Basic and clinical study on the antithrombotic mechanism of glycosaminoglycan extracted from sea cucumber.
OBJECTIVE
To investigate the antithrombotic mechanism of glycosaminoglycan (GAG) extracted from sea cucumber.


METHODS
We studied the effects of GAG on the coagulant pathway by measuring cloting time. The antithrombin mechanism of GAG was checked by assaying its effects on the thrombin activity in normal human pooled plasma, purified human heparin cofactor II system and antithrombin III system. The effects of GAG on the assembly, dispersion, and structure of fibrin gels as well as on the activity of plasmin were studied by means of turbidimetry, electron microscopy, and chromogenic substrate assay. We studied the effect of GAG on the expression and transcription of tissue factor (TF) and thrombomodulin (TM) in LPS (lipopolysaccharide)-stimulated human umbilical vein endothelial cells (HUVECs), and used heparin as a control. HUVECs were treated with different concentrations of GAG (1 microgram/ml, 5 micrograms/ml, and 10 micrograms/ml respectively) and 5 micrograms/ml heparin as a control together with LPS (1 microgram/ml). After incubation for 6 hours, TF and TM were investigated by ELISA and the mRNA study was carried out by RT-PCR. In a clinical trail, a series of variables were observed before and after treatment with GAG in patients recovering from cerebral ischemic stroke or suffering from ischemic heart disease.


RESULTS
The TT and APTT were significantly prolonged by GAG (0.1 microgram/ml). GAG inhibited thrombin activity in the presence of HCII with a second order rate constant of 1.14 x 10(7) m-1.min-1, which was 4.6 times higher than that of ATIII. GAG significantly inhibited the polymerization of fibrin monomer and enhanced the activity of plasmin in a concentration dependent manner. GAG could impair TF mRNA expression and up-regulate TM mRNA expression. The result of clinical trail showed that the fat metabolism was enhanced in addition to the anticoagulant and the blood viscosity reducing effects. No side-effect was found.


CONCLUSIONS
GAG mainly affected on the intrinsic pathway of blood coagulation. GAG was similar to dermatan sulfate both in the efficiency and in the mechanism of antithrombin. The acceleration of colt lysis by GAG depended on its ability to increase the activity of plasmin, to inhibit the polymerizing of fibrin monomer, and consequently, to alter the architecture of the fibrin net work. This effect on HUVECs appears to be at a transcriptional level and might be relevant for the antithrombotic action of GAG. GAG possess anticoagulant activity in vivo and it is a promising drug for antithrombotic therapy.
Li
Z
Z
Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Wang
H
H
Li
J
J
Zhang
G
G
Gao
C
C
D000818:Animals; D000991:Antithrombins / Q000494:pharmacology*; D002545:Brain Ischemia / Q000188:drug therapy; D006025:Glycosaminoglycans / Q000302:isolation & purification* / Q000494:pharmacology / Q000627:therapeutic use; D006801:Humans; D017202:Myocardial Ischemia / Q000188:drug therapy; D012616:Sea Cucumbers / Q000737:chemistry*
D000991:Antithrombins; D006025:Glycosaminoglycans