Recombinant human soluble thrombomodulin delivers bounded thrombin to antithrombin III: thrombomodulin associates with free thrombin and is recycled to activate protein c.
Recombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (Kd = 2.6 nM) or PC activation (Kd = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (Kd < 0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.
Aritomi
M
M
Institute for Life Science Research, Asahi Chemical Industry, Fuji City, Japan.
Watanabe
N
N
Ohishi
R
R
Gomi
K
K
Kiyota
T
T
Yamamoto
S
S
Ishida
T
T
Maruyama
I
I
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D011486:Protein C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism*
D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D000990:Antithrombin III; D013917:Thrombin