AV513 Promotes Tissue Factor-Mediated Coagulation in Fresh Human Whole Blood from Severe and Moderate Hemophilia a Subjects.

Abstract

The sulfated polysaccharide extract AV513 accelerates effective clotting of plasma from severe hemophilia A and B subjects, primarily through the extrinsic pathway by inhibiting TFPI activity. AV513 is efficacious in normalizing bleeding times in hemophilia A dogs when administered orally or subcutaneously, either alone or in the presence of very low levels of factor VIII. In this report, we have examined the effects on hemostasis of different concentrations of AV513 (0.25 to 5 ug/mL) added to fresh whole blood from eight severe (FVIII <1 %) and four moderate (FVIII 3–5 %) hemophilia A subjects. Blood was drawn on two separate occasions, 30 days apart, from each subject after receiving no FVIII infusion for 5 days and analyzed using tissue factor-triggered whole blood thromboelastography (TEG) and plasma based thrombin generation assay. In the tissue factor-induced TEG assay, AV513 reduced the whole blood R time (time to initial clot formation) of severe and moderate human hemophilia A blood in a dose dependent manner. The R time values were corrected to the normal range (≤ 27 min) at AV513 concentrations of 0.25 to 1 ug/mL in blood from both severe and moderate hemophilia A subjects. The TEG EC50 (AV513 concentration that reduces R time by 50% from baseline) values were approximately 0.7 ug/mL in both cohorts. Based on two TEG determinations for each subject, the intra-subject baseline variability of whole blood R time had a range of 3–19 % (mean: 10 %) for severe and 2–16 % (mean: 8 %) for moderate subjects. The intersubject baseline R time value varied from 29 to 91 min in the severe group and from 30 to 43 min in the moderate hemophilia A group. Further, an AV513 dose dependent increase in thrombin generation was observed in hemophilia A plasma with maximal thrombin peaks of 52 to 216 nM (mean: 110 nM). For all subjects, we observed a 2–4 fold maximum increase in thrombin peak and a 2–3 fold reduction of both the lag phase time and time to peak thrombin generation.. The EC50 values for peak thrombin generation were 0.2 ± 0.15 ug/mL and 0.7 ± 0.3 ug/mL of AV513 in severe and moderate hemophilia A plasma, respectively. These results demonstrate that AV513 in whole blood from severe and moderate hemophilia A subjects effectively shortened R time in the TEG assay to within the normal range, and that AV513 markedly enhanced thrombin generation in plasma from these subjects. These studies suggest that AV513 may prove useful to promote clotting and prevent spontaneous bleeding in clinical trials for patients with hemophilia A.