Nagase H, Enjyoji K, Shima M, Kitazato K, Yoshioka A, Saito H, Kato H
Taiho Pharmaceutical Co., Ltd., Kawauchi-cho, Tokushima.
J Biochem. 1996 Jan;119(1):63-9. doi: 10.1093/oxfordjournals.jbchem.a021217.
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.
我们之前的研究表明，海参糖胺聚糖解聚产物（DHG）在血液凝固级联反应中具有两种不同的抑制活性：依赖肝素辅因子II的凝血酶抑制作用；以及对内在因子X酶复合物的抗凝血酶III和肝素辅因子II非依赖性抑制作用[长濑等人（1995年），《血液》85卷，第1527 - 1534页]。在本研究中，使用纯化的人源成分检测了DHG对凝血酶激活因子VIII和因子V的影响。DHG在浓度超过80 nM时抑制凝血酶对因子VIII的激活，但在浓度高达8 μM时不抑制凝血酶对因子V的激活。在蛋白质印迹分析中，DHG在浓度超过0.8 μM时抑制因子VIII轻链的裂解。使用DHG - 纤维素柱检测了DHG与因子VIII、V和凝血酶之间的相互作用。DHG对因子V和凝血酶具有强亲和力，但对因子VIII的亲和力较弱。使用异硫氰酸荧光素标记的DHG分析了DHG与凝血酶的相互作用。1摩尔DHG结合2摩尔凝血酶，解离常数（Kd）为3.04×10⁻⁶ M。这些结果表明，DHG可能通过凝血酶的阴离子结合外位点II形成二元复合物，从而干扰凝血酶与因子VIII之间的相互作用。
