Study On The Antiadhesive Effect Of Holothurian Glycosaminoglycan On Platelet-endothelial Cell Adhesion And Involved Mechanisms

ObjectivesThe aim of this study was to assess the influence of holothurian glycosaminoglycan (GAG) on the adherence of thrombin-induced platelets to human umbilical vein endothelial cells (HUVECs) in a flow chamber system. Adhesive molecules expressed on platelets and prothrombotic factors produced and secreted by HUVECs were used as the targets of this research to explore the possible mechanisms involved in the antiadhesive effect of GAG .Methods1 Culture of the primary human umbilical vein endothelial cells (HUVECs) was done. Cell identification was verified by morphological , immunological and histochemical criteria.2 MTT assay was applied to test the effect of GAG on cell viability.3 Washed platelets and HUVECs were pre-induced by thrombin with or without particular concentrations of GAG Fluorescence-labelled platelet suspensions which were pushed by a syringe pump in a flow chamber system were applied to simulate flowing circumstance in vivo and cultured HUVECs in vitro were used to simulate monolayer of vessel wall. The effect of various concentrations of GAG on the adhesion of platelets to HUVECs under different fluid shear rate conditions was explored in this model. The process of platelets-endothelial cells adhesion was observed and recorded by fluorescence microscope-TV system and the photographs of adhesion were obtained through Laser Scan Confocal Microscope (LSCM).4 Acute thromboembolism in mice was induced by the injection of thrombin. The effect of GAG on the mortality rates of mice was observed. The expression of GPⅡ_b/ Ⅲ_a complex on platelets in mice pre-administered with or without GAG was detected by flow cytometry. We also activated human platelets with thrombin in vitro and investigated the effect of GAG on the expression of GP Ⅰ_b on thrombin-induced platelets by flow cytometry.5 Several particular concentrations of GAG were co-cultured with HUVECs whichwere pre-induced by thrombin. vWF antigen and PAI-1 antigen which were produced and secreted into the supernatants by activated HUVECs were tested by ELISA. HUVECs were then treated with TritonX-100 to obtain cell extract after the collection of the supernatant. vWF antigen remaining in cells was also measured by ELISA. The effect of GAG on vWF mRNA and PAI-1 mRNA expression in activated endothelial cells was tested by real-time quantitative RT-PCR technique (RQ-PCR).Results1 Primary culture and confirmation of HUVECs HUVECs were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein and cultured in M199-20% defined fetal calf serum-2mM glutamine. They might become confluent monolayer 7 to 10 days later. The cultured cells were verified to be HUVECs by typically large, closely opposed, polygonal appearance in inverted microscope, the ability to produce and secret vWF and positive immunofluorescent staining by antibody to factorⅧ antigen. Cells were harvested with 0.25% trypsin-0.02% EDTA and the firstly subcultured cells were used in all assays.2 The effect of GAG on cell viability Ranging from 1mg/L to 5mg/L, GAG had no evident effect on cell viability. But the viabilities of endothelial cells decreased significantly with increased doses of GAG, following patches of loss of endothelial cells examined by invert microscopy when GAG was at the concentration of 40mg/L. This led to our determination that the doses of GAG in the following studies were below 10mg/L.3 The effect of GAG on platelet-endothelial cell adhesion We produced flow shear rates of 1100s^-1 and 300s^-1 by altering flow rates of platelet suspensions in a flow chamber system. After both pre-induced with thrombin, the adhesion rates of platelets to HUVECs under two flow shear rates were much higher than those without thrombin. GAG (1 mg/L ～ 10mg/L ) inhibited platelet-endothelial adhesion under both high and low fluid shear rates. When GAG was at the concentration of 5mg/L, the inhibitory activity was most significant and the adhesion rates were only about 35% and 40% of those in thrombin stimulating group, respectively (P<0.001, P<0.01) . These results indicated that GAG had an antiadhesive effect on the adhesion of activated platelets to endothelial cells, especially under high flow shear rate. Heparin also had a supprssive influence on platelet-endothelial cell adhesion, butbeing weaker than that of the same dose of GAG.4 The effect of GAG on the expression of platelet adhesion molecules Administration of GAG or heparin could decrease the mortality rates of mice in acute thromboembolism model. Ten minutes afer the injection of thrombin, the expression of platelets GP Ⅱ_b/Ⅲ_a complex in mice was much lower in GAG group and heparin group than in control group (P<0.001, P<0.001 ) . The inhibitory activity on the expression of GP Ⅱ_b/Ⅲ_a complex was slightly evident in GAG group, but no significant differences were found between heparin group and GAG group (P= 0.06). Forty minutes later, an increasing tendency was seen on the expression of platelets GP Ⅱ_b/Ⅲ_a complex in control and GAG group but a decresing one in heparin group. About threefold and fourfold of increases in the expression of GPⅠ_b on thrombin-induced platelets could be tested if the platelets were co-cultured with GAG or heparin. Heparin up-regulated the expression of GP Ⅰ_b more markedly, but the differences were statistically significant both in heparin and GAG group compared with that in control group (P<0.001, P<0.001 ) .5 The effect of GAG on the expression of vWF and PAI-1 vWF antigen in the supernatant increased significantly if the cells were stimulated with thrombin. GAG could down-regulate the release of vWF antigen from thrombin induced HUVECs. Ranging from 1mg/L to 5mg/L, the inhibitive activity of GAG rose gradually, reaching its highest level at the dose of 5mg/L, and then showed a weakening tendency with following increasd doses. Although the contents of vWF antigen inside endothelial cells which were co-cultured with GAG were higher than those inside the cells being stimulated by thrombin alone, the total contents of vWF antigen (in supernatant plus intra-cellular) were still the lowest in GAG 5mg/L group. Similar to the effect on vWF, GAG also restrained the expression of PAI-1 antigen in activated HUVECs, most significantly when GAG was at the concentration of 5mg/L. Compared with heparin, the same dose of GAG had higher depressive activity on the expression of both antigens. GAG could reduce vWF mRNA expression in thrombin induced HUVECs. Compared with thrombin group, the differences had statistical significance when GAG was at the doses of 4mg/L and 5mg/L (P<0.05, P<0.01 ) . GAG could also statistically diminish PAI-1 mRNA expression at the concentration of 5mg/L (P<0.05) . Heparin at 5mg/L suppressed the mRNA expression of both antigens as well, but the differences were not statistically significant on PAI-1 mRNA expression.ConclusionsGAG can lower the mortality rates of mice with acute thromboembolism and inhibit the adhesion of thrombin-induced platelets to endothelial cells through down-regulating the release of vWF antigen and PAI-1 antigen and their mRNA expression in stimulated HUVECs and modulating the expression of adhesive molecules GPⅡ_b/Ⅲ_a complex and GP Ⅰ_b on activated platelets. So it might be an effective antiadhesive agent.