AV513 Enhances Tissue Factor Activity by Inhibiting TFPI in Hemophilia A Plasma: Potential TFPI Target Sites and Potentiation of Factor VIII Activity.

AV513, a sulfated polysaccharide prepared from brown seaweed and enriched for fucoidan accelerates clotting in hemophilia A and B plasma. Prior studies have demonstrated that AV513 functions mainly by inhibiting TFPI activity. In this report, we have further studied AV513 mechanism of action using human plasma thromboelastography (TEG) model and a thrombin generation assay in FVIII deficient plasma. In the TEG assay, AV513 enhanced tissue factor (TF) induced clotting in human hemophilia A pooled plasma in a dose-dependent manner. A dose-dependent potentiation effect by AV513 was observed with reduction of TEG R time to normal levels at 0.5 μg/mL of AV513. An AV513 dose dependent increase in thrombin generation was observed in hemophilia A pooled plasma with a 3-fold increase in peak thrombin generation at 2.5 μg/mL AV513 (basal: 40 nM vs. treated: 140 nM). When TFPI was immunodepleted from hemophilia A plasma (FVIII/TFPI deficient), sub-optimal concentrations of TF enhanced thrombin generation which was not further enhanced by addition of AV513. The increase in thrombin generation in FVIII /TFPI deficient plasma was inhibited by 93% after the addition of exogenous full-length TFPI or by 47% after the addition of truncated rTFPI (amino acids 1–161) that lacks the Kunitz 3 domain and the C-terminal tail. AV513 at low concentrations reversed exogenous full length TFPI activity almost completely in FVIII/TFPI-deficient plasma but had only a small effect on truncated TFPI. In a human plasma TEG combination study of AV513 and low concentrations of rFVIII (ReFacto), AV513 at a concentration of 0.25 μg/mL reduced the TEG R time by 35% in severe hemophilia A plasma compared to 10 mU/mL of rFVIII alone. These results indicate that AV513 functions as a pro-coagulant by inhibiting TFPI at least in part via targeting the C-terminal region. Moreover, AV513, in the presence of low rFVIII (ReFacto) levels, improves severe hemophilia A plasma clotting - as measured by TEG - beyond that observed with either agent alone.