Thrombin-induced cell proliferation and platelet-derived growth factor-AB release from A172 human glioblastoma cells.

BACKGROUND: In a previous study, we found that thrombin induced proliferation of TM-1 and T98G human glioma cells and that the mitogenic effect was abolished by hirudin. OBJECTIVES: We investigated thrombin's effects on the proliferation of A172 human glioblastoma cells and the induction of growth factors. Furthermore, we examined whether or not the expression of heparin cofactor II (HCII) in A172 cells using adenovirus vector could suppress thrombin's effects. METHODS: The effect of thrombin on cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. The amount of growth factors in the conditioned medium was measured by enzyme-linked immunosorbent assay. The level of platelet-derived growth factor (PDGF)-B mRNA was assessed by reverse transcriptase-polymerase chain reaction analysis. RESULTS: Thrombin-induced proliferation of A172 cells primarily depended on the enhanced secretion of PDGF-AB by thrombin. The action of thrombin depended on its proteolytic activity. However, thrombin-induced PDGF-AB secretion was not abolished by anti-protease-activated receptor (PAR) antibody. The PAR-1 agonist peptide had no effect on cell growth and PDGF-AB levels. Thrombin did not increase PDGF-B gene expression. Expression of HCII effectively suppressed thrombin-induced PDGF-AB release. CONCLUSIONS: These results indicate that thrombin may play an important role in the proliferation of A172 cells by inducing PDGF-AB secretion and that thrombin's action is mediated by its proteolytic activity. Inhibition of thrombin's proteolytic activity may be a new therapeutic method for gliomas.

Adenovirus-mediated expression of heparin cofactor II inhibits thrombin-induced cellular responses in fibroblasts and vascular smooth muscle cells.

Heparin cofactor II functions as a physiological inhibitor of thrombin activity. The rate of inactivation of thrombin by heparin cofactor II is increased in the presence of dermatan sulfate, which is produced by fibroblasts or smooth muscle cells. To elucidate the role of heparin cofactor II in the extravascular cells, we induced expression of heparin cofactor II in cultured human fibroblasts or vascular smooth muscle cells using adenovirus-mediated gene transfer. After infection of adenovirus vector, these cells secreted heparin cofactor II protein into culture medium. The expressed heparin cofactor II formed the complex with exogenous thrombin and inhibited the proteolytic activity of thrombin. Expression of heparin cofactor II by infection of adenovirus vector inhibited thrombin-induced tissue-type plasminogen activator and interleukin-6 releases from fibroblasts and thrombin-induced interleukin-6 release from vascular smooth muscle cells. These findings show that fibroblasts and vascular smooth muscle cells expressing heparin cofactor II are resistant to thrombin-induced cellular responses.

Further studies on the mechanism for the antithrombotic effects of naroparcil, an orally active thiozyloside compound.

The antithrombotic beta-D-xyloside, naroparcil, has previously been shown to induce a dose-related increase of circulating glycosaminoglycans (GAGs) together with an antithrombin activity (anti-IIa) via heparin cofactor II (HCII) in the rabbit. In order to go further in the mechanisms, the relationship between the antithrombotic activity, the HCII-mediated anti-IIa activity and the plasma GAG content was investigated. We showed that the in vitro specific activity on the inhibition of thrombin by HCII of the plasma GAG extract from naroparcil-treated rabbits was increased by a factor of 60 when compared to controls. In addition, the fractionation of the plasma GAG extract by affinity chromatography on immobilized HCII led to a more potent material whereas the low-affinity fraction was shown to be inactive in thrombin inhibition by HCII. The qualitative analysis of GAGs showed the presence of the deltaDi-4S DS disaccharide, undetectable in control, which accounted for 22% in the unfractionated GAG extract and for 60% in the high affinity fraction. In vitro experiments using immuno-depleted plasma in antithrombin III (ATIII), HCII or both, indicated that the anti-IIa activity of the plasma GAG extract from naroparcil-treated rabbits was mainly due to HCII potentialisation. The unfractionated GAG extract and the high affinity fraction were shown to be antithrombotic in a Wessler-based model in the rat, giving ED80 values of 610 UA/kg and 56 UA/kg respectively whereas the low-affinity fraction was devoid of any antithrombotic activity. These results show that the antithrombotic activity of naroparcil is dependent on modification in the plasma GAG profile which inactivates thrombin via the HCII.

Heparin cofactor II as a novel vascular protective factor against atherosclerosis.

Heparin cofactor II (HCII) specifically inhibits thrombin action at the site of vascular wall injury. We encountered a congenital HCII deficiency patient with advanced multiple atherosclerotic lesions. This patient led us to conduct clinical studies to examine the role of HCII against atherosclerosis. We found that the incidence of in-stent restenosis after percutaneous coronary intervention, severity of carotid atherosclerosis and prevalence of peripheral arterial disease are inversely associated with plasma HCII activity. In order to clarify the vascular protective action of HCII, we generated HCII- deficient mice by gene targeting. In contrast to a previous study, HCII(-/-) mice were embryonically lethal. In HCII(+/-) mice, accelerated intimal hyperplasia and frequent thrombosis were observed after cuff or wire injury of femoral arteries. The number of protease-activated receptor-1 (PAR-1) -positive cells and the gene expression levels of inflammatory cytokines and chemokines were increased in the thickened vascular walls of HCII(+/-) mice. The accelerated intimal hyperplasia in HCII+/- mice with vascular injury was attenuated by human HCII administration. Furthermore, HCII deficiency exaggerated aortic plaque formation with increased oxidative stress in apolipoprotein E(-/-) mice. These results demonstrate that HCII protects against thrombin-induced vascular remodeling in both humans and mice and suggest that HCII is a predictive biomarker and therapeutic target for atherosclerosis.

Evaluation of the haemostatic potential of factor VIII-heparin cofactor II hybrid proteins in a mouse model.

We constructed factor VIII-heparin cofactor II (FVIII-HCII) hybrid molecules, which are more readily activated by thrombin in vitro than the respective wild-type molecules. The hybrid proteins were tested in a murine model of haemophilia A to investigate their haemostatic efficacy in vivo. Bleeding characteristics, measured using standard tail-tip cutting techniques, were total blood loss, bleeding time and survival rate. FVIII-HCII hybrids were found to be effective in preventing bleeding in FVIII knockout mice. While in vitro experiments showed that the chimaeric molecules had higher haemostatic functions than the wild-type proteins, the variables analysed in vivo were similar for both proteins.

Effect of covalent serpin-heparinoid complexes on plasma thrombin generation on fetal distal lung epithelium.

Extravascular coagulation within the lung airspace is a hallmark of respiratory distress syndrome (RDS) in premature infants. We previously showed that covalent antithrombin-heparin complex (ATH) is superior to noncovalent antithrombin (AT) + heparin (H) mixtures at inhibiting plasma thrombin generation on rat fetal distal lung epithelium (FDLE) in vitro. However, heparin cofactor II (HC) has been shown to selectively inhibit thrombin, which may be advantageous if other enzyme activities are present in the airspace. We compared the abilities of ATH, covalent HC-heparin complex (HCH), and covalent HC-dermatan sulfate (HCD) to inhibit thrombin generation on FDLE in plasmas from either adults or newborns. In the presence of ATH, peak free thrombin generation in adult plasma on the cell surface was reduced by 92% compared with controls (no anticoagulant). However, whereas HCH reduced peak free thrombin generation in adult plasma by 81%, HCD was only able to reduce activity by 33%. All covalent complexes caused a greater decrease in thrombin activity compared with that with the corresponding noncovalent serpin + heparinoid mixtures. Experiments in plasma from newborns resulted in peak free thrombin that was less than or equal to that in adult plasma when covalent conjugates were added. Relative peak free thrombin was proportional to rate of prothrombin consumption and amount of thrombin-inhibitor complexes formed. In vivo, experiments in newborn rats showed that a greater percentage of intratracheally instilled ATH and HCH could be recovered in lung lavage fluid compared withwith that for HCD. In summary, ATH, HCH, and HCD are inhibitors of thrombin generation on FDLE superior to the corresponding noncovalent mixtures, with ATH and HCH being more potent than HCD. Covalent conjugates of AT or HC with H may be preferred in treatment of extravascular coagulation.