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.
Nagase
H
H
Taiho Pharmaceutical Co., Ltd., Kawauchi-cho, Tokushima.
Enjyoji
K
K
Shima
M
M
Kitazato
K
K
Yoshioka
A
A
Saito
H
H
Kato
H
H
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D000990:Antithrombin III / Q000494:pharmacology; D001667:Binding, Competitive; D005165:Factor V / Q000187:drug effects / Q000378:metabolism*; D005169:Factor VIII / Q000187:drug effects / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism* / Q000494:pharmacology*; D015844:Heparin Cofactor II / Q000494:pharmacology; D006801:Humans; D012616:Sea Cucumbers / Q000737:chemistry; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*
D000925:Anticoagulants; D006025:Glycosaminoglycans; D015844:Heparin Cofactor II; D000990:Antithrombin III; D005165:Factor V; D005169:Factor VIII; D013917:Thrombin