activated FXa protease renders it inadequate as a therapeutic for acute bleeding.

Here, we investigate FXa^I16L,a recently described variant of coagulation FXa engineered to overcome these limitations. The FXa^I16L variant has an isoleucine (I) to leucine (L) substitution at amino acid 16 (based on chymotrypsin numbering). FXa^I16L exhibits zymogen-like properties with both reduced activity and sensitivity toward plasma inhibitors. In the presence of its cofactor, FVa, FXa^I16L activity is restored. We assessed the hemostatic activity of FXa^I16L in an acute tail bleeding model that results in severe bleeding in normal mice. Ex vivo pharmacodynamic parameters in plasma and whole blood were also measured. FXa^I16L was administrated intravenously to normal male C57BL/6J mice at doses of 1, 10, 25, 50, 100, or 200 μg/kg. Control mice received vehicle only. Two minutes post administration, a 3 mm tail transection was made. Tails were immediately immersed in tubes containing pre-warmed phosphate buffered saline for blood collection over a ten minute period. Bleeding times were recorded and volume of blood loss was determined by measurement of the hemoglobin content in the collected blood. Following administration of FXa^I16L, a dose dependent reduction in bleeding was observed. Mice dosed with FXa^I16Lshowed a decrease in blood loss of 12% (1 μg/kg), 16.6% (10 μg/kg), 26.7% (25 μg/kg), 45.3% (50 μg/kg), 62.9% (100 μg/kg), and 69.6% (200 μg/kg) compared to vehicle-dosed mice. The estimated ED₅₀ was 46 μg/kg.

Following infusion of FXa^I16L (25 μg/kg) or vehicle into normal male CD-1 mice, we measured the ex vivo activity in plasma using an activated partial thromboplastin time (aPTT) clotting assay and a thrombin generation assay (TGA). Plasma collected from FXa^I16L-dosed animals at 2 minutes post-injection displayed a 67% reduction in aPTT compared to vehicle-dosed mice. Dosing of FXa^I16L at 25 μg/kg also enhanced thrombin generation, as reflected by a shortened lag phase, increased peak thrombin, increased endogenous thrombin potential and higher velocity index compared to vehicle treated mice. We also measured thromboelastography (TEG) parameters of whole blood collected from mice infused with FXa^I16L. At a 10 μg/kg intravenous dose of FXa^I16L, the TEG R-value and K-value measures of clotting time decreased, while TEG alpha angle and maximum amplitude increased compared to vehicle treated mice.

We conclude that administration of FXa^I16L in normal mice enhances hemostasis, decreasing bleeding in an injury model. Together, these studies suggest that FXa^I16L may provide a new and unique way to achieve hemostasis in clinical situations of uncontrolled bleeding.