Effect of depolymerized holothurian glycosaminoglycan (DHG) on the activation of factor VIII and factor V by thrombin

Our previous study has shown that depolymerized holothurian glycosaminoglycan (DHG) has two different inhibitory activities in the blood coagulation cascade: heparin cofactor II-dependent thrombin inhibition; and antithrombin III- and heparin cofactor II-independent inhibition of the intrinsic factor Xase complex [Nagase et al. (1995) Blood 85, 1527-1534]. In the present study, the effect of DHG on the activation of factor VIII and factor V by thrombin was examined with purified human components. DHG inhibited the activation of factor VIII by thrombin at concentrations exceeding 80 nM, but not the activation of factor V by thrombin at concentrations of up to 8 mu M. On Western blot analysis, DHG inhibited the cleavage of factor VIII light chain at concentrations exceeding 0.8 mu M. The interaction between DHG and factors VIII and V and thrombin was examined with a DHG-cellulofine column. DHG had strong affinity for factor V and thrombin, but slight affinity for factor VIII. The interaction of DHG with thrombin was analyzed, using fluorescein isothiocyanate-labeled DHG. One mole of DHG bound 2 mol of thrombin, with a dissociation constant (Kd) of 3.04 x 10(-6) M. These results suggest that DHG interferes with the interaction between thrombin and factor VIII, probably by making a binary complex through the anionic binding exosite II of thrombin.