Increased anticoagulant activity of recombinant thrombomodulin modified with glycosaminoglycan.
Thrombomodulin (TM) is a thrombin receptor on the endothelial cell surface, effective as an anticoagulant by changing procoagulant thrombin to an anticoagulant one. As rabbit TM with glycosaminoglycan (GAG) has a more potent anticoagulant activity than that without GAG, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C-127 cells. The effect of an additional GAG chain on anticoagulant activity was investigated in comparison with unmodified recombinant UTM (r-UTM). In vitro, the activity of cleavage of fibrinogen by thrombin or prothrombinase activity was more potently depressed by GAG-UTM than by r-UTM, and the generation of activated protein C by TM-thrombin complex was accelerated by GAG modification. The acceleration of antithrombin III-dependent anticoagulant activity was shown only by GAG-UTM. Parameters like thrombin time, prothrombin time and activated partial thromboplastin time in human plasma were prolonged by GAG-UTM more than by r-UTM. In vivo, the effect of GAG-UTM and r-UTM in endotoxin-induced disseminated intravascular coagulation (DIC) rats was investigated using hematological parameters. GAG-UTM and r-UTM significantly reduced the decrease in fibrinogen and platelet number induced by endotoxin at the dosage of 0.1 and 1.0 mg/kg/h, respectively, suggesting that the antithrombotic effect of GAG-UTM in endotoxin-induced DIC rats was 10-fold as potent as that of r-UTM. GAG-UTM reduced the prolongation of the bleeding time induced by endotoxin, while r-UTM accelerated it. These results suggest that the addition of a GAG chain may increase availability as an anticoagulant.
Edano
T
T
Tokyo Research Laboratories, Kowa Co., Ltd., Higashimurayama, Japan.
Inoue
K
K
Yoshizaki
H
H
Yamamoto
S
S
Komine
N
N
Tabunoki
H
H
Sawada
H
H
Koshi
T
T
Murakami
A
A
Wada
Y
Y
Ohkuchi
M
M
D000805:Angiotensin III / Q000502:physiology; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry* / Q000494:pharmacology*; D001772:Blood Cell Count / Q000187:drug effects; D001777:Blood Coagulation / Q000187:drug effects; D004247:DNA / Q000096:biosynthesis; D004211:Disseminated Intravascular Coagulation / Q000139:chemically induced; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D008297:Male; D016133:Polymerase Chain Reaction; D011486:Protein C / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D018180:Thrombomodulin / Q000737:chemistry*; D013925:Thromboplastin / Q000037:antagonists & inhibitors
D000925:Anticoagulants; D004791:Enzyme Inhibitors; D006025:Glycosaminoglycans; D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D000805:Angiotensin III; D004247:DNA; D013925:Thromboplastin