Depolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicusSelenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor Vila by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor Vila inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitroactivity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.
References
1
Kitazato K,
Kitazato TK,
Nagase H,
Minamiguchi K.
DHG, a new depolymerized holothurian glycosaminoglycan, exerts an antithrombotic effect with less bleeding than unfractionated or low molecular weight heparin in rats. Thromb Res 1996; 84: 111-120
2
Suzuki N,
Kitazato K,
Takamatsu J,
Saito H.
Antithrombotic and anticoagulant activity of depolymerized fragment of the glycosaminoglycan extracted from Stichopus japonicusSelenka. Thromb Haemost 1991; 65: 369-373
3
Nagase H,
Enjyoji K,
Minamiguchi K,
Kitazato TK,
Kitazato K,
Saito H,
Kato H.
Depolymerized holothurian glycosaminoglycan (DHG) is an anticoagulant with two different inhibitory activities: One, heparin cofactor II-dependent inhibition and the other, antithrombin III-and heparin cofactor II-independent inhibition of factor X activation by factor IXa-factor Villa complex. Blood 1995; 85: 1527-1534
4
Nagase H,
Enjyoji K,
Shima M,
Kitazato K,
Yoshioka A,
Saito H,
Kato H.
Effect of depolymerized holothurian glycosaminoglycan (DHG) on the activation of factor VIII and factor V by thrombin. J Biochem 1996; 119: 63-69
8
Ofosu FA,
Sié P,
Modi GJ,
Fernandes F,
Buchanan MR,
Blajchman MA,
Boneu B,
Hirsh J.
The inhibition of thrombin-dependent positive feedback reaction is critical to the expression of the anticoagulant effect of heparin. Biochem J 1987; 243: 579-588
9
Minamiguchi K,
Nagase H,
Kitazato KT,
Kitazato K.
Interaction of DHG, a new depolymerized holothurian glycosaminoglycan, with proteins in human plasma. Thromb Res 1996; 83: 253-264
11
Broze GJ,
Novotny WF,
Higuchi DA,
Girard TJ,
Miletich JP.
The lipoprotein-associated coagulation inhibitor that inhibits the factor Vlla-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action. Blood 1988; 71: 335-343
14
Bregengaard C,
Nordfang O,
Ostergaad P,
Petersen JGL,
Meyn G,
Diness V,
Svendsen O,
Hedner U.
Pharmacokinetics of full length and two-domain tissue factor pathway inhibitor in combination with heparin in rabbits. Thromb Haemost 1993; 70: 454-457
16
Jesty J,
Wun TC,
Lorenz A.
Kinetics of the inhibition of factor Xa and the tissue factor-factor Vila complex by the tissue factor pathway inhibitor in the presence and absence of heparin. Biochemistry 1994; 33: 12686-12694
17
Enjyoji K,
Miyata T,
Kamikubo Y,
Kato H.
Effect of heparin on the inhibition of factor Xa by tissue factor pathway inhibitor: A segment, Gly212-Phe243, of the third Kunitz domain is a heparin binding site. Biochemistry 1995; 34: 5725-5735
18
Yoshida K,
Minami Y,
Nemoto H,
Numata K,
Yamanaka E.
Structure of DHG, a depolymerized glycosaminoglycan from sea cucumber, Stichopus japonicus. Tetrahedron Lett 1992; 33: 4959-4962
21
Lindahl AK,
Jacobsen PB,
Sandset PM,
Abildgaard U.
Tissue factor pathway inhibitor with high anticoagulant activity is increased in post-heparin plasma and in plasma from cancer patients. Blood Coagulation Fibrinol 1991; 2: 713-721
22
Kokawa T,
Enjyoji K,
Kumeda K,
Kamikubo Y,
Harada-Shiba M,
Koh H,
Tsushima M,
Yamamoto A,
Kato H.
Measurement of the free form of TFPI antigen in hyperlipidemia. Arterio Thromb Vase Biol 1996; 16: 802-808